Split (1)

train · 9.37k rows

id int64 7 5.24k	url stringlengths 48 63	content stringlengths 567 149k
4,532	https://bio-protocol.org/en/bpdetail?id=4532&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Staphylococcus aureus 30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis MB Margarita Belinite IK Iskander Khusainov SM Stefano Marzi Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4532 Views: 1322 Reviewed by: Alba BlesaKristin L. ShinglerNathan Rhys James Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Frontiers in Molecular Biosciences Nov 2021 Abstract The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K+ and Mg2+, polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of Staphylococcus aureus is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S–mRNA complex from S. aureus at 3.6 Å resolution. The 30S–mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract: Keywords: 30S ribosomal subunit Translation Staphylococcus aureus Toeprinting assay Cryo-EM Background Protein synthesis is performed by the ribosome, which is composed of two dynamic subunits. These are made by RNAs and proteins that interact with several ligands and adopt several conformations during the translation process (Frank et al., 2007; Agirrezabala et al., 2008; Zhang et al., 2009). Structural and functional studies have revealed that ions impact the stability and dynamics of the ribosome (Nierhaus, 2014; Akanuma, 2021). Optimal ions and polyamine concentrations are necessary for maintaining both structural integrity and intrinsic flexibility. The small subunit (30S), responsible for the translation initiation process and the mRNA decoding, is particularly structurally dynamic and, thus, more challenging for structural studies. This subunit is composed of two main structural domains, the head and the body, which undergo continuous rearrangement (swiveling and nodding, respectively), heavily influencing the position of the mRNA in the decoding channel (Fahnestock et al., 1977; Hussain et al., 2016; Belinite et al., 2018). For this reason, specific ionic conditions have been used to stabilize the position of the mRNA for structural studies. Ribosome structure and dynamics are especially affected by the presence of cations, notably Mg2+, Zn2+, K+, NH4+, and polyamines (Gordon and Lipmann 1967; Weiss et al., 1973; Stahli and Noll 1977; Kim et al., 2007; Wohlgemuth et al., 2010; Nierhaus, 2014; Rozov et al., 2019). For instance, recent studies have demonstrated that different magnesium concentrations drastically affect the conformation of the 30S, and high temperatures help induce some conformational changes in Escherichia coli (Jahagirdar et al., 2020). In addition, spermidine has been shown to stabilize the active conformation of Staphylococcus aureus 30S by interacting with rRNA (Belinite et al., 2021); indeed, the correct folding of the decoding center at the top of the helix 44 (h44) in the 30S body can be achieved by maintaining a certain concentration of spermidine along the 30S purification process (Belinite et al., 2021). Spermidine and spermine are polyamines, organic polycations present in all cell types (Cohen and Lichtenstein, 1960; Stevens et al., 1969; Weiss and Morris, 1970; Cohen, 1971; Hardy and Turnock, 1971; Turnock and Birch, 1973), including in S. aureus (Herbst et al., 1958; Hamana and Satake 1995; Rosenthal and Dubin, 1962). These have been shown to associate with the ribosome (Cohen and Lichtenstein, 1960) and contribute to both the efficiency and fidelity of protein synthesis (Echandi and Algranati, 1975; Igarashi et al., 1980; Igarashi and Kashiwagi, 2018; McMurry and Algranati, 2018). They are known to stimulate in vitro translation (Hershko et al., 1961; Martin and Ames, 1962; Takeda, 1969; Igarashi et al., 1973; Konecki et al., 1975; Hetrick et al., 2010), lessening the Mg2+ requirement (Rheinberger and Nierhaus, 1987; Bartetzko and Nierhaus, 1988). In this study, we describe in detail the protocols for the efficient 30S purification from S. aureus and the complex preparation with a natural S. aureus mRNA, spa mRNA, which encodes the virulence factor protein A. The proper conformation of the h44, full accommodation of the mRNA in the channel, and reduced flexibility of certain parts of the 30S were achieved by maintaining spermidine at a proper concentration throughout the whole process, starting from the 30S subunit isolation. The 30S subunit was obtained by separating the 70S ribosome under dissociative conditions and analyzed for high-molecular-weight contaminants and rRNA integrity. Activity of the isolated 30S was tested using a toeprinting assay (Fechter et al., 2009), while its structure was obtained by cryo-electron microscopy (cryo-EM). This technique has also been used to test different buffer conditions, thanks to the development of methods to rapidly get multiple structures (Cressey and Callaway, 2017; Nakane et al., 2020). The final S. aureus 30S–mRNA structure was obtained with focused refinement of the head and body at 3.6 and 3.4 Å resolution, respectively. Our previous work demonstrated the requirement of spermidine for the rRNA stabilization and its use in the cryo-EM studies (Belinite et al., 2021). Here, we show that the 30S subunits are stable in these conditions, confirming that spermidine not only contributes to their structural integrity, but also assures their required flexibility. Hence, protocols described in this article can be used for the future investigations of S. aureus functional complexes. Materials and Reagents Cells Staphylococcus aureus RN6390 strain (Khusainov et al., 2016) 70S ribosome purification Polycarbonate 50 mL centrifuge tubes (NalgeneTM, catalog number: 3117-0500) Open-top thinwall ultra-clear tube, 38.5 mL (Beckman Coulter, catalog number: 344058) Blood agar plates (BDTM Columbia agar with 5% sheep blood, catalog number: PA-254005.06) Brain–heart infusion broth (BHI, Sigma-Aldrich, catalog number: 53286) Lysostaphin (Sigma-Aldrich, catalog number: L7386) DNase I (Roche, catalog number: 4716728001) Protease inhibitor cocktail without ethylenediaminetetraacetic acid (EDTA, Thermo ScientificTM, catalog number: 78429) 30% PEG 20000 (Hampton Research, HR2-609) Buffer A (see Recipes) Buffer B (see Recipes) 5× buffer E (see Recipes) Dissociation buffer (see Recipes) 30S subunit purification Open-top thinwall ultra-clear tube, 13.2 mL (Beckman Coulter, catalog number: 344059) Amicon ultra-15 centrifugal filter unit (Merck Millipore, catalog number: UFC910008) Liquid nitrogen 30S storage buffer (see Recipes) mRNA purification RNaseZap (Thermo ScientificTM, catalog number: AM9780) NucleoSpin Plasmid, mini kit for plasmid DNA (Macherey-Nagel, catalog number: 740588.50) NucleoSpin Gel and PCR Clean-up, mini kit for gel extraction and PCR clean-up (Macherey-Nagel, catalog number: 740609.50) BamHI enzyme (NEB, catalog number: R0136S) CutSmart® buffer (NEB, catalog number: B7204S) MEGAscript kit with DNase turbo (Ambion, catalog number: AM1333) ROTI® phenol/chloroform/isoamyl alcohol, 250 mL, pH 7.5–8.0 (Carl Roth, catalog number: A156.1) ROTI® Aqua-P/C/I, 250 mL, pH 4.5–5.0 (Carl Roth, catalog number: X985.1) 3 M sodium acetate Buffer ELU (see Recipes) 30S and mRNA quality analysis PageRulerTM protein ladder (Thermo ScientificTM, catalog numbers: 26616 and 26619) InstantBlue® coomassie protein stain (Abcam, catalog number: ab119211) ROTI® aqua-phenol, 250 mL, pH 4.5–5.0 (Carl Roth, catalog number: A980.1) Chloroform (Thermo ScientificTM, catalog number: AC158210010) Agarose (SeaKemTM, catalog number: 11590717) 1 M potassium thiocyanate (Sigma, catalog number: P3011) Ethidium bromide (Thermo ScientificTM, catalog number: 15585011) 100% formamide (Sigma, catalog number: F9037) RiboRuler low-range RNA ladder (Thermo ScientificTM, catalog number: SM1831) 4% stacking gel (see Recipes) 15% resolving gel (see Recipes) 4× blue buffer (see Recipes) 10× running buffer (see Recipes) 6% polyacrylamide gel (see Recipes) 10× TBE (see Recipes) Toeprinting assay 10 μM Toe primer (5′ CCTGCAGGTCGACTC 3′) 10× PNK buffer (Thermo ScientificTM, catalog number: EK 0032) 10 U/μL T4 PNK (Thermo ScientificTM, catalog number: EK 0032) [γ-32P]-ATP (370 MBq/mL, 10 mCi/mL, 185 MBq) Micro bio-spin chromatography columns (Bio-Rad, catalog number: 732-6200) 20 U/μL AMV reverse transcriptase, isolated from Avian Myeloblastosis Virus (QBIOgene, catalog number: EMAMV200) 10× AMV buffer (QBIOgene, catalog number: EMAMV200) 10 mM dATP, dCTP, dGTP, and dTTP (Roche, catalog numbers: 11051440001, 11051458001, 11051466001, and 11051482001) 0.3 M Na acetate (Sigma, catalog number: S2889) Phenol/chloroform, pH 4.5–5.0 (Carl Roth, catalog number: X985.1) Chloroform/isoamyl alcohol (Carl Roth, catalog number: X984.1) 100% cold ethanol 80% cold ethanol 8 M urea (Sigma, catalog number: U5128) 0.1% bromophenol blue (Sigma, catalog number: B0126) 0.1% xylene cyanol (Sigma, catalog number: X4126) 100 µM ddATP, ddGTP, ddCTP, and ddTTP (Roche, catalog number: 3732738001) 3 M KOH (Sigma, catalog number: 221473) 1 M tris-HCl, pH 8.0 (Sigma, catalog number: T1503) 10% SDS (Bio-Rad, catalog number: 1610416) Acetic acid (Sigma, catalog number: A6283) 10× toeprinting buffer (see Recipes) 30S–mRNA complex preparation for cryo-EM Liquid ethane Liquid nitrogen Quantifoil R2/2300-mesh holey carbon grids Note: All buffers are prepared the day before and stored at 4 °C. Equipment 70S ribosome purification 2 L flasks Microbiological incubator (Thermo ScientificTM, catalog number: 50125882 or equivalent) Cell culture shaker at 30 °C Class II biosafety cabinet Floor centrifuges (Beckman, Avant J20 XP and/or Avanti J-E or equivalent) JA-25.50 rotor (Beckman, catalog number: 363055) JLA-16.250 rotor (Beckman, catalog number: 363930) Ultracentrifuge (Beckman, OPTIMA XE90 or equivalent) Type 45 Ti rotor (Beckman, catalog number: 339160) SW28 rotor (Beckman, catalog number: 342204) Gradient MasterTM base unit (Biocomp, catalog number: B108-2) Gradient MasterTM device (BioComp Instruments, Fredericton, Canada) Fraction collector (Bio-Rad, model: 2110) NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA or equivalent) 30S subunit purification Ultracentrifuge (Beckman, OPTIMA XE90 or equivalent) SW41 Ti swinging-bucket rotor (Beckman, catalog number: 331362) Floor centrifuges (Beckman, Avanti J-E or equivalent) JLA-16.250 rotor (Beckman, catalog number: 363930) mRNA purification Cell culture shaker at 37 °C Benchtop centrifuge (Eppendorf MiniSpin® Plus personal microcentrifuge with 12-place rotor or equivalent) Eppendorf thermomixer R, dry block heating and cooling shaker or equivalent 30S and mRNA quality analysis Mini-PROTEAN® Tetra Handcast systems (Bio-Rad or equivalent) PowerPacTM basic power supply (Bio-Rad or equivalent) Wide mini-sub cell GT cell (Bio-Rad or equivalent) GelDoc Go Gel imaging system (Bio-Rad) Toeprinting assay Benchtop centrifuge (Eppendorf MiniSpin® Plus personal microcentrifuge with 12-place rotor or equivalent) Phosphor imaging or autoradiography (Typhoon or equivalent) 30S–mRNA complex preparation cryo-EM data acquisition Thermo Scientific Vitrobot Mark IV Talos Arctica equipped with Falcon III direct electron detector (Thermo Fisher) Microscope control PC with installed EPU software Software MotionCor2 (https://emcore.ucsf.edu/ucsf-software/) Gctf (https://www2.mrc-lmb.cam.ac.uk/research/locally-developed-software/zhang-software/) Relion 3.0 (https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page/) ResMap (http://resmap.sourceforge.net/) Chimera (https://www.cgl.ucsf.edu/chimera/) Procedure 70S ribosome purification Streak Staphylococcus aureus’ RN6390 cells on a blood agar plate and grow at 37 °C for 20 h. Inoculate a single colony in 50 mL of sterile BHI medium in a 250 mL flask and grow for 17 h at 37 °C and 180 rpm. Add 5 mL of overnight culture to four 2 L flasks, each filled with 500 mL of sterile BHI medium, and grow for 3 h at 37 °C and 180 rpm until OD600 = 1. Harvest cells by centrifugation at 4,400 × g for 15 min at 4 °C. Resuspend the pellet in 50 mL of buffer A. Collect the pellet by centrifugation at 4,400 × g for 15 min. Repeat the previous steps (5–6) two times. Weigh cells and keep at -80 °C. Note: The typical yield is 2.8 g of cells from 2 L of culture. Thaw the cells (2.8 g) on ice for 15–20 min and resuspend in 6.25 mL of buffer A supplemented with 0.7 mg lysostaphin, 1× protease inhibitor cocktail, 1 mM EDTA pH 7.5, and 6 µl DNase I per 1 gram of cell weight. Incubate for 1 h at 37 °C. Spin down the lysate at 21,500 × g (JA-25.50 rotor) for 90 min at 4 °C. Adjust the PEG20K concentration in the collected supernatant to 2.8% and leave the reaction on ice under agitation for 5 min. Collect the supernatant by centrifugation at 20,000 × g for 5 min (JA-25.50 rotor). Increase the PEG20K concentration to 4.2% and leave the reaction on ice under agitation for an additional 10 min. Collect the supernatant by centrifugation at 20,000 × g for 10 min (JA-25.50 rotor). Resuspend each pellet in 17.5 mL buffer A with 1 mM EDTA, lay on a 25 mL sucrose cushion (buffer B), and spin at 235,000 × g for 17 h at 4 °C (Type 45 Ti rotor). Rinse the walls of each tube with 5 mL of buffer E to remove excess sucrose and resuspend the pellet in 1 mL of buffer E. Lay the obtained sample on twelve 7%–30% sucrose gradients and spin at 83,000 × g for 16.5 h (SW28 rotor). Fractionate sucrose gradients (Figure 1A-I), pool fraction of 70S, and adjust the magnesium acetate to 25 mM and PEG20K to 4.5%. Leave on ice under agitation for 10 min. Pellet down the ribosomes by centrifugation at 20,000 × g for 13 min (JA-25.50 rotor) (Khusainov et al., 2016). Wash the tube walls with the dissociation buffer to remove the excess of sucrose and PEG. Resuspend the pellet in 1.5 mL of dissociation buffer. Note: The usual yield is 14 mg of 70S with at least 95% purity. Check the purity of 70S by 15% polyacrylamide gel (Figure 1A-II) and by mass-spectrometry analysis (Figure 1A-III). 30S subunit purification Load 14 mg of 70S in the dissociation buffer directly on 0%–30% sucrose gradients equilibrated in the same buffer and spin at 109,000 × g for 14.5 h (SW28 rotor). Fractionate the sucrose gradients (Figure 1B-I) and adjust the magnesium acetate of pooled fractions to 10 mM and spermidine to 2.5 mM. Concentrate the 30S sample to 2–3 mg/mL in the Amicon ultra centrifugal filter. Note: By using two filters in parallel, it takes 1 h to concentrate the sample. Make aliquots, flash freeze them in liquid nitrogen, and store at -80 °C. Note: The volume of the aliquots should not be less than 3 μL to prevent evaporation of the sample. Moreover, the size of the aliquots should be small (3–5 μL), as a large amount of sample is not required for subsequent experiments. Figure 1. The 30S ribosomal subunits purification. The 30S (B) were obtained from 70S (A) by running through sucrose gradients twice (I and IV, 35 mL is the total volume of sucrose gradients). The bold blue line represents the fraction used for further experiments. Both 70S and 30S fractions were run on SDS-PAGE to check the purity of the samples (II and V). 70S ribosome was sent to mass spectrometry analysis to assess the sample purity (III). 16S was obtained from 30S by phenol/chloroform extraction and run on agarose gel in denaturing conditions to represent its integrity (VI). spa mRNA purification The plasmid used in this study was previously described in Benito et al. (2000) and Huntzinger et al. (2005). The pUT7 plasmid contains a T7 promoter for T7 in vitro transcription and ampicillin resistance. spa mRNA sequence with a strong Shine–Dalgarno AGGGGG (underlined) and UUG start codon (bold) is shown below: ACAAAUACAUACAGGGGGUAUUAAUUUGAAAAAGAAAAACAUUUAUUCAAUUCGUAAACUAGGUGUAGGUAUUGCAUCUGUAACUUUAGGUACAUUACUUAUAUCUGGUGGCGUAACACCUGCUGCAAAUGCUGCGCAACACGAUGAAGCUCAACAAAAUGCUUUUUAUCAAGUCUUAAAUAUGCCUAACUUAAACGCUGGGAUCCUCUAGAGUCGACCUGCAGG Note: Before starting to work with the mRNA, wipe the bench and pipettes with a liquid that removes RNase contamination (e.g., RNaseZap). Transform XL1 Escherichia coli cells with the pUT7-spa plasmid. Grow cells in 250 mL of LB medium with 100 μg/mL ampicillin at 37 °C for 16 h. Purify the plasmid using a NucleoSpin Plasmid mini kit and linearize it (up to 50 μg of plasmid per reaction) with BamHI enzyme in a 1× CutSmart buffer for 2 h at 37 °C. Note: The usual yield is 10 μg of plasmid per one reaction. One linearization reaction is enough to proceed with the experiments. Extract the linearized plasmid DNA from 1% agarose gel using a NucleoSpin Gel and PCR Clean-up mini kit. Extract the DNA with phenol/chloroform: Add an equal volume of phenol, vortex well, and spin down at 17,000 × g for 1 min. Keep the upper phase. Add an equal volume of chloroform, vortex well, and spin down at 17,000 × g for 1 min. Keep the upper phase. Precipitate the DNA with 2.5 volumes of 100% cold ethanol and 1/16 volume of 3 M sodium acetate. Wash the pellet with 500 μL of 80% cold ethanol. In vitro transcribe the mRNA for 3 h at 37 °C using a MEGAscript kit. Stop the reaction with 2 U/μL DNase turbo and incubate for 15 min at 37 °C. Extract the mRNA with phenol/chloroform and precipitate it with 100% cold ethanol (similar to steps 5 and 6). Wash the pellet with 500 μL of 80% cold ethanol. Add 50% formamide to the sample and heat for 3 min at 90 °C. Separate the mRNA by 6% polyacrylamide midi-size gel and elute with the buffer ELU for 16 h at 4 °C. Extract the mRNA with phenol/chloroform and precipitate it with 100% cold ethanol (similar to steps 5 and 6). Wash the pellet with 200 μL of 80% cold ethanol. Resuspend in 10–30 μL mQ water. Store the aliquots at -20 °C. 30S and mRNA quality analysis The absence of high-molecular-weight non-ribosomal proteins and the presence of all ribosomal proteins are detected on a 15% polyacrylamide gel (Figure 1B-V). The positions of all 70S ribosomal proteins on the SDS-PAGE are identified in Khusainov et al. (2016) (Figure 1A-II). The ribosomal RNA integrity is checked by 0.8% agarose gel in denaturing conditions (Figure 1B-VI). Protein polyacrylamide gel Mix all buffer components for the 15% resolving gel and leave to polymerase for 15–20 min. Repeat the same procedure for 4% stacking gel. Add 1× blue buffer to the 30S samples (0.15 OD per lane) and heat for 3 min at 95 °C. Load 5 μL of protein ladder and 5 μL of 30S samples in the blue buffer. Run the gel in a 1× running buffer at 120 V for 30 min and then at 150 V for 1 h. Stain the gel in InstantBlue® coomassie for 20 min and destain in water for 1 h. Extracting the ribosomal RNA Add an equal volume of phenol to the 0.15 OD 30S, mix well by vortexing, and spin down at 17,000 × g for 1 min. Add an equal volume of chloroform to the upper phase, mix well by vortexing, and spin down at 17,000 × g for 1 min. Repeat the two last steps. Precipitate the ribosomal RNA with 1/10 volume 3 M sodium acetate pH 5.3 and 2.5 volumes of 100% cold ethanol. Keep the sample overnight at -20 °C or for 1 h at -80 °C and spin down at 17,000 × g for 15 min at 4 °C. Wash the pellet with 70% cold ethanol and dry it for 5 min in a speed vac. Resuspend the obtained sample in 10 μL of mQ water. RNA agarose gel Melt 0.8% agarose in 1× TBE buffer in the microwave and then cool down to 60 °C. Add 100 mM potassium thiocyanate and ethidium bromide to the mixture. Leave the gel for 40 min to polymerase. Load 0.015 OD of the sample on the gel together with a high-range RNA ladder. Run the gel in a 1× TBE buffer at 120 V for 45 min. RNA polyacrylamide gel The mRNA purity and integrity are checked by 6% polyacrylamide gel stained in ethidium bromide (Figure 2C). The sequence of spa mRNA is shown in Figure 2D. Mix all components for 6% polyacrylamide gel and leave to polymerize for 15–20 min. Load 4 μL of low-range RNA ladder in 1× RNA loading dye close to the 100 ng of mRNA in 50% formamide preheated at 90 °C for 2 min. Run the gel at 200 V for 30 min. Stain the gel in ethidium bromide for 10 min. Toeprinting assay Mix 1 μM Toe primer in PNK buffer, 10 U/μL T4 PNK, and 5 μL of [γ-32P]-ATP, incubate at 37 °C for 1 h, and then pass through the micro bio-spin column pre-equilibrated in mQ water. Incubate 0.5 μM of spa mRNA with 169,916 cpm/μL Toe primer in a toeprinting buffer at 90 °C for 1 min, put on ice for 1 min, and then add MgCl2 to a 10 mM final concentration. Leave for refolding/annealing at 20 °C for 15 min. The final volume of the reaction mixture is 20 μL. Mix 33,983 cpm/μL of mRNA-Toe primer with 0.025 μM, 0.05 μM, and 0.2 μM 30S small ribosomal subunits. Incubate at 37 °C for 10 min and with 0.1 μM itRNA for an additional 5 min (final MgCl2 concentration should be 8 mM). The final volume of the reaction mixture is 13 μL. Incubate obtained samples with 0.3 U/μL of AMV reverse transcriptase and 0.3 mM dNTPs in AMV buffer for 30 min at 37 °C, extract with phenol/chloroform, and resuspend in urea blue buffer. For sequencing lanes, incubate 0.33 μM mRNA with 135,932 cpm/μL Toe primer, 1.7 μM nucleotides, and 0.27 U/μL AMV reverse transcriptase in AMV buffer at 37 °C for 30 min. Add 237 mM KOH, 26 mM tris-HCl pH 8, 0.26 % SDS, and 3.9 mM EDTA to the samples for the sequencing lanes and incubate at 90 °C for 3 min and at 37 °C for 1 h. Precipitate RNA in 0.3 M sodium acetate and 100% cold ethanol and resuspend in urea blue buffer. Migrate both samples on a 10%, 40 cm, 0.5 mm denaturing polyacrylamide gel. Obtain the data by autoradiography (Figure 2A and 2B). Figure 2. Toeprinting assay of the 30S–mRNA–itRNA complex. (A) Scheme of the toeprinting assay. (B) Toeprint analysis of the 30S–mRNA–itRNA complex formation with increasing 30S concentration. (C) The intactness of mRNA was checked by polyacrylamide gel in denaturing conditions. (D) Sequence of spa mRNA with the start codon in bold and Shine–Dalgarno sequence underlined; the +16 nucleotide is shown by the black arrow. 30S–mRNA complex preparation for cryo-EM Combine 450 nM of spa mRNA with 90 nM of 30S in a storage buffer, apply immediately on the Quantifoil R2/2300-mesh holey carbon cryo-EM grids, and plunge-freeze in liquid ethane using a Vitrobot Mark IV device (4 °C and 100% humidity) under the following conditions: 4 μL of complex on the grid, 3 s blotting time, blot force of 5, and waiting time of 30 s. Clip the grids into the cartridges if you intend to use an Autoloader equipped microscope. Keep in liquid nitrogen until data acquisition. Data acquisition and analysis Collect the data on a 200 kV Talos Arctica instrument with a Falcon III direct electron detector under the focus range of -0.5 to -2.7 μm with a 0.2 step. Note: For our purposes, the 120,000× magnification was used, yielding a pixel size of 1.24 Å. Transfer the obtained data to the workstation or cluster for data processing. Use the RELION 3.0 software for data processing (Zivanov et al., 2018). Perform drift and gain correction and dose weighting with MotionCor2 (Zheng et al., 2017). Use a dose-weighted average image of the whole stack to determine the contrast transfer function with the software Gctf (Zhang et al., 2016). Pick the particles using a Laplacian of Gaussian function (min diameter 180 Å, max diameter 290 Å). Extract the particles with a box size of 270 pixels and divide into several classes for 3D classification. Merge classes (if necessary), extract the original size, and continue with 3D refinement. The obtained 30S–mRNA structure has been refined to an overall 3.6 Å resolution with individual focused refinement of the head and the body leading to the resolution of 3.6 and 3.4 Å, respectively (Figure 3B and 3C). Estimate the local resolution in RELION 3.0 (Zivanov et al., 2018). Analyze structures in Chimera (Pettersen et al., 2004) or ChimeraX (Goddard et al., 2018) (Figure 3A). Figure 3. Scheme of the cryo-EM data processing. Electron movies were obtained from sample collection on the Talos microscope. After motion correction and contrast transfer function (CTF) estimation, the particles were picked and extracted with a minimum diameter of 180 Å and maximum diameter of 290 Å. The next steps were 2D and 3D classification, followed by 3D refinement and post-processing. The final 30S–mRNA structure was obtained at 3.6 Å with focus refinement on the head and body at 3.4 and 3.6 Å, respectively. Recipes 70S ribosome purification Buffer A 100 mM NH4Cl 21 mM Mg acetate 20 mM HEPES-KOH, pH 7.5 1 mM dithiothreitol (DTT) Buffer B 1.05 M sucrose 0.5 M KCl 10.5 mM Mg acetate 0.5 mM EDTA 20 mM HEPES-KOH, pH 7.5 1 mM DTT 5× buffer E 500 mM KCl 50 mM Mg acetate 2.5 mM EDTA 50 mM HEPES-KOH, pH 7.5 5 mM DTT Dissociation buffer 1 mM Mg acetate 30 mM NH4Cl 10 mM HEPES-KOH, pH 7.5 1 mM DTT 30S subunit purification 30S storage buffer 10 mM Mg acetate 30 mM NH4Cl 10 mM HEPES-KOH, pH 7.5 1 mM DTT 2.5 mM spermidine mRNA purification Buffer ELU 16% (v/v) phenol pH 4.5–5.0 50 mM ammonium acetate 1 mM EDTA, pH 8.0 30S and mRNA quality analysis 4% stacking gel 4% (v/v) ROTIPHORESE® NF-acrylamide/bis-solution 30 (37.5:1) 125 mM tris-HCl, pH 6.8 0.1% (w/v) sodium dodecyl sulfate 0.1% (v/v) ammonium persulfate 1/500 V TEMED 15% resolving gel 15% (v/v) ROTIPHORESE® NF-acrylamide/bis-solution 30 (37.5:1) 375 mM tris-HCl, pH 8.8 0.1% (w/v) sodium dodecyl sulfate 0.1% (v/v) ammonium persulfate 1/1000 V TEMED 4× blue buffer 0.25 M tris-HCl, pH 6.8 9.2% (w/v) sodium dodecyl sulfate 40% (v/v) glycerol 0.1% (w/v) bromophenol blue 100 mM DTT 10× running buffer 30 g tris base 144 g glycine 10 g sodium dodecyl sulfate in 1,000 mL of mQ water, pH 8.3 6% polyacrylamide gel 6% (v/v) ROTIPHORESE® NF-acrylamide/bis-solution 40 (19:1) 1× TBE 8 M urea 10× TBE 108 g tris base 55 g boric acid 40 mL 0.5 M EDTA, pH 8.0 in 1,000 mL of mQ water, pH 8.3 1× TBE is a 1:10 dilution of the 10× TBE (from step 6) Toeprinting assay 10× toeprinting buffer 100 mM tris-HCl, pH 7.5 300 mM KCl 5 mM DTT 10% polyacrylamide gel 10% (v/v) acrylamide/bis-solution 40 (19:1) 1× TBE 8 M urea Urea blue buffer 0.025% bromophenol blue xylene cyanol in 7.6 M urea Acknowledgments This work, of the Interdisciplinary Thematic Institute IMCBio+, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS, and Inserm, was supported by the CNRS, by the Agence Nationale de la Recherche (ANR, grant ANR-21-CE12-0030-01 (SM), IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT’US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. We thank Dr. Marat Yusupov, Dr. Yaser Hashem, Dr. Pascale Romby, and Dr. Angelita Simonetti for useful discussions and critical advice, and Dr. Yaser Hashem and Dr. Armel Bézault for help in Data acquisition on the Talos Arctica microscope. This protocol was adapted from Belinite et al. (2021). Competing interests The authors declare no competing interests. References Agirrezabala, X., Lei, J., Brunelle, J. L., Ortiz-Meoz, R. F., Green, R. and Frank, J. (2008). Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome. Mol Cell 32(2): 190-197. Akanuma, G. (2021). Diverse relationships between metal ions and the ribosome. Biosci Biotechnol Biochem 85(7): 1582-1593. Bartetzko, A. and Nierhaus, K. H. (1988). Mg2+/NH4+/polyamine system for polyuridine-dependent polyphenylalanine synthesis with near in vivo characteristics. Methods Enzymol 164: 650-658. Belinite, M., Simonetti, A., Marzi, S., Khusainov, I., Romby, P., Yusupov, M. and Hashem, Y. (2018). Initiation translation complex of Staphylococcus aureus ribosome. FEBS OpenBio 8: 433. Belinite, M., Khusainov, I., Soufari, H., Marzi, S., Romby, P., Yusupov, M. and Hashem, Y. (2021). Stabilization of Ribosomal RNA of the Small Subunit by Spermidine in Staphylococcus aureus. Front Mol Biosci 8. Benito, Y., Kolb, F. A., Romby, P., Lina, G., Etienne, J. and Vandenesch, F. (2000). Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of protein A expression. RNA 6(5): 668-679. Cohen, S. S. (1971). Introduction to the Polyamines. Englewood cliffs, New Jersey: Prentice-Hall. Cohen, S. S. and Lichtenstein, J. (1960). Polyamines and ribosome structure. J Biol Chem 235: 2112-2116. Cressey, D. and Callaway, E. (2017). Cryo-electron microscopy wins chemistry Nobel. Nature 550(7675): 167. Echandi, G. and Algranati, I. D. (1975). Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli. Biochem Biophys Res Commun 67(3): 1185-1191. Fahnestock, S. R. (1977). Reconstitution of active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis. Arch Biochem Biophys 182(2): 497-505. Fechter, P., Chevalier, C., Yusupova, G., Yusupov, M., Romby, P. and Marzi, S. (2009). Ribosomal initiation complexes probed by toeprinting and effect of trans-acting translational regulators in bacteria. Methods Mol Biol 540: 247-263. Frank, J., Gao, H., Sengupta, J., Gao, N. and Taylor, D. J. (2007). The process of mRNA-tRNA translocation. Proc Natl Acad Sci U S A 104(50): 19671-19678. Goddard, T. D., Huang, C. C., Meng, E. C., Pettersen, E. F., Couch, G. S., Morris, J. H. and Ferrin, T. E. (2018). UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci 27(1): 14-25. Gordon, J. and Lipmann, F. (1967). Role of divalent ions in poly U-directed phenylalanine polymerization. Journal of Molecular Biology 23(1): 23-33. Hamana, K. and Satake, S. (1995). ABSENCE OF CELLULAR POLYAMINES IN GRAM-POSITIVE ANAEROBIC COCCI AND LACTIC ACID BACTERIA. J Gen Appl Microbiol 41(2): 159-163. Hardy, S. J. and Turnock, G. (1971). Stabilization of 70S ribosomes by spermidine. Nat New Biol 229(1): 17-19. Herbst, E. J., Weaver, R. H. and Keister, D. L. (1958). The gram reaction and cell composition: Diamines and polyamines. Arch Biochem Biophys 75(1): 171-177. Hershko, A., Amoz, S. and Mager, J. (1961). Effect of polyamines and divalent metals on in vitro incorporation of amino acids into ribonucleoprotein particles. Biochem Biophys Res Commun 5: 46-51. Hetrick, B., Khade, P. K., Lee, K., Stephen, J., Thomas, A. and Joseph, S. (2010). Polyamines accelerate codon recognition by transfer RNAs on the ribosome. Biochemistry 49(33): 7179-7189. Huntzinger, E., Boisset, S., Saveanu, C., Benito, Y., Geissmann, T., Namane, A., Lina, G., Etienne, J., Ehresmann, B., Ehresmann, C., et al. (2005). Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. The EMBO Journal 24(4): 824-835. Hussain, T., Llácer, J. L., Wimberly, B. T., Kieft, J. S. and Ramakrishnan, V. (2016). Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation. Cell 167(1): 133-144.e113. Igarashi, K., Hikami, K., Sugawara, K. and Hirose, S. (1973). Effect of polyamines on polypeptide synthesis in rat liver cell-free system. Biochim Biophys Acta 299(2): 325-330. Igarashi, K. and Kashiwagi, K. (2018). Effects of polyamines on protein synthesis and growth of Escherichia coli. J Biol Chem 293(48): 18702-18709. Igarashi, K., Kishida, K. and Hirose, S. (1980). Stimulation by polyamines of enzymatic methylation of two adjacent adenines near the 3' and end of 16S ribosomal RNA of Escherichia coli. Biochem Biophys Res Commun 96(2): 678-684. Jahagirdar, D., Jha, V., Basu, K., Gomez-Blanco, J., Vargas, J. and Ortega, J. (2020). Alternative conformations and motions adopted by 30S ribosomal subunits visualized by cryo-electron microscopy. RNA 26(12): 2017-2030. Khusainov, I., Vicens, Q., Bochler, A., Grosse, F., Myasnikov, A., Ménétret, J. F., Chicher, J., Marzi, S., Romby, P., Yusupova, G., et al. (2016). Structure of the 70S ribosome from human pathogen Staphylococcus aureus. Nucleic Acids Res 44(21): 10491-10504. Kim, H. D., Puglisi, J. D. and Chu, S. (2007). Fluctuations of Transfer RNAs between Classical and Hybrid States. Biophys J 93(10): 3575-3582. Konecki, D., Kramer, G., Pinphanichakarn, P. and Hardesty, B. (1975). Polyamines are necessary for maximum in vitro synthesis of globin peptides and play a role in chain initiation. Arch Biochem Biophys 169(1): 192-198. Martin, R. G. and Ames, B. N. (1962). THE EFFECT OF POLYAMINES AND OF POLY U SIZE ON PHENYLALANINE INCORPORATION. Proc Natl Acad Sci U S A 48(12): 2171-2178. McMurry, L. M. and Aglranati, I. D. (1986). Effect of polyamines on translation fidelity in vivo. Eur J Biochem 155(2): 383-390. Nakane, T., Kotecha, A., Sente, A., McMullan, G., Masiulis, S., Brown, P. M. G. E., Grigoras, I. T., Malinauskaite, L., Malinauskas, T., Miehling, J., et al. (2020). Single-particle cryo-EM at atomic resolution. Nature 587(7832): 152-156. Nierhaus, K. H. (2014). Mg2+, K+, and the ribosome. J Bacteriol 196(22): 3817-3819. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and Ferrin, T. E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25(13): 1605-1612. Rheinberger, H.-J. and Nierhaus, K. H. (1987). The Ribosomal E Site at Low Mg2+: Coordinate Inactivation of Ribosomal Functions at Mg2+ Concentrations Below 10 mM and its Prevention by Polyamines. J Biomol Struct Dyn 5(2): 435-446. Rosenthal, S. M. and Dubin, D. T. (1962). Metabolism of polyamines by Staphylococcus. J Bacteriol 84(4): 859-863. Rozov, A., Khusainov, I., El Omari, K., Duman, R., Mykhaylyk, V., Yusupov, M., Westhof, E., Wagner, A. and Yusupova, G. (2019). Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction. Nat Commun 10(1): 2519. Stahli, C. and Noll, H. (1977). Structural dynamics of bacterial ribosomes. Molecular and General Genetics MGG 153(2): 159-168. Stevens, L. (1969). The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus. Biochem J 113(1): 117-121. Takeda, Y. (1969). Polyamines and protein synthesis. I. The effect of polyamines on cell free polyphenylalanine synthesis in Escherichia coli. J Biochem 66(3): 345-349. Turnock, G. and Birch, B. (1973). Binding of putrescine and spermidine to ribosomes from Escherichia coli. Eur J Biochem 33(3): 467-474. Weiss, R. L., Kimes, B. W. and Morris, D. R. (1973). Cations and ribosome structure. 3. Effects on the 30S and 50S subunits of replacing bound Mg2+ by inorganic cations. Biochemistry 12(3): 450-456. Weiss, R. L. and Morris, D. R. (1970). The inality of polyamines to maintain ribosome structure and function. Biochim Biophys Acta 204(2): 502-511. Wohlgemuth, I., Pohl, C. and Rodnina, M. V. (2010). Optimization of speed and accuracy of decoding in translation. Embo j 29(21): 3701-3709. Zhang, K. (2016). Gctf: Real-time CTF determination and correction. J Struct Biol 193(1): 1-12. Zhang, W., Dunkle, J. A. and Cate, J. H. (2009). Structures of the ribosome in intermediate states of ratcheting. Science 325(5943): 1014-1017. Zheng, S. Q., Palovcak, E., Armache, J.-P., Verba, K. A., Cheng, Y. and Agard, D. A. (2017). MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nature Methods 14(4): 331-332. Zivanov, J., Nakane, T., Forsberg, B. O., Kimanius, D., Hagen, W. J., Lindahl, E. and Scheres, S. H. (2018). New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7: e42166. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biochemistry > Protein > Isolation and purification Biophysics > Microscopy > Cryogenic microscopy Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase Aisha Mahboob [...] Afzal Husain Apr 5, 2024 912 Views Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages Juan C. Sanchez [...] Elizabeth R. Wright Jul 20, 2024 1021 Views Optimizing Transmembrane Protein Assemblies in Nanodiscs for Structural Studies: A Comprehensive Manual Fernando Vilela [...] Dorit Hanein Nov 5, 2024 562 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,533	https://bio-protocol.org/en/bpdetail?id=4533&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Analysis of the Ubiquitination and Phosphorylation of Vangl Proteins DF Di Feng * ZH Ziwei He * BG Bo Gao (*contributed equally to this work) Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4533 Views: 1980 Reviewed by: Gal Haimovich Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Version history Bio-protocol journal peer-reviewed Oct 20, 2022 \| This version Preprint Mar 22, 2022 Original Research Article: The authors used this protocol in Science Advances May 2021 Abstract The core planar cell polarity (PCP) protein Vang/Vangl, including Vangl1 and Vangl2 in vertebrates, is indispensable during development. Our previous studies showed that the activity of Vangl is tightly controlled by two important posttranslational modifications, ubiquitination and phosphorylation. Vangl is ubiquitinated through an endoplasmic reticulum-associated degradation (ERAD) pathway and is phosphorylated by casein kinase 1 (CK1) in response to Wnt. Here, we present step-by-step procedures to analyze Vangl ubiquitination and phosphorylation, including cell culture, transfection, sample preparation, and signal detection, as well as the use of newly available phospho-specific antibodies to detect Wnt-induced Vangl2 phosphorylation. The protocol described here can be applicable to the analysis of posttranslational modifications of other membrane proteins. Keywords: Planar cell polarity (PCP) Wnt Wnt/PCP Vangl1 Vangl2 Ubiquitination Phosphorylation Background As a conserved cellular mechanism from invertebrates to vertebrates, planar cell polarity (PCP) refers to the asymmetric pattern of a group of cells within a tissue plane, and it controls the polarized cell behaviors and tissue morphogenesis during embryonic development (Yang and Mlodzik, 2015). PCP function is carried out by a set of core PCP proteins, including Fzd-Dvl/Dgo, Vangl-Pk, and Fmi complexes, which have mutually exclusive localizations within cells. Deletion or mutation of any of the core PCP proteins causes disintegration of the PCP asymmetry, and, thus, developmental defects, such as disorientation of bristles and hairs in Drosophila, convergent extension (CE) impairment in zebrafish, and neural tube defects in mammals (Butler and Wallingford, 2017). Of all the core PCP components, Vangl proteins are more dedicated to PCP signaling, with Vangl2 being developmentally more important than its homolog Vangl1. Disruption and aberrant activation of Vangl lead to a variety of developmental defects and cancer malignancy, respectively (Butler and Wallingford, 2017; Humphries and Mlodzik, 2018; Wang et al., 2021). Vangl is a four-pass transmembrane protein with both N-terminal and C-terminal in the cytosol. Our previous studies have shown that phosphorylation and ubiquitination, two posttranslational modifications, are interrelated in the regulation of Vangl protein homeostasis (Gao et al., 2011; Yang et al., 2017; Feng et al., 2021). Phosphorylation mainly occurs at two clusters in the N-terminus. Casein kinase 1 (CK1), particularly CK1δ and CK1ϵ, first induces basal phosphorylation of Vangl in the endoplasmic reticulum (ER), leading to its stabilization and trafficking to the plasma membrane, where Vangl is further phosphorylated by CK1 for its normal PCP function. Wnt5a can induce Vangl phosphorylation via its receptor Ror2. On the other hand, Vangl undergoes ubiquitination mainly at two lysine sites (K300/K306) in the C-terminus (Feng et al., 2021; Gao et al., 2011). The E3 ubiquitin ligase CUL3-KBTBD7 and the AAA+ ATPase p97/VCP mediate Vangl poly-ubiquitination and subsequent ER-associated degradation (ERAD), leading to its destruction in the proteasome (Feng et al., 2021). We also found that phosphorylation of Vangl prevents its ubiquitination and ERAD (Feng et al., 2021). The functional significance of Vangl and Vangl phosphorylation in establishing PCP has been validated in various animal models, ranging from Drosophila, Xenopus, and zebrafish to mouse (Gao et al., 2011; Ossipova et al., 2015; Kelly et al., 2016; Yang et al., 2017; Strutt et al., 2019; Chuykin et al., 2021). However, the role of Vangl ubiquitination is only recently emerging (Feng et al., 2021; Radaszkiewicz et al., 2021). Unlike the canonical Wnt/ß-catenin signaling that can be measured by a number of biochemical assays, the noncanonical Wnt/PCP signaling lacks such tools. As Vangl phosphorylation is induced by Wnt5a, but ubiquitination is inhibited by Wnt5a (Feng et al., 2021), the analysis of Vangl ubiquitination and phosphorylation may provide a unique approach to analyzing Wnt/PCP signaling. Hence, here we describe the ubiquitination assay of Vangl proteins by providing two different methods with detailed information regarding reagents, procedure, and analysis. We also introduce the new method for Vangl phosphorylation assay by using the recently available site-specific phospho-Vangl2 monoclonal antibodies, which can sensitively detect the CK1-mediated Vangl2 phosphorylation induced by Wnt. The protocols described below for detection of the ubiquitination and phosphorylation of Vangl may facilitate the future studies of Wnt/PCP signaling. Materials and Reagents Materials Cell culture dish, 100 mm diameter (Corning, catalog number: 9380H79) Cell culture dish, 60 mm diameter (Corning, catalog number: 9380H77) 12-well plate (Fisher Scientific, catalog number: 08-100-241) 15 mL centrifuge tube (Sigma-Aldrich, catalog number: CLS430791) 10 mL pipet (Corning, catalog number: 4488) 1.5 mL Eppendorf tube (Axygen, catalog number: MCT-150-C) 1,000 µL blue tip (Axygen, catalog number: T-1000-B) 200 µL yellow tip (Axygen, catalog number: T-200-Y) 10 µL clear tip (Axygen, catalog number: T-300) Eppendorf pipettes (Eppendorf, catalog number: 2231300002) Electronic pipette (Thermo Scientific, catalog number: 9501) 0.22 µm filter (Fisher Scientific, catalog number: SLGP033RS) Cell scraper (Fisher Scientific, catalog number: 08-100-241) Cell lines and plasmids HEK293T (Human Embryonic Kidney 293T) cells (ATCC, catalog number: CRL-3216) CHO (Chinese Hamster Ovary) cells (ATCC, catalog number: CCL-61) HA-Vangl2 plasmid (described previously in Gao et al., 2011) His-Ubiquitin plasmid (described previously in Feng et al., 2021) FLAG-Wnt5a plasmid (described previously in Gao et al., 2011) FLAG-Ror2 plasmid (described previously in Gao et al., 2011) Myc-CK1δ plasmid (described previously in Gao et al., 2011) Antibodies Anti-HA antibody (Roche, catalog number: 11867431001, 1:5,000 dilution) Anti-His antibody (Abcam, catalog number: ab18184, 1:5,000 dilution) Anti-FLAG antibody (Sigma-Aldrich, catalog number: F1804, 1:5,000 dilution) Anti-Myc antibody (Santa Cruz Biotechnology, catalog number: sc-40, 1:2,000 dilution) Anti-Ubiquitin antibody (FK2) (Enzo Life Sciences, catalog number: ENZ-ABS840-0100, 1:1,000 dilution) Anti-Vangl2-Phospho-T78/S79/S82 antibody (ABclonal, catalog number: AP1206, 1:1,000 dilution) Anti-Vangl2-Phospho-S79/S82/S84 antibody (ABclonal, catalog number: AP1207, 1:1,000 dilution) Anti-Actin antibody (Sigma-Aldrich, catalog number: A2228, 1:5,000 dilution) Anti-GAPDH antibody (Santa Cruz Biotechnology, catalog number: sc-47724, 1:5,000 dilution) Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen, catalog number: 31430) Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Invitrogen, catalog number: 31460) Goat anti-Rat IgG (H+L) Secondary Antibody, HRP (Invitrogen, catalog number: 31470) Reagents Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, catalog number: 12-800-017) Fetal Bovine Serum (FBS) (Gibco, catalog number: 10-099-141) Penicillin-Streptomycin (PS) (Gibco, catalog number: 15140122) 0.25% Trypsin-EDTA (Gibco, catalog number: 25-200-056) Proteasome inhibitor MG132 (Abcam, catalog number: ab141003) Lysosome inhibitor chloroquine (CQ) (Sigma-Aldrich, catalog number: C6628) CK1 inhibitor D4476 (Abcam, catalog number: ab120220) Deubiquitinase inhibitor N-Ethylmaleimide (NEM) (Thermo Fisher Scientific, catalog number: 23030) cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche, catalog number: 11836153001) Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 78420) Polyethylenimine (PEI) (Sigma-Aldrich, catalog number: 765090) Opti-MEMTM I Reduced Serum Medium (Gibco, catalog number: 31985070) Ni-NTA Agarose (Qiagen, catalog number: 30210) Protein A/G Plus-agarose (Santa Cruz Biotechnology, catalog number: sc-2003) Tris (Thermo Scientific, catalog number: J75825) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888) Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S9763) Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S0751) Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911) Sodium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379) Glycine (Affymetrix, catalog number: 16407) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 436143) Imidazole (Sigma-Aldrich, catalog number: 12399) Guanidine hydrochloride (Sigma-Aldrich, catalog number: 50950) 4x Laemmli Sample Buffer (Bio-Rad, catalog number: 1610747) β-Mercaptoethanol (Bio-Rad, catalog number: 1610710) 30% Acrylamide/Bis Solution, 29:1 (Bio-Rad, catalog number: 1610156) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281) PageRuler Prestained Protein Ladder (Thermo Scientific, catalog number: 26616) Immobilon-P membrane (PVDF) (Merck, catalog number: IPVH00010) Methanol (VWR International, catalog number: BDH1135-1LP) 2-Propanol (VWR International, catalog number: BDH1131-1LP) Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A3912) IGEPAL CA-630 (Sigma-Aldrich, catalog number: 18896) Triton X-100 (Sigma-Aldrich, catalog number: 11332481001) Tween-20 (Sigma-Aldrich, catalog number: P1379) Deoxycholic acid (DCA) (Selleck Chemicals, catalog number: S4689) SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, catalog number: PI34096) PBS (see Recipes) RIPA lysis buffer (see Recipes) Buffer A (see Recipes) Buffer TI (see Recipes) Buffer B (see Recipes) 8% Resolving Gel (see Recipes) 5% Stacking Gel (see Recipes) WB Running Buffer (see Recipes) WB Transfer Buffer (see Recipes) WB Blocking Buffer (see Recipes) WB Washing Buffer (see Recipes) Equipment 37 °C, 5% CO2 forced-air incubator (Thermo Scientific, catalog number: 4110) Warm water bath Ultrasonic cell disruptor (Costar, catalog number: 3513) Dry bath (Heater) (VWR International, catalog number: SH-1001) Microcentrifuge (Hitachi, catalog number: CT15RE) Shaker (VWR, catalog number: 97109-890) Rotator (VWR, catalog number: 10136-084) SDS-PAGE gel casting apparatus (Bio-Rad, catalog number: 1658008) Protein electrophoresis and blotting instruments (Bio-Rad, catalog number: 1656019) ChemiDoc MP imaging system (Bio-Rad, catalog number: 17001402) Software Image Lab 6.1 Software (Bio-Rad) ImageJ (NIH) Part I: For Vangl2 ubiquitination assay Procedure Cell culture, transfection, and MG132 treatment HEK293T cells are maintained in DMEM complete medium (DMEM supplemented with 10% FBS and 1% PS) in a humid incubator at 37 °C and 5% CO2. Culture HEK293T cells in a 100 mm diameter culture dish until 90% confluency. Pass cells at the ratio of 1:6 into 60 mm diameter cell culture dishes. Incubate cells at 37 °C, 5% CO2 for ~20 h until 50%–70% confluency. Replace with 4 mL of fresh medium 4 h before transfection. Transfect cells with plasmids expressing HA-Vangl2 and/or His-Ubiquitin, using 1 mg/mL polyethylenimine (PEI) at the ratio of 1:4 (µg DNA: µg PEI). For a 60 mm cell culture dish, transfect 4 µg plasmids in total (2 µg HA-Vangl2 + 2 µg empty vector, 2 µg His-Ubiquitin + 2 µg empty vector, or 2 µg HA-Vangl2 + 2 µg His-Ubiquitin). Incubate the cells for 12 h. Replace with 4 mL of fresh medium 12 h post transfection. Culture the transfected cells for 48 h in total. Add MG132 to a final concentration of 10 µM, and incubate for 4 h before harvesting the cells. To ensure the ubiquitination signal is exclusively from Vangl2 rather than its interacting proteins, the ubiquitination assay is performed under denaturing conditions, using either 2% SDS lysis buffer (Choo and Zhang, 2009) or 6 M guanidine lysis buffer (Wang et al., 2017). We provided two methods for analysis of the ubiquitination of a protein of interest because they are both commonly used, and we have verified them in the previous studies. Therefore, here we provide the two methods to detect the ubiquitination of Vangl2. We suggest that readers choose one of the two methods to start their experiments. Ubiquitination assay under 2% SDS denaturing condition Gently aspirate media, and rinse cells with cold PBS three times immediately before cell harvest. Lyse cells in a 60 mm dish with 100 µL of RIPA lysis buffer containing 2% SDS, 10 mM deubiquitinase inhibitor NEM, protease and phosphatase inhibitors. Incubate at room temperature (RT) for 5 min before scraping and collecting the cell lysates into 1.5 mL Eppendorf tubes. Boil at 95 °C for 10 min. Sonicate with the ultrasonic cell disruptor at 3s ON/7s OFF with 30% amplitude, until the solution is clear. Dilute the supernatants with nine times (900 µL) of RIPA lysis buffer, to reduce the SDS concentration to 0.2%, and make the final volume of lysates 1 mL. Incubate on ice for 15 min. Centrifugate at 16,000 × g, 4 °C for 15 min, and collect the supernatants. Prepare the input sample by collecting 1/20 of the total supernatants (50 µL). Add 17 µL of 4× Laemmli Sample Buffer containing 10% 2-Mercaptoethanol to the supernatants. Boil at 95 °C for 10 min. Store at -20 °C until use as input sample. Incubate the remaining 19/20 of total supernatants (950 µL) with anti-HA antibody with rotation, at 4 °C overnight. Next day, pre-wash the Protein A/G PLUS-Agarose beads twice with cold PBS, and once with cold RIPA lysis buffer. Add 20 µL of pre-washed Protein A/G PLUS-Agarose beads to the supernatants for further incubation at 4 °C for 2 h. Wash protein-bound Protein A/G PLUS-Agarose beads four times in RIPA lysis buffer. For each wash, add 1 mL of RIPA lysis buffer to the beads, mix well, and incubate for 2 min. Centrifugate at 2,000 × g, 4 °C for 2 min, and discard the supernatants. Prepare the immunoprecipitation (IP) sample. After the final wash, leave 75 µL of buffer in the Eppendorf tube and add 25 µL of 4× Laemmli Sample Buffer containing 10% 2-Mercaptoethanol to the beads. Boil at 95 °C for 10 min. Store at -20 °C until use as IP sample. Ubiquitination assay under 6 M guanidine denaturing condition Gently aspirate media, and rinse cells with cold PBS three times immediately before cell harvest. Collect the cells using 1 mL of cold PBS. Pipette the cells in PBS several times until they detach from the culture dish, and then pipette them into an Eppendorf tube. Separate the cells into two Eppendorf tubes, with 1/20 of total cells in one tube and the remaining 19/20 in the other tube. Centrifugate at 1,500 × g, 4 °C for 3 min to collect the two individual parts of cells. Discard the supernatants. Prepare the input sample. Lyse 1/20 of the cells in 50 µL of RIPA lysis buffer containing protease and phosphatase inhibitors. Incubate on ice for 20 min. Centrifugate at 16,000 × g, 4 °C for 15 min, and collect the supernatants. Add 17 µL of 4× Laemmli Sample Buffer containing 10% 2-Mercaptoethanol to the supernatants. Boil at 95 °C for 10 min. Store at -20 °C until use as input sample. Lyse the remaining 19/20 of total cells using 1 mL of Buffer A containing 6 M guanidine, 10 mM NEM, protease and phosphatase inhibitors (see Recipes). Sonicate with the ultrasonic cell disruptor at 3s ON/7s OFF with 30% amplitude, until the solution is clear. Centrifugate at 16,000 × g for 15 min, and collect the supernatants. During centrifugation, pre-wash the nickel-nitrilotriacetic acid (Ni-NTA) beads with Buffer A three times. Add 20 µL of pre-washed Ni-NTA beads to the supernatants and incubate with gentle rotation at RT for 3 h. Wash protein-bound Ni-NTA beads using Buffer A, Buffer B, and Buffer TI shown below. For each wash, add 1 mL of buffer to the beads, mix well, and incubate for 2 min, centrifugate at 2,000 × g for 2 min, and discard the supernatants. Wash the beads once in Buffer A (see Recipes). Wash the beads twice in Buffer B (see Recipes). Wash the beads twice in Buffer TI (see Recipes). Prepare the pulldown (PD) sample. After the final wash, leave 75 µL of Buffer TI in the Eppendorf tube, and add 25 µL of 4× Laemmli Sample Buffer containing 10% 2-Mercaptoethanol to the beads. Boil at 95 °C for 10 min. Store at -20 °C until use as PD sample. After sample preparation from either B or C, perform the following detection and analysis procedures. Ubiquitination analysis by western blotting (WB) Set an 8% SDS-PAGE gel. Assemble the SDS-PAGE gel into an SDS-PAGE electrophoresis chamber. Fill the chamber with WB Running Buffer (see Recipes). Load 1 µg protein ladder and 30 µg input or IP/PD samples in the wells of the stacking gel. Run for ~20 min using constant 80 V when the samples are in the stacking gel, and for ~120 min in the resolving gel at 120 V for a better separation of proteins. Rinse PVDF membrane in methanol for 15s for activation. Transfer the gel onto the activated membrane in WB Transfer Buffer (see Recipes) using constant 300 mA at 4 °C for 120 min. Block the transferred membrane in WB Blocking Buffer (see Recipes) with gentle shaking at RT for 1 h. Dilute primary antibodies in WB Blocking Buffer (see Recipes) at the indicated dilution. Incubate the membrane with primary antibodies with gentle shaking at 4 °C overnight. Next day, wash the membrane in WB Washing Buffer (see Recipes, TBST Buffer) at RT 5 × 10 min. Dilute HRP-conjugated secondary antibodies in WB Blocking Buffer at 1:5,000 dilution. Incubate the membrane with secondary antibodies with gentle shaking at RT for 1 h. Wash the membrane in WB Washing Buffer (TBST Buffer) at RT 5 × 10 min. Mix Solutions A and B at 1:1 from SuperSignal West Femto Maximum Sensitivity Substrate kit, and use the mixed solution to cover the membrane. Incubate for an appropriate time for chemiluminescent signal development. Perform image acquisition using the ChemiDoc MP imaging system, following the manufacturer’s instructions. Edit gel images using ImageJ, and present the results. Data analysis Here is a representative result showing the ubiquitination of Vangl2 under 2% SDS denaturing condition (Figure 1). Figure 1. Detection of Vangl2 ubiquitination under 2% SDS denaturing condition. HEK293T cells were transfected with HA-Vangl2 plasmid for 48 h, and treated with proteasome inhibitor MG132 (10 µM) or lysosome inhibitor chloroquine (CQ, 25 μM) for 4 h before cell harvest. HA antibody conjugated Protein A/G agarose beads were used for immunoprecipitation (IP) of HA-Vangl2 and its covalently bound endogenous ubiquitin, under 2% SDS denaturing condition. HA-Vangl2 and endogenous ubiquitin from input and IP samples were subjected to immunoblotting with the indicated antibodies. Endogenous ubiquitin was examined by total ubiquitin FK2 antibody. GAPDH serves as a loading control. The 55kDa bands in the HA IP portion indicate the IgG heavy chains. This result is from Feng et al. (2021). Here is a representative result showing the ubiquitination of Vangl2 under 6 M guanidine denaturing condition (Figure 2). Figure 2. Detection of Vangl2 ubiquitination under 6 M guanidine denaturing condition. HEK293T cells were transfected with HA-Vangl2 and/or His-Ubiquitin (His-Ub) plasmids for 48 h and treated with proteasome inhibitor MG132 (10 µM) for 4 h before cell harvest. Ni-NTA beads were used to pull down His-Ubiquitin and its covalently bound proteins under 6 M guanidine denaturing condition. HA-Vangl2 and His-Ubiquitin from Input and Pulldown samples were subjected to immunoblotting by the indicated antibodies. GAPDH serves as a loading control. The band in the leftmost pulldown sample should be the background. This result is from Feng et al. (2021). Part II: For Vangl2 phosphorylation assay Procedure HEK293T cells are commonly used cell line for protein ubiquitination studies, and Vangl2 ubiquitination is easily detected under our investigation. Based on our experimental experience, Wnt5a/CK1 could induce more robust phosphorylation of Vangl proteins in CHO cells than in HEK293T cells (the band shift is more remarkable in CHO cells), so we switch from HEK293T cells to CHO cells for better demonstration of Vangl phosphorylation assay. To analyze the phosphorylation of Vangl2, we transfect CHO cells with plasmids expressing Wnt5a/Ror2 or CK1δ, to induce Vangl2 phosphorylation (Feng et al., 2021; Gao et al., 2011; Yang et al., 2017). The phosphorylation is detected by western blotting analysis with HA and Vangl2 phospho-specific antibodies. Procedures of cell culture, transfection, harvest, sample preparation, and western blotting analysis are similar to those in the ubiquitination assay. CHO cells are maintained in DMEM complete medium (DMEM supplemented with 10% FBS and 1% PS) in a humid incubator at 37 °C and 5% CO2. Culture CHO cells in a 100 mm diameter culture dish until 90% confluency. Pass cells at the ratio of 1:30 into a 12-well cell culture plate. Incubate the cells at 37 °C, 5% CO2 for ~20 h until 50%–70% confluency. Replace with 1 mL of fresh medium 4 h before transfection. Transfect cells with plasmids expressing HA-Vangl2, and/or FLAG-Wnt5a/Ror2 and Myc-CK1δ, using 1 mg/mL PEI at the ratio of 1:4 (µg DNA: µg PEI). For each well of a 12-well culture plate, transfect 1 µg plasmids in total (0.5 µg HA-Vangl2 + 0.5 µg empty vector, 0.5 µg HA-Vangl2 + 0.5 µg FLAG-Wnt5a/Ror2, or 0.5 µg HA-Vangl2 + 0.5 µg Myc-CK1δ). Incubate the cells for 12 h. Replace with 1 mL of fresh medium 12 h post transfection. Culture the transfected cells for 48 h in total. Gently aspirate media, and rinse cells with cold PBS three times immediately before cell harvest. Collect cells as follows: detach cells using trypsin, and inactivate trypsin using DMEM complete medium. Pipette cells into an Eppendorf tube for centrifugation, and resuspend the cell pellet using 200 µL of cold PBS. Centrifugate at 1,500 × g, 4 °C for 3 min, to collect the cells. Discard the supernatants. Prepare the sample for western blotting. Lyse the cells in 75 µL of RIPA lysis buffer containing protease and phosphatase inhibitors. Incubate on ice for 20 min. Centrifugate at 16,000 × g, 4 °C for 15 min, and collect the supernatants. Add 25 µL of 4× Laemmli Sample Buffer containing 10% 2-Mercaptoethanol to the supernatants. Boil at 95 °C for 10 min. Store at -20 °C until use. Detect and analyze the phosphorylation of Vangl2 by western blotting. Run an 8% Tris/Bis SDS-PAGE gel at 120 V for ~2 h, until the 55 kDa marker band runs to the bottom of the gel. Use HA and Vangl2 phospho-specific antibodies for detection. Data analysis Here is a representative result showing that Wnt5a/Ror2 and CK1δ markedly promote phosphorylation, especially the hyperphosphorylation of Vangl2 (Figure 3). Figure 3. Detection of Vangl2 phosphorylation. Wnt5a/Ror2 and CK1δ largely promote the phosphorylation of HA-Vangl2. CHO cells were transfected with HA-Vangl2 and/or co-transfected with FLAG-Wnt5a/Ror2 and Myc-CK1δ plasmids for 48 h before cell harvest, in the presence or absence of CK1 inhibitor D4476 (100 μM) for 6 h. The cell lysates were subjected to immunoblotting by the indicated antibodies. For detection of the phosphorylation of Vangl2, HA and two site-specific phospho-Vangl2 antibodies (p-T78/S79/S82 and p-S79/S82/S84) were used. Actin serves as a loading control. The lower-black, middle-blue, and upper-red arrows denote unphosphorylated, basally phosphorylated, and hyperphosphorylated Vangl2, respectively. Recipes Phosphate Buffered Saline (PBS) Reagent Concentration NaCl 137 mM KCl 2.7 mM Na2HPO4 10 mM KH2PO4 1.8 mM Adjust the pH to 7.4. RIPA Lysis Buffer Reagent Concentration Tris [pH 7.4] 50 mM NaCl 150 mM IGEPAL CA-630 1% (v/v) Deoxycholic acid 0.25% (v/v) SDS 0.1% (m/v) For cell lysis, add protease and phosphatase inhibitors (final concentration 1×) immediately before use. Buffer A Reagent Concentration Guanidine 6 M Na2HPO4/NaH2PO4 0.1 M Imidazole 10 mM Adjust the pH to 8.0, by preparing 1 L of 0.1 M Na2HPO4/NaH2PO4 buffer (mix 93.2 mL of 1 M Na2HPO4 and 6.8 mL of 1 M NaH2PO4). For cell lysis before the ubiquitination assay, add deubiquitinase inhibitor NEM (10 mM), and protease and phosphatase inhibitors (final concentration 1×) immediately before use. Buffer TI Reagent Concentration Tris 25 mM Imidazole 20 mM Adjust the pH to 6.8. Buffer B A mixture of Buffer A: Buffer TI with a volume ratio of 1:3. 8% Tris/Bis SDS-PAGE Gel An 8% Tris/Bis SDS-PAGE gel contains the lower resolving gel (8%) and the upper stacking gel (5%). 8% Resolving Gel Reagent Volume (mL) per 10 mL resolving gel H2O 4.6 30% Acrylamide/Bis solution 29:1 2.7 1.5 M Tris (pH 8.8) 2.5 10% SDS 0.1 10% APS 0.1 TEMED 0.006 5% Stacking Gel Reagent Volume (mL) per 2 mL stacking gel H2O 1.4 30% Acrylamide/Bis solution 29:1 0.33 1.0 M Tris (pH 6.8) 0.25 10% SDS 0.02 10% APS 0.02 TEMED 0.002 WB Running Buffer Reagent Concentration Tris-HCl [pH 7.6] 25 mM Glycine 192 mM SDS 0.1% (m/v) WB Transfer Buffer Reagent Concentration Tris-HCl [pH 7.6] 25 mM Glycine 192 mM Methanol 10% (v/v) WB Blocking Buffer (5% BSA in TBST Buffer) Reagent Concentration Tris 19 mM NaCl 137 mM KCl 2.7 mM Tween-20 0.1% (v/v) BSA 5% (m/v) WB Washing Buffer (TBST Buffer) Reagent Concentration Tris 19 mM NaCl 137 mM KCl 2.7 mM Tween-20 0.1% (v/v) Acknowledgments Funding: This research was supported by grants from the Hong Kong Research Grants Council (ECS_27115317), (GRF_17122119), and (GRF_17118120) to B.G., and grants from the National Natural Science Foundation (general program_31771561 and 32170711) to B.G. We appreciate our original research papers where this protocol was derived from: Feng et al. (2021), Gao et al. (2011), and Yang et al. (2017). Competing interests The authors declare that they have no competing interests. References Butler, M. T. and Wallingford, J. B. (2017). Planar cell polarity in development and disease. Nat Rev Mol Cell Biol 18(6): 375-388. Choo, Y. S. and Zhang, Z. (2009). Detection of protein ubiquitination. J Vis Exp (30): 1293. Chuykin, I., Itoh, K., Kim, K. and Sokol, S. Y. (2021). Frizzled3 inhibits Vangl2-Prickle3 association to establish planar cell polarity in the vertebrate neural plate. J Cell Sci 134(24): jcs258864. Feng, D., Wang, J., Yang, W., Li, J., Lin, X., Zha, F., Wang, X., Ma, L., Choi, N. T., Mii, Y., et al. (2021). Regulation of Wnt/PCP signaling through p97/VCP-KBTBD7-mediated Vangl ubiquitination and endoplasmic reticulum-associated degradation. Sci Adv 7(20): eabg2099. Gao, B., Song, H., Bishop, K., Elliot, G., Garrett, L., English, M. A., Andre, P., Robinson, J., Sood, R., Minami, Y., et al. (2011). Wnt signaling gradients establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2. Dev Cell 20(2): 163-176. Humphries, A. C. and Mlodzik, M. (2018). From instruction to output: Wnt/PCP signaling in development and cancer. Curr Opin Cell Biol 51: 110-116. Kelly, L. K., Wu, J., Yanfeng, W. A. and Mlodzik, M. (2016). Frizzled-Induced Van Gogh Phosphorylation by CK1epsilon Promotes Asymmetric Localization of Core PCP Factors in Drosophila. Cell Rep 16(2): 344-356. Ossipova, O., Kim, K. and Sokol, S. Y. (2015). Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling. Biol Open 4(6): 722-730. Radaszkiewicz, T., Noskova, M., Gomoryova, K., Vondalova Blanarova, O., Radaszkiewicz, K. A., Pickova, M., Vichova, R., Gybel, T., Kaiser, K., Demkova, L., et al. (2021). RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy. Elife 10: e65759. Strutt, H., Gamage, J. and Strutt, D. (2019). Reciprocal action of Casein Kinase Iepsilon on core planar polarity proteins regulates clustering and asymmetric localisation. Elife 8: e45107. Wang, B., Jie, Z., Joo, D., Ordureau, A., Liu, P., Gan, W., Guo, J., Zhang, J., North, B. J., Dai, X., et al. (2017). TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling. Nature 545(7654): 365-369. Wang, J., Feng, D. and Gao, B. (2021). An Overview of Potential Therapeutic Agents Targeting WNT/PCP Signaling. Handb Exp Pharmacol 269: 175-213. Yang, W., Garrett, L., Feng, D., Elliott, G., Liu, X., Wang, N., Wong, Y. M., Choi, N. T., Yang, Y. and Gao, B. (2017). Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development. Cell Res 27(12): 1466-1484. Yang, Y. and Mlodzik, M. (2015). Wnt-Frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt). Annu Rev Cell Dev Biol 31: 623-646. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > General technique > Biochemical assays Biochemistry > Protein > Modification Cell Biology > Cell signaling > Phosphorylation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology Ran Wang and Sumaira Z. Hasnain Jul 20, 2017 18826 Views Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis Koshi Imami and Tomoharu Yasuda Apr 20, 2019 8236 Views Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging Kristina Seiler [...] Mario P. Tschan Jul 20, 2023 896 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,534	https://bio-protocol.org/en/bpdetail?id=4534&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Single-molecule Force Spectroscopy on Biomembrane Force Probe to Characterize Force-dependent Bond Lifetimes of Receptor–ligand Interactions on Living Cells TZ Tongtong Zhang CA Chenyi An WH Wei Hu WC Wei Chen Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4534 Views: 1323 Reviewed by: Willy R Carrasquel-UrsulaezChen Fan Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Cell Research Aug 2021 Abstract The transmembrane receptor–ligand interactions play a vital role in the physiological and pathological processes of living cells, such as immune cell activation, neural synapse formation, or viral invasion into host cells. Mounting evidence suggests that these processes involve mechanosensing and mechanotransduction, which are directly mediated by the force-dependent transmembrane receptor–ligand interactions. Some single-molecule force spectroscopy techniques have been applied to investigate force-dependent kinetics of receptor–ligand interactions. Among these, the biomembrane force probe (BFP), a unique and powerful technique, can quantitatively and accurately determine the force-dependent parameters of transmembrane receptor–ligand interactions at the single-molecule level on living cells. The stiffness, spatial resolution, force, and bond lifetime range of BFP are 0.1–3 pN/nm, 2–3 nm, 1–103 pN, and 5 × 10-4–200 s, respectively. Therefore, this technique is very suitable for studying transient and weak interactions between transmembrane receptors and their ligands. Here, we share in detail the in situ characterization of the single-molecule force-dependent bond lifetime of transmembrane receptor–ligand interactions, based on a force-clamp assay with BFP. Keywords: Biomembrane force probe Receptor–ligand interaction Single molecule Force-dependent kinetics In situ Background Traditional biochemical methods, such as co-immunoprecipitation, surface plasmon resonance, bio-layer interferometry, or isothermal titration calorimetry, are widely employed to determine the kinetics of receptor–ligand interactions (Cole et al., 2007; Hui et al., 2017; Marasco et al., 2020; Maruhashi et al., 2022). These binding kinetics—on-rate kon, off-rate koff, and equilibrium dissociation constant KD—are detected at a static equilibrium state in solution. Remarkably, however, transmembrane receptors are restricted to the plasma membrane of the cells. Their anchor pattern, orientation, and diffusion rate on the plasma membrane can directly tune the accessibility and association possibility to affect in situ kinetics of receptor–ligand interactions (Dustin et al., 2001; Huang et al., 2004; Hu et al., 2019; Zhang et al., 2021). Therefore, the determination of in situ receptor–ligand binding kinetics on living cells is urgent for understanding the biological functions induced by transmembrane receptor–ligand interactions. Transmembrane receptors and their ligands also experience mechanical forces on the plasma membrane, which mainly derive from living cell’s movement, cell–cell contact, and dynamic changes to the plasma membrane or cytoplasmic cytoskeleton (Zhu et al., 2019). Many studies have shown that mechanical forces can act on transmembrane receptors and ligands to dynamically regulate their binding kinetics by altering their conformations. For example, SARS-CoV-2 viral invasion induces host cell membrane bending, which generates the tensile force to prolong the bond lifetime of spike/ACE2 interaction, fostering viral entry (Hu et al., 2021). Also, mechanical forces directly enhance the interaction of T-cell receptors (TCR) and agonist peptide-loaded major histocompatibility complex, which can accurately determine TCR antigen recognition (Liu et al., 2014; Wu et al., 2019). From the molecular mechanism, the mechanical force could allosterically regulate the conformation of the transmembrane receptors and ligands, generate force-induced intermediate binding states, and govern dissociation pathway selection to impede their dissociation (Sarangapani et al., 2004; Hong et al., 2015; Wu et al., 2019; Zhu et al., 2019). Also, a sliding-rebinding mechanism is applied to explain force-enhanced transmembrane receptor–ligand binding strength (Lou and Zhu, 2007). Single-molecule force spectroscopy techniques—such as atomic force microscopy, optical tweezers, magnetic tweezers, or biomembrane force probe (BFP)—have become important for studying the force-dependent kinetics of receptor–ligand interactions (Marshall et al., 2003; Kim et al., 2010; Chen and Zhu, 2013; Kong et al., 2013). Among these, BFP can quantitatively and accurately determine the force-dependent bond lifetime of transmembrane receptor–ligand interactions at the single-molecule level on living cells. The BFP technique uses soft human red blood cells (RBC, which have a proper elasticity for pN-level force detection) attached to microbeads as force sensors (Figure 1). When the transmembrane receptor expressed on the target cell binds to recombinant ligand–coated microbeads, the magnitude of the mechanical force is calculated according to the deformation of RBC, and the bond lifetime is recorded by a high-speed camera. The stiffness, spatial resolution, force, and bond lifetime range of BFP are 0.1–3 pN/nm, 2–3 nm, 1–103 pN, and 5 × 10-4–200 s, respectively (Evans et al., 1995; Chen et al., 2008; An et al., 2020). Therefore, BFP has the advantages of ultra-high mechanical detection accuracy of optical tweezers and magnetic tweezers, and also the high dynamic response characteristics of atomic force microscopy, being very suitable for studying transient and weak interactions between transmembrane receptors and their ligands in situ. Figure 1. Schematic diagram of BFP setup. The left micropipette holds the force probe, which contains a soft RBC attached to a ligand-coated bead. The right micropipette aspirates a transmembrane receptor-expressing target cell. Materials and Reagents 6-well plate (JetBioFil, catalog number: TCP011006) 0.45 μm filter (JetBioFil, catalog number: FPV403013) Micropipette tips [Crystalgen, catalog numbers: 23-5104 (10 μL); 23-3346 (200 μL); 23-3150 (1,000 μL)] 1.5 mL tubes (Crystalgen, catalog number: 23-2052) 1.5 mL low surface tension tubes (Simport, catalog number: T330-7LST) 15 mL tube (Crystalgen, catalog number: 232266) 50 mL tube (Crystalgen, catalog number: 203102) 0.5 μm capillary glass tube (West China Medical University Instrument Factory, catalog number: 10032512166199) 22 × 40 mm microscope cover glass (Fisherbrand, catalog number: 12-545C) Micro injector (World Precision Instruments, catalog number: MF28G67-5) Glass vials (Hamag Technology Co., Ltd., catalog number: HM-4455A) 10 cm dish (JetBioFil, catalog number: MCD110090) Twist lancet (HURHONG, catalog number: IR28100200) 3-mercapto-propyl-trimethoxysilane (MPTMS) (United Chemical Technologies, Inc., catalog number: M8500) Biotin-PEG 3500-SGA (JenKem, catalog number: 62717) Streptavidin-maleimide (SA-MAL) (Sigma, catalog number: S9415) 30% bovine serum albumin (BSA) (Sigma, catalog number: A0336) Nystatin (Sigma, catalog number: N4014) Borosilicate glass beads (Duke Scientific, catalog number: 9002) Streptavidin (Sangon Biotech, catalog number: A1004970-0001) PBS (Genom, catalog number: GNM20012-2) Mineral oil (Fisher Scientific, catalog number: BP2629-1) pMD2.G plasmid (Addgene, catalog number: 12259) psPAX2 plasmid (Addgene, catalog number: 12260) Polyethylenimine linear (PEI) (Yeasen, catalog number: 40816ES02) Trypsin (Yeasen, catalog number: 40127ES60) RPMI 1640 medium (BasalMedia, catalog number: L220KJ) DMEM medium (BasalMedia, catalog number: L110KJ) 293T (From Sun Qiming’s Laboratory) FBS (Yeasen, catalog number: 40130ES76) Lentiviral plasmid (From Sun Qiming’s Laboratory) Argon (Shanghai Wugang Gas Co., Ltd) Phosphate buffer (see Recipes) C buffer (see Recipes) N2 buffer (see Recipes) CR buffer (see Recipes) Cleaning buffer I (see Recipes) SA-MAL stock solution (see Recipes) PEI solution (see Recipes) Nystatin stock solution (see Recipes) Equipment 50 mL beaker Micropipettes 20, 200, 1000 (Eppendorf) Centrifuge (Eppendorf, catalog number: 5424R) 4 °C refrigerator (Melng, catalog number: YC-300L) Z2 cell counter (Beckman, catalog number: AY52487) Micropipette holder (Narishige, catalog number: HI-7) Micro forge (Narishige, catalog number: MF-900) Rotator (Qilinbeier, catalog number: BE-1100) Micropipette puller (Sutter Instrument, catalog number: P-1000) Biomembrane force probe (BFP, built by our laboratory) Desiccator (Yue Cheng Trading, catalog number: PC-150mm PC-150) Lancing device (HURHONG, catalog number: 116B015002) Vacuum pump (JINTENG, catalog number: GM-0.33A) Cell incubator (Heal Force, catalog number: HF90) Flow cytometer (Beckman, catalog number: CytoFLEX S) Cover glass (Fisherbrand, catalog number: 12545C) Light source microscope (Nikon, catalog number: Eclipse Ti) Electric-thermostatic water bath (Shang Hai Jing Hong, catalog number: XMTD 8222) Software LabVIEW 14.0 Development System Prism 8 Microsoft Excel 2019 BFP program (written by our laboratory) Procedure Figure 2. Scheme of the functionalization of the beads and RBC SH-group modification of borosilicate glass beads Weight and suspend 50 mg glass beads into 500 μL of dH2O in a 1.5 mL tube. Add the glass beads to 5 mL of boiled cleaning buffer I and boil for 5 min in a 50 mL beaker; mix the solution every minute (Figure 2A). Warning: Handle this compound with gloves in the chemical hood. After boiling, cool for 15 min at room temperature (RT), transfer the solution into a 15 mL tube, and then centrifuge at 17,000 × g for 5 min. Remove the supernatant and resuspend glass beads with 15 mL of fresh dH2O in a clean 50 mL beaker, then continue to boil for 5 min. After boiling, transfer the bead suspension to a 15 mL tube and centrifuge at 17,000 × g for 5 min. Repeat steps A2–A5 three times. Resuspend the glass beads with 50 mL of CR buffer and rotate for 3 h at RT (Figure 2A). After the reaction, centrifuge at 17,000 × g for 5 min to discard the supernatant. Wash glass beads once with fresh methanol and discard the supernatant. Resuspend glass beads with 500 μL of methanol. Divide the glass beads into a set of 20 dry clean glass vials. Evaporate the methanol by a jet of dry argon (approximately 0.05 MPa, controlled by a pressure valve) at a 45° angle inside the vial with gently horizontal rotation for 5 min. Place the vials into a preheated drying oven at 120 °C for 5 min. After heating, take the vials out of the drying oven and quickly place them in a vacuum desiccator filled with dry argon until cooled completely for 15 min. Note: The desiccator is vacuumed with a vacuum pump and then flushed with dry argon. The MPTMS glass beads in the vial desiccated in an argon environment may be stored for up to three months at RT in a dry and dark storage box. Preparation of recombinant ligand–coated beads Take out and resuspend one vial of MPTMS glass beads with phosphate buffer (pH = 6.8) into a 1.5 mL tube, centrifuge at 17,000 × g for 5 min, discard the supernatant, and wash two more times with phosphate buffer. Resuspend the MPTMS beads with 200 μL of phosphate buffer and count the concentration of MPTMS beads with the Z2 cell counter (e.g., 3 × 108 beads/mL). Mix 200 μL of MPTMS beads with 20 μL of 4 mg/mL SA-MAL stock solution (Figure 2A). Incubate the mixture with rotation overnight at RT. Then, wash three times with phosphate buffer containing 0.5% BSA, centrifuge at 17,000 × g for 5 min, resuspend into 200 μL of phosphate buffer containing 0.5% BSA, and store at 4 °C after counting with Z2 cell counter. Mix the desired biotinylated recombinant ligand (add a series of titrations in the different tubes, such as 1, 0.1, 0.01, 0.001, and 0.0001 μg) with 2 × 105 streptavidin (SA) beads in 200 μL of PBS containing 0.5% BSA in the 1.5 mL low surface tension tube (Figure 2A). Note: For the BFP assay, the adhesion frequency should be <20% by adjusting the surface density of desired biotinylated recombinant ligand on the microspheres to ensure approximately 90% single-bond event. Incubate the mixture with rotation for 30 min at RT; then, wash three times with PBS containing 0.5% BSA, resuspend into 100 μL of PBS containing 0.5% BSA, and store at 4 °C for the BFP assay. Preparation of biotinylated RBC Take the biotin-PEG 3500-SGA out of the -20 °C fridge and leave it at RT for 30 min. Then, take out one vial (approximately 10 mL) of C buffer and one vial (approximately 15 mL) of N2 buffer from the 4 °C fridge. Preheat (37 °C) the electric-thermostatic water bath for subsequent experiments. Prepare a 1.5 mL tube containing 1 mL of C buffer. Add a big drop of blood (approximately 100 μL) to the vial from a volunteer using a finger prick, lancing device, and twist lancet (Figure 2B). Wash and centrifuge blood two times with 1 mL of C buffer for 2.5 min at 2,500 × g, and resuspend with 500 μL of C buffer. Calculate the concentration of RBC with the Z2 cell counter and prepare 3 mg/mL of biotin-PEG 3500-SGA by dissolving 3 mg biotin-PEG 3500-SGA into 1 mL of C buffer (Figure 2B). Mix 500 μL of 7.5 × 107/mL RBC suspension with 117 μL of 3 mg/mL biotin-PEG 3500-SGA in a 1.5 mL tube, add 383 μL of C buffer to get a 1 mL incubate volume, then incubate with rotation for 30 min in a 50 mL tube at RT. After the incubation, centrifuge RBC suspension for 2.5 min at 2,500 × g, wash and centrifuge two more times with 1 mL of N2 buffer for 2.5 min at 2,500 × g, and resuspend RBC with 200 μL of N2 buffer. Add 5 μL of nystatin stock solution to 500 μL of N2 buffer, add 20 μL of RBC suspension from step C6, mix thoroughly, and incubate with rotation for 1 h at 4 °C in a 1.5 mL tube (Figure 2B). Centrifuge the RBC suspension for 2.5 min at 2,500 × g, wash two times with 1 mL of N2 buffer containing 0.5% BSA (37 °C preheat), and resuspend RBC with 200 μL of N2 buffer containing 0.5% BSA. Add 5 μL of RBC solution into approximately 500 μL of receptor–ligand interaction buffer (such as RPMI-1640) and observe RBC shape (Figure 3). Note: An oval shape of RBC is appropriate, which could be adjusted by the concentration of nystatin in step C7. Figure 3. Example of non-ideal and ideal RBC shapes. Scale bar = 2 μm. Establishment of transmembrane receptor–expressing cell line Culture 293T cells in a 10 cm dish to 80%–90% confluence, wash the cells with 2 mL of PBS, add 1 mL of trypsin to trypsinize the cells for 1 min, stop trypsinization with 5 mL of DMEM with 10% FBS, and collect the cells by centrifuging at 300 × g for 3 min. Remove the supernatant, resuspend with 2 mL of PBS, and wash once. Resuspend with 5 mL of DMEM medium and count to adjust the concentration of 293T cells to 2.5 × 106/mL. Add 2 mL of DMEM medium to each well of a 6-well plate, transfer 0.2 mL of 293T cells from step D2 to the plate, and shake well. Place the plate in a cell incubator and incubate at 37 °C with 5% CO2 overnight. After the cells are completely adherent, prepare them for transfection. Add 1 μg of lentiviral plasmid, 1 μg of psPAX2 plasmid, and 1 μg of pMD2.G plasmid to 250 μL of DMEM medium without FBS in a 1.5 mL tube. Incubate for 5 min. At the same time, add 6 μL of PEI (1 mg/mL) to another 1.5 mL tube of 250 μL of DMEM medium without FBS and incubate for 5 min. Slowly add the PEI solution to the mixed plasmid solution and incubate for 15 min. Add the mixed solution from step D7 to the 6-well plate with 293T cells and incubate at 37 °C with 5% CO2. After 48 h, collect the lentivirus in the medium, filter with a 0.45 μm filter, and store it in a -20 °C refrigerator. Prepare target cells (such as 293T or U937; the cell type can be determined according to each experiment) to be infected and add 2 mL of cells (2.5 × 106/mL) to a 6-well plate. Wait for cells to be completely adherent for approximately 24 h and add 500 μL of prepared lentivirus (for suspension cells, directly add 500 μL of prepared lentivirus). Incubate at 37 °C with 5% CO2 for 48 h and detect the expression level of the transmembrane receptor by flow cytometer. Sort the receptor-expressing cells to obtain a cell line and prepare for BFP experiment. Operation of BFP assay Put a 0.5 mm glass capillary on the glass cutter, select program, and click “start” button. The glass capillary can be heated into two capillaries with sharp tips. Through the selection of the program, obtain the appropriate capillary tips: 6–8 mm taper and 0.1–0.5 μm tip. Use the micropipette forge to melt the capillary tip and insert the tip in the glass sphere, which has been heated. Cool down the glass sphere. The capillary can break from the surface of the glass sphere. Repeat this procedure until the capillary tip orifice is appropriate (RBC is 2.0–2.2 μm and beads are approximately 1.5 μm; the capillary tip orifice depends on the size of cell diameter). Use a glass cutter to cut the cover glass into the required width (usually one-third of the glass width) and glue the cut glass to the top and bottom sides of the chamber holder, to form a parallel-coverslip cell chamber (Figure 4A). Use a 200 μL pipette to inject buffer (such as DMEM or PBS; can be changed according to experiments) in the cell chamber. Inject 5 μL of prepared target cells (approximately 104 cells), 5 μL of prepared beads, and 5 μL of prepared RBC into the cell chamber. Inject the mineral oil into the two sides of the cell chamber to prevent the volatilization of the buffer during the long-time experiment from affecting the experimental results (Figure 4B). Figure 4. Cell chamber for BFP experiments. (A) The cover glass is cut and glued to the top and bottom of the chamber holder. (B) Inject the buffer to fill the chamber, then sequentially inject the target cell, bead, and RBC; finally, seal the two sides of the cell chamber with mineral oil. Turn on the light source microscope and place the cell chamber on the microscope stage. Backfill the treated capillaries with dH2O with a micro injector and put the capillaries into the pipette holder. This process should be done quickly, ensuring that no air bubble gets into the pipette holder during the injection. Put the pipette holder on the micro-manipulator (Figure 5B). Push the micropipette towards the cell chamber, take the capillary tips into the chamber, and adjust the micropipette position to find the capillary tips under the microscope field of view. Control the water tower height of the RBC micropipette to change the pressure inside the micropipette, adjust the zero point of micropipette (no suction or blowing force to the RBC), and correct it. Move the chamber holder stage and adjust the position of the micropipette to find the target cell, bead, and RBC. Aspirate them by changing the pressure inside the micropipette, and switch the microscope field to the program’s vision field (Figure 5C and Video 1). Adjust the height of RBC and bead to ensure that they are on a uniform plane, then move the bead slowly to adhere to the RBC. Adjust the pressure and position to move the bead micropipette away (Figure 5D and Video 1). Align the target cell on the same plane as RBC and bead (Figure 5E and Video 1). Figure 5. Experimental setup for the BFP. (A) Setup of BFP system. (B) Move three micropipettes into the chamber. (C) Aspirate RBC, bead, and target cell. (D) Build up the force probe by attaching the bead to the RBC. (E) Adjust the position of target cell to align with force probe. Select the BFP program to measure the respective diameter of micropipette, RBC, and bead, and the contact area between the RBC and bead (Figure 6A and Video 2). Draw a horizontal line in the contact area between the RBC and bead, which will yield a curve in the adjacent window to adjust the signal during the experiment. If the curve is not sharp—meaning that the RBC and bead are not aligned (Figure 7)—start again. Select the force clamp assay experiment mode, set the parameters (such as Clamp force and Contact time, Figure 6B) as desired, and click “start.” Then, the program will cyclically drive the movement of the target cell by pipetting to contact with the bead for 0.1 s and retract. Meanwhile, the position of the bead is recorded in real time (Video 2). After 500–1000 events, click “stop” to stop the BFP experiment and save recorded data. Note: Approximately 100 bond lifetime curves are generated. Figure 6. Measurement and parameter settings for BFP experiments. (A) Measure the parameters of force probe to calibrate the force in system in the following order: 1: micropipette; 2: RBC; 3: contact area of the RBC and bead; 4: bead. (B) Program interface in the BFP system. Figure 7. Bead tracking in the BFP system. Draw a horizontal line (red) to track the border of bead and obtain a tracking curve (right). The sharp curve ensures a stable signal during BFP cycle. Data analysis Open BFP data analysis program and select the bond lifetime mode to open the data (Figure 8). Inspect each cycle of force and bond lifetime, record which cycles contain an adhesion event and which do not (Figure 9A and 9B), and calculate the adhesion frequency. During each cycle of the above inspection, record the bond lifetime parameters (bond lifetime and average force). Collect all lifetime events under a range of force. Using the BFP data autobin program, group all events to make a “lifetime-force” curve (Figure 9). Figure 8. Graphical user interface of the BFP data analysis program Figure 9. Example of BFP data (from Hu et al., 2021). (A) Raw data curve of adhesion (recorded and collected the bond lifetime). (B) No adhesion event. (C) Lifetime force curve obtained from above data collection. Notes When the micropipette aspirates RBC, the RBC tail in the micropipette cannot exceed the radius of the micropipette to ensure the accuracy of the detection force during the experiment. Data with an adhesion frequency exceeding 20% cannot be collected into the final data. When the bead tracking is unstable, the experiment should be stopped immediately and a new group should be started. The continuous collision events of the same target cell and bead should not exceed 1,000 times. The bond lifetimes are usually from >20 target cells–bead pairs in a single lifetime force curve. Ensure that the target cells are healthy to avoid non-specific adhesion, which will affect the experimental results. Recipes Phosphate buffer (pH = 6.8) 200 mM NaH2PO4·H2O Adjust to pH = 6.8 with 200 mM Na2HPO4 C buffer (pH = 8.5) 100 mM NaHCO3 Adjust to pH = 8.5 with 100 mM Na2CO3 N2 buffer (pH = 7.2) 280 mM KCl 40 mM NaCl 1 μM KH2PO4 8 mM Na2HPO4 28 mM sucrose Adjust to pH = 7.2 with HCl CR buffer 46.6 mL methanol 0.4 mL acetic acid (glacial 17.5 M) 1.85 mL dH2O 1.15 mL MPTMS Cleaning buffer I (pH = 10.9) 10% H2O2 Adjust to pH = 10.9 with NH3·H2O 4 mg/mL SA-MAL stock solution Dissolve 2 mg of SA-MAL in 500 μL of phosphate buffer (pH = 6.8). Add 10 μL per tube and store at -80 °C. Avoid repeated freezing and thawing. 1 mg/mL PEI solution Dissolve 1 mg of PEI directly in 1 mL of dH2O. Adjust to pH = 7.4 with NaOH. Nystatin stock solution Dissolve in DMSO to a concentration of 5 mg/mL. Acknowledgments We thank Evan Evans from Boston University, Cheng Zhu from Georgia Institute of Technology, Jizhong Lou from Institute of Biophysics, and Chinese Academy of Sciences for their help with BFP system setup. We also thank Zhejiang University School of Medicine for providing resources and technical supports. Competing interests There are no conflicts of interest or competing interests. Ethics The use of human RBC in this study was authorized by the ethics committee of Zhejiang University. References An, C., Hu, W., Gao, J., Ju, B. F., Obeidy, P., Zhao, Y. C., Tu, X., Fang, W., Ju, L. A. and Chen, W. (2020). Ultra-stable Biomembrane Force Probe for Accurately Determining Slow Dissociation Kinetics of PD-1 Blockade Antibodies on Single Living Cells. Nano Lett 20(7): 5133-5140. Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J. and Zhu, C. (2008). Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods. Cell Mol Bioeng 1(4): 276-288. Chen, W. and Zhu, C. (2013). Mechanical regulation of T-cell functions. Immunol Rev 256(1): 160-176. Cole, D. K., Pumphrey, N. J., Boulter, J. M., Sami, M., Bell, J. I., Gostick, E., Price, D. A., Gao, G. F., Sewell, A. K. and Jakobsen, B. K. (2007). Human TCR-binding affinity is governed by MHC class restriction. J Immunol 178(9): 5727-5734. Dustin, M. L., Bromley, S. K., Davis, M. M. and Zhu, C. (2001). Identification of self through two-dimensional chemistry and synapses. Annu Rev Cell Dev Biol 17: 133-157. Evans, E., Ritchie, K. and Merkel, R. (1995). Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys J 68(6): 2580-2587. Hong, J., Persaud, S. P., Horvath, S., Allen, P. M., Evavold, B. D. and Zhu, C. (2015). Force-Regulated In Situ TCR-Peptide-Bound MHC Class II Kinetics Determine Functions of CD4+ T Cells. J Immunol 195(8): 3557-3564. Hu, W., Zhang, Y., Fei, P., Zhang, T., Yao, D., Gao, Y., Liu, J., Chen, H., Lu, Q., Mudianto, T., et al. (2021). Mechanical activation of spike fosters SARS-CoV-2 viral infection. Cell Res 31(10): 1047-1060. Hu, W., Zhang, Y., Sun, X., Zhang, T., Xu, L., Xie, H., Li, Z., Liu, W., Lou, J. and Chen, W. (2019). FcgammaRIIB-I232T polymorphic change allosterically suppresses ligand binding. Elife 8: e46689. Huang, J., Chen, J., Chesla, S. E., Yago, T., Mehta, P., McEver, R. P., Zhu, C. and Long, M. (2004). Quantifying the effects of molecular orientation and length on two-dimensional receptor-ligand binding kinetics. J Biol Chem 279(43): 44915-44923. Hui, E., Cheung, J., Zhu, J., Su, X., Taylor, M. J., Wallweber, H. A., Sasmal, D. K., Huang, J., Kim, J. M., Mellman, I., et al. (2017). T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science 355(6332): 1428-1433. Kim, J., Zhang, C. Z., Zhang, X. and Springer, T. A. (2010). A mechanically stabilized receptor-ligand flex-bond important in the vasculature. Nature 466(7309): 992-995. Kong, F., Li, Z., Parks, W. M., Dumbauld, D. W., Garcia, A. J., Mould, A. P., Humphries, M. J. and Zhu, C. (2013). Cyclic mechanical reinforcement of integrin-ligand interactions. Mol Cell 49(6): 1060-1068. Liu, B., Chen, W., Evavold, B. D. and Zhu, C. (2014). Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell 157(2): 357-368. Lou, J. and Zhu, C. (2007). A structure-based sliding-rebinding mechanism for catch bonds. Biophys J 92(5): 1471-1485. Marasco, M., Berteotti, A., Weyershaeuser, J., Thorausch, N., Sikorska, J., Krausze, J., Brandt, H. J., Kirkpatrick, J., Rios, P., Schamel, W. W., et al. (2020). Molecular mechanism of SHP2 activation by PD-1 stimulation. Sci Adv 6(5): eaay4458. Marshall, B. T., Long, M., Piper, J. W., Yago, T., McEver, R. P. and Zhu, C. (2003). Direct observation of catch bonds involving cell-adhesion molecules. Nature 423(6936): 190-193. Maruhashi, T., Sugiura, D., Okazaki, I. M., Shimizu, K., Maeda, T. K., Ikubo, J., Yoshikawa, H., Maenaka, K., Ishimaru, N., Kosako, H., et al. (2022). Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity. Immunity 55(5): 912-924 e918. Sarangapani, K. K., Yago, T., Klopocki, A. G., Lawrence, M. B., Fieger, C. B., Rosen, S. D., McEver, R. P. and Zhu, C. (2004). Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan. J Biol Chem 279(3): 2291-2298. Wu, P., Zhang, T., Liu, B., Fei, P., Cui, L., Qin, R., Zhu, H., Yao, D., Martinez, R. J., et al. (2019). Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition. Mol Cell 73(5): 1015-1027 e1017. Zhang, T., Hu, W. and Chen, W. (2021). Plasma Membrane Integrates Biophysical and Biochemical Regulation to Trigger Immune Receptor Functions. Front Immunol 12: 613185. Zhu, C., Chen, W., Lou, J., Rittase, W. and Li, K. (2019). Mechanosensing through immunoreceptors. Nat Immunol 20(10): 1269-1278. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biophysics > Force spectroscopy Molecular Biology > Protein > Protein-protein interaction Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Spatial Centrosome Proteomic Profiling of Human iPSC-derived Neural Cells Fatma Uzbas and Adam C. O’Neill Sep 5, 2023 788 Views Proximity Labelling to Quantify Kv7.4 and Dynein Protein Interaction in Freshly Isolated Rat Vascular Smooth Muscle Cells Jennifer van der Horst and Thomas A. Jepps Mar 20, 2024 506 Views Determination of Ligand-Target Interaction in vitro by Cellular Thermal Shift Assay and Isothermal Dose-response Fingerprint Assay Danyu Du [...] Jing Xiong Aug 5, 2024 705 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,535	https://bio-protocol.org/en/bpdetail?id=4535&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Novel Method of Inducible Directed Evolution to Evolve Complex Phenotypes IA Ibrahim S. Al’Abri * ZL Zidan Li * DH Daniel J. Haller * NC Nathan Crook (*contributed equally to this work) Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4535 Views: 1932 Reviewed by: Alessandro DidonnaVasudevan AchuthanChhuttan L MeenaRita Marie Celine Meganck Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nucleic Acids Research Jun 2022 Abstract Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for most existing methods. Since many important microbial phenotypes are encoded by multigene pathways, they are usually improved via adaptive laboratory evolution (ALE), which while straightforward to implement can suffer from off-target and hitchhiker mutations that can adversely affect the fitness of the evolved strain. We have therefore developed a new directed evolution method (Inducible Directed Evolution, IDE) that combines the specificity and throughput of recent continuous directed evolution methods with the ease of ALE. Here, we present detailed methods for operating Inducible Directed Evolution (IDE), which enables long (up to 85kb) DNA sequences to be mutated in a high throughput manner via a simple series of incubation steps. In IDE, an intracellular mutagenesis plasmid (MP) tunably mutagenizes the pathway of interest, located on the phagemid (PM). MP contains a mutagenic operon (danQ926, dam, seqA, emrR, ugi, and cda1) that can be expressed via the addition of a chemical inducer. Expression of the mutagenic operon during a cell cycle represses DNA repair mechanisms such as proofreading, translesion synthesis, mismatch repair, and base excision and selection, which leads to a higher mutation rate. Induction of the P1 lytic cycle results in packaging of the mutagenized phagemid, and the pathway-bearing phage particles infect naïve cells, generating a mutant library that can be screened or selected for improved variants. Successive rounds of IDE enable optimization of complex phenotypes encoded by large pathways (as of this writing up to 36 kb), without requiring inefficient transformation steps. Additionally, IDE avoids off-target genomic mutations and enables decoupling of mutagenesis and screening steps, establishing it as a powerful tool for optimizing complex phenotypes in E. coli. Graphical abstract: Figure 1. Overview of Inducible Directed Evolution (IDE). Pathways of interest are cloned into a P1 phagemid (PM) backbone and transformed into a strain ofE. coli containing MP (diversification strain). The mutagenesis plasmid is induced to generate mutations. Phage lysate is produced and used to infect a strain that expresses the phenotype of interest (screening/selection strain). The resulting strain library is screened to identify those with improved properties. Narrowed-down libraries can then go through another IDE cycle by infecting a fresh diversification strain. Keywords: Directed Evolution Inducible Systems Complex Phenotypes Pathways Gene Clusters P1 Phage Mutagenesis Background Complex phenotypes are defined as those that result from the combined action of multiple genes. Frequently, the genes that most limit these phenotypes are difficult to determine. Improvement of such phenotypes therefore requires methods that can diversify large pathways and screen or select for improved variants in a quick manner. Adaptive Laboratory Evolution (ALE) provides genome-wide diversification that is usually used to study the mechanisms and changes that can drive a microbial strain to adapt under a specific pressure. However, accumulation of genomic hitchhiker mutations complicates interpretation of ALE results. Directed evolution methods restrict mutations to a defined DNA sequence, avoiding hitchhikermutations, yet most methods are unable to evolve the large sequences of DNA necessary to optimize complex phenotypes, either due to poor transformation efficiency of large plasmids or low packaging limits for M13-phage–based methods, such as PACE (Esvelt et al., 2011). Here, we introduce Inducible Directed Evolution (IDE), a method for evolving large DNA pathways using the P1 phage. IDE combines key components of previous phage-based directed evolution methods, including an inducible mutagenesis plasmid, as well as a phage that transfers mutagenized DNA to unmutated cells, producing a screenable mutant library that is often larger than can be obtained using chemical or electrical transformation methods at similar effort. Instead of the M13 phage (which has a packaging limit <5 kb), IDE employs the P1 phage, whose large packaging limit (5–100 kb) enables much larger DNA sequences to be shuttled between cells. IDE avoids the troublesome hitchhiker mutations associated with ALE and CRISPR-based methods (Wang et al., 2009; Crook et al., 2019), decouples mutagenesis and screening steps, and allows large pathways to be evolved (up to 36 kb has been published thus far). IDE’s workflow is both fast and simple to adapt. First, the diversification strain is prepared by cloning the desired DNA sequence into the phagemid backbone (see section Construction of phagemid). Once the phagemid is constructed, it is transformed to the diversification strain (see section Construction of phagemid). The diversification strain containing the P1 phage, mutagenesis plasmid, and phagemid goes through mutagenesis (see section Induction of Mutagenesis). After the mutagenesis step is completed, diversified phagemids can be produced for screening/selection in an appropriate strain (see sections Phage production, Phage infection, and Screening/selection). This cycle is described in Figure 1 and can be repeated until improvement ceases, or the desired phenotypes are obtained. Materials and Reagents 0.22 µm filter (Genesee Scientific, catalog number: 25-227 or equivalent), store at room temperature PCR tubes (Thermo Fisher Scientific, Eppendorf, catalog number: E0030124286 or equivalent), store at room temperature 14 mL culture tubes (Thermo Fisher Scientific, Falcon, catalog number: 14-959-1B or equivalent), store at room temperature 50 mL tubes (Thermo Fisher Scientific, Genesee Scientific Corporation, catalog number: NC1259986 or equivalent), store at room temperature 50 mL reactor tubes (Greiner Bio-One CELLSTAR® CELLreactor Conical Bottom Polypropylene Filter Top Tube 227245 or equivalent) 1.7 mL microtubes (Thermo Fisher Scientific, Genesee Scientific Corporation, catalog number: NC2045332 or equivalent), store at room temperature Serological pipets (Thermo Fisher Scientific, Genesee Scientific Corporation, catalog number: NC0631030 or equivalent), store at room temperature Shake flask (Thermo Fisher Scientific, PYREX, catalog number: 09-552-32 or equivalent), store at room temperature Petri dishes (Genesee Scientific, catalog number: 32-107G or equivalent), store at room temperature 96-deep-well plates (VWR International, catalog number: 10755-248 or equivalent), store at room temperature Pipette tips (Genesee Scientific, catalog number: 23-130RL & 24-150R & 24-160RS or equivalent), store at room temperature Electroporation cuvette 1 mm (Genesee Scientific, catalog number: 40-100), store at room temperature Nuclease-free water (Thermo Fisher Scientific, catalog number: J71786.K2 or equivalent), store at room temperature Virkon (VirkonTM S Broad Spectrum Disinfectant, Chemours), store at room temperature L-Arabinose (Teknova, catalog number: A2010 or equivalent), store at room temperature Chloroform (Thermo Fisher Scientific, catalog number: J67241.AP or equivalent), store at room temperature Anhydrotetracycline hydrochloride (Thermo Fisher Scientific, catalog number: J66688.MB or equivalent), store at -20 °C Dextrose (D-Glucose) (Thermo Fisher Scientific, catalog number: D16-1 or equivalent), store at room temperature OneTaq 2× Master Mix with Standard Buffer (New England Biolabs, catalog number: M0482S or equivalent), store at -20 °C Q5 Site-Directed Mutagenesis Kit (New England Biolabs, catalog number: E0554S or equivalent), store at -20 °C Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs, catalog number: M0494S or equivalent), store at -20 °C SGI-DNA Gibson Assembly® (GA) HiFi 1-Step Kit (CODEX DNA, Inc., catalog number: GA1200), store at -20 °C NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, catalog number: E2621S), store at -20 °C NEB® 5-alpha Competent E. coli (New England Biolabs, catalog number: C2987H or equivalent). Store at -80 °C. NEB® 10-beta Competent E. coli (New England Biolabs, catalog number: C3019H or equivalent). Store at -80 °C. LB broth (Thermo Fisher Scientific, BD Difco, catalog number: DF0446-07-5 or equivalent), store at room temperature Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166-25G or equivalent), store at room temperature Chloramphenicol (VWR, catalog number: 97061-244or equivalent), store at room temperature Kanamycin monosulfate (TCI America, catalog number: K0047 or equivalent), store at room temperature Magnesium chloride hexahydrate (VWR, catalog number: BDH9244-500G or equivalent), store at room temperature Calcium chloride dihydrate (Thermo Fisher Scientific, catalog number: C79-500 or equivalent), store at room temperature TAE buffer (Tris-Acetate-EDTA) 50× (Thermo Fisher Scientific, catalog number: BP133220 or equivalent), store at room temperature Gel loading dye (New England Biolabs, catalog number: B7024S or equivalent), store at room temperature SYBRTM Safe DNA Gel Stain (Thermo Fisher Scientific, Invitrogen, catalog number: S33102 or equivalent), store at room temperature Quick-Load® 1 kb Plus DNA Ladder (New England Biolabs, catalog number: N0469S or equivalent), store at room temperature DpnI (New England Biolabs, catalog number: R0176S or equivalent), store at -20 °C Agarose (Thermo Fisher Scientific, Lonza Inc, catalog number: BMA50004 or equivalent), store at room temperature Plasmid Miniprep Kit (Zyppy Plasmid Miniprep Kit, Zymo Research, catalog number: D4019 or equivalent), store at room temperature DNA Library Prep Master Mix (New England Biolabs, catalog number: E6040S or equivalent), store at -20 °C QIAamp DNA Kit (Qiagen, catalog number: 56304), store at room temperature DNA Clean and Concentrator-5 kit (Zymo Research, catalog number: D4004 or equivalent), store at room temperature 1× DPBS (Thermo Fisher Scientific, Gibco, catalog number: 14-190-136 or equivalent), store at room temperature Glycerol (Thermo Fisher Scientific, catalog number: G33-4 or equivalent), store at room temperature Sodium citrate tribasic dihydrate (Sigma-Aldrich, catalog number: C7254-1KG or equivalent), store at room temperature Super Optimal Broth (Thermo Fisher Scientific, Bioworld, catalog number: 50-254-370 or equivalent), store at room temperature Phagemid backbone (Addgene, catalog number: 40782) MP6 plasmid (Addgene, catalog number: 69669) aTc inducible promoter (Addgene, catalog number: 108529) aTc-MP6 plasmid (Addgene, catalog number: 189935) Primer #1: Forward primer for amplifying phagemid backbone: aggatccgaggcttggattc Primer #2: Reverse primer for amplifying phagemid backbone: atgagatctctatgctactc Primer #3: Mutagenic operon Forward primer: acccgtttttttggacgcgt Primer #4: Mutagenic operon Reverse primer: agtcaaaagcctccgaccgg Primer #5: CloDF13 Forward primer: tacgtgccaagccaaatagg Primer #6: CloDF13 Reverse primer: ggctgacttcaggtgctaca Primer #7: pTet and KanR Forward primer: cagtggaacgaaaaatcaat Primer #8: pTet and KanR Reverse primer: tcagtatctctatcactgat Primer #9: TetR Forward primer: ctcggtaccaaattccagaa Primer #10: TetR Reverse primer: ctcacttccctgttaagtat Note: Primers/Oligos can be ordered from Eurofins Genomics or equivalent supplier. Ampicillin sodium salt (100 mg/mL) (see Recipes) Chloramphenicol (34 mg/mL) (see Recipes) Kanamycin monosulfate (50 mg/mL) (see Recipes) 10% glycerol (see Recipes) LB media (see Recipes) PLM (see Recipes), store at room temperature ePLM (see Recipes), store at room temperature Super Optimal Broth with glucose (SOC) containing 200 mM sodium citrate (see Recipes) Equipment iSeq 100 or equivalent (Illumina, catalog number: 20021532) -80 °C freezer (Thermo Fisher Scientific, Revco RLE Series, model number: RLE50086A) -20 °C freezer (Thermo Fisher Scientific, Isotemp, model number: 20LFEEFSA) 4 °C refrigerator (Jordon Refrigeration) Microplate reader (Agilent BioTek, Synergy H1, catalog number: 11-120-535) I26 shaking incubator (New Brunswick Scientific, Eppendorf, part number: M1324-000) Convection incubator (VWR, Symphony, part number: 414004-610) Incubating microplate shaker (Thermo Fisher Scientific, catalog number: 02-217-759) Centrifuge (Thermo Fisher Scientific, Sorvall Legend X1R, catalog number: 75004261) Microcentrifuge (Thermo Fisher Scientific, Sorvall Legend Micro 21R, catalog number: 75-002-446) Mini Centrifuge (Thermo Fisher Scientific, catalog number: 05-090-100) Thermocycler (Eppendorf, Mastercycler nexus eco, catalog number: 6330000021) Gradient Thermocycler (Eppendorf, Mastercycler nexus gradient, catalog number: 6331000025) Single channel adjustable pipettors, 0.01–1,000 µL (Eppendorf) Multichannel adjustable pipettors 1–1,000 µL (Eppendorf) Flow cytometer (BD Biosciences, AccuriTM C6 Plus, catalog number: 660517) Digital dry bath heat block (Benchmark Scientific, model number: BSH1001) MicroPulser Electroporator (Bio-Rad, catalog number: 1652100) Spectrophotometer (Thermo Fisher Scientific, NanoDropTM 2000c, catalog number: ND-2000C) DNA Electrophoresis Cell (Bio-Rad, Mini-Sub Cell GT Tray, catalog number: 1664400EDU) DNA Electrophoresis Power Supply (Bio-Rad, PowerPacTM Basic, catalog number: 1645050) Gel Imager (Bio-Rad, Gel DocTM EZ Imager, catalog number: 170-8270) Software Samtools (BSD, MIT, http://www.htslib.org/) BCFtools (BSD, MIT, http://www.htslib.org/) VarScan ( http://varscan.sourceforge.net/) Bowtie ( http://bowtie-bio.sourceforge.net/index.shtml) Benchling ( https://www.benchling.com) or equivalent plasmid management software Procedure Construction of phagemid (PM) Use Gibson assembly (or any appropriate cloning technique) to construct the phagemid containing the pathway of interest. The phagemid (Addgene #40782) is used as the backbone for all phagemid cloning because without an insert it turns E. coli colonies purple due to the production of violacein. Primer #1: Forward primer for amplifying phagemid backbone: aggatccgaggcttggattc Primer #2: Reverse primer for amplifying phagemid backbone: ctatgctactccatcgagcc Use tmcalculator.neb.com to calculate annealing temperature. Design primers to amplify the insert that introduce at least 20 bp homology between the fragments (see NEB Gibson Assembly protocol for instructions). Amplify the backbone and DNA insert (20–30 ng of template) using NEB Q5 Hot Start High-Fidelity 2× Master Mix, and run the gel to check the size of the amplified fragments. Phagemid backbone amplification is 6.5kbp. Use tmcalculator.neb.com to calculate annealing temperature. Add 1 μL of DpnI to each PCR reaction, and incubate at 37 °C for 2 h. This reaction will digest template DNA. Purify PCR reactions using a Clean and Concentrator-5 kit according to the manufacturer’s instructions, and measure the concentration using a NanoDrop spectrophotometer. Assemble the backbone and the insert together using NEBuilder HiFi DNA Assembly Master Mix or SGI-DNA Gibson Assembly according to the manufacturer’s instructions, except for incubating samples in a thermocycler at 50°C for 30 min instead of 15 min. Transform the Gibson reactions to NEB 5-alpha or 10-beta Competent E. coli cells according to the manufacturer’s instructions, and grow overnight using chloramphenicol selection. Colony PCR white colonies (purple colonies are phagemid backbone) to verify successful cloning. Colony PCR examines successful cloning before plasmid extraction. Single colonies are mixed in a PCR reaction according to themanufacturer’s instructions. The PCR reaction is prepared as the following: 0.5 µL of 10 µM forward primer, 0.5 µL of 10 µM reverse primer, 12.5 µL of OneTaq 2× Master mix, and11.5 µL nuclease-free water. Use tmcalculator.neb.com to calculate annealing temperature of the primers. Amplify junctions with NEB OneTaq Hot Start High-Fidelity 2× Master, and run PCR reactions in gel to verify amplification size. Successful amplifications are sentfor Sanger sequencing. Verified constructs with Sanger sequencing are then cultured in 2 mL of LB with appropriate antibiotics overnight in a shaking incubator (37 °C, 250 rpm). Plasmids from overnight culture wereextracted using Zyppy Plasmid Miniprep Kit according to the manufacturer’s instructions. Prepped plasmids can then be sequenced using whole plasmid sequencing services, such as plasmidsaurus (www.plasmidsaurus.com/). Construction of Mutagenesis Plasmid (MP) –aTc-MP6 plasmid (Addgene #189935) An Anhydrotetracycline-Inducible Mutagenesis Plasmid (aTc-MP) was constructed as follows: Amplify the mutagenic operon (danQ926, dam, seqA, emrR, ugi, and cda1) and origin of replication (CloDF13) from the L-arabinose–inducible MP6 plasmid (Addgene #69669). Primer #3: Mutagenic operon Forward primer: acccgtttttttggacgcgt Primer #4: Mutagenic operon Reverse primer: agtcaaaagcctccgaccgg Primer #5: CloDF13 Forward primer: tacgtgccaagccaaatagg Primer #6: CloDF13 Reverse primer: ggctgacttcaggtgctaca Amplify the anhydrotetracycline-inducible system (pTet and TetR) and kanamycin resistance gene (KanR) from pAJM.011 (Addgene #108529). Primer #7: pTet and KanR Forward primer: cagtggaacgaaaaatcaat Primer #8: pTet and KanR Reverse primer: tcagtatctctatcactgat Primer #9: TetR Forward primer: ctcggtaccaaattccagaa Primer #10: TetR Reverse primer: ctcacttccctgttaagtat Use NEBuilder HiFi to assemble the fragments according to manufacturer’s instructions. Transform assembly reactions to NEB 10-beta Competent E. coli cells according to the manufacturer’s instructions. Grow transformations on LB agar plates containing kanamycin and 1% glucose. Glucose is used to repress the mutagenic operon. Colony PCR (see section Construction of phagemid) 5–10 colonies to verify successful cloning. Amplify junctions with NEB OneTaq Hot Start High-Fidelity 2× Master, and run PCR reactions in gel to verify amplification size. Successfulamplifications are sent for Sanger sequencing. Verified constructs with Sanger sequencing are then cultured in LB containing kanamycin and 1% glucose overnight in a shaking incubator (37 °C, 250 rpm). Plasmids fromovernight culture were extracted using Zyppy Plasmid Miniprep Kit according to the manufacturer’s instructions. Prepped plasmids can then be sequenced using whole plasmid sequencing services, such as plasmidsaurus(www.plasmidsaurus.com/). Sequence-verified aTc-MP6 can then be transformed into E. coli C600 containing the P1 phage (available upon request from Crook Lab). E. coli C600 containing both P1 phage and aTc-MP is the starting point for the construction of the diversification strain. Preparation and transformation of electrocompetent cells to generate Diversification Strain Inoculate the selected diversification strain (E. coli C600 strain containing mutagenesis plasmid and P1 phage) into 2 mL of LB media containing 50 µg/mL of kanamycin and grow overnight in a shaking incubator (37 °C, 250 rpm). Dilute 500 μL of overnight culture into 50 mL of LB media (1:100 dilution), and grow in a shaking incubator (37 °C, 250 rpm) to OD600 = 0.8. Transfer culture to a 50 mL tube and chill on ice for 10–15 min. Spin down cells at 3,000 × g and 4 °C for 5 min. Remove supernatant and resuspend pellets in 25 mL of ice-cold 10% glycerol. Spin down cells at 3,000 × g and 4 °C for 5 min. Repeat steps 5–6 two times (total 2 washes). Repeat more times if having trouble with arcing during electroporation. Resuspend washed cells in 1 mL of 10% glycerol in microcentrifuge tubes, and spin at 3,000 × gand 4 °C for 3 min. Resuspend pellets in 0.5 mL of 10% glycerol. Place the electroporation cuvette on ice for 2 min. Transfer 50 μL of the cells to a chilled microcentrifuge tube. Add 1 μL of the phagemid DNA (>50 ng). Carefully transfer the cell/DNA mix into the chilled cuvette without introducing bubbles, and make sure that the cells deposit across the bottom of the cuvette. Electroporate cuvette with the transformation mixture on an electroporator (e.g., Bio-Rad EC1). Time constant should be 5.0–6.0 ms for successful transformation. Immediately add 1 mL of Super Optimal Broth (SOC) medium to the electroporation cuvette, mix by pipetting up and down, and transfer to a 1.5 mL centrifuge tube. Place the transformation reaction in a shaking incubator (37 °C, 250 rpm) for 1 h. Centrifuge the cells at 3,000 × g for 2 min and resuspend cells in 100 μL of DPBS. Spread cells onto a prewarmed selective plate (e.g., LB + kanamycin + chloramphenicol), and incubate at 37 °C overnight. Induction of mutagenesis Subinoculate overnight culture of diversification strain in LB with 1% glucose, kanamycin, and chloramphenicol into 2 mL of LB containing kanamycin and chloramphenicol (no glucose, 1:100 dilution) in a culture tube. Induce mutagenesis by adding 200 ng/μL of aTc (2 μL of 100 ng/μL of aTc) to the subinoculated culture. Grow induced culture in a shaking incubator (37 °C, 250 rpm) for 8–16 h depending on desired mutation rate (3.4 × 10–7 substitutions per bp per generation). For the first cycle of IDE, start with one colony for overnight cultures. For cycles 2 and beyond of IDE, start with a mixed population from overnight outgrowth after infection of the diversification strain. Phage production Inoculate 2 μL of strains after the mutagenesis step (containing P1, aTc-MP, and the phagemid) or after the screening/selection step (containing P1 and the phagemid) into 2 mL of LB media containing chloramphenicol, andgrow overnight in a shaking incubator (37 °C, 250 rpm). Dilute 150 μL of O/N culture into 15 mL of ePLM (1:100 dilution) in 50 mL reactor tubes, and grow in the shaking incubator (37 °C, 250 rpm) to OD600 = 0.8–1.0. Add 150 μL of 20% L-arabinose (1/100 culture volume), and place back in the shaking incubator (37 °C, 250 rpm) for 2 h. Cultures should clarify as inFigure 2. Transfer the cultures to a 50 mL centrifuge tube, and add 400 μL of chloroform (2.5:100 volume). Place the tubes on ice for 5 min with gentle mixing or pipetting every 1 min. Centrifuge at 3,000 × g and 4 °C for 10 min, and transfer the supernatant to sterile tubes for storage. Phage lysate is stable at 4 °C for 1 year and indefinitely at -80 °C. Figure 2. Cell cultures before and after phage production. Top: Strains after the mutagenesis step or after the screening/selection step are subinoculated into fresh ePLM and grown to OD600 = 1.0. Bottom: Cell cultures are lysed after 2 h of induction with L-arabinose. Phage infection Inoculate the screening/selection strain containing P1 into 2 mL of LB media for the screening step or LB media containing kanamycin for diversification step, and grow overnight in a shaking incubator (37 °C, 250 rpm). Subinoculate overnight culture into ePLM (1:100 dilution), and grow in a shaking incubator (37 °C, 250 rpm) to OD600 = 1.0. Centrifuge at 3,000 × gand 4 °C for 5 min, and resuspend the pellet in 1/3 volume of fresh ePLM. Mix 1 mL of cells with 1 mL of phage lysate in a 14 mL culture tube. Place the infection mixture in a standing incubator (37 °C) for 20 min and then for another 20 min in a shaking incubator (37 °C, 250 rpm). Add 1 mL of SOC containing 200 mM of sodium citrate to the infection reaction to quench the infection. Place the reaction in a shaking incubator (37 °C, 250 rpm) for 1 h. To eliminate the uninfected cells, transfer the infection reaction to 50 mL of LB media containing kanamycin and chloramphenicol for the diversification step or LB agar plates containing chloramphenicol for the screening step. To count the library size of the infection, spot plate on LB agar plates containing the appropriate antibiotics. In a 96-well plate, perform serial dilutions of the infection reaction with ePLM. Perform five dilutions, from 1:10 to 1:105. Add 180 μL of ePLM to rows A-E. In row A, add 20 μL of the infection reaction.Preform the rest of the dilutions by adding 20 μL of the previous dilution to the next row. Make sure to mix well and change pipette tips after each dilution. This step can be done with multichannel pipettors. Using a multichannel pipettor, plate 5 μL of each infection reaction dilution on LB agar plates containing kanamycin and chloramphenicol for the diversification step or LB agar plates containing chloramphenicol forthe screening step. Incubate spotted plates overnight in a standing incubator (37 °C). Count CFU/mL in the morning to determine library size. Remember to multiply the number of colonies counted by the dilution factor corresponding to that row to obtain CFU/mL. To check whether phage production and infection steps were carried correctly, run the following controls: Infect desired strains with another phage lysate that has already generated an expected library size. This control will determine if phage production or infection failed. Plate uninfected cells on LB agar plates containing chloramphenicol or kanamycin to test whether the infected strain has resistance to either antibiotic. Introduction of P1 phage into P1-free strains for desired screening strains Produce phage lysate from a strain containing P1 and phagemid. Induce phage production (see section: Phage production). Infect the strain of interest (see section: Phage infection). Streak 10 μL of infection reaction on standard size LB agar plates to obtain single colonies. Screen 10–20 colonies for P1 using P1-specific primers (fragment size 296 bp): Forward primers: ACGACCATGAAAGCTCTTCACCCGTAG Reverse primers: GCTTATTCGCACCTTCCCTAAAAACAAAGATGTTTGTAG Note: To increase P1 infection efficiency to a new strain, use 1:10 cells:phage lysate by volume. Note that this ratio differs for infection with phagemid-containing particles that were discussed previously. Screening/Selection Freshly infected screening/selection strains are directly used from the prior step. Screening/selection steps are different based on the application. Example 1: Growth-based selection for increasing the utilization of a specific sugar. Ensure that the screening/selection strain achieves a low, but nonzero growth rate in minimal media containing the sugar as the sole carbon source. Select improved variants by culturing the library in minimal media containing the sugar as the sole carbon source. Subinoculate culture 2–5 times to select rapidly-growing strains before transfer back to the diversification strain. Repeated subinoculations without transfer run the risk of selecting for strains withgenomic mutations. Example 2: Biosensor-enabled screening for increasing the production of a molecule or protein. Include a biosensor in the screening/selection strain that couples productivity to fluorescence, where wild-type productivity remains in the linear range of the biosensor. Culture the screening/selection strain under conditions that favor biomolecule production. Screen for high-producing variants via Fluorescence-Activated Cell Sorting (FACS). As above, isolated variants can be grown and re-sorted via FACS several times before transfer back to the diversification strain. Mutagenesis, Phage production, Phage infection, and Screening/Selection steps are repeated until a satisfactory variant is found. After screening is complete Plasmids may be extracted from screened/selected libraries and used for next-generation sequencing to measure the frequencies of common mutations. Illumina iSeq 100 was used to achieve 80–100× sequencing depth.Other sequencing machines and services that can reach similar sequencing depth can be used. Illumina whole-genome sequencing kits and IDT® for Illumina® DNA/RNA UD Indexes are used for librarypreparation and sequencing. Equivalent NGS primers can be used to index different samples. Plasmid or amplicon-based sequencing via plasmidsaurus (www.plasmidsaurus.com/) can also be used. Trim reads using Trimmomatic. Align trimmed reads using Bowtie2. Identify mutations using VarScan. A sample computational pipeline is provided below: #!/bin/bash ##trimming reads java -jar trimmomatic-0.38.jar PE Mel1_R1.fastq R2.fastq R1_F_P.fastq R1_F_U.fastq R1_R_P.fastq R1_R_U.fastq ILLUMINACLIP:NexteraPE-PE.fa:2:30:10:2:keepBothReads LEADING:3 TRAILING:3 MINLEN:36 SLIDINGWINDOW:4:15 ##Mapping to our Genome bowtie2-build -f PM.fasta plasmidseq bowtie2 --very-sensitive --end-to-end -x plasmidseq -1 R1_F_P.fastq -2 R1_R_P.fastq -U R1_F_U.fastq,R1_R_U.fastq -S R1.sam samtools sort -m 10G -o R1.bam -T R1 R1.sam samtools index -b R1.bam samtools mpileup -f PM.fasta -d 0 R1.bam -o variants.pileup ##Calling variants java -jar VarScan.v2.3.9.jar mpileup2snp variants.pileup --min-var-freq 0.0001 > snps.txt java -jar VarScan.v2.3.9.jar mpileup2indel variants.pileup --min-var-freq 0.0001 > indels.txt Plasmids may be extracted from individual isolates and used for whole-plasmid sequencing. Align whole-plasmid sequencing results to find mutations in isolated variants against the wild-type sequence using https://www.benchling.com or equivalent tools. Note that the phagemid backbone may accumulate mutations during screening, potentially affecting its copy number. To directly compare evolved pathways to the wild type, evolved pathways may be cloned into a fresh phagemid backbone,or both evolved, and wild-type pathways may be integrated into the genome. The performance of individual isolates may be compared to the screening/selection strain containing the wild-type phagemid using additional assays (e.g., growth in 96-well plates or productivity in shake flasks, followed byHPLC analysis). The most productive variants may be used as is or subjected to additional rounds of IDE. Appendix 1: Running gels Confirm cloning and PCR amplifications using a 1% agarose gel. Dissolve 300 mg of agarose in 30 mL of 1× TAE buffer by heating until boiling. Add 3 μL of SYBR Safe DNA gel stain to the liquid gel. Cast the gel by pouring into gel trays with combs in place and allow to solidify. Run the loaded gel with 5 μL of Quick-Load 1 kb Plus DNA Ladder at 120 V for 25–30 min. Power is supplied by BioRad PowerPac, and the gel is run in a BioRad DNA electrophoresis chamber. Image the gel using a Gel Doc EZ Imager or equivalent. Appendix 2: Best practices for handling phage: All steps containing phage lysate should be conducted in the BSL2 hood to avoid contaminating other E. coli experiments. Quarantine your P1 work as much as possible from your other projects. This can be done by designating a separate bench space and materials for projects involving phages. Use filter tips when pipetting P1 phage lysate. Use Virkon to wipe off all surfaces and tools after handling phages. Store phage lysate in screw-top 15 or 50 mL centrifuge tubes. Infections and phage production are also done in these tubes. If possible, store P1 lysates in a 4 °C fridge that is not used for other E. coli experiments. Do not keep old plates of bacteria containing P1 as phages can initiate lysis in stationary colonies (<1 week in 4 °C). Recipes Ampicillin sodium salt (100 mg/mL) Dissolve 5 g of ampicillin into a 50 mL Falcon tube of ddH2O, and filter through a 0.22 µm filter to sterilize. Aliquot 1 mL and store at -20 °C (use at 1:1000 dilution in LB or LB-Agar). Chloramphenicol (34 mg/mL) Dissolve 1.7 g of chloramphenicol to 30 mL of 100% ethanol in a 50 mL Falcon tube, and bring volume to 50 mL with 100% ethanol. Aliquot 1 mL into microcentrifuge tubes, and store at -20 °C (use at 1:1,000 dilution in LB or LB-Agar). Kanamycin monosulfate (50 mg/mL) Add 2.5 g of kanamycin monosulfate to 30 mL of DI water in a 50 mL Falcon tube, and then bring volume to 50 mL with DI water. Filter through a 0.22 µm filter to sterilize. Aliquot 1 mL into microcentrifuge tubes, and store at-20 °C (use at 1:1,000 dilution in LB or LB-Agar). 10% glycerol Reagent Final concentration Amount Glycerol 10% 10 mL Nuclease-free H2O n/a 90 mL Total n/a 100 mL LB media Dissolve 25 g/L of LB broth in DI water, and autoclave the final solution at 121 °C for 20 min. Tighten the caps, and store at room temperature once fully cooled. PLM Autoclave the final solution at 121 °C for 20 min. Reagent Final concentration Amount LB broth n/a 25 g MgCl·6HO 100 mM 20.3 g CaCl2·2H2O 5 mM 0.7 g H2O n/a 100 mL Total n/a 100 mL ePLM Autoclave the final solution at 121 °C for 20 min. Reagent Final concentration Amount LB broth n/a 25 g MgCl·6HO 140 mM 28.4 g CaCl2·2H2O 7 mM 1 g H2O n/a 100 mL Total n/a 100 mL Super Optimal Broth with glucose (SOC) containing 200 mM of sodium citrate Reagent Final concentration Amount Super Optimal Broth n/a 28 g Sodium citrate tribasic dihydrate 200 mM 58.8 g H2O n/a 1 L Total n/a 1 L Autoclave once dissolved. Allow the solution to cool to 50 °C or below before adding 20 mL of 1 M MgSO4 and 20 mL of 1 M glucose. Store at room temperature. Acknowledgments We thank the labs of Dr Christopher Anderson (UC Berkeley) for phagemid constructs (Addgene #40782), Dr. David R. Liu (Harvard University) for the MP6 plasmid (Addgene #69669), and Dr. Chase Beisel for wild-type and engineered P1 bacteriophages. This protocol has been adapted from Al’Abri et al. (2022). Competing interests I.S.A. and N.C. have a patent pending related to this work. References Al'Abri, I. S., Haller, D. J., Li, Z. and Crook, N. (2022). Inducible directed evolution of complex phenotypes in bacteria. Nucleic Acids Res 50(10): e58. Crook, N., Ferreiro, A., Gasparrini, A. J., Pesesky, M. W., Gibson, M. K., Wang, B., Sun, X., Condiotte, Z., Dobrowolski, S., Peterson, D., et al. (2019). Adaptive Strategies of the Candidate Probiotic E. coli Nissle in the Mammalian Gut. Cell Host Microbe 25(4): 499-512. Esvelt, K. M., Carlson, J. C. and Liu, D. R. (2011). A system for the continuous directed evolution of biomolecules. Nature 472(7344): 499-503. Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R. and Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature 460(7257): 894-898. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biological Engineering > Synthetic biology Molecular Biology > DNA > Mutagenesis Molecular Biology > DNA > Transfection Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes Cecilia Di Genova [...] Nigel Temperton Nov 5, 2021 4005 Views VirScan: High-throughput Profiling of Antiviral Antibody Epitopes Ellen L. Shrock [...] Stephen J. Elledge Jul 5, 2022 5412 Views A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus Ryotaro Amatsu [...] Ken-ichi Yoshida Sep 5, 2022 871 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,536	https://bio-protocol.org/en/bpdetail?id=4536&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Negative Staining Transmission Electron Microscopy of HIV Viral Particles Permeabilized with PFO and Capsid Stabilized with IP6 DL Derrick Lau CM Chantal L. Márquez MP Michael W. Parker TB Till Böcking Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4536 Views: 1086 Reviewed by: Kristin L. ShinglerMaria L CagigasSvetlana Kurilova Download PDF Ask a question Favorite Cited by Version history Bio-protocol journal peer-reviewed Oct 20, 2022 \| This version Preprint Jun 08, 2022 Original Research Article: The authors used this protocol in eLIFE Jun 2018 Abstract The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM. Graphical abstract: Keywords: Electron microscopy Negative staining Virus HIV Pore-forming proteins Capsid Inositol-6-phosphate Transmission electron microscopy Ultrastructure Background The human immunodeficiency virus 1 (HIV-1) enters target cells by membrane fusion and releases the viral capsid containing HIV genomic RNA and associated proteins to the cytoplasm. The capsid is a conical protein container consisting of approximately 1,500 copies of the viral capsid protein (CA) that assemble into a closed lattice of hexamers and pentamers. This lattice is stabilized by the cellular metabolite inositol hexakisphosphate (IP6) that binds to a highly conserved ring of arginine residues in the center of CA hexamers. The disassembly of the HIV-1 capsid, also known as uncoating, needs to occur at the right place and time for a productive infection to occur. To study the properties of the HIV capsid and its interactions without the need for low-yielding capsid isolation procedures, we have recently developed a method for permeabilizing the membrane of HIV particles with perfringolysin O (PFO). This cholesterol-dependent cytolysin assembles into ring-shaped oligomers on cholesterol-containing membranes, to form large transmembrane pores with a diameter of approximately 30–40 nm (Dang et al., 2005). These pores serve as windows that allow the passage of proteins in and out of the viral particle, facilitating real-time imaging of the interactions between the capsid and host proteins and of capsid uncoating, by single-molecule fluorescence microscopy. The same approach has also been used for visualizing the capsid by negative staining transmission electron microscopy (TEM) and by cryogenic electron microscopy. Here, we describe the protocol for permeabilizing HIV-1 particles with PFO, followed by negative staining TEM. Our images reveal that PFO forms rings on the membrane of these particles containing IP6-stabilized capsids. The HIV-1 particles used in this protocol lack the viral envelope protein and are non-infectious. Alternatively, virus-like particles prepared using lentiviral packaging plasmids could be used. Permeabilization can be achieved using other pore-forming proteins in place of PFO (e.g., streptolysin O, which is commercially available). Materials and Reagents HIV-1 particle preparation T25 cell culture flask (Corning, catalog number: 430639, or equivalent) Microwell plates (Cytiva, catalog number: 28403943) 10 cm2 culture dishes (BD Biosciences, catalog number: 353803) Greiner SensoplateTM poly-L-lysine-coated glass bottom (175 μm thickness) 96-well plates (Sigma, catalog number: M4187) HEK-293T cells (ATCC, catalog number: CRL-3216) Dulbecco’s modified eagle medium (DMEM) (Life Technologies, Invitrogen, catalog number: 11965-092) Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442-500ML) 1× PBS (Gibco, catalog number: 10010031) 1× trypsin–EDTA (Gibco, catalog number: 15400054) Plasmid, psPAX2 (NIH AIDS Reagent Program, catalog number: 11348) Plasmid, pNL4.3-iGFP-∆Env (Hübner et al., 2007; Aggarwal et al., 2012) Polyethylenimine (PEI Max) reagent (Polysciences, catalog number: 9002-98-6) Sodium chloride solution 0.9% w/v (Sigma-Aldrich, catalog number: S8776) HEPES (Sigma-Aldrich, catalog number: H3375-250G) NaCl (Chem Supply, catalog number: SA046-5KG) Absolute ethanol (Chem Supply, catalog number: EA043-500M) to prepare storage solution for the column Negative staining Carbon Type B electron microscope grid (Ted Pella, catalog number: 01811) Corning Cell-Tak cell tissue adhesive (Corning, catalog number: 354240) Amicon concentrator Ultra-4, 3K MWCO (Millipore, catalog number: UFC800324) 1 M NaOH (Chem Supply, catalog number: SA178-5KG) 0.1 M sodium bicarbonate (Chem Supply, catalog number: SA001-500G) Uranyl formate 1% (Electron Microscopy Science, catalog number: 22450) or uranyl acetate 1% w/v (BDH Chemicals Ltd, catalog number: 10288) supplied by UNSW Electron Microscope Facility Phytic acid sodium salt hydrate, also known as inositol-6-phosphate (IP6) (Sigma Aldrich, catalog number: P8810-10G) Perfringolysin O (PFO, 20 µM in HBS) supplied by lab of Michael W. Parker Whatman filter paper (Whatman, catalog number: 1440-090) cut into pizza slices Bacterial petri dish (Bio-strategy, catalog number: BDAA351029) Parafilm (Labtek, catalog number: 155448) EM grid box (e.g., reuse the boxes from Ted Pella, catalog number: 01811) General 1.5 mL microcentrifuge tubes (Interpath, catalog number: 616201, or equivalent) Milli-Q Water (from Millipore Milli-Q Integral 5 water purification system, or equivalent) 0.22 µm syringe filters (Merck, catalog number: SLGP033RS) Pipettes (Eppendorf Research series, or equivalent) Refrigerated tabletop centrifuge (Beckman Coulter, model: X-15R, or equivalent) equipped with an SX4750 swinging-bucket rotor for concentrating viral particles Tabletop centrifuge (Eppendorf Centrifuge, model: 5417R, or equivalent) Pipette tips (Edwards, catalog numbers: 1036-260-000, 1032-260-000, 1045-960-008) Water bath (PolyScience, or equivalent) Medium Cell culture media (see Recipes) HEPES buffered saline, pH 7.5 (HBS, see Recipes) Equipment HIV-1 particle preparation 10 mL super loop (GE Healthcare, catalog number: 18-1113-81) Tissue culture incubator, humidity-, temperature-, and CO2-regulated (Thermo Fisher Scientific, model: 3110, or equivalent) Biosafety cabinet (Thermo Fisher Scientific, model: 1323TS, or equivalent) HiPrep 16/60 Sephacryl S-500 HR column (GE Healthcare, catalog number: 28-9356-06) Fast protein liquid chromatography (FPLC) system including injector, one pump, UV-detector, and fraction collector (GE Healthcare, ÄKTA pure, or equivalent) Neubauer cell counter (Blaubrand, or equivalent) 4 °C refrigerator -20 °C freezer -80 °C freezer Negative electron microscopy staining and imaging Pelco easiGlow 91000 Discharge Cleaning System or equivalent plasma cleaner Pelco easiGlow TEM grid holder blocks (ProSciTech, PEL16820-81) TEM grid holder block (Pelco, PEL16820-81) Reverse action electron microscope grid tweezers (Electron Microscope Sciences Style 3X) or similar Water bath set at 37 °C with floaties FEI Tecnai G2 20 TEM operating at 200 kV Procedure Part I: HIV-1 viral particle preparation Note: This protocol is reproduced from Márquez et al. (2019). Producing HIV-1 viral particles (2.5 days) Note: The combination of plasmids used in this protocol results in the production of viral particles that lack envelope proteins and are non-infectious. Procedures should be done in accordance with local biosafety regulations for producing virus-like particles. All the following steps are performed in a biosafety cabinet. Culture HEK-293T cells as follows: Thaw a vial of HEK-293T cells by gently agitating in a 37 °C water bath for 2 min or until completely thawed. Remove the vial from the water bath, decontaminate the tube by spraying with 70% ethanol, and transfer the vial to the interior of a biosafety cabinet to work in aseptic conditions. Transfer the thawed cells to a centrifuge tube containing 9 mL of cell culture media (see Recipes). Spin at 125 × g for 10 min and remove the supernatant. Resuspend the cell pellet with 5 mL of culture media and transfer to a T25 cell culture flask. Incubate the cells at 37 °C and 5% CO2 in the tissue culture incubator. Passage the cells when confluency reaches approximately 80%–90% by performing a dilution of 1:10 per passage. This should be done every two to three days. Note: Do not exceed 20 passages. In a 15 mL conical tube, prepare DNA solution for transfection by mixing 6.6 μg of pNL4.3-iGFP-∆Env plasmid, 3.3 μg of psPAX2 plasmid, and 60 μL of 1 mg/mL PEI Max solution in a final volume of 500 μL of 0.9% (w/v) sodium chloride. Incubate the mixture for 30 min at room temperature to allow formation of DNA/PEI complexes. Split the HEK-293T cell culture as follows: Heat 1× PBS, 1× trypsin–EDTA, and culture media to 37 °C. Remove the culture media and wash the cell monolayer with 1× PBS. Add 1× trypsin–EDTA (typically 1 mL for a T25 cell culture flask) and incubate for 5 min at 37 °C. Stop the trypsin digestion by adding new culture media and transfer the cell suspension to a 15 mL conical tube. Take a sample to count cells using the Neubauer cell counter and determine the total number of cells in the tube. Centrifuge the cell suspension at 300 × g for 5 min at room temperature to pellet the cells and discard the supernatant. Resuspend the cells in the appropriate volume of fresh culture media to obtain a concentration of 7 × 106 cells/mL. Gently add 1 mL of cell suspension to the DNA/PEI mix and incubate for 5 min at room temperature. Plate the cells/DNA/PEI mixture drop by drop in a 10 cm2 culture dish containing 6.5 mL of culture media. Slightly shake the dish to distribute the cells homogeneously. Incubate at 37 °C and 5% CO2 for 48 h. Collect the virus-containing supernatant in a 15 mL conical tube and centrifuge at 2,100 × g for 10 min at 4 °C to remove cellular debris. Collect the supernatant and transfer to a new conical tube. The final volume of the cleared virus-containing medium should be approximately 7 mL. Notes: Handling of viral particles must always be done wearing gloves and appropriate personal protective equipment, which should be discarded according to local biosafety regulations. To verify the presence of fluorescent HIV particles in the supernatant, dilute the supernatant 1 in 100 in PBS and then spinoculate 200 μL of the virus preparation onto a poly-L-lysine-coated glass bottom plate well at 1,200 × g and 4 °C for 60 min. Inspect for fluorescent dots with a 60× objective in a fluorescence microscope. The number of viral particles per surface area can be determined by counting the number of fluorescent dots in at least four fields of view. The particle concentration can then be obtained by multiplying the number of particles per surface area by the surface area of the well (to obtain the number of particles per well) and then dividing this number by the volume of supernatant added to the well (0.2 mL). Purifying the HIV-1 viral particles by size exclusion chromatography (1 day) Connect a HiPrep 16/60 Sephacryl S-500 HR size exclusion chromatography column to the FPLC system. All solutions used for FPLC should be passed through a filtration membrane with 0.2 μm pore size for the removal of particulates and to degas the solutions. Replace the storage solution (usually 20% ethanol) with 2 column volumes (CV) of purified water and then equilibrate the column with 2 CV of HBS pH 7.5. Monitor the absorption at 280 nm. Flow rate: 1 mL/min. Note: This step should be started the day before collecting the viral particles, as it takes approximately 8 h for the column to be equilibrated. Load the viral particles (approximately 6.5 mL) onto the column using a 10 mL super loop. Monitor the absorption at 280 nm. Flow rate: 0.5 mL/min. Elute the sample with HBS pH 7.5 and collect 1 mL fractions until a total of 1.5 CV is reached (approximately 6 h). A representative elution profile is shown in Figure 1. Combine the fractions corresponding to the first small peak that contains the viral particles (typically around fractions 34–39 or C10–D3 on a microwell plate, see Figure 1). Purified viral particles can be used within seven days if they are stored at 4 °C. Alternatively, make 200–500 μL aliquots and store at -80 °C (no flash freezing in liquid nitrogen needed). Frozen samples can be thawed on ice before use. Wash the column with 2 CV of purified water and then 2 CV of storage solution. Figure 1. Representative elution profile of HIV-1 viral particles by size exclusion chromatography. Chromatographic separation of HIV-1 viral particles from culture media proteins. The first small peak (fractions C10–D3, marked with a red asterisk) corresponds to the HIV-1 viral particles. Reproduced from Márquez et al. (2019). Part II: Negative staining of viral particles stabilized with IP6 Preparing reagents for negative staining (1 h) Thaw viral particles and concentrate the particles 40–60-fold using the Amicon concentrator Ultra-4 at 4,000 × g and 4 °C, at 10 min per spin (Figure 2A1). Resuspend after each cycle to prevent clumping until the final volume is achieved. Note: For example, the pooled SEC fractions C10–D3 (approximately 9.5 mL) in Figure 1 were concentrated to a final volume of 160 μL. Thaw a solution of uranyl formate (1% w/v in water) and centrifuge at 18,000 × g and room temperature for 30 min to remove the precipitate. Transfer the supernatant to a clean Eppendorf tube wrapped in aluminum foil to protect uranyl formate from light (Figure 2A2). Note: Uranyl formate can be replaced by uranyl acetate. Prepare the Corning Cell-Tak cell tissue adhesive solution (100 μL) by mixing 3.34 μL of 1.5 mg/mL of Cell-Tak tissue adhesive, 93 μL of 0.1 M sodium bicarbonate, and 1.73 μL of 1 M NaOH (Figure 2A3). Dilute perfringolysin O in HBS to a final concentration of 20 µM and keep at room temperature. Passivating the electron microscope (EM) grid with Cell-Tak (0.5 h) Pick up a Carbon Type B electron microscope grid with reverse action tweezers and place them onto the Pelco easiGlow TEM grid holder block with the carbon (dark) side facing up. Insert the grid holder block into the Pelco easiGlow 91000 Discharge Cleaning System and glow discharge the grid on AUTO setting. Apply 5 μL of the Cell-Tak adhesive solution to a glow-discharged EM grid on the carbon (dark) side of the grid and invert the grid on a layer of parafilm so that the grid floats upside down. Alternatively, a drop of Cell-Tak solution can be placed onto parafilm followed by inverting the grid onto the drop for incubation. Incubate the Cell-Tak on the EM grid for 5 min (Figure 2B). Negative staining of viral particles (0.5 h) Prepare viral particle solution by mixing 9.5 μL of concentrated viral particles, 0.25 μL of IP6 (4 mM), and 0.25 μL of PFO (20 µM). The final concentration of PFO is 500 nM and of IP6 is 1 mM. This is sufficient for the preparation of two grids. Note: The IP6 is prone to precipitation in the presence of divalent cations. If necessary, the IP6 concentration can be lowered to 0.1 mM, or it can be replaced by mellitic acid (hexacarboxybenzene) to stabilize the capsids. Incubate the viral particle solution in a water bath (37 °C) for 10 min for PFO to form pores on the membrane (Figure 2C1). Wick dry a Cell-Tak-coated EM grid with a small piece of filter paper. Apply 5 μL of the incubated viral particle solution to the side coated with Cell-Tak and incubate at room temperature for 1 min (Figure 2C2). Wick dry the sample using filter paper. Apply 5 μL of uranyl formate (1%, supernatant, free from aggregates) and wick dry immediately with filter paper (Figure 2C3). Repeat step 6 twice. Allow the sample to air dry and store at room temperature in a 100 mm bacterial Petri dish lined with a filter paper for 10 min; then, transfer the grid to the EM grid box. Load sample onto the FEI Tecnai G2 and image at condenser aperture of 3 and objective aperture of 1 or 3, at 19,500× magnification (Figure 2C4). Figure 2. Schematic of the negative staining procedures. (A1) Concentrate HIV-1 particles. (A2) Prepare uranyl formate stain. (A3) Mix and prepare the Cell-Tak solution. (B) Coat the TEM grid with Cell-Tak solution. (C1) Incubate HIV-1 particles with PFO to induce membrane permeabilization. (C2) Apply permeabilized HIV-1 particles to Cell-Tak-coated TEM grid. (C3) Negative staining of the TEM grid with uranyl formate stain. (C4) Air dry and image on the electron microscope. Data analysis Quantitative analysis, such as measuring the diameter of particles, length of objects, or distance between two features, can be performed as follows: Capture all images from the microscope using the same magnification. Include the scale bar in each image if this option is available. Download and install the Fiji (distribution of ImageJ) image analysis software package (Schneider et al., 2012) from https://imagej.net/software/fiji/downloads. Open a TEM image in the Fiji application by dragging the corresponding file from your folder to the menu bar (Figure 3A). Alternatively, the image can be opened from the menu bar using File > Open and selecting the file for the TEM image. Adjust the image contrast using the B & C tool (Figure 3A, available from the menu bar under Adjust > Brightness/Contrast). Use Auto or adjust Minimum and Maximum values by dragging the corresponding sliders, or use the Set button to define minimum and maximum intensity values for the TEM image. Select the Line Drawing tool (Figure 3A) and drag the mouse cursor while holding the left mouse button to draw a straight line from one end of the scale bar to the other. Note: Holding the shift key on the keyboard ensures that a horizontal line is drawn. Set the scale of the image after drawing the straight line by selecting Analyze > Set scale. The Distance in pixels is defined by the straight line that was drawn in the previous step. Enter the distance and unit represented by the scale bar into the text boxes labeled Known distance and Unit of length, respectively (500 nm in the example in Figure 3). Tick the checkbox labeled Global if analyzing multiple images with the same magnification and pixel dimensions to avoid the need to define the scale for every TEM image. Note: Use the +/- keys on the keyboard to zoom in and out; left click and hold to navigate around the image. To measure the length of an object in the TEM image, use the Line Drawing tool to draw a straight line along the object. Open the ROI Manager (Analyze > Tools > ROI Manager) and click the Add button to add the particle to the list (Figure 3B). Repeat this step until all features of the image are added. Particles selected for analysis will have a yellow line displayed on the particle. Particle numbers are displayed when the checkboxes labeled Show All and Labels are ticked in the ROI Manager. Select all the particles in the ROI Manager by clicking one of the particles and pressing Control + A on the keyboard. Press Measure to display the length values for the selected ROI in the panel labeled Results (Figure 3B). Copy and paste these measurements into other software (e.g., Excel, GraphPad Prism, or Origin) for further analysis and to generate histograms. The diameter of the non-permeabilized virus-like particles and the diameter of PFO pores are plotted in Figure 3C–D. Note: The result panel columns can be customized in Results, and then Set measurements. In the original paper (Márquez et al., 2018), TEM characterization was used to confirm the presence of intact viral capsids and to determine the diameter of the PFO pores. Before permeabilization, negatively stained viral particles appeared as shiny spheres (Figure 4A-B) with an average diameter of approximately 150 nm, consistent with previous measurements (Briggs et al., 2003). Upon incubation with PFO, viral particles were no longer spherical, and PFO pores with an average diameter of approximately 35 nm (Figure 3D) were observed on the membrane. The pores allowed the entry of uranyl formate into the particle to stain the conical viral capsid. In the presence of IP6, a higher fraction of particles contained a capsid, as expected for the IP6-mediated stabilization of the capsid lattice (Figure 4C–D). Figure 3. Analysis of virus-like particles in TEM images using Fiji. (A) The contrast of the image can be adjusted using the B & C tool. The straight line drawn along the scale bar with the Line Drawing Tool is used to calibrate the scale of the image. (B) Particle selection using the ROI tool. (C) Diameters of viral-like particles in the absence of PFO [148 ± 25 nm (µ ± SD), N = 141]. (D) Diameters of PFO pores [35.0 ± 5.4 nm (µ ± SD), N = 346] (Márquez et al., 2018). Figure 4. Representative electron micrographs of negatively stained HIV particles. (A-B) A typical field of view at 19,500× magnification of HIV particles negatively stained in the absence of PFO (A) or treated with PFO and IP6 (B). (C) Selected HIV particles in the absence of PFO. (D) Selected HIV-1 particles following incubation with PFO and IP6. The particles are no longer spherical after treatment with PFO. Ring- and arc-shaped PFO pores are visible on the viral membrane (small arrows). The HIV-1 capsid (long arrow) inside the permeabilized viral particle is stabilized by IP6. Recipes Cell culture media DMEM supplemented with 10% FBS. HEPES buffered saline pH 7.5 HBS, 50 mM HEPES, pH 7.5, 100 mM NaCl Weigh out 11.9 g of HEPES. Weigh out 5.8 g of NaCl. Dissolve HEPES and NaCl in 800 mL of Milli-Q water with a stir bar in a beaker. Adjust the pH of the solution to 7.5 using a pH probe (Thermo Fisher Scientific Eutech pH 5+) and a solution of HCl (3 M). Adjust volume to 1 L with Milli-Q water. Acknowledgments We acknowledge Nicholas Ariotti and all technical staff at the UNSW Electron Microscope Facility for the training and maintenance of the FEI G2 Tecnai transmission electron microscope. We thank Sara Lawrence (St. Vincent’s Institute) for the expression and purification of recombinant PFO. This protocol is a detailed version of the original research paper published in Márquez et al. (2018). The authors acknowledge the National Health and Medical Research Council (NHMRC APP110071 and APP1194263), Australian Research Council (ARC DP DP160101874), Australian Centre for HIV and Hepatitis Virology Research, where this work was derived from the original paper. Competing interests The corresponding author declares that there were no financial or non-financial competing interests on behalf of all authors. CM and DL received an Australian Government Research Training Program Scholarship. References Aggarwal, A., Iemma, T. L., Shih, I., Newsome, T. P., McAllery, S., Cunningham, A. L. and Turville, S. G. (2012). Mobilization of HIV spread by diaphanous 2 dependent filopodia in infected dendritic cells. PLoS Pathog 8(6): e1002762. Briggs, J. A., Wilk, T., Welker, R., Kräusslich, H. G. and Fuller, S. D. (2003). Structural organization of authentic, mature HIV-1 virions and cores.EMBO J 22(7): 1707-1715. Dang, T. X., Hotze, E. M., Rouiller, I., Tweten, R. K. and Wilson-Kubalek, E. M. (2005). Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy. J Struct Biol 150(1): 100-108. Hübner, W., Chen, P., Del Portillo, A., Liu, Y., Gordon, R. E. and Chen, B. K. (2007). Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. J Virol 81(22): 12596-12607. Márquez, C. L., Lau, D., Walsh, J., Faysal, K. M. R., Parker, M. W., Turville, S. G. and Böcking, T. (2019). Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro. Bio-protocol 9(13): e3297. Márquez, C. L., Lau, D., Walsh, J., Shah, V., McGuinness, C., Wong, A., Aggarwal, A., Parker, M. W., Jacques, D. A., Turville, S. and Böcking, T. (2018). Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis. Elife 7: e34772. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675. Article Information Copyright Lau et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Microbiology > Microbial biochemistry Biological Sciences > Biological techniques > Microbiology techniques Biophysics > Microscopy Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages Fernando L. Gordillo Altamirano and Jeremy J. Barr Apr 20, 2021 7321 Views Intrathoracic Inoculation of Zika Virus in Aedes aegypti Irma Sanchez-Vargas [...] Ken E. Olson Sep 20, 2021 1798 Views Simple and Fail-safe Method to Transform Miniprep Escherichia coli Strain K12 Plasmid DNA Into Viable Agrobacterium tumefaciens EHA105 Cells for Plant Genetic Transformation Beenzu Siamalube [...] Steven Runo Jan 5, 2025 369 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,537	https://bio-protocol.org/en/bpdetail?id=4537&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos MU Madoka K. Utsumi * TA Taichi Akahoshi * KO Kotaro Oka KH Kohji Hotta (*contributed equally to this work) Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4537 Views: 989 Reviewed by: Sunanda Marella Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Version history Bio-protocol journal peer-reviewed Oct 20, 2022 \| This version Preprint Jun 08, 2022 Original Research Article: The authors used this protocol in Science Advances Dec 2021 Abstract When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light–oxygen–voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A (Ciona robusta), a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera. Graphical abstract: Keywords: Ascidian Tunicate Motor neuron miniSOG2 Reactive oxygen species Photoablation Microscopy Background To understand neuronal function during development of specific neural circuits, several inhibitors of neurotransmitters or the knock-down/knock-out (KD/KO) of the neuron-specific gene have been used in previous ascidian research. However, inhibitors suppress the function of not only the targeted neuron but also the same types of other neurons, and the gene KD/KO method cannot artificially regulate the timing of inhibition. Therefore, specific ablation of the targeted neuron in the appropriate timeframe and area has been considered to be difficult. On the other hand, the mini singlet oxygen generator, or miniSOG2 (Makhijani et al., 2017), can be genetically introduced to the specific neurons and then ablate them at a given time using laser irradiation. Combined with the various tissue- and cell-specific promoters in Ciona (https://www.aniseed.cnrs.fr/aniseed/gene/?choice=find_cisreg) provides general information for ascidian-specific promoters), it was possible to ablate different neuron subtypes even at a single cell level. This method will be a strong tool for understanding not only the function of neurons but also the function of non-neuronal cells during ascidian development. Materials and Reagents Glass-based Petri dish (glass diameter ϕ12 mm or ϕ27 mm, IWAKI) MiniSOG2 construct driven by your chosen promoter, e.g., pSP-Neurog::miniSOG2-CAAX. Plasmid coding miniSOG2 (pcDNA3.1_miniSOG2 T2A H2B-EGFP) (Addgene, catalog number: 87410). The DNA sequence of miniSOG2-CAAX is shown in Table 1. Arbitrarily chosen fluorescent protein driven by the same promoter used for identifying miniSOG2 positive cells, such as pSP-Neurog::mCherry-CAAX. DNA sequence of mCherry-CAAX is shown in Table 1. Egg and sperm dissected from Ciona matured adults Natural sea water Table 1. Sequences for miniSOG2-CAAX and mCherry-CAAX Equipment Confocal laser scanning microscope (CLSM), inverted type (OLYMPUS, Olympus fv1000) Optical power meter (HIOKI, optical power meter 3664) Optical sensor (HIOKI, optical sensor 9742) High-speed camera (WRAYMER, WRAYCAM-VEX230M) Stereoscopic microscope (OLYMPUS, Olympus SZX12) Software Olympus fv1000 viewer (OLYMPUS) Procedure Electroporate the miniSOG2 construct together with a fluorescence marker into Ciona fertilized egg (Christiaen et al., 2009). In the case of photoablating MN2R and MN2L, pSP-Neurog::miniSOG2-CAAX can be used to express miniSOG2 at MN2/A10.64, and pSP-Neurog::mCherry-CAAX to label the position of MN2 and determine the region of interest (ROI) for laser irradiation (Figure 1). Tip: In the case of Ciona, electroporation should be conducted 30 min after fertilization. Both constructs will be observable after the embryo develops to mid-tailbud II (St. 22) (Akahoshi et al., 2021). Place embryo on a glass-based Petri dish with natural sea water. To evaluate the influence on photoablation of the targeted single neuron, image embryos with a high-speed camera (200 to 500 fps) attached to a stereoscopic microscope, before performing laser irradiation. Place your embryo under a CLSM (Olympus fv1000). Set the ROI for laser irradiation of your targeted neuron. Select the target neuron from the neurons labeled with fluorescence protein (e.g., mCherry. Excite the red fluorescence of mCherry with a 559-nm laser). To avoid photoablating other neurons, carefully check whether the ROI is restricted to your targeted neuron only, before conducting laser irradiation. Either Olympus 20× or 40× oil immersion lenses can be used as objectives. Measure the 440-nm laser power density with an optical power meter and an optical sensor. Optimize dichroic filters or other optical equipment in your microscope so that the laser power density is at least more than 15 µW/cm2. Perform 440-nm laser irradiation of the ROI from the embryo’s lateral side (Figures 1, 2) for 10 min. Tip: Every minute, turn the 440-nm laser off and ensure that embryo movement does not shift the ROI out of range from its original position. If the embryo moved during irradiation, reset the ROI to the targeted neuron again, and restart laser irradiation. Figure 1. miniSOG2-transduced Ciona embryo. Lateral view of the fluorescence image (left) of a Ciona embryo (St. 22) electroporated with pSP-Neurog::mCherry-CAAX and pSP-Neurog::miniSOG2-CAAX. The image merged with differential interference contrast (DIC) is shown in the right panel. The signal of mCherry-CAAX seen in MN2 is indicated with a white arrowhead. ROI for laser irradiation is indicated with a yellow circle. Figure 2. MiniSOG2-transduced Ciona embryo before and after laser irradiation. Lateral view of the fluorescence image merged with differential interference contrast (DIC) of Ciona embryo (St. 24) electroporated with pSP-Neurog::mCherry-CAAX and pSP-Neurog::miniSOG2-CAAX. The mCherry-CAAX signal seen in MN2 is indicated with a white arrowhead. ROI for laser irradiation is indicated with yellow circles. Left; before laser irradiation. Right; after laser irradiation. As a negative control, photoablate other labeled neurons, or the same neuron only transduced with mCherry, not miniSOG2. Thirty minutes after irradiation, image the irradiated embryos with a high-speed camera again. Check whether any changes occured in their behavior due to photoablation of the targeted neuron, and compare these changes with the embryos irradiated as control (Video 1). Video 1. Behavior of control or miniSOG2-transduced Ciona embryos before and after laser irradiation. Tail movements of both a control (pSP-Neurog::mCherry-CAAX) embryo and a miniSOG2-transduced (pSP-Neurog::mCherry-CAAX & pSP-Neurog::miniSOG2-CAAX) embryo, before and after 10 min of laser irradiation. Videos were captured with a high-speed camera. Tail movement of the control embryo occurred every 30 sec, and there was no significant change after laser irradiation. However, the miniSOG2-transduced embryo became immobile after laser irradiation. Acknowledgments This protocol was adapted from Makhijani et al. (2017) and Akahoshi et al. (2021). Competing interests The authors declare that they have no competing interests. References Akahoshi, T., Utsumi, M. K., Oonuma, K., Murakami, M., Horie, T., Kusakabe, T. G., Oka, K. and Hotta, K. (2021). A single motor neuron determines the rhythm of early motor behavior in Ciona. Sci Adv 7(50): eabl6053. Christiaen, L., Wagner, E., Shi, W. and Levine, M. (2009). Electroporation of transgenic DNAs in the sea squirt Ciona. Cold Spring Harb Protoc 2009(12): pdb prot5345. Makhijani, K., To, T. L., Ruiz-Gonzalez, R., Lafaye, C., Royant, A. and Shu, X. (2017). Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System. Cell Chem Biol 24(1): 110-119. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Development > Electroporation Developmental Biology Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation Mariko Kinoshita-Kawada [...] Jane Y. Wu Sep 20, 2019 5059 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,538	https://bio-protocol.org/en/bpdetail?id=4538&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Measuring Intracellular H2O2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7 LJ Lianne J. H. C. Jacobs MH Michaela N. Hoehne JR Jan Riemer Published: Vol 12, Iss 20, Oct 20, 2022 DOI: 10.21769/BioProtoc.4538 Views: 1806 Reviewed by: David PaulVsevolod Belousov Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The EMBO Journal Apr 2022 Abstract Depending on its local concentration, hydrogen peroxide (H2O2) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H2O2 are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H2O2 in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H2O2 dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H2O2 sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H2O2 dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader. Graphical abstract: Overview of HyPer7 sensor function and measurement results. Keywords: H2O2 measurement HyPer7 Real-time imaging Subcellular resolution pH-independent Background Aerobic life comes at a price due to the formation of reactive oxygen species (ROS) as an unavoidable by-product of essential metabolic processes. ROS is a collective term for reactive chemical species containing oxygen, of which hydrogen peroxide (H2O2) is the most relevant member due to its long half-life and comparably high cellular concentration. H2O2 has both beneficial and deleterious effects on cells depending on its levels, flux, and the functional state of the cell (Sies and Jones, 2020). Consequently, cellular H2O2 levels are tightly controlled. The subcellular dynamics of H2O2 are poorly understood in intact mammalian cells. To explore these dynamics, biosensors that are targetable, sensitive, specific, and reversible are required. Research previously relied on small chemical probes (mostly irreversible and therefore not measurements of dynamic fluxes of H2O2) or comparably insensitive genetically encoded sensors (reversible but insensitive) (Roma et al., 2018; Kalinovic et al., 2019; Liao et al., 2020; Plecita-Hlavata et al., 2020). In recent years, more sensitive genetically encoded fluorescent H2O2 sensors were developed, including HyPer7 (Pak et al., 2020). HyPer7 consists of a circular permutated yellow fluorescent protein (cpYFP) that is integrated into the H2O2-sensing regulatory domain of Neisseria meningitidis OxyR (Oxy-RD). OxyR is a natural H2O2 sensor and transcription factor. OxyR from different organisms have different sensitivities; the specific Oxy-RD domain from N. meningitidis renders HyPer7 particularly sensitive and allows measurement of close to baseline H2O2 levels in otherwise unperturbed mammalian tissue cell lines. Oxy-RD contains two cysteine residues that react in a sensitive and highly specific manner with H2O2 and consequently form a disulfide bond. This disulfide bond in Oxy-RD puts a strain on the fused cpYFP, thus changing the cpYFP excitation spectrum. cpYFP has two excitation maxima at ca 400 nm and 499 nm, respectively, and emits at 516 nm. Changes in the sensor oxidation state result in ratiometric changes with decreases in the excitation maximum at 400 nm and increases at 499 nm upon Oxy-RD oxidation (Pak et al., 2020). Notably, targeted mutations in the cpYFP backbone result in pH insensitivity of HyPer7. This is a clear advantage compared with previous HyPer sensor variants (e.g., HyPer3) that were all pH sensitive in the physiological pH range and required laborious pH control experiments. Sensors for different biomolecules can be combined with different treatments of cells, with different genetic backgrounds, or with genetic engineering tools. One such genetically encoded tool is D-amino acid oxidase (DAO), an enzyme that converts D-amino acids to the corresponding α-keto acid and generates as a by-product H2O2 (Matlashov et al., 2014). Since DAO can be targeted to different regions within a cell, it can be employed for the localized generation of H2O2. Here, we describe a protocol to measure local H2O2 dynamics in cytosol and mitochondria. Combining this method with genetically engineered tools, such as the DAO system or CRISPR-cas9–mediated gene editing, to specifically remove parts of the antioxidative system allows detailed investigations of subcellular H2O2 dynamics. Materials and Reagents Cultivation of HEK293 cells 1.5 mL Eppendorf tube (Diagonal, catalog number: 02-023-0100) Sterile filter pipette tips: 10 µL (Greiner Bio-One, Sapphire, catalog number: 772353) 100 µL (Greiner Bio-One, Sapphire, catalog number: 774353) 1,250 µL (Greiner Bio-One, Sapphire, catalog number: 778353) 96-well plate (µClear, Greiner Bio-One, catalog number: 655090) 15 mL Falcon tube (VWR, catalog number: 734-0451) LUNA reusable cell counting slide (Logos Biosystems, catalog number: L12011) Autoclaved disposable glass Pasteur pipettes without cotton pad (VWR, catalog number: HECH40567001) Sterile, individually packed serological pipettes: 5 mL pipettes (Sarstedt, catalog number: 86.1253.001) 10 mL pipettes (Sarstedt, catalog number: 86.1254.001) 10% Fetal calf serum (FCS, Sigma-Aldrich, catalog number: F0804) 1% Penicillin/streptomycin (P/S, Sigma-Aldrich, catalog number: P0781-100ML) Dulbecco's Modified Eagle Medium high glucose (DMEM, Thermo Fisher, Gibco, catalog number: 41965062) Reagent reservoir (VWR, catalog number: 613-1175) Flp-In T-Rex HEK293 cells (Human embryonic kidney 293, Invitrogen, catalog number: R78007) Poly-L-lysine (Sigma-Aldrich, catalog number: P4832-50mL) Dulbecco's Phosphate Buffered Saline powder (DPBS, Sigma-Aldrich, catalog number: D5652) 10× Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4174) Trypan Blue (Logos Biosystems, catalog number: T13001) DMEM medium-complete (Gibco, catalog number: 41965039) (see Recipes) DPBS (see Recipes) Trypsin-EDTA solution (see Recipes) Transfection of HEK293 cells Plasmids: pCS2+HyPer7-NES [addgene: plasmid #136467 (Pak et al., 2020)] pCS2+MLS-HyPer7 [addgene: plasmid #136470 (Pak et al., 2020)] Polyethylenimine (PEI) (Polysciences, catalog number: 23966-1) Polyethylenimine (PEI) 1 mg/mL (see Recipes) Dulbecco’s Modified Eagle Medium medium-pure (DMEM, Thermo Fisher, Gibco, catalog number: 41965062) (see Recipes) Induced expression of DAO in HEK293 cells Doxycycline (DOX) (AppliChem, catalog number: A2951,0005) H2O2 measurement D-Alanine (Sigma-Aldrich, catalog number: 338-69-2) L-Alanin BioChemica (Applichem, catalog number: A3690,0100) H2O2 (Sigma-Aldrich, catalog number: 216763-100ML) Minimal media (see Recipes): NaCl (Roth, catalog number: 7647-14-5) KCl (Roth, catalog number: 7447-40-7) MgCl2 (Roth, catalog number: 2189.2) CaCl2 (Merck, catalog number: 23.891.000) HEPES (VWR, catalog number: 7365-45-9) Glucose (CIL, catalog number: 110187-42-3) 10% Fetal calf serum (FCS, Sigma-Aldrich, catalog number: F0804) Equipment Cultivation of HEK293 cells Laminar flow hood class II (ENVAIR eco) CO2 incubator (New Brunswick) Vacuum pump (laboport) Microscope (motic AE2000) Multichannel pipette (VWR) Cell counter – LUNA-II (Logos Biosystems, catalog number: L40002) Equipped cell culture laboratory containing, e.g., a laminar flow hood class II (ENVAIR eco), a CO2 incubator for cultivation of cells (with cultivation set at 37 °C and 5% CO2), a vacuum pump for removal of medium using sterile glass Pasteur pipettes, a microscope, a cell counter, and a cooling centrifuge. Transfection of HEK293 cells In addition to A. Vortex shaker H2O2 measurement Cytation 3 (Agilent, BioTek) CO2 control Injection system (optional) 390LED Rev H (Agilent, BioTek, catalog number: 1225009) roGFPsmall filterblock ex.390 em. 525 Rev D (Agilent, BioTek, catalog number: 1225108) 465LED Rev I (Agilent, BioTek, catalog number: 1225001) GFP filterblock ex. 469 em. 525 Rev I (Agilent, BioTek, catalog number: 1225101) Minimal media [HEPES buffer (HBSS) solution from (Poburko et al., 2011)] (see Recipes) Complete minimal media [HEPES buffer (HBSS) solution from (Poburko et al., 2011)] (see Recipes) Software Redox Ratio Analysis (RRA) [Dr M.D. Fricker, https://markfricker.org/77-2/software/redox-ratio-analysis/ (Fricker, 2016)] R (https://www.r-project.org/) Rstudio (https://www.rstudio.com/) Procedure An overview of the procedure is depicted in Figure 1. Figure 1. Schematic representation of the steps in the HyPer7 measurement protocol. Cells are seeded in 96-well plates. We describe two different experiments, one where the response upon addition of external bolus H2O2 is assessed (experiment A), and one where H2O2 is locally generated by DAO (optional experiment B). Data are acquired on a Cytation 3 automated multi-well microscope setup that allows culturing cells at 37 °C in the presence of 5% CO2. After data acquisition, data must be processed (raw data analysis) and then further analyzed for presentation. Cultivation of HEK293 cells (day 1) (see Note 1) Flp-In T-Rex HEK293 cells are cultured in high-glucose DMEM medium-complete containing FCS and a penicillin/streptomycin antibiotic mixture. HEK293 cells are cultured on 100 mm dishes in 10 mL of DMEM medium-complete until 90% confluency. For the described experiment, HEK293 cells are plated on a poly-L-lysine–coated 96-well plate in 100 µL of DMEM medium-complete. Transfer a 96-well plate from the package together with the poly-L-lysine to a surface-sterilized laminar flow hood class II. Take a 10 mL pipette, and dropwise add poly-L-lysine until the surface is fully covered. Let it sit at room temperature for 5 min. Remove the poly-L-lysine carefully using a 1 mL pipette, as it can be reused up to approximately five times. Transfer the coated 96-well plate to an incubator at 37 °C and 5% CO2 and let it dry for a minimum of 1 h. Cultivate HEK293 cells on 100 mm dishes in 10 mL of DMEM medium-complete in an incubator at 37 °C and 5% CO2 until they reach 90% confluency. Transfer the sterile DMEM medium-complete, sterile DPBS, and sterile Trypsin-EDTA to a surface-sterilized laminar flow hood class II. Preheat all solutions to 37 °C. Before seeding, transfer the coated 96-well plate to the laminar flow hood. Wash the plate three times carefully with preheated DPBS. Transfer the dishes to the laminar flow hood. Remove the media using an autoclaved disposable glass Pasteur pipette and a vacuum pump. Wash the cells carefully by adding 5 mL of sterile DPBS at the edge of the dish using a serological pipette and a pipette boy. Remove the DPBS using an autoclaved disposable glass Pasteur pipette and a vacuum pump. Add 1 mL of sterile Trypsin-EDTA onto the cells using a 1 mL sterile filter pipette tip. Incubate the dish at 37 °C and 5% CO2 for 5 min until the cells detach. Flick the dish with the palm of your hand. Transfer the dish back into the laminar flow hood, and add 4 mL of DMEM medium-complete using a 5 mL serological pipette. Singularize the cells using a 5 mL serological pipette and a pipette boy. Transfer the cell suspension to a 15 mL Falcon tube, and transfer 20 µL of this cell suspension to a 1.5 mL Eppendorf tube for counting. Determine the concentration of cells using the small aliquot of cell suspension with a cell counter or hemocytometer using Trypan Blue. Dilute the cell suspension so that they are at a concentration of 4,000 cells/100 µL. Of this solution, 100 µL needs to be seeded per well. Total volume depends on the number of wells needed. Try to avoid seeding the outer wells due to evaporation. Seed the diluted cell suspension using a reservoir, sterile filter pipette tips, and a multichannel pipette. Incubate the plate in an incubator at 37 °C and 5% CO2. Transfection of HEK293 cells (day 2) (see Notes 2 and 3) The day after seeding, cells are transfected with the sensor of interest. If more wells are transfected with the same sensor, a transfection master mix is prepared in a 1.5 or 2 mL Eppendorf tube (dependent on the number of wells). Transfer the sterile DMEM medium-pure and sterile DMEM medium-complete to a surface-sterilized laminar flow hood class II. Preheat all solutions to 37 °C. Thaw the plasmid as well as the Polyethylenimine [PEI (1 mg/mL)]. Mix 0.05 μg/well of plasmid DNA with 10 μL/well of DMEM medium-pure and incubate at room temperature for 5 min. After the incubation, add 0.15 μL/well of PEI (1 µg/mL final concentration), and vortex for 10 s. Incubate the transfection mix for 10 min at room temperature to ensure the formation of transfection complexes. Transfer the seeded 96-well plate to the laminar flow hood. Add 40 μL/well of DMEM medium-complete to the transfection solution. Add 50 µL of plasmid transfection solution to each well. Incubate the plate in an incubator at 37 °C and 5% CO2. Induce expression of DAO in HEK293 cells [day 3; only if inducible system is used (experiment B)] (see Note 4) Induce the protein expression of the mitochondrial-targeted DAO (mtDAO), which was stably transfected using the Flp-In T-Rex system. DAO will generate H2O2 upon reacting with D-alanine. It does not react with L-alanine. Transfer the sterile DMEM medium-complete to a surface-sterilized laminar flow hood class II. Preheat all solutions to 37 °C. Thaw the doxycycline (DOX). Prepare 1 mL of DMEM medium-complete with DOX 1:100 diluted (from 1 mg/mL stock, final concentration 10 µg/mL) using sterile filter pipette tips. Transfer the 96-well plate to the laminar flow hood. Transfer the prepared DOX solution to a reservoir. Add 10 µL of this solution to each well of the 96-well plate using sterile filter pipette tips and a multichannel pipette. Incubate the plate in an incubator at 37 °C and 5% CO2. H2O2 measurement (day 4) (see Note 5) Measure the response of the HyPer7 to the addition of bolus H2O2 (experiment A), or if the DAO is expressed by treating with D-alanine or L-alanine (experiment B). Check the filters before starting up the Cytation 3. HyPer7 is measured at excitation levels of 390 nm (±9) and 469 nm (±17.5). Preheat the Cytation 3 to 37 °C with 5% CO2, and set up the measuring protocol accordingly. Experiment A: 60 min steady-state (acquire a picture every 2 to 3 min); injection/addition by hand of 30 µL of H2O2 (range for the initial experiment of 2.5–20 µM of H2O2 final concentration); 60 min measurement (acquire a picture every 1 to 1.5 min); injection/addition by hand of 20 µM of H2O2 (to fully oxidize the HyPer7 sensor); and 30 min measurement (acquire a picture every 1 to 1.5 min). Experiment B: 60 min steady-state (acquire a picture every 2 to 3 min); injection/addition by hand of 30 µL of D/L-alanine (range for the initial experiment of 1–8 mM D-alanine final concentration); 60 min measurement (acquire a picture every 1 to 1.5 min); injection/addition by hand of 20 µM H2O2 (to fully oxidize the HyPer7 sensor); and 30 min measurement (acquire a picture every 1 to 1.5 min). Prepare the H2O2 (experiment A) and D-alanine and L-alanine (experiment B) dilutions in minimal media (without 10% FCS). Consider that further dilution will take place, due to the already present 50 µL of complete minimal media (with 10% FCS) in each well. Prime the injection system with the H2O2, D-alanine, or L-alanine dilution. Transfer the 96-well plate to a working bench, and add distilled water (dH2O) to all the empty outer wells to prevent the evaporation of media. Remove the media of the wells that will be measured. Do not remove media from wells that will be measured at a later point during the day. With these Cytation 3 settings, it is possible to measure up to 18 wells in the same measurement. Add 50 µL of complete minimal media (with 10% FCS) to each well. Place the 96-well plate in the Cytation 3, and start setting the beacons for each well. After setting the beacons and saving the settings, start the measurement. Add between 10–30 µL of treatment solution. Do not add less volume to ensure that the solutions will properly mix. Data analysis In this protocol, we describe the use of the ratiometric sensor HyPer7. Using two excitation peaks at approximately 405 and 488 nm, the redox state of the sensor and thus indirectly the levels of H2O2 in its surroundings can be assessed. Forming the ratio of the intensities at these peaks reduces errors that result from differences in sensor concentrations (Pak et al., 2020). Data can be analyzed using standard software like excel. For the waste amount of single cell data, we employ a semi-automatized software package, the RRA (redox ratio analysis) program (Fricker, 2016). The data exported from this program are further analyzed using R. As the first step of the analysis, we use the RRA program to extract the fluorescence intensities for both channels and each single cell. For this, load the images into RRA, and stack all images of one well. As the 469 nm channel is much brighter, make sure to first select this one and afterwards the 390 nm channel (roGFPsmall); it will improve the alignment. Select a region of interest (ROI) and align the images. This is necessary to ensure that the cells are at the exact same spot over time and their signal can be extracted properly. To remove background noise, continue the “advanced ratio analysis” mode, and filter the background. Afterwards, select every cell present in the ROI, and export the data as an excel file. As the second step, we perform downstream analysis in R. Load the excel file into R, where you will clean the data and remove potential outliers, such as floating dead cells. These outliers are easily identified as ROIs having ratio values of + 6000 as well as ×10x. Merge the data of the replicates, and calculate the mean of all the single cells measured. Represent each cell individually as points, together with the calculated mean as a line using ggplot2. This way, we can see the heterogeneity of the individual cell responses, as well as the cell population mean. Export the graphs. Notes Cell seeding: Cells are seeded at a low density to ensure the measurement on a single-cell basis with a 70% confluency. It is important to score how fast different cell lines used in this experiment grow to adjust the seeding of cells on day 1. Cell density often has effects on the handling of the applied treatment by the cells (e.g., a higher number of cells might more rapidly remove H2O2 from the medium compared to a lower number). If the cells are too dense on the day of the measurement, this can cause the presence of more floating cells during your measurement and thus hamper proper automated data analysis. For the same reason, we also advise precoating the 96-well plate with poly-L lysine to increase cell attachment. When comparing multiple experiments, it is important to seed the same number of cells, transfect at the same time, and perform the experiment in a similar manner. Transfection: We always perform measurements 48 h after transfection. This time can be varied as the transiently transfected sensor often is detectable for up to 5 days. A high transfection efficiency is important for the subsequent single-cell measurements. The imaging parameters used in this protocol (390 and 469 nm) are not the most optimal. The maxima of the sensor excitation peaks are at 405 and 488 nm; however, the commercially available filters used are the best fitting ones for the Cytation setup that we are using. Sensor targeting to different compartments: Biosensors can be genetically targeted to different (sub-)compartments including cytosol (equipment with a nuclear export signal), mitochondrial matrix and intermembrane space (IMS), outer mitochondrial membrane and plasma membrane, and nucleus. For each localization, sensor measurements must be established from scratch. For example, “small” compartments like the IMS might harbor only a small number of sensor molecules, thus giving rise to only small fluorescence signals (especially of the 405 nm channel). Moreover, the mechanisms of sensor reversibility often rely on the presence of enzymes. In the case of HyPer7, the sensor is oxidized by H2O2 directly but requires reduction by the thioredoxin system. Thus, if this system is not present in sufficient amounts in the compartment of choice, results might be affected. Conversely, biosensor expression might also affect its local surroundings. For HyPer7, it has to be considered that it scavenges H2O2 when it “detects” H2O2, and thus it might interfere with redox signaling. Inducible expression systems: Instead of transient transfections, inducible expression systems might be used. We often combine the transient expression of biosensors with the inducible stable expression of genetic engineering tools like the mtDAO system. It is important to carefully titrate expression levels of these genetic tools (using different expression times and DOX amounts) and confirm (like for the biosensors) their correct localization. To confirm functionality of the engineering tool, it is advisable to directly assess its function. In the case of mtDAO, cotransfection with HyPer7 to the mitochondrial matrix and addition of D-alanine could serve as control. Measurements: Minimal medium: We often observe a strong interference of colored medium (e.g., medium containing phenol red) with the measurements. To ensure no interference, we use minimal medium. This medium can also be easily adapted to additional needs of the experimental regime, such as incubation of cells with different nutrient sources or depletion of cells from those nutrients. Sensors: For a multiplexing/multiparameter measurement approach, it would be highly interesting to combine different biosensors within the very same cell. This would also help to explore the strong heterogeneity between cells that is observed in single-cell measurements. It is, however, important to consider that biosensors with similar fluorescence spectrum features cannot be combined. Steady-state measurements: Like with most H2O2 sensors, assessing the steady state of HyPer7 can require a long time. The sensor is initially more oxidized due to the contact of the cells with oxygenated medium. The loss of oxygen from the medium reduces HyPer7. A steady signal is often reached after 40 min. This steady-state signal often corresponds to almost fully reduced HyPer7 indicating that experiments are most often performed to assess oxidizing insults. Heterogeneity in sensor responses: When assessing single cells, we observe a strong heterogeneity in biosensor responses between cells. On the one hand, this necessitates the presentation of results on the level of single cells and the performance of the experiment with many cells to indicate a reliable mean for the experiment. It also indicates that multiparameter experiments should best be done with different sensors within the same cell to identify correlations. Recipes Dulbecco’s Modified Eagle Medium (DMEM) medium-complete Add 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S) to a fresh bottle of high glucose DMEM. Store at 4 °C and prewarm to 37 °C before use. Dulbecco’s Modified Eagle Medium (DMEM) medium-pure No fetal calf serum (FCS) and no penicillin/streptomycin (P/S) are added. Store at 4 °C and prewarm to 37 °C before use. Dulbecco's Phosphate Buffered Saline (DPBS) Dissolve one bottle of DPBS powder in double-distilled water (ddH2O) according to the manufacturer’s description. Sterilize by autoclaving. Store at 4 °C and prewarm to 37 °C before use. Trypsin-EDTA solution Dilute 10× Trypsin-EDTA solution 1:10 with sterile PBS. Store at 4 °C and prewarm to 37 °C before use. Aliquots can be kept frozen at -20 °C. Polyethylenimine (PEI) 1 mg/mL Dissolve PEI in ddH2O. While stirring, add HCl to increase the pH to 7.0. Sterile filter the solution. Store in small aliquots at -20 °C for short-term storage; for long-term storage store at -80 °C. Minimal media [HEPES buffer (HBSS) solution from Poburko et al. (2011)] 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM HEPES and 10 mM glucose in distilled water. Store at 4 °C. Complete minimal media [HEPES buffer (HBSS) solution from Poburko et al. (2011)] 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM HEPES and 10 mM glucose in distilled water. Add 10% FCS; aliquots can be kept frozen at -20 °C. Preheat an aliquot of the Minimal media to 37 °C. Acknowledgments The Deutsche Forschungsgemeinschaft (DFG) funds research in the Laboratory of JR (RI2150/2-2 – project number 251546152, RI2150/5-1 – project number 435235019, CRC1218 / TP B02 – project number 269925409, and RTG2550/1 – project number 411422114). The protocol was used in the following original research paper (Hoehne et al., 2022). Competing interests The authors declare that they have no competing interests. References Fricker, M. D. (2016). Quantitative Redox Imaging Software. Antioxid Redox Signal 24(13): 752-762. Hoehne, M. N., Jacobs, L., Lapacz, K. J., Calabrese, G., Murschall, L. M., Marker, T., Kaul, H., Trifunovic, A., Morgan, B., Fricker, M., et al. (2022). Spatial and temporal control of mitochondrial H2O2 release in intact human cells. EMBO J 41(7): e109169. Kalinovic, S., Oelze, M., Kroller-Schon, S., Steven, S., Vujacic-Mirski, K., Kvandova, M., Schmal, I., Al Zuabi, A., Munzel, T. and Daiber, A. (2019). Comparison of Mitochondrial Superoxide Detection Ex Vivo/In Vivo by mitoSOX HPLC Method with Classical Assays in Three Different Animal Models of Oxidative Stress. Antioxidants (Basel) 8(11). Liao, P. C., Franco-Iborra, S., Yang, Y. and Pon, L. A. (2020). Live cell imaging of mitochondrial redox state in mammalian cells and yeast. Methods Cell Biol 155: 295-319. Matlashov, M. E., Belousov, V. V. and Enikolopov, G. (2014). How much H2O2 is produced by recombinant D-amino acid oxidase in mammalian cells? Antioxid Redox Signal 20(7): 1039-1044. Pak, V. V., Ezerina, D., Lyublinskaya, O. G., Pedre, B., Tyurin-Kuzmin, P. A., Mishina, N. M., Thauvin, M., Young, D., Wahni, K., Martinez Gache, S. A., et al. (2020). Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function. Cell Metab 31(3): 642-653 e646. Plecita-Hlavata, L., Engstova, H., Holendova, B., Tauber, J., Spacek, T., Petraskova, L., Kren, V., Spackova, J., Gotvaldova, K., Jezek, J., et al. (2020). Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic beta Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+ Ratio. Antioxid Redox Signal 33(12): 789-815. Poburko, D., Santo-Domingo, J. and Demaurex, N. (2011). Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations. J Biol Chem 286(13): 11672-11684. Roma, L. P., Deponte, M., Riemer, J. and Morgan, B. (2018). Mechanisms and Applications of Redox-Sensitive Green Fluorescent Protein-Based Hydrogen Peroxide Probes. Antioxid Redox Signal 29(6): 552-568. Sies, H. and Jones, D. P. (2020). Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21(7): 363-383. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cell Biology > Cell imaging > Live-cell imaging Biochemistry > Other compound > Reactive oxygen species Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Acutely Modifying Phosphatidylinositol Phosphates on Endolysosomes Using Chemically Inducible Dimerization Systems Wei Sheng Yap [...] Maxime Boutry Oct 5, 2024 320 Views Preparing Chamber Slides With Pressed Collagen for Live Imaging Monolayers of Primary Human Intestinal Stem Cells Joseph Burclaff and Scott T. Magness Nov 20, 2024 280 Views Versatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells Angélique Pipier and David Monchaud Feb 5, 2025 199 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,539	https://bio-protocol.org/en/bpdetail?id=4539&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Motion-capture Analysis of Mice Using a Video Recorded on an iPhone Camera RN Ryo Nakamichi HA Hiroshi Asahara Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4539 Views: 1128 Reviewed by: Julie WeidnerHSIU CHUN CHUANGJordi Boix-i-Coll Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Translational Medicine Jun 2022 Abstract When focusing on quick movements in the analysis of animal behavior, a high-speed camera can be used as a powerful tool. There are many options for high-speed cameras to record movement. In recent years, the quality and sophistication of videos captured on cell phones have evolved so much that the iPhone’s slow-motion video system can function as a tool for behavior analysis. Here, we describe a method to analyze the movement of the ankle joint and jump speed during the jumping action of mice, using an iPhone. Keywords: Mouse Jump Speed Ankle angle Motor function iPhone Background Behavioral analysis is commonly performed in animals (Hart et al., 2009; Charles et al., 2016), and some studies have conducted gait analysis in mice, focusing on ankle motion for musculoskeletal research (Iwata et al., 2010; Suzuki et al., 2016; Suzuki et al., 2021). A camera is essential for this kind of analysis, and a high-speed camera is specifically useful for capturing quick movements (Iwata et al., 2010; Druelle et al., 2019; Pfeffer et al., 2019). Preparatory behavior just before a jump affects the jumping performance of animals, and analyzing the former is important for evaluating the latter (Bosco et al., 1982; Komi, 1984; Bosco et al., 1987; Bobbert and Casius, 2005). iPhone, a popular cell phone, has a high-speed camera mode (slow-motion video support). This mode can function as a tool for behavior analysis (Pittman and Ichikawa, 2013). In this protocol, we have introduced a method to evaluate the mouse jumping motion using an iPhone. Materials and Reagents Laboratory-bred mice Note: Both male and female mice were used for the test. The conditions described in this study (bar size and distance from the camera to bar) were suitable for over 12-week-old mice. Mice were housed in groups of 3–5 per cage and kept in a room with controlled temperature (~23 °C) and humidity (50–60%) under a 12-h light/dark cycle (lights on at 8:30 AM) with ad libitum access to food and water. Equipment iPhone 8 (MQ842J/A, Apple) Notes: Slow-motion video support for 1,080 pixel at 120 or 240 frames per second (fps), video stabilization, and continuous autofocus video. The 240 fps slow-motion video support is available on almost all iPhone 6 and later models; therefore, it can be applied to models other than the iPhone 8. Tripod Hair removal cream Black marker pen Steel jump bar with carbon plates Note: Dimensions: H: 0.8 cm (0.4 cm + 0.4 cm) × W: 4.5 cm × L: 100 cm. Plastic ruler Anesthesia machine with sevoflurane Note: This was used for a short time to mark the landmarks described below. Software Tracker (https://physlets.org/tracker/) ImageJ software (https://imagej.nih.gov/ij/) Microsoft Excel (Microsoft) Procedure Note: This analysis was conducted to evaluate the jumping motion of mice. The mice were subjected to a training session. Five minutes before the beginning of the session, the mice were placed in the landing place (cage) for habituation. Then, at the beginning of the training session, the beam was placed in such a way that it touched the cage, and the mice were placed on the beam such that they could simply walk into the cage. After three successful entries at each distance, the beam was slowly moved further away from the cage in 5-cm increments. Through this process, mice could jump straight from the bar to the landing space (Video 1). The method for this long jump test has been described previously (Mittal et al., 2015). Video 1. Representative movie of a mouse jumping. This is an overview of the equipment setup. C57BL6J male mice jumped 35 cm from the bar into the landing space. For video camera analysis, the iPhone camera on the tripod was placed in front of the bar, as shown in Figure 1. Figure 1. Schematic image of a video recorded using an iPhone camera (Created with BioRender.com) Transport the mice from the breeding room to the laboratory in cages. Under anesthesia, shave the right hindlimbs of the mice using a hair removal cream, to analyze the motion of the ankle joint. Mark three anatomical landmarks (the lower third of the tibia, fibula epiphysis, and fifth metatarsal head) with a black ink marker (Charles et al., 2016) (Figure 2). After this procedure, rest mice for 1 h to remove the effects of anesthesia. Figure 2. Representative image of a mouse jumping. Yellow arrows indicate the three ankle markers and tail ridge. The image on the right is an enlarged view of the hind limb (red dotted line). Set up the jump bar 80 cm above the ground, and adjust the height of the camera vertically at the edge of the jump bar, such that the lens is at the same height as the jump bar. Adjust the distance between the jump bar and the camera, depending on the area to be photographed. For this analysis, we set the distance to 25 cm (Figure 2). Set the iPhone to slow-motion video mode (Tap “setting” icon and select “General” → ”Camera” → 1080p/240 fps). Let the mice jump from the jump bar to the landing space (Figure 1) (Mittal et al., 2015). Jumping is performed three times. Film all jumps, allowing a 3-min interval between jumps. Note: The movie is excluded from evaluation if the mouse did not jump in a straight line from the jump bar to the cage. Data analysis Analyze the recorded videos using the Tracker software. Use a reference length in the movie as the calibration scale Note: The thickness of the carbon plate was 4 mm, which was used as the calibration scale. Manually define the coordinate positions of the markers for each image. The tracker displays the X- and Y-values of the marked coordinate positions. Reflect this information onto an Excel file, and analyze the data (Figure 3). Figure 3. Representative image of marker identification. The red, blue, and purple points indicate the ankle markers. The white points indicate the tail ridge. Calculate the changes in the angle and angular velocity of the ankle joint, from the positional information of the three coordinates. Calculate the incident velocity and angle of incidence, based on the coordinates of the tail ridge and jump bar. Analyze the centroid using ImageJ software by tracing the edge of the trunk and measuring the centroid to evaluate the change in potential energy (Figure 4). The tail is not included in the trace area, as the mice do not move their tails in the preparation phase. Note: How to use ImageJ: Briefly, open “Set measurement” and set “Centroid,” then open “Analyze” and select “measure.” Figure 4. Representative image of centroid calculation. The red line indicates the traced edge of the trunk in the preparation phase. Representative data Based on the video analysis data, graph the variation of ankle joint angle over time (Figure 5), and calculate the changes in angular velocity. Figure 5. Represents the ankle angle of 12-week-old C57/BL6J mice. It shows the change in ankle angle from the preparation phase of jumping to the take-off. Acknowledgments This protocol was modified from that of previous studies (Iwata et al., 2010; Mittal et al., 2015). The project received financial support from AMED-CREST (Japan Agency for Medical Research and Development) (JP21gm0810008 to H.A.), JSPS KAKENHI (grant Nos. 20H05696 and 21K19403 to H.A.), and NIH (grant Nos. AR050631 and AR065379 to H.A.). This protocol was performed in our original research paper (Nakamichi et al., 2022). Competing interests The authors declare that they have no competing interests. Ethics All animal experiments were performed with the approval of the Scripps Institutional Animal Care and Use Committee (protocol No. IACUC-09-0029). References Bobbert, M. F., Casius, L. J. (2005). Is the effect of a countermovement on jump height due to active state development? Med Sci Sports Exerc 37(3): 440-6. Bosco, C., Montanari, G., Ribacchi, R., Giovenali, P., Latteri, F., Iachelli, G., Faina, M., Colli, R., Dal Monte, A., La Rosa, M., et al. (1987). Relationship between the efficiency of muscular work during jumping and the energetics of running. Eur J Appl Physiol Occup Physiol 56(2):138-43. Bosco, C., Viitasalo, J. T., Komi, P. V. and Luhtanen, P. (1982). Combined effect of elastic energy and myoelectrical potentiation during stretch-shortening cycle exercise. Acta Physiol Scand 114(4): 557-565. Charles, J. P., Cappellari, O., Spence, A. J., Hutchinson, J. R., Wells, D. J. (2016). Musculoskeletal Geometry, Muscle Architecture and Functional Specialisations of the Mouse Hindlimb. PLoS One 11(4): e0147669. Druelle, F., Goyens, J., Vasilopoulou-Kampitsi, M. and Aerts, P. (2019). Compliant legs enable lizards to maintain high running speeds on complex terrains. J Exp Biol 222(Pt 6): jeb195511. Hart, P. C., Bergner, C. L., Dufour, B. D., Smolinsky, A. N., Egan, R. J., et al. (2009). Analysis of Abnormal Repetitive Behaviors in Experimental Animal Models. Transl Neurosci 71-82. Hwang, S. H., Chang, E. H., Kwak, G., Jeon, H., Choi, B. O., Hong, Y. B. (2021). Gait parameters as tools for analyzing phenotypic alterations of a mouse model of Charcot-Marie-Tooth disease. Anim Cells Syst (Seoul) 25(1): 11-18. Iwata, A., Fuchioka, S., Hiraoka, K., Masuhara, M. and Kami, K. (2010). Characteristics of locomotion, muscle strength, and muscle tissue in regenerating rat skeletal muscles. Muscle Nerve 41(5): 694-701. Komi, P. V. (1984). Physiological and biomechanical correlates of muscle function: effects of muscle structure and stretch-shortening cycle on force and speed. Exerc Sport Sci Rev 12: 81-121. Mittal, N., Pan, J., Palmateer, J., Martin, L., Pandya, A., Kumar, S., Ofomata, A., Hurn, P. D., Schallert, T. (2015). So you think you can jump? A novel long jump assessment to detect deficits in stroked mice. J Neurosci Methods 256: 212-9. Nakamichi, R., Ma, S., Nonoyama, T., Chiba, T., Kurimoto, R., Ohzono, H., Olmer, M., Shukunami, C., Fuku, N., Wang, G., et al. (2022). The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance. Sci Transl Med 14(647): eabj5557. Pfeffer, S. E., Wahl, V. L., Wittlinger, M. and Wolf, H. (2019). High-speed locomotion in the Saharan silver ant, Cataglyphis bombycina. J Exp Biol 222(Pt 20): jeb198705. Pittman, J. T. and Ichikawa, K. M. (2013). iPhone® applications as versatile video tracking tools to analyze behavior in zebrafish (Danio rerio). Pharmacol Biochem Behav 106: 137-42. Suzuki, H., Ito, Y., Shinohara, M., Yamashita, S., Ichinose, S., Kishida, A., Oyaizu, T., Kayama, T., Nakamichi, R., Koda, N., et al. (2016). Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis. Proc Natl Acad Sci U S A 113(28): 7840-7845. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Mechanobiology Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,540	https://bio-protocol.org/en/bpdetail?id=4540&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed OxiDIP-Seq for Genome-wide Mapping of Damaged DNA Containing 8-Oxo-2'-Deoxyguanosine FG Francesca Gorini GS Giovanni Scala SA Susanna Ambrosio BM Barbara Majello SA Stefano Amente Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4540 Views: 1265 Reviewed by: Istvan Boldogh Avinash Chandra Pandey Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nucleic Acids Research Apr 2022 Abstract 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is considered to be a premutagenic DNA lesion generated by 2'-deoxyguanosine (dG) oxidation due to reactive oxygen species (ROS). In recent years, the 8-oxodG distribution in human, mouse, and yeast genomes has been underlined using various next-generation sequencing (NGS)–based strategies. The present study reports the OxiDIP-Seq protocol, which combines specific 8-oxodG immuno-precipitation of single-stranded DNA with NGS, and the pipeline analysis that allows the genome-wide 8-oxodG distribution in mammalian cells. The development of this OxiDIP-Seq method increases knowledge on the oxidative DNA damage/repair field, providing a high-resolution map of 8-oxodG in human cells. Keywords: 8-oxo-7,8-dihydro-2′-deoxyguanosine 8-oxodG DNA oxidation DNA damage OxiDIP-Seq Background DNA, as a highly dynamic molecule, is constantly exposed to mutation events (Lindahl, 1993). Among DNA mutant factors, reactive oxygen species (ROS) are the most prominent source of DNA modifications that may trigger processes such as neurodegeneration (Kim et al., 2015), ageing (Beckman and Ames, 1998), and cancer (Klaunig and Kamendulis, 2004). The 2'-deoxyguanosine (dG) is the most ROS-targeted nucleobase due to its lower oxidation potential (Steenken and Jovanovic, 1997; Baik et al., 2001). The best characterized product of dG oxidation induced by ROS is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG; Figure 1A) (Baik et al., 2001; Cooke et al., 2003; Evans and Cooke, 2004; van Loon et al., 2010). This altered base is chemically generated by C8 oxidation and subsequent addition of a hydrogen atom on the N7 in the imidazole ring of deoxyguanosine. The 8-oxodG is considered a premutagenic DNA lesion that may bond 2′-deoxyadenosine, thus causing a dC:dG to dA:dT premutagenic transversion (Shibutani et al., 1991; Maga et al., 2007; Batra et al., 2010; Koga et al., 2013; Boiteux et al., 2017). Human cells can survive this mutational phenomenon through the base excision repair (BER), a multi-layer defence machinery (Lindahl, 1990; Lindahl and Barnes, 2000). The OGG1 glycosylase/AP(apurinic/apyrimidinic) lyase initiates the BER pathway, specifically recognizing and removing the 8-oxodGs from the sugar backbone. The OGG1 activity creates an abasic site, which is subsequently incised by either the OGG1 intrinsic AP lyase activity, or by the AP endonuclease 1, APE1. The lyases’ activity generates a single strand DNA (ssDNA) break. Then, the short patch BER carries on with the gap filling, mediated by the DNA polymerase beta, while the long patch BER generates a 5′ overhanging flap that is removed by FEN1 (flap structure–specific endonuclease 1). Finally, the DNA ligase I (short patch) or DNA ligase III (long patch) complete the repair process by fixing the nicked strand (Frosina et al., 1996; Fortini et al., 1999; van Loon et al., 2010). Several next-generation sequencing (NGS)–based strategies have recently been developed by different laboratories to provide a high-resolution mapping of 8-oxodG in human and mouse genomes (Ding et al., 2017; Poetsch et al., 2018; Wu et al., 2018; Amente et al., 2019; Liu et al., 2019, 2019; Cao et al., 2020; Fang and Zou, 2020; Poetsch, 2020). The previous study developed a highly sensitive methodology named OxiDIP-Seq to isolate and map oxidized DNA fragments in mammalian cells (Amente et al., 2019; Gorini et al., 2020; Scala et al., 2022). OxiDIP-Seq combines the immuno-precipitation of single-stranded DNA through specific anti-8-oxodG antibodies with NGS, with a resolution of approximately 200–300 bp (Amente et al., 2019; Gorini et al., 2020; Scala et al., 2022). Briefly, with this methodology, genomic DNA containing 8-oxodG residues were first extracted and then fragmented by sonication. The DNA fragments were then denatured at 95 °C to expose the 8-oxodG on the single-stranded DNA. Then, the single-stranded DNA containing 8-oxodG residues was immunoprecipitated using a specific antibody targeting 8-oxodG. The so formed immunocomplexes were then pulled down through specific magnetic beads. The eluted DNA was enriched in fragments containing 8-oxodGs that can be analyzed by qPCR and/or sequenced by high-throughput sequencing (Figure 1B). In this protocol, crucial steps were carried out in low light conditions and in the presence of free radical scavengers to preserve the oxidized state of DNA extracted from cells and to prevent the introduction of possible new nonspecific 8-oxodGs during DNA handling. Figure 1. OxiDIP-Seq technique. (A) ROS oxidation of 2′-deoxyguanosine generates 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodG). (B) Schematic protocol of the OxiDIP-Seq technique. Sequencing was performed adopting the SBS (sequencing by synthesis) chemistry and using the Illumina HiSeq 2000 platform. Reads with an average length of 50 bp were obtained. Raw sequenced data were collected in a FASTQ file and subjected to further analyses. With the OxiDIP-Seq approach, it has been demonstrated that in the genome of normal human cells 42% of the identified 8-oxodG peaks map within gene loci, specifically in the promoter and gene body regions (Amente et al., 2019). Moreover, it has been revealed that 8-oxodG regions show a specific G4-enrichment and that there is a complex relationship between 8-oxodG and guanine–cytosine (GC) content. In particular, the promoter regions with high (>47%) GC content display low levels of 8-oxodG (Gorini et al., 2020). This suggests that other mechanisms, such as the epigenetic regulation of transcription and replication, may be involved in the accumulation of 8-oxodG (Amente et al., 2019; Gorini et al., 2020). Moreover, a set of oxidized enhancers in human epithelial cells have been recently identified and characterized, which could be classified as super-enhancers. Specifically, it has been demonstrated that these oxidized enhancers are associated with bidirectional-transcribed enhancer RNAs and DNA damage response activation. Additionally, it has been revealed that the oxidized enhancer is physically associated with promoter regions in specific CTCF-mediated chromatin loops (Scala et al., 2022). In conclusion, the OxiDIP-Seq technique allowed us to demonstrate that 8-oxodG accumulation in enhancers–promoters regions occur in a transcription-dependent manner, providing novel mechanistic insights on the intrinsic fragility of chromatin loops containing oxidized enhancers–promoters interactions (Scala et al., 2022). Materials and Reagents 100 mm2 plate for cell culture (Corning, catalog number: CC430167) 96-well plate for StepOnePlusTM Real-Time PCR System Primo® filter pipette tips, 0.5–10 μL (Euroclone, catalog number: ECTD00010) Corning® filtered polypropylene IsoTipTM pipet tips, 1–200 μL (Corning, catalog number: 4810) Corning® filtered polypropylene IsoTipTM pipet tips, 100–1,000 μL (Corning, catalog number: 4809) 1.5 mL Eppendorf safe-lock tubes (Eppendorf, catalog number: 0030 120.086) InvitrogenTM QubitTM assay tubes (Invitrogen, catalog number: Q32856) PBN, N-tert-butyl-α-phenylnitrone (Sigma-Aldrich, catalog number: B7263) DNeasy Blood & Tissue kit (QIAGEN, catalog number: 69504) Ethylenediaminetetraacetic acid (EDTA) solution, 0.5 M in H2O (Sigma-Aldrich, catalog number: E7889) Agarose low EEO (agarose standard) (AppliChem, catalog number: A2114) 8-hydroxydeoxyguanosine antibody (Millipore, catalog number: AB5830) Dynabeads® magnetic beads protein G (Thermo Fisher Scientific, catalog number: 10003D) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: B6917-100MG) Mini Elute® PCR purification kit (QIAGEN, catalog number: 28004) Random primers DNA labeling system (Thermo Fisher Scientific, catalog number: 18187-013) QubitTM dsDNA HS assay kit (Invitrogen, catalog number: Q32851) Nuclease-free water (not DEPC-treated) (Ambion, catalog number: AM9930) Proteinase K, recombinant, PCR grade (Thermo Fisher Scientific, catalog number: EO0492) TruSeq ChIP library preparation kit (Illumina) N-acetyl cysteine (see Recipes) NaPi buffer (1 M, pH 7.4) (see Recipes) Adjust buffer (see Recipes) Tris EDTA (TE) buffer (see Recipes) IP buffer (see Recipes) Washing buffer (see Recipes) Elution buffer (see Recipes) Notes: Pipette tips used in this protocol should be low-retention, RNase-free, and DNase-free, with aerosol filter. All steps have to be performed using these tips. PCR tubes and microtubes used in this protocol should be low-retention, RNase-free, and DNase-free. All steps have to be performed using these tubes. Optional: MCF10A cell line (ATCC, catalog number: CRL-10317) Dulbecco's modified Eagle medium (DMEM) (Euroclone, catalog number: ECM0728L) Ham's nutrient mixture F-12 without L-glutamine (Euroclone, catalog number: ECB7502L) Horse serum (Euroclone, catalog number: ECS0090L) Hydrocortisone (Millipore, catalog number: 3867) Cholera toxin (Sigma-Aldrich, catalog number: C8052) Insulin (Sigma-Aldrich, catalog number: I5500) Recombinant human epidermal growth factor (Thermo Fisher Scientific, catalog number: PHG0311) Penicillin/streptomycin 100× (Euroclone, catalog number: ECB3001D) Trypsin-EDTA 1× in PBS w/o calcium w/o magnesium w/o phenol red (Euroclone, catalog number: ECB3052D) Dulbecco's phosphate (PBS) buffer saline w/o calcium w/o magnesium (Euroclone, catalog number: ECB4004L) N-acetyl cysteine (Sigma-Aldrich, catalog number: A7250) DNA oligos (Integrated DNA Technologies) Luna® Universal qPCR master mix (BIOLABS, catalog number: M3003) Growth medium for the MCF10A cell line (see Recipes) Equipment Qubit® 2.0 fluorometer (Thermo Fisher) Eppendorf micro centrifuge (Eppendorf, model: 5418 R) Diagenode Bioruptor® Plus B01020001 (Diagenode, model: UCD-300TM) Eppendorf® Thermomixer® R dry block heating and cooling shaker (Eppendorf, catalog number: T3442) Wide mini-sub cell GT system (Bio-Rad, catalog number: 1704405EDU) Wide mini-sub cell GT mini handcasting kit (Bio-Rad, catalog number: 1704497) PowerPac HC power supply (Bio-Rad, catalog number: 1645052EDU) Vortex mixer (IKA 3340000) PureProteomeTM magnetic stand (Millipore, catalog number: LSKMAGS08) SavantTM SpeedVacTM DNA 130 integrated vacuum concentrator system (Thermo Fisher Scientific, catalog number: 15819206) StepOnePlusTM Real-Time PCR system upgrade (Applied BiosystemsTM, catalog number: 4379216) Nanodrop microvolume spectrophotometers (Thermo Fisher Scientific) (Optional) UV crosslinker (Stratalinker, model: 1800) Software Fastqc Trimmomatic Burrows-Wheeler Aligner (BWA, version: 0.7.12-r1039, http://bio-bwa.sourceforge.net/, free) Samtools (v1.7, http://www.htslib.org/, free) Bedtools (v2.17.0, https://bedtools.readthedocs.io/en/latest/, free) Deeptools (https://deeptools.readthedocs.io/en/develop/, free) Homer (http://homer.ucsd.edu/homer/ngs/peaks.html, free) Procedure This protocol can be easily applied to any cell line. The current study also includes the optimized procedure for preparation and analysis of untreated and UV- and N-acetyl cysteine (NAC)–treated MCF10A OxiDIP-Seq samples. UV- and NAC-treated MCF10A cells could be used as a positive and negative control, respectively, as described in Amente et al. (2019), Gorini et al. (2020), and Scala et al. (2022). Compared to untreated cells, a significantly higher level of 8-oxodG has been detected in UV-treated and a lower level of 8-oxodG in NAC-treated MCF10A cells, which demonstrates the sensibility and specificity of our method to detect 8-oxodG. Please refer to the above mentioned original papers for more information. Day I Optional: Treat MCF10A cells with 1 mM NAC for 1 h, as negative control. Irradiate MCF10A cells as positive control, as follows: Remove the medium from the 100 mm2 plate and put it in a new 15 mL tube. Insert plate into the Stratalinker® UV crosslinker equipped with a 254 nm UV light. Irradiate cells by setting 40 J/m2 on the instrument. After irradiation, add the medium recovered at step 2a on the irradiated cells and incubate cells at 37 °C for 30 min. Extraction of genomic DNA Trypsinize untreated and (optionally) treated cells (at least 5 × 106 cells; optimal cell number should be experimentally determined) by adding 1 mL of 0.05% trypsin to a 100 mm2 plate. Incubate in a 5% CO2 humidified incubator at 37 °C for 10 min. Add 5 mL of growth medium to neutralize trypsin and transfer the cells to a 15 mL centrifuge tube. Centrifuge the cells at 300 × g and room temperature (RT) for 3 min. Carefully aspirate the supernatant, resuspend the cells in 200 μL PBS of 1× PBS, and add 20 μL of Proteinase K. Add 200 μL of buffer AL (see DNeasy Blood & Tissue kit), mix thoroughly by vortexing, and incubate samples at 37 °C for 4 h. Add 200 μL of ethanol 100%. Mix thoroughly by vortexing. Pipet the mixture into a DNeasy mini spin column (see DNeasy Blood & Tissue kit) placed in a 2 mL collection tube. Centrifuge at 6,000 × g (8,000 rpm) for 1 min. Discard the flow-through and collection tube. Place the DNeasy mini spin column in a new 2 mL collection tube. Add 500 μL of buffer AW1 (see DNeasy Blood & Tissue kit). Centrifuge at 6,000 × g (8,000 rpm) for 1 min. Discard the flow-through and collection tube. Place the spin column in a new 2 mL collection tube. Add 500 μL of buffer AW2 (see DNeasy Blood & Tissue kit). Centrifuge at 6,000 × g (8,000 rpm) for 1 min. Discard the flow-through and collection tube. Repeat step A12. Transfer the DNeasy mini spin column to a new 1.5 mL microcentrifuge tube. Elute the DNA by adding 50 μL of sterile nuclease-free water. Wait for 5 min and centrifuge at 6,000 × g (8,000 rpm) for 1 min. Elute the remaining DNA by adding another 50 μL of sterile nuclease-free water. Wait for 5 min and centrifuge at 6,000 × g (8,000 rpm) for 1 min. Pool together the DNA eluted from steps A15 and A16. Note: Work under low luminosity throughout as much as possible (by turning off the lights and covering the windows with a blackout panel in the laboratory), as we experienced that light might reduce the yield of 8-oxodG IP. Also, add PBN (stock 28 mM in nuclease-free water; working 0.07 mM) to each solution indicated in steps A4–A16 just before use to preserve the oxidized state of DNA extracted from cells and to prevent the introduction of possible new nonspecific 8-oxodGs during DNA handling. Moreover, it is suggested reading the recommendations and troubleshooting guide displayed in the DNeasy Blood & Tissue kit handbook. Sonication of genomic DNA Note: Make sure that the Bioruptor is cold. Quantify the eluted DNA using the Nanodrop. Transfer 5 μg of quantified DNA, in a final volume of 100 μL of TE buffer (see Recipe 3), to a new 1.5 mL tube. Sonicate using 15–20 cycles (30 s ON and 30 s OFF) in high power mode. Note: The optimal number of cycles must be determined experimentally. Use 400 ng of sonicated DNA to test the sonication on agarose gel (1.5%); a good sonication should produce fragments ranging in size between 200 and 800 bp. Keep samples on ice before and after sonication. Note: Work under low luminosity throughout as much as possible and add PBN (stock 28 mM in nuclease-free water; working 0.07 mM) to each solution indicated in steps B1–B3. Immunoprecipitation of 8-oxodG-containg ssDNA Dilute 4 μg of sonicated DNA in 500 μL of IP buffer (see Recipe 6). Take 3% as Input (15 μL), collect it in a new tube, and store it at -20 °C until step D7. Denature the samples for 5 min at 95 °C on the thermo-shaker (except for the Input that must be stored at -20 °C). Then transfer to ice for 15 min. Add 4 μL of polyclonal antibody against 8-hydroxydeoxyguanosine to the sample and incubate overnight at 4 °C under constant rotation. Block magnetic beads with BSA as follow: Put 30 μL/sample in a new 1.5 mL microcentrifuge tube. Wash the magnetic beads with 1 mL of IP buffer. Resuspend the magnetic beads in 1 mL IP buffer + BSA (0.5 mg/mL) and incubate overnight at 4 °C under constant rotation. Day II Wash the magnetic beads with 1 mL of cold IP buffer twice. Resuspend the beads in a final volume of 30 μL/sample of cold IP buffer. Add 30 μL of BSA-blocked magnetic beads to the sample of step C4 and incubate under constant rotation for 3 h in a cold room (at 4 °C). Note: Work under low luminosity throughout as much as possible for step C1–C6 and add PBN (stock 28 mM in nuclease-free water; working 0.07 mM) to each solution indicated in steps C1–C5. Wash the beads–antibody–ssDNA complex with 1 mL of washing buffer (see Recipe 7) as follows: Put the sample on the magnetic rack for 5 min. Discard the flow-through. Add 1 mL of washing buffer. Take the sample under constant rotation for 10 min in a cold room (at 4 °C). Repeat step 7 four times. Note: From step 7 onwards, work under normal light condition in the laboratory. Purification of 8-oxodG-containg ssDNA and Input DNA When washes are finished, discard the flow-through and resuspend the beads with 250 μL of elution buffer (see Recipe 8). Incubate O/N at 37 °C on the thermo-shaker (800 rpm). Day III Increase temperature to 52 °C and leave for 1 extra hour. Spin the sample for 3 min at 13,000 rpm, capture beads on magnetic rack, and transfer supernatant to new 2 mL tubes. Resuspend beads in 100 μL of elution buffer (see Recipe 8) again and re-incubate at 52 °C for 10 min on the thermo-shaker (800 rpm). Spin the sample for 3 min at 13,000 rpm, capture beads on magnetic rack, and transfer supernatant into the same 2 mL tubes previously used in step D4. Note: Elution is performed in one tube per sample to have 8-oxodG-containing ssDNA sample (IP) in a total 350 μL volume. Thaw Input DNA sample (previously stored at -20 °C, step C2) and add 160 μL of elution buffer to reach the total volume of 175 μL. Purify both IP and Input samples using Qiagen mini elute PCR purification kit: Add 1,750 μL and 875 μL of PB buffer (without pH indicator, see Qiagen mini elute PCR purification kit) to IP and Input, respectively. Vortex and spin briefly. Transfer the samples to MiniElute columns (see Qiagen mini elute PCR purification kit). Note: Only for the IP sample, as its volume is higher than what can be loaded on a single MiniElute column, split IP sample into two MiniElute columns (1,050 μL each) by loading 525 μL of IP sample on the same column twice. Spin samples for 1 min at 13,000 rpm. Discard the flow-through. Wash the columns with 750 μL of PE buffer (wash buffer containing ethanol, see Qiagen mini elute PCR purification kit). Spin samples for 1 min at 13,000 rpm. Discard the flow-through and spin again for 1 min to dry the columns as much as possible. Elute the DNA in a total volume of 30 μL of nuclease-free water by performing two elutions with 15 μL for each column. Note: For IP sample, collect the eluted DNA from the two MiniElute columns in one 1.5 mL tube (so the final volume of each IP sample is 60 μL); for Input sample, add further 30 μL nuclease-free water to reach the same IP final volume (60 μL). Optional: qPCR quality test of UV- and NAC-treated MCF10A samples Design DNA oligonucleotides using IDT (Integrated DNA Technologies) platform considering both positive and negative regions (see Table 1). Table 1. Primers for qPCR Name Primer Positive control (qPCR probe #6, Amente et al., 2019) FW_pos_ctrl CCAACATCTTAAATTTGTCAACTCTC RV_pos_ctrl TGCTGGCAGAAGTGTGATTT Negative control (qPCR probe C1, Amente et al., 2019) FW_neg_ctrl AGACACAGCCTGGGAAACC RV_neg_ctrl CATCCGTCGTGCAGACCT Load 17 μL of master mix (see Table 2) and 3 μL of sample in a 96-well plate. Notes: Perform each reaction three times to obtain a statistically significant value. We recommended StepOneTM Real-Time PCR system instrument and the proper thermocycling protocol. Table 2. Master mix Master mix components Final Concentration Amount SYBER GREEN (Luna universal) 1× 10 μL Oligos 0.2 μM 0.8 μL Nuclease-free water Up to 17 μL Double stranding (using the Invitrogen random labelling kit) Note: Only before NGS library prep. Not required if samples will be analyzed by qPCR. Vacuum dry each IP sample to a final volume of 30 μL by using Speed-vac at RT. Boil for 5 min at 95 °C and transfer to ice for 1 min. Make the following double-stranding (D-S) master mix (1×) using reagents provided by the Invitrogen random labelling kit: Reagent Amount dATP solution 0.5 mM 2.0 μL dCTP solution 0.5 mM 2.0 μL dGTP solution 0.5 mM 2.0 μL dTTP solution 0.5 mM 2.0 μL Random primers buffer mixture 1.5 μL Klenow fragment 1.0 μL dH2O 9.5 μL Total 20 μL Add 20 μL of D-S master mix to IP sample of step F2. Incubate for 3 h at 25 °C with gently mixing (usually 450 rpm). Add 5 μL of stop buffer (see Invitrogen random labelling kit) and put on ice. Purify using Mini Elute PCR purification kit following manufacturer’s instructions. Quantify dsDNA using Qubit dsDNA HS assay kit. Briefly, mix 1 μL of purified DNA (from step F7) and 199 μL of Qubit® working solution (Qubit dsDNA HS reagent 200× and Qubit dsDNA HS buffer) in a clean Qubit assay tube. Incubate at RT for 2 min and then measure the concentration in a Qubit 2.0 fluorometer. Usually, the Qubit dsDNA HS assay requires two standards. Each standard tube requires 10 µL of each Qubit standard and 190 µL of Qubit working solution. The assay is highly selective for double-stranded DNA (dsDNA) and is accurate for initial sample concentrations from 10 pg/µL to 100 ng/µL. Library preparation and sequencing Prepare NGS Library using 1–2 ng of IP or Input DNA with TruSeq ChIP sample prep kit. Perform sequencing with a throughput of 40–50 million of 50 bp single-end reads per sample using Illumina platform according to standard operating procedures. Data analysis Analyze the OxiDIP-Seq (Figure 2) as indicated in Amente et al. (2019), Gorini et al. (2020), and Scala et al. (2022). Briefly: Necessary files GRCh37 (hg19) reference fasta file GRCh37 (hg19) bwa index Filtering and mapping The following steps need to be executed for each sequenced (IP and Input) sample (Figure 2A). Perform reads quality check using FastQC. Perform reads trimming using Trimmomatic with the following parameters: -phread33 ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:30. Perform quality check of trimmed reads using FastQC. Map reads to GRCh37 (hg19) with BWA-backtrack method using “bwa aln” with default parameters. Perform SAM to BAM format conversion with SAM-tools view module with default parameters. Sort BAM file using SAM-tools sort with default parameters. Index BAM file using SAM-tools index with default parameters. Filter out unmapped reads and alignments with MPAQ smaller than 1 using SAM-tools view with the following parameters: -F 4 -q 1 -b. Filter standard chromosomes using SAM-tools view with standard chromosomes id list as input. Remove PCR duplicates using SAM-tools rmdup with default parameters. Sort BAM file using SAM-tools sort with default parameters. Generate BAM file index using SAM-tools index with default parameters. Peak calling This step needs to be executed for each sample starting from the OxiDIP and the corresponding DNA Input alignments (Figure 2B): Perform peak calling using MACS2 with default parameters. Perform peak annotation starting from narrowpeak file (step 1) using HOMER annotatePeaks.pl and specifying hg19 as genome. Genomic signal processing Signals normalization and correlation analyses are performed for each sample using DeepTools suite (Figure 2C). Estimate GC bias using DeepTools computeGCbias with default parameters on the IP and Input BAM file. If the estimation performed in step D1 reveals the presence of bias, perform GC content bias correction using DeepTools correctGCbias with default parameters on the BAM files to produce a GC corrected BAM file and proceed to step D3. Generate normalized 8-oxodG signal over the corresponding genomic input (log2 8-oxodG/Input ratio) using the DeepTools bamCompare with SES method as scaling factor and visualize the output bigwig file on a genome browser. Additionally, generate the bigwig files containing normalized reads count signal for IP and Input samples using DeepTools bamCoverage on the BAM file and choosing CPM (count per millions) as normalization method, and visualize the two signals (IP and Input) on a genome browser. Perform metagene analysis using the DeepTools computeMatrix on the bigwig files produced in steps D3–D4 with default parameters. Generate metagene heatmap using DeepTools plotHeatmap on the matrices computed in the previous step with default parameters. Only if you performed the optional steps D1 and D2 for NAC- and UV-treated cells: Perform a correlation analysis between the samples in the two experimental conditions (UV/NAC and Untreated) and between biological replicates using the DeepTools multiBamSummary command on and choosing “bins” as sub-command. Plot the correlation between the samples in the two experimental conditions (UV/NAC and Untreated) and between biological replicates using the DeepTools plotCorrelation command. Generate the bigwig-containing signal comparison between 8-oxodG signals in UV- or NAC- treated MCF10A cells using DeepTools bamCompare on the corresponding BAM files with exactScaling method as scaling factor and visualize the track on a genome browser. Perform metagene analysis using the DeepTools computeMatrix on the bigwig files produced in step D7 with default parameters. Figure 2. Workflow of bioinformatics analysis. The scheme represents sequential steps of data analysis. Recipes Growth medium for MCF10A cells Reagent Final concentration Amount DMEM/HAM’S F12 Horse serum Recombinant human EGF (100 μg/mL in sterile H2O) Hydrocortisone (10 mg/mL in ethanol) Cholera toxin (1 mg/mL in sterile H2O) n/a n/a n/a n/a n/a 95 mL 5 mL 20 μL 50 μL 10 μL Insulin (10 mg/mL in sterile H2O with 1% glacial acetic acid) n/a 100 μL N-acetyl cysteine Reagent Final concentration Amount N-acetyl cysteine n/a 100 mg Nuclease-free water n/a 1 mL TE buffer Reagent Final concentration Amount Tris-HCl (1 M, pH 8) EDTA (0.5 M, pH 8) 10 mM 1 mM 500 μL 100 μL Nuclease-free water n/a 49.4 mL Total n/a 50 mL NaPi buffer (1 M, pH 7.4) Reagent Final concentration Amount NaH2PO4 (1 M) n/a 43 mL Na2HPO4 (1 M) n/a 57 mL Total n/a 100 mL Adjust buffer Reagent Final concentration Amount NaPi buffer (1 M, pH 7.4) 110 mM 110 μL NaCl (5 M) Triton X-100 (20%) Nuclease-free water 1,540 mM 0.5% n/a 308 μL 25 μL 557 μL Total n/a 1 mL IP buffer Reagent Final concentration Amount Adjust buffer TE buffer n/a n/a 1 mL 9 mL Total n/a 10 mL Washing buffer Reagent Final concentration Amount NaPi buffer (1 M, pH 7.4) NaCl (5 M) Triton X-100 (20%) 10 mM 150 mM 0.05% 100 μL 300 μL 25 μL Nuclease-free water n/a 9.575 mL Total n/a 10 mL Elution buffer Reagent Final concentration Amount Tris-HCl (1 M, pH 8) EDTA (0.5 M, pH 8) SDS (10%) PK (20 mg/mL) 50 mM 10 mM 1% 0.5 mg/mL 250 μL 100 μL 500 μL 125 μL Nuclease-free water n/a 4,025 μL Total n/a 5,000 μL Acknowledgments Funding: POR Campania FESR 2014-2020 “SATIN” grant to S.A. AIRC IG23066 grant to B.M. GS acknowledges support from PON AIM 2014–2020 E69F19000070001. The original papers have been published in Amente et al. (2019) (DOI: 10.1093/nar/gky1152), Gorini et al. (2020) (DOI: 10.1093/nar/gkaa175), and Scala et al. (20220) (DOI: 10.1093/nar/gkac143). Competing interests The authors declare that they have no other competing interests. References Amente, S., Di Palo, G., Scala, G., Castrignano, T., Gorini, F., Cocozza, S., Moresano, A., Pucci, P., Ma, B., Stepanov, I., et al. (2019). Genome-wide mapping of 8-oxo-7,8-dihydro-2'-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells. Nucleic Acids Res 47(1): 221-236. Baik, M.-H., Silverman, J. S., Yang, I. V., Ropp, P. A., Szalai, V. A., Yang, W. and Thorp, H. H. (2001). Using Density Functional Theory To Design DNA Base Analogues with Low Oxidation Potentials. J Physical Chemistry B 105(27): 6437-6444. Batra, V. K., Beard, W. A., Hou, E. W., Pedersen, L. C., Prasad, R. and Wilson, S. H. (2010). Mutagenic conformation of 8-oxo-7,8-dihydro-2'-dGTP in the confines of a DNA polymerase active site. Nat Struct Mol Biol 17(7): 889-890. Beckman, K. B. and Ames, B. N. (1998). The free radical theory of aging matures. Physiol Rev 78(2): 547-581. Boiteux, S., Coste, F. and Castaing, B. (2017). Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: Properties and biological roles of the Fpg and OGG1 DNA N-glycosylases. Free Radic Biol Med 107: 179-201. Cao, B., Wu, X., Zhou, J., Wu, H., Liu, L., Zhang, Q., DeMott, M. S., Gu, C., Wang, L., You, D. et al. (2020). Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage. Nucleic Acids Res 48(12): 6715-6725. Cooke, M. S., Evans, M. D., Dizdaroglu, M. and Lunec, J. (2003). Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17(10): 1195-1214. Ding, Y., Fleming, A. M. and Burrows, C. J. (2017). Sequencing the Mouse Genome for the Oxidatively Modified Base 8-Oxo-7,8-dihydroguanine by OG-Seq. J Am Chem Soc 139(7): 2569-2572. Evans, M. D. and Cooke, M. S. (2004). Factors contributing to the outcome of oxidative damage to nucleic acids. Bioessays 26(5): 533-542. Fang, Y. and Zou, P. (2020). Genome-Wide Mapping of Oxidative DNA Damage via Engineering of 8-Oxoguanine DNA Glycosylase. Biochemistry 59(1): 85-89. Fortini, P., Parlanti, E., Sidorkina, O. M., Laval, J. and Dogliotti, E. (1999). The type of DNA glycosylase determines the base excision repair pathway in mammalian cells. J Biol Chem 274(21): 15230-15236. Frosina, G., Fortini, P., Rossi, O., Carrozzino, F., Raspaglio, G., Cox, L. S., Lane, D. P., Abbondandolo, A. and Dogliotti, E. (1996). Two pathways for base excision repair in mammalian cells. J Biol Chem 271(16): 9573-9578. Gorini, F., Scala, G., Di Palo, G., Dellino, G. I., Cocozza, S., Pelicci, P. G., Lania, L., Majello, B. and Amente, S. (2020). The genomic landscape of 8-oxodG reveals enrichment at specific inherently fragile promoters. Nucleic Acids Res 48(8): 4309-4324. Kim, G. H., Kim, J. E., Rhie, S. J. and Yoon, S. (2015). The Role of Oxidative Stress in Neurodegenerative Diseases. Exp Neurobiol 24(4): 325-340. Klaunig, J. E. and Kamendulis, L. M. (2004). The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol 44: 239-267. Koga, Y., Taniguchi, Y. and Sasaki, S. (2013). Synthesis of the oligoribonucleotides incorporating 8-oxo-guanosine and evaluation of their base pairing properties. Nucleosides Nucleotides Nucleic Acids 32(3): 124-136. Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362(6422): 709-715. Lindahl, T. (1990). Repair of intrinsic DNA lesions. Mutat Res 238(3): 305-311. Lindahl, T. and Barnes, D. E. (2000). Repair of endogenous DNA damage. Cold Spring Harb Symp Quant Biol 65: 127-133. Liu, Z. J., Martinez Cuesta, S., van Delft, P. and Balasubramanian, S. (2019). Sequencing abasic sites in DNA at single-nucleotide resolution. Nat Chem 11(7): 629-637. Maga, G., Villani, G., Crespan, E., Wimmer, U., Ferrari, E., Bertocci, B. and Hubscher, U. (2007). 8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins. Nature 447(7144): 606-608. Poetsch, A. R. (2020). The genomics of oxidative DNA damage, repair, and resulting mutagenesis. Comput Struct Biotechnol J 18: 207-219. Poetsch, A. R., Boulton, S. J. and Luscombe, N. M. (2018). Genomic landscape of oxidative DNA damage and repair reveals regioselective protection from mutagenesis. Genome Biol 19(1): 215. Scala, G., Gorini, F., Ambrosio, S., Chiariello, A. M., Nicodemi, M., Lania, L., Majello, B. and Amente, S. (2022). 8-oxodG accumulation within super-enhancers marks fragile CTCF-mediated chromatin loops. Nucleic Acids Res 50(6): 3292-3306. Shibutani, S., Takeshita, M. and Grollman, A. P. (1991). Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349(6308): 431-434. Steenken, S. and Jovanovic, S. V. (1997). How Easily Oxidizable Is DNA? One-Electron Reduction Potentials of Adenosine and Guanosine Radicals in Aqueous Solution. J Am Chem Soc 119(3): 617-618. van Loon, B., Markkanen, E. and Hubscher, U. (2010). Oxygen as a friend and enemy: How to combat the mutational potential of 8-oxo-guanine. DNA Repair (Amst) 9(6): 604-616. Wu, J., McKeague, M. and Sturla, S. J. (2018). Nucleotide-Resolution Genome-Wide Mapping of Oxidative DNA Damage by Click-Code-Seq. J Am Chem Soc 140(31): 9783-9787. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > Genome instability & mutation > Genetics Molecular Biology > DNA > DNA damage and repair Molecular Biology > DNA > Chromatin accessibility Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 1 Q&A The levels of 8-oxoG will vary between samples/treatments, but what amount of IP DNA should you expect out of the 4ug? 0 Answer 9 Views Feb 23, 2023 Related protocols Unscheduled DNA Synthesis at Sites of Local UV-induced DNA Damage to Quantify Global Genome Nucleotide Excision Repair Activity in Human Cells Paula J. van der Meer [...] Martijn S. Luijsterburg Feb 5, 2023 704 Views Quantification of Chromosomal Aberrations in Mammalian Cells Inés Paniagua and Jacqueline J. L. Jacobs Aug 20, 2023 1217 Views Conditional Depletion of STN1 in Mouse Embryonic Fibroblasts Sara Knowles and Weihang Chai Apr 20, 2024 2682 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,541	https://bio-protocol.org/en/bpdetail?id=4541&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource This protocol has been corrected. See the correction notice. Peer-reviewed Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli PZ Phillip Zhu RM Ryan A. Mehl RC Richard B. Cooley Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4541 Views: 2031 Reviewed by: Petru-Iulian TrasneaSrujana Samhita YadavalliNoelia Foresi Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in ACS Chemical Biology Jul 2019 Abstract This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer to the media will not affect expression yields. For “truncation-free” expression and improved yields with high flexibility of construct design, it is preferred to use the Release Factor-1 (RF1) deficient strain B95(DE3) ∆A ∆fabR ∆serB, though use of the standard RF1-containing BL21(DE3) ∆serB is also described. Both of these strains are serine auxotrophs and will not grow in standard minimal media. This protocol uses rich auto-induction media for streamlined and maximal production of homogeneously modified protein, yielding ~100–200 mg of single pSer-containing sfGFP per liter of culture. Using this genetic code expansion (GCE) approach, in which pSer is installed into proteins during translation, allows researchers to produce milligram quantities of specific phospho-proteins without requiring kinases, which can be purified for downstream in vitro studies relating to phosphorylation-dependent signaling systems, protein regulation by phosphorylation, and protein–protein interactions. Graphical abstract: Keywords: Genetic code expansion Orthogonal translation system Phosphoserine Amber suppression Protein phosphorylation Recombinant protein Background Cellular signaling pathways are tightly regulated by serine phosphorylation, yet, historically, it has been very challenging to produce site-specifically and homogenously phosphorylated proteins in sufficient quantity for biochemical, biophysical, and structural characterization. Aspartate or glutamate have often been used to mimic the negative charge of phosphoserine (pSer) and phosphothreonine; however, due to the differences in charge and structure of Asp and Glu compared to pSer, both Asp and Glu commonly fail to recapitulate the effects of authentic phosphorylation. Kinases can be used to install phosphates on proteins, but the complexities of kinase specificity, as well as their need to be phosphorylated to be active, limit their utility to the few that can be recombinantly expressed in an active form and have well-defined substrates. Further discussion of these challenges is provided in our earlier publication, on which we base the protocol described here (Zhu et al., 2019). Genetic code expansion (GCE) technologies have been developed for the translational installation of pSer in response to amber (TAG) stop codons. These technologies require a phosphoserine amino-acyl tRNA synthetase (SepRS), its cognate tRNA with an anti-codon that can suppress TAG codons (Sep-tRNACUA), and an EFTu variant (EFSep) engineered to deliver the pSer-amino-acylated tRNA to the ribosome (Park et al., 2011; Rogerson et al., 2015). Additionally, all pSer expression hosts have their serB gene knocked out, which causes free pSer amino acid to build up inside the cell to levels sufficient to feed the GCE machinery (Park et al., 2011). A few such ∆serB strains have been developed, and the choice of which to use is an important factor in the success of pSer protein expression. We recently created a healthy Release Factor-1 (RF1—the protein responsible for terminating translation at TAG codons) deficient strain of E. coli for pSer protein expression, called B95(DE3) ∆A ∆fabR ∆serB (Zhu et al., 2019). As reported by Zhu et al. (2019), by coupling this expression host with the efficient pSer GCE machinery created by Chin and colleagues (Rogerson et al., 2015), this expression system produces pSer proteins at higher yields than when using the parent RF1-containing BL21(DE3) ∆serB strain, does so without buildup of truncated protein, and has been shown to express proteins with up to five pSer residues incorporated. Because little or no truncation is observed, target proteins can be expressed with N-terminal affinity and solubility tags (e.g., SUMO, GST, MBP) without co-purification of truncated protein. This pSer protein expression chassis grows healthily (doubling time of ~40–45 min at 37 °C), and avoids misincorporation of natural amino acids caused by near-cognate suppression seen in other RF1 deficient pSer protein expression systems. Target proteins are expressed from the well-established T7 promoter expression system for high levels of protein production, and expression is compatible with auto-induction media. Hydrolysis of the pSer moiety by endogenous E. coli phosphatases during protein expression can be an issue, depending on target protein and site of incorporation, and so expression conditions and times may require additional optimization. Described below is the general workflow for expressing pSer proteins with B95(DE3) ∆A ∆fabR ∆serB, as well as with BL21(DE3) ∆serB, should users decide to use this strain. Materials and Reagents Expression strain options B95(DE3) ∆A ∆fabR ∆serB (Zhu et al., 2019). Available upon request. BL21(DE3) ∆serB (Addgene # 34929) (Park et al., 2011; Rogerson et al., 2015) Neither strain has any plasmids in them, and they are sensitive to all antibiotics. The B95 ∆A ∆fabR ∆serB strain is RF1-deficient, and so little or no truncated protein is produced. Thus, N-terminal purification tags can be used with minimal concern of co-purifying truncated protein. This strain is highly preferred when (i) expressing homomultimeric proteins, (ii) C-terminal affinity tags hinder protein function, (iii) N-terminal solubility enhancing domains are needed (e.g., SUMO, GST, MBP, etc.), or (iv) seeking to achieve improved yields of multi-site pSer incorporation. These cells grow modestly, slower than the BL21 strain (described next), but yields from this strain are at least equivalent and in many cases higher than those of the BL21 strain (Zhu et al., 2019). The BL21 (DE3) ∆serB strain contains Release Factor 1 (RF1), so truncated protein is produced along with full-length phospho-protein. To avoid co-purification of truncated protein, C-terminal rather than N-terminal purification tags are strongly recommended (Rogerson et al., 2015). Furthermore, for proteins that self-assemble to homomultimers (dimers, trimers, etc.), purification can be problematic due to the possible co-purification of truncated forms that are incorporated as subunits in the assembly. This is especially a concern when the TAG codon is near the C-terminus. These strains can be made chemically competent using the Inoue method (Green and Sambrook, 2020). Aliquots of chemically competent cells can be stored at -80 °C for at least 3 years. Do not re-freeze aliquots. The C321 ∆A strain, a different RF1-deficient strain of E. coli that has been used for recombinant pSer protein expression (Pirman et al., 2015), grows more slowly, is not compatible with T7-based protein expression systems or auto-induction media, and therefore should not be used with this protocol. Plasmids pKW2-EFSep (Addgene #173897) Machinery plasmid for pSer incorporation, chloramphenicol resistance/pBR322 origin of replication (Rogerson et al., 2015). The pKW2-EFSep machinery plasmid has the same origin of replication as standard pET/pBad/pGEX vectors. Therefore, these vectors cannot be used to express target proteins with pSer, due to origin incompatibility with pKW2-EFSep. Target proteins must be expressed from vectors with a different origin of replication, such as p15a, CloDF, or RSF. The pRBC plasmid described below is recommended for target protein expression. pRBC-sfGFP (Addgene #174075) Expresses sfGFP wild-type (wt) control protein with C-terminal His6 tag, under a T7 transcriptional promoter, and ampicillin resistance/p15a origin of replication (Zhu et al., 2019). pRBC-sfGFP 150TAG (Addgene #174076) Same as above (including the C-terminal 6x-His tag), except for the sfGFP gene containing a TAG amber stop codon at site N150. pRBC-[x] wild type (you must create) Expresses your protein of interest [x] or wild type. It is important, at least for initial tests, to clone your wild-type protein into the pRBC plasmid, to ensure it will express as expected. pRBC-[x] TAG (you must create) Your protein of interest [x] with a TAG codon at the intended site (or sites) of pSer incorporation. Media reagents and materials LB/agar media (see Recipes) ZY media (see Recipes) Non-inducing ZY Media (see Recipes) Auto-inducing ZY media (see Recipes) SOC media (see Recipes) 25× M-salts (see Recipes) 1 M MgSO4 (see Recipes) 40% (w/v) glucose (see Recipes) 50× 5052 solution (see Recipes) 5,000× Trace metals solution (optional, see Recipes) Tryptone (e.g., VWR, catalog number: 97063-386) Yeast Extract (e.g., VWR, catalog number: 97064-368) NaCl (e.g., VWR, catalog number: 97061-274) Agar (e.g., VWR, catalog number: 97064-336) MgSO4·7H2O (e.g., VWR, catalog number: 97062-134) Na2HPO4 (sodium dibasic) (e.g., VWR, catalog number: AA13437-30) KH2HPO4 (potassium monobasic) (e.g., VWR, catalog number: BDH9268) NH4Cl (e.g., VWR, catalog number: 97062-050) Na2SO4 or (NH4)2SO4 (e.g., VWR, catalog number: BDH9302 or BDH9216) α-D-glucose (e.g., VWR, catalog number: 97061-168) α-D-lactose (e.g., VWR, catalog number: 36218.A3) Glycerol (e.g., VWR, catalog number: BDH24388.320) Ampicillin (e.g., VWR, catalog number: 97061-442) Chloramphenicol (e.g., VWR, catalog number: 97061-244) Ethanol (e.g., VWR, catalog number: 89125-188) Antifoam B (e.g., J.T. Baker, catalog number: B531-05) Sodium Fluoride (e.g., VWR, catalog number: 470302-540) Sodium pyrophosphate (e.g., VWR, catalog number: JT3850-1) Sodium orthovanadate (e.g., VWR, catalog number: BT219470-25G) Phos-tag acrylamide (e.g., VWR, catalog number: 101974-086) Phosphatase inhibitor cocktail mix (e.g., Sigma, catalog number: P2850) Phos-tag agarose resin (e.g., Wako Chemicals, catalog number: AG-501) 1.7 mL Eppendorf tubes (e.g., VWR, catalog number: 87003-294) 100 mm plates (e.g., VWR, catalog number: 470210-568) 50 mL conical tubes (e.g., VWR, catalog number: 89039-656) 250 mL baffled flasks (e.g., VWR, catalog number: 89095-266) 2.8 L baffled Fernbach flasks (e.g., Sigma, catalog number: CLS44232XL) Equipment Autoclave capable of sterilizing liquid media and culturing materials at 121 °C, and of a saturated steam pressure of 15 PSI. Expression Static incubator for growing LB/agar plates (set to 37 °C) (e.g., VWR, catalog number: 97025-630) Shaker incubator for growing liquid cultures (e.g., New Brunswick I26R, Eppendorf, catalog number: M1324-0004) Shaker should be able to rotate at 200–250 rpm Refrigeration is necessary for expressions below room temperature (< 25 °C) Shaker deck should have clamps to hold 250 mL and 2.8 L Fernbach flasks Optical density 600 nm spectrophotometer (e.g., Ultrospec 10, Biochrome, catalog number: 80-2116-30) Cell Harvesting Centrifuge capable of speeds of at least 5,000 rcf and able to hold volumes commensurate with culture sizes (e.g., Beckman Coulter, model: Allegra 25R) Centrifuge bottles that can withstand centrifugal forces and hold the volume of cultures grown for harvesting cells. The type of bottles depends on the centrifuge and rotor used. Freezer (-80 °C), if cells are to be stored before protein is purified (e.g., VIP ECO ULT freezer, VWR, catalog number: 76305-596). 42 °C water bath (e.g., VWR, catalog number: 76308-830) Fluorometer capable of reading sfGFP fluorescence (excitation 488 nm/emission 512 nm). Handheld fluorometers work well for routine fluorescence reads (e.g., PicoFluor from Turner Biosystems). Procedure 4-Day expression process Day 1: Transformation Notes: Fresh double plasmid transformations must be performed for each expression. Never freeze expression cells containing plasmids; after freezing for storage, the cells will grow with the antibiotics, but they will not express protein. Do not sequentially transform plasmids. To standardize this protocol in your lab, run control expressions with sfGFP wt and sfGFP-150TAG first, to verify you can achieve the reference expression benchmarks, and ensure that pSer suppression of the TAG site is functioning as expected with a model protein. The protocol below contains information for a standard 50-mL test expression, as well as a 1-L expression, as is often used for target proteins once the system is known to be working. Expression volumes can be scaled as needed. Ensure a water bath is available at 42 °C. A water bath is important to ensure rapid heat transfer for the heat shock step. For each expression, add 2 μL each of pKW2-EFSep and the desired pRBC plasmid into a 1.7-mL Eppendorf tube. This should be ~50–200 ng of each plasmid. Place tube on ice for 5 min to pre-chill. Thaw aliquot(s) of B95(DE3) ∆A ∆fabR ∆serB or BL21(DE3) ∆serB competent cells. Add 50 μL of competent cells to each tube with plasmids, gently mix cells with plasmids, and place back on ice for 30 min. After 30 min on ice, heat shock cells by placing the tube in the water bath at 42 °C for exactly 45 s. Immediately place tube back on ice, and incubate for 2 min. Add 1 mL of SOC media. Note: make sure SOC is not contaminated from prior use. Allow cells to recover with shaking at >200 rpm at 37 °C. It is convenient to simply tape Eppendorf tubes horizontally to shaker deck. For B95(DE3) ∆A ∆fabR ∆serB, recover for 120 min. For BL21(DE3) ∆serB cells, recover for 90 min. Note: Longer recovery times for the B95(DE3) ∆A ∆fabR ∆serB are suggested, due to the slightly slower growth rates and metabolism of this strain. Plate the recovered culture onto LB/agar/ampicillin/chloramphenicol plates appropriate for the chosen expression strain: 25 μg/mL ampicillin + 7 μg/mL chloramphenicol for B95(DE3) ∆A ∆fabR ∆serB, and 100 μg/mL ampicillin + 25 μg/mL chloramphenicol for BL21(DE3) ∆serB cells. To ensure a sufficient number of colonies, plate all cells. To do this, centrifuge cells at 3000 rcf for 3 min, remove the top 900 μL of supernatant, resuspend the cell pellet by gentle pipetting in the remaining 100 μL of supernatant, then plate and spread the fully resuspended cells. Let plate(s) dry with lid partially open for ~20 min near a flame (maintaining sterility), and then incubate plate(s) upside down at 37 °C overnight. Day 2: Starter culture Notes: Volumes of media can be changed depending on the scale of expression desired. Described below are volumes for a 50-mL sfGFP test expression and a 1-L target protein expression. This protocol requires overnight non-inducing starter cultures to reach stationary phase. Starter cultures that do not reach stationary phase may not express target protein appropriately in auto-inducing media. Remove LB/agar plates from the incubator at 37 °C, and place them at room temperature or 4 °C for the day. ~20–100 colonies should have grown for B95(DE3) ∆A ∆fabR ∆serB cells (Figure 1). If no colonies grew, check that the correct antibiotic concentrations were used (25 μg/mL ampicillin, 7 μg/mL chloramphenicol). If correct concentrations were used, the cells may not have been made sufficiently competent. Note: Concentrations of antibiotics for this strain are adjusted to facilitate faster-growth on LB/agar plates. These concentrations were chosen to be approximately one quarter (25%) that used with standard strains. ~100–500 colonies should have grown for BL21(DE3) ∆serB cells (Figure 1). Figure 1. Representative LB/agar plates after co-transformation of pRBC-sfGFP and pKW2-EFSep into B95 ∆A ∆fabR ∆serB (left) and BL21(DE3) ∆serB strains and growth at 37 °C overnight (~18 h). Note that the B95 ∆A ∆fabR ∆serB strain contains ~10-fold fewer colonies, due to the lower transformation efficiency of this strain compared to BL21(DE3) ∆serB. At the end of the day (~3–5 p.m.), prepare ZY-NIM starter culture: Prepare 50 mL of ZY-NIM with appropriate antibiotics. For B95(DE3) ∆A ∆fabR ∆serB, use 50 μg/mL ampicillin and 13 μg/mL chloramphenicol. Note: Concentrations of antibiotics for this strain in liquid media are adjusted to be approximately one half (50%) of that used with standard strains. For BL21(DE3) ∆serB, use 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. Scrape several dozen colonies from overnight LB/agar plate, and use them to inoculate: For a ≥ 1 L expression: 50 mL of ZY-NIM starter culture in a 250-mL baffled flask. Add 1–2 drops of antifoam B. For a 50 mL expression: 5 mL of ZY-NIM in a 15-mL sterile culture tube. Grow starter culture(s) with shaking at ~250 rpm at 37 °C overnight. Day 3: Expression Prepare expression culture: Note: Baffled flasks are highly recommended, to ensure sufficient aeration for auto-induction cultures. Overnight ZY-NIM should have grown to saturation overnight (OD600 ~3–8). If it did not reach OD > 3, either culture did not grow for long enough or media/antibiotic concentrations may not have been correct. Prepare ZY-AIM according to the recipe below. Note: It is recommended that you make all media required in a single batch and then divide into appropriate culture flasks, to ensure all expressions contain the same media. For a 1-L expression: prepare ZY-AIM in a baffled 2.8-L Fernbach flask. For a 50-mL test expression: prepare ZY-AIM in a 250-mL baffled flask. Add antibiotics For B95(DE3) ∆A ∆fabR ∆serB, use 50 μg/mL ampicillin and 13 μg/mL chloramphenicol. For BL21(DE3) ∆serB, use 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. Add Antifoam B: ~5–7 drops for a 1-L culture, or 1–2 drops for a 50-mL culture. Add ~1% inoculum of ZY-NIM culture to the ZY-AIM culture (e.g., 10 mL into 1 L, or 0.5 mL into 50 mL). Grow at 200–250 rpm and 37 °C until OD600 is ~1.5. This should take about 3–4 h for BL21 cells, or 4–5 h for B95 cells. Note: The linear range of spectrophotometers is 0.1–1.0 when reading OD600, so a 5- or 10-fold dilution of the culture will be necessary for accurate measurements of cell density, once they grow above 1.0. Once OD600 reaches ~1.5, add more antifoam: ~5 drops for a 1-L culture; or ~1–2 drops for a 50-mL culture. Reduce temperature as needed for effective target protein expression. For sfGFP and sfGFP-150TAG expressions, cultures can be kept at 37 °C. For B95(DE3) ∆A ∆fabR ∆serB, expressions can go as low as 18 °C, but 22–25 °C may be preferable for “cold temperature” expressions. Temperatures as high as 37 °C are acceptable. For BL21(DE3) ∆serB, expressions as low as 18 °C and as high as 37 °C are acceptable. Continue shaking at 200–250 rpm for ~16–20 h. Day 4: Expression analysis and harvesting of cells sfGFP control protein expression analysis. Approximately 16–20 h after cells reached OD ~1.5; the control culture cells should be visibly green. Measure OD600 and fluorescence of both sfGFP and sfGFP-150TAG cultures using a fluorometer. Since sfGFP chromophore formation requires synthesis of full-length protein, fluorescence of whole cells provides a convenient strategy to evaluate the efficiency of sfGFP TAG codon suppression, and therefore pSer incorporation. Fluorescence can be measured with any fluorimeter capable of detecting sfGFP fluorescence (ex/em: 488/510nm). Diluting the culture 1:10 to 1:100 in a buffer (e.g., 100 μL of culture + 1,900 μL of PBS for a 1:20 dilution) prior to fluorescence measurements may be necessary to obtain a signal within the detection limits of the fluorometer. The OD600 values will vary depending on target protein. Normal values will range from ~2.5 to 15. Final OD600 values below 2 are indicative of poor cell growth or toxicity due to target protein expression. Test Expression Benchmarks: Carrying out 50-mL test expressions for the wild-type sfGFP and the sfGFP-150TAG constructs ensures that the protocol is properly implemented. The benchmark yields listed in Table 1 are based on 50-mL culture volumes, expressed exactly as described in this protocol (37 °C expression temperature, ZY-AIM). Table 1. Benchmarks for sfGFP-WT and 150TAG protein expression yields, based on culture fluorescence Strain Fluorescence of culture# mg of sfGFP per liter culture* OD600 wt sfGFP B95 9,550 (6,000–10,000) 550 (340–600) 8.0 sfGFP 150TAG B95 2,900 (2,000–3,000) 180 (120–200) 8.5 wt sfGFP BL21 8,500 (6,000–10,000) 480 (340–600) 11.0 sfGFP 150TAG BL21 1,300 (1,000–2,500) 80 (60–150) 12.1 #The range indicated in parenthesis is considered normal depending on the day and reagent preparation. Fluorescence values reported here were obtained on a hand-held PicoFluor fluorometer (Turner Biosystems) by diluting cells directly from the culture into PBS (1:20). Fluorescence values are arbitrary and will depend on the fluorometer used. Important is that the relative ratio of sfGFP-150TAG and sfGFP-WT culture fluorescence is consistent with the above values (i.e., the fluorescence of the sfGFP-150TAG protein expression cultures should be ~1/4 to 1/2 that of WT sfGFP). Yield of sfGFP in milligrams per liter was determined using a fluorescence standard curve of purified sfGFP. Harvest cells by centrifugation if OD600 > ~2 at 5,000 rcf for 15 min. Pour off culture supernatant and resuspend in appropriate buffer. The choice of buffer depends on the protein of interest, the downstream purification strategy, and the application, and should be determined by the user. Adding a cryoprotectant [e.g., 10% (v/v) glycerol] to this buffer can help to minimize adverse effects associated with freezing for sensitive or unstable proteins. The addition of phosphatase inhibitors (see Recipes) to the buffer is recommended for all steps of purification. Cells can be flash frozen in liquid nitrogen and stored at -80 °C, or you can proceed with purification. For His6 tagged proteins to be purified via TALON resin, a recommended resuspension/lysis buffer would be 50 mM Tris pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 5 mM imidazole, 50 mM NaF, 5 mM sodium pyrophosphate, and 1 mM sodium orthovanadate. Data analysis Analysis of Protein Phosphorylation For each expression, it is important to confirm faithful incorporation of pSer into the target protein. While E. coli does not harbor the extensive network of protein phosphatases found in eukaryotic cells, they do have phosphatases that can hydrolyze the target protein during protein expression, resulting in a mixture of phosphorylated and non-phosphorylated protein. The susceptibility of pSer hydrolysis depends greatly on the target protein, site of pSer incorporation, and culturing conditions. When expressing sfGFP-150TAG using the above-described methods at 37 °C, the purified protein should be 90–95% phosphorylated (see Figure 2 and Zhu et al., 2019). Several methods can be used to evaluate the phosphorylation status of the purified target protein. Phos-tag electrophoresis: Phos-tag gel electrophoresis is a modified form of SDS-PAGE, in which phosphorylated proteins migrate with attenuated electrophoretic mobility compared to their non-phosphorylated counterparts; the more sites of phosphorylation on a protein, the slower it will migrate, allowing one to distinguish between no, single-, and multi-pSer containing proteins (Kinoshita et al., 2006). Further, by measuring the density of the phosphorylated vs. non-phosphorylated protein bands, one can qualitatively evaluate the percentage of protein that is phosphorylated. Other advantages of Phos-tag include the ability to evaluate multiple samples at once, requiring only a standard SDS-PAGE electrophoresis setup, and being economical. Important to note is that wild-type (non-phosphorylated) protein must be run side-by-side with the phosphorylated protein, to observe relative shifts in electrophoretic mobility. Do not run molecular weight markers on Phos-tag gels, as they contain EDTA, which is not compatible with Phos-tag. See Figure 2, as well as Zhu et al. (2019), for examples of sfGFP control protein SDS-PAGE and Phos-tag gels. Figure 2. Assessing sfGFP phosphorylation status by Phos-tag gel electrophoresis. Phos-tag (top) and SDS-PAGE (bottom) gels of purified WT sfGFP and sfGFP-150pSer proteins using the above described expression methods at 37 °C. Because they migrate differently on Phos-tag gels, phosphorylated and non-phosphorylated proteins can be resolved from one another and the fraction of phosphorylated protein can be easily evaluated. In this example, the 150pSer-sfGFP protein samples are >90% phosphorylated, having only trace amounts of non-phosphorylated protein that migrate identically as the wild-type sfGFP protein. All proteins migrate identically on SDS-PAGE, indicating the electrophoretic shifts observed in Phos-tag are due to phosphorylation status and not differences in protein size. These samples were incubated at 95 °C for 5 min in standard Laemmli buffer prior to their loading on gel. Mass spectrometry: Whole-protein mass spectrometry (MS) can be the most convincing methodology for confirming protein phosphorylation, if facilities are available and the target protein is amenable to MS analysis. Important note: tryptic-digest followed by MS fragmentation analysis (i.e., “bottom up” MS/MS analysis) of phospho-proteins is useful for confirming pSer incorporation and the site of incorporation, but it should not be used to evaluate or make conclusions regarding the degree of phosphorylation. Troubleshooting tips if the purified target protein is partially hydrolyzed. Increase concentrations of phosphatase inhibitors in purification buffers, or consider a more extensive phosphatase inhibitor cocktail mix (e.g., Sigma #P2850). Lower temperature of expression to 18–22 °C. Shorten expression time from 16–20 h to 10–12 h, though care should be taken to ensure culture reached OD600 > 2.5 for sufficient time to allow auto-induction and expression to occur. This will result in less, but potentially better-quality protein. Filter all purification buffers through a 0.2 μm filter, to ensure they are clean and free of contaminating bacterial growth. Perform purifications at 4 °C. Develop purification strategies to separate phosphorylated protein from non-phosphorylated protein, e.g., via anion-exchange chromatography or Phos-tag agarose resin (e.g., Wako Chemicals #AG-501). Consider incorporating the non-hydrolyzable mimic of phospho-serine containing a carbon-phosphorus bond instead of the labile oxygen-phosphorus bond. This is a distinct GCE expression system, and its methodology is outside the scope of this protocol (Zhu et al., 2021). Recipes Note: Ampicillin antibiotic stocks can be made at 100 mg/mL in water. Chloramphenicol stocks can be made at 25 mg/mL in ethanol. Store in 1-mL aliquots at -20 °C. LB/agar (1 L) 10 g Tryptone, 5 g yeast extract, 5 g NaCl, 15 g agar After mixing thoroughly, autoclave on the standard liquid setting. After autoclaving, gently swirl the bottle to ensure melted agar is evenly mixed. Notes: Store LB/agar bottle in a 55 °C oven and pour plates on an as-needed basis. LB/agar can be stored in molten form for ~2 weeks, if sterility is maintained. If an oven is not available, plates can be poured with antibiotics once LB/agar is sufficiently cooled to touch. Plates can be stored at 4 °C for 3–5 days. For B95(DE3) ∆A ∆fabR ∆serB plates, use 25 μg/mL ampicillin (for pRBC plasmid) and 7 μg/mL chloramphenicol (for pKW2-EFSep plasmid). For BL21(DE3) ∆serB plates, use 100 μg/mL ampicillin (for pRBC plasmid) and 25 μg/mL chloramphenicol (for pKW2-EFSep plasmid). Starter culture media ZY-non inducing media (ZY-NIM), adapted from Studier (2005). Each component in Table 2 below should be prepared separately and sterilized separately. Once sterilized, each component can be stored separately at room temperature indefinitely, provided sterility is maintained. Components should only be mixed when at room temperature and immediately before use. Table 2. Y-NIM formulation For 100 mL ZY media 94.5 mL 1 M MgSO4 0.2 mL 25× M-Salts 4.0 mL 40% (w/v) α-D-glucose 1.25 mL Trace Metals solution (5,000×) 20 µL Ampicillin (100 mg/mL stock) 50 μL for B95 cells, 100 μL for BL21 cells Chloramphenicol (25 mg/mL stock) 50 μL for B95 cells, 100 μL for BL21 cells Trace metals are not essential, but can help when expressing proteins that require metal co-factors. ZY media (1 L) 10 g Tryptone, 5 g yeast extract Autoclave 1 M MgSO4(50 mL) 12.3 g of heptahydrate salt, add MilliQ H2O to 50 mL total volume Autoclave or sterile filter 25× M-salts (1 L) 0.625 M Na2HPO4 (sodium dibasic), 88.73 g anhydrous salt 0.625 M KH2HPO4 (potassium monobasic), 85.05 g anhydrous salt 1.25 M NH4Cl, 66.86 g 0.125 M Na2SO4, 17.75 g anhydrous salt Autoclave NOTE: If you do not have sodium sulfate but you do have ammonium sulfate, use the following alternate recipe: 0.625 M Na2HPO4 (sodium dibasic), 88.73 g 0.625 M KH2HPO4 (potassium monobasic), 85.05 g 1.0 M NH4Cl, 53.49 g 0.125 M (NH4)2 SO4, 16.52 g Autoclave 40% (w/v) glucose (50 mL) 20 g α-D-glucose, add MilliQ H2O to 50 mL total volume Autoclave or sterile filter. 5,000× Trace metals (see Table 3) Table 3. Trace metal mix formulation Amount for 30 mL stock solution Amount of 30 mL stock solution to make 50 mL of a 5,000× solution 1× media concentration CaCl2·2H2O (8.82 g) 500 μL 4 μM MnCl2·4H2O (5.93 g) 500 μL 2 μM ZnSO4·7H2O (8.62 g) 500 μL 2 μM CoCl2·6H2O (1.32 g) 500 μL 0.4 μM CuCl2 (807 mg) 500 μL 0.4 μM NiCl2 (777 g) 500 μL 0.4 μM Na2MoO4·2H2O (1.45 g) 500 μL 0.4 μM Na2SeO3 (1.03 g) 500 μL 0.4 μM H3BO3 (371 mg) 500 μL 0.4 μM FeCl3 (486 mg) 25 mL 10 μM Dilute to 50 mL with sterile water Expression media ZY-auto inducing media (ZY-AIM), adapted from (Studier, 2005). Each component in Table 4 below should be prepared separately and sterilized separately. Once sterilized, each component can be stored separately at room temperature indefinitely, provided sterility is maintained. Components should only be mixed when at room temperature and immediately before use. Note that recipes for all components, except the 50× 5052 solution, are in the section above describing “starter culture media.” Table 4. ZY-AIM formulation For 1 L ZY media 940 mL MgSO4 (1M) 2 mL 25× M-Salts 40 mL 50× 5052 solution 20 mL Trace Metals solution (5,000×) 200 µL Ampicillin (100 mg/mL stock) 500 μL for B95 cells, 1 mL for BL21 cells Chloramphenicol (25 mg/mL stock) 500 μL for B95 cells, 1 mL for BL21 cells *Trace metals are not essential but can help when expressing proteins that require metal co-factors. 50× 5052 solution (500 mL) 2.5% (w/v) α-D-glucose, 12.5 g 10% (w/v) lactose, 50 g 25% (v/v) glycerol, 125 mL of glycerol or 250 mL of a 50% solution Note: The lactose will not dissolve immediately. Heating solution in a microwave for ~2–3 min will warm solution sufficiently to dissolve lactose. One dissolved, lactose will stay in solution indefinitely. Lactose must be in solution before autoclaving. Sterilize by autoclaving Note: Due to the high viscosity of pure glycerol, it is easier to first make a 50% (v/v) glycerol solution as follows: Add an appropriately sized, clean stir bar to a 500-mL graduated cylinder. Add 250 mL of MilliQ H2O to the graduated cylinder. Place on stir plate and begin stirring water in the graduated cylinder. Slowly pour 100% glycerol into the graduated cylinder while stirring, until final volume is 500 mL. Continue stirring for 5–10 min until glycerol is thoroughly mixed. SOC media (1 mL needed for each transformation) Prepare the following individual sterile components 2× YT media: 16 g tryptone, 10 g yeast extract, 5 g NaCl per liter (autoclave) 40% (w/v) glucose (see above) 1 M MgSO4 (see above) Combine the following for 50 mL of SOC 49 mL of 2× YT 450 μL of 40% glucose (20 mM final concentration) 500 μL of 1 M MgSO4 (20 mM final concentration) Note: It is very easy to contaminate SOC. We suggest breaking this into 5 × 10-mL aliquots before use, or make smaller batches. If sterility is maintained, SOC can be stored at room temperature indefinitely. It can also be stored at -20 °C, but avoid repeated freeze/thaw cycles. Phosphatase inhibitors for protein purification STOCK SOLUTIONS 1 L of 1 M NaF (sodium fluoride) Dissolve 42 g of NaF with 900 mL of MilliQ H2O with stirring. Once dissolved, adjust to 1 L final volume. This solution is stable indefinitely at room temperature. 250 mM sodium pyrophosphate pH 7.5 (NaPPi) Dissolve 27.7 g NaPPi (dibasic, MW = 221.9) in 450 mL. pH adjust to 7.5 with 5 M NaOH or 5 M HCl. Adjust total volume to 500 mL. Aliquot 45 mL into 50 mL conicals, store frozen at -20 °C until use. 200 mM Sodium orthovanadate (Na3VO4) Add 1.85 g Na3VO4 to 45 mL of MilliQ water and dissolve with stirring. Slowly add 1 M HCl dropwise with stirring, to adjust pH to 10. Adding HCl will make the solution yellow, indicative of vanadate oligomers forming. Bring solution to a gentle boil by heating in a microwave. After boiling for 5–15 sec, the solution will become clear and colorless. Cool (on ice will speed up the process) until the solution reaches room temperature. At this point, the pH might be greater than 10. Add a small amount (several drops, with stirring) of 1 M HCl to adjust solution back to pH 10. If solution turns yellow, repeat steps (iii–v). After several cycles of boiling, cooling, and adjusting pH, the solution should reach a point where the pH stabilizes at ~10. Make 1-mL aliquots in 1.5 mL Eppendorf tubes and store activated (colorless and monomeric) Na3VO4 at -20 °C. WORKING SOLUTION Dilute inhibitor stocks into your buffers at the following working concentrations: 20–50 mM NaF 5–10 mM NaPPi 0.5–1 mM orthovanadate Acknowledgments This protocol was adapted from previous work by Zhu et al. ( 2019). This research was funded in part by the GCE4All Biomedical Technology Development and Dissemination Center, supported by National Institute of General Medical Science grant RM1-GM144227 as well as National Institutes of Health grant 1R01GM131168-01. Competing interests The authors declare no conflicts of interest or competing interests. References Green, M. R. and Sambrook, J. (2020). The Inoue Method for Preparation and Transformation of Competent Escherichia coli: "Ultracompetent" Cells. Cold Spring Harb Protoc 2020(6): 101196. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. and Koike, T. (2006). Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5(4): 749-757. Park, H. S., Hohn, M. J., Umehara, T., Guo, L. T., Osborne, E. M., Benner, J., Noren, C. J., Rinehart, J. and Söll, D. (2011). Expanding the genetic code of Escherichia coli with phosphoserine. Science 333(6046): 1151-1154. Rogerson, D. T., Sachdeva, A., Wang, K., Haq, T., Kazlauskaite, A., Hancock, S. M., Huguenin-Dezot, N., Muqit, M. M., Fry, A. M., Bayliss, R., et al. (2015). Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog. Nat Chem Biol 11(7): 496-503. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234. Zhu, P., Franklin, R., Vogel, A., Stanisheuski, S., Reardon, P., Sluchanko, N. N., Beckman, J. S., Karplus, P. A., Mehl, R. A. and Cooley, R. B. (2021). PermaPhos (Ser) : autonomous synthesis of functional, permanently phosphorylated proteins. bioRxiv. doi: 10.1101/2021.10.22.465468. Zhu, P., Gafken, P. R., Mehl, R. A. and Cooley, R. B. (2019). A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins. ACS Chem Biol 14(7): 1564-1572. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biological Engineering > Synthetic biology Biological Engineering > Biomedical engineering Biochemistry > Protein > Posttranslational modification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols A Co-purification Method for Efficient Production and Src Kinase-mediated Phosphorylation of Aplysia Cortactin Sherlene L. Brown [...] Seema Mattoo Sep 20, 2021 2159 Views A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS Sree I. Motipally [...] Saravanan Kolandaivelu Apr 20, 2023 1042 Views Preparation of Protein Lysates Using Biorthogonal Chemical Reporters for Click Reaction and in-Gel Fluorescence Analysis Yaxin Xu and Tao Peng Nov 20, 2024 363 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,542	https://bio-protocol.org/en/bpdetail?id=4542&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Fluorescence-based Approach Utilizing Self-labeling Enzyme Tags to Determine Protein Orientation in Large Unilamellar Vesicles LP Laura Charlotte Paweletz * SV Sarina Veit * TP Thomas Günther Pomorski (*contributed equally to this work) Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4542 Views: 885 Reviewed by: David PaulKumiko Okazaki Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in ACS Applied Materials & Interfaces Jun 2022 Abstract Reconstitution of membrane proteins into large unilamellar vesicles is an essential approach for their functional analysis under chemically defined conditions. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, and its assessment is important prior to functional measurements. Common approaches for determining the orientation of a membrane-inserted protein are based on limited proteolytic digest, impermeable labeling reagents for specific amino acids, or membrane-impermeable quenchers for fluorescent proteins. Here, we describe a simple site-specific fluorescent assay based on self-labeling enzyme tags to determine the orientation of membrane proteins after reconstitution, exemplified on a reconstituted SNAP-tag plant H+-ATPase. This versatile method should benefit the optimization of reconstitution conditions and the analysis of many types of membrane proteins. Graphical abstract: Keywords: Proteoliposomes Sidedness SNAP-tag Reconstitution Membrane protein Fluorescence Background Reconstitution of purified proteins into model membranes is an essential approach for investigating membrane protein function under defined conditions outside the complex cellular environment (Rigaud and Lévy, 2003; Murray et al., 2014; Amati et al., 2020). Typically, proteins are reconstituted with phospholipids into large unilamellar vesicles with 100–200 nm diameters. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, including the type of protein, the lipid composition, and the reconstitution procedure (Tunuguntla et al., 2013; Amati et al., 2020). Knowledge of the orientation distribution of the protein is of crucial significance for interpreting its function in the proteoliposomal in vitro system and for the design of proteoliposome experiments. A common approach for determining the orientation of a membrane-inserted protein is the protease-mediated cleavage (e.g., Schuette et al., 2004; Serek et al., 2004; Islam et al., 2013; Eisinger et al., 2018). This assay is based on the principle that an externally added protease can cleave only the accessible portions of the membrane-inserted protein, which requires several critical steps such as limited proteolytic digest, stopping the proteolytic reaction, and fragment analysis (e.g., by SDS-PAGE). Alternative approaches are based on impermeable labeling reagents for specific amino acids or membrane-impermeable quenchers for fluorescent proteins (e.g., Zhang et al., 2003; Marek et al., 2011; Deutschmann et al., 2022). The protocol described here takes advantage of the SNAP-tag technology (Keppler et al., 2004; Cole, 2013), whereby a protein can be labeled site-specific after expression or purification with a diversity of dyes, which are either membrane-permeable or impermeable (Figure 1). This approach can be extended to other self-labeling enzyme tags including SNAP-tag, HALO-tag, or CLIP-tag and, thus, can be easily customized for the protein of interest. In contrast to other labels such as cysteine labeling, side-specific, stoichiometric 1:1 labeling without changing the amino acid sequence is ensured. Compared to protease-based approaches, labeling with dyes of different permeability allows analysis under nondestructive conditions for the vesicle and the protein. The presented strategy will be useful for the optimization of reconstitution conditions and the analysis of many types of membrane proteins. Thus, this new assay expands the toolbox of fluorescence-based methods for the estimation of membrane protein orientation in liposomes. Figure 1. SNAP-labeling process. The membrane protein of interest is expressed with SNAP-tag enabling site-specific labeling. Based on the human DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase, the SNAP enzyme undergoes self-labeling via a covalent bond with O6-benzylguanine derivatives. Depending on the characteristics of the derivative, the labeling substrate is either membrane-permeable like SNAP-647-SIR or membrane-impermeable like SNAP-Alexa488 [chemical structures based on Lukinavičius et al. (2013) and Wilhelm et al. (2021), and drawn with https://chem-space.com]. Things to consider before starting Choice of labeling substrate Here, we used a SNAP-tagged fused membrane protein and benzylguanine-derived substrates. Labeling of the SNAP-tag is irreversible and quantitative, and thus well suited for the detection and quantitation of labeled proteins via in-gel fluorescence scanning of SDS-PAGE gels. If alternative labeling systems based on, for example, CLIP-tag or ACP-tag, are used, the according substrate has to be chosen as basis for the labeling dye. Choice of self-labeling fluorophore The assay relies on the use of a membrane-impermeable dye in combination with a membrane-permeable dye. In addition, both dyes should be selected for low spectral overlap and based on the imaging system available. Choice of lipid environment Here, we describe labeling conditions optimized for proteins reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) (9:1 mol/mol) vesicles. In other lipid environments, labeling duration and label concentrations might need adjustment. Additives in buffer A reducing agent (e.g., DTT) is added to the labeling buffer to increase labeling efficiency, due to a reactive cysteine in the SNAP-tag. However, if its presence is affecting the protein, the experiment could be tested without DTT, with optimized labeling time and label concentrations. However, under oxidizing conditions, labeling might not be achieved efficiently. The presence of chelating reagents such as EDTA should be avoided as the SNAP-tag protein contains a structural Zn2+ ion. Biological materials The exemplary detergent-solubilized SNAP-tagged membrane protein is a C-terminally truncated version (∆73) of the Arabidopsis thaliana auto-inhibited H+-ATPase isoform 2 (AHA2-SNAP) with additional StrepII and hexahistidine N-terminal tags. AHA2-SNAP was heterologously expressed in the Saccharomyces cerevisiae strain RS-72 (MATa, ade1-100 his4-519 leu2-3,112; endogenous proton pump PMA1 gene is under control of GAL1 promoter) (Cid et al., 1987) and purified via the His-tag resulting in 5–10 mg/mL protein in storage buffer (see Recipe 3), stored at -80 °C (Lanfermeijer et al., 1998). Note: The protocol can also be applied to purified membrane proteins prepared differently in combination with liposomal reconstitution procedures not based on preformed liposomes, as described here. However, these membrane proteins may have different preferences for lipids, with respect to the phospholipid headgroup and the lipid fatty acid composition. Materials and Reagents All catalog numbers provided below shall serve as guide; alternative sources can be used as well. Materials 2 mL disposable syringe (Henry Schein, catalog number: 9003017) Pipette tips 10 µL, 200 µL, and 1,000 µL (Sarstedt, catalog numbers: 70.760.002, 70.3030.020, and 70.3050.020) Reaction tubes 1.5 mL, 2 mL, 15 mL, and 50 mL (Sarstedt, catalog numbers: 72.690.001, 72.691, 62.554.502, and 62.547.254) Glass beads, 3 mm (Supelco, catalog number: 1040150500) Round bottom glass tube (Roth, catalog number: NY90.1) Screw cap, ND8 (Roth, catalog number: NL96.1) Screw neck ND8 vial (Roth, catalog number: KE27.1) Sterican disposable 26 gauge needle, 0.45 × 12 mm (Braun, catalog number: 4665457) Chemicals Chloroform, ethanol-stabilized and certified for absence of phosgene and HCl (Roth, catalog number: 7331.2) Methanol (VWR, catalog number: 20847.307) 4-Morpholinepropanesulfonic acid, 3-(N-Morpholino)propanesulfonic acid (MOPS) (Sigma, catalog number: M3183) Dimethylsulfoxide (DMSO) (Sigma, catalog number: 34943) KOH (Fisher Chemical, catalog number: P/5640/60) K2SO4 (VWR, catalog number: 26997.293) Sodium acetate (Sigma, catalog number: S2889) 2-(N-Morpholino)-ethane sulphonic acid (MES) (Roth, catalog number: 4256.2) Glycerol (VWR, catalog number: 24388.295) KCl (Honeywell, catalog number: 31248) Ethylenediaminetetraacetic acid (EDTA) (Sigma, catalog number: E6758) threo-1,4-dimercapto-2,3-butanediol (DTT) (Sigma, catalog number: 43819) Liquid nitrogen N-dodecyl β-maltoside (DDM) (Glycon, catalog number: D97002) N-octyl β-D-glucopyranoside (OG) (Glycon, catalog number: D97001) Sephadex G50, fine (Cytiva, catalog number: 17004201) BioBeads (Bio-Rad, catalog number: 152-3920). Bromophenol blue (Roth, catalog number: A512.3) Tris(hydroxymethyl)aminomethan (Tris) (Sigma, catalog number: T1503) Sucrose (Fisher Chemical, catalog number: S/8600/60) N,N,N′,N′-tetramethyl ethylenediamine (TEMED) (Merck, catalog number: 8.08742.0250) Ammonium peroxydisulfate (APS) (Roth, catalog number: 9592.2) Sodium dodecyl sulfate (SDS) (Sigma, catalog number: L3771) Aluminum sulfate hydrate (Roth, catalog number: 3731.1) Ethanol absolute ≥99.8% (VWR, catalog number: 20821.321) Acrylamide (Roth, catalog number: 3029.1) Orthophosphoric acid (VWR, catalog number: 20624.295) Coomassie brilliant blue G-250 (Serva, catalog number: 35050) PageRulerTM Plus Prestained Protein Ladder, 10–250 kDa (Thermo Scientific, catalog number: 26619) Reconstitution buffer (see Recipes) Protein storage buffer (see Recipes) 1 M OG stock (see Recipes) 0.5 M DTT stock (see Recipes) Sephadex G50 fine slurry (see Recipes) Pre-washed BioBeads (see Recipes) Laemmli buffer (see Recipes) 20% SDS stock (see Recipes) SDS-PAGE running buffer (see Recipes) SDS gel (see Recipes) 10% APS stock (see Recipes) 1 M Tris (see Recipes) Colloidal Coomassie staining solution (see Recipes) Colloidal Coomassie destaining solution (see Recipes) Lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) (Avanti Polar Lipids, catalog number: 840457) SNAP dyes in DMSO SNAP-Surface Alexa488 (New England BioLabs Inc., catalog number: S9129S) SNAP-Cell 647-SiR (New England BioLabs Inc., catalog number: S9102S) Note: The assay has also been successfully performed using SNAP-Surface 647 (New England BioLabs Inc., catalog number: S9136S) and SNAP-Cell Oregon Green (New England BioLabs Inc., catalog number: S9104S) Equipment Analytical balance (Sartorius Entris-i II, 220 g/0.1 mg, Buch Holm, catalog number: 4669128) Avanti mini extruder set (Avanti Polar Lipids, catalog number: 610000) Filter supporter for extruder (Polyester Drain Disc, 10 mm; Cytiva, catalog number: 230300) Membrane 200 nm for extruder (Cytiva, catalog number: 10417004) 1,000 μL gas-tight syringe (Avanti Polar Lipids, catalog number: 610017) Centrifuge with rotor for 15 and 50 mL polypropylene tubes (Eppendorf 5810 R; Wesseling, Germany) Chemidoc MP imaging system (Bio-Rad) with illumination at 460–490 nm and 520–545 nm with 530/28 nm and 695/55 nm emission filters, respectively. Eppendorf Research® plus pipettes P2.5, P20, P200, and P1000 (Eppendorf, catalog numbers: 3123000012, 3123000039, 3123000055, and 3123000063) Glass desiccator Boro 3.3 with socket in lid, 20 cm, including stopcock (BRAND GmbH, catalog number: 65238) Hamilton 700 Series syringes 25 µL, 100 µL, and 1,000 µL (Hamilton Company, Nevada, USA) Head-over-head turning device (neoLab, catalog number: 7-0045) Mini-PROTEAN® Tetra cell system (Bio-Rad, catalog number: 1658000) Vacuum Pump V-100 with interface I-100 and rotary evaporator Rotavapor® R-100, SJ29/32, V, 220–240V (Buchi, catalog numbers: 11593636, 11593655D and 11100V111, 11061895) Vortex mixer (Scientific Industries Inc., model: Vortex Genie 2, catalog number: SI-0236) Water bath (Julabo, CORIO C-BT5, catalog number: 9011305) Software ImageLab software version 5.2.1 (Bio-Rad) Procedure When using other liposomal reconstitution procedures not based on preformed liposomes as described here, skip sections A and B and start at section C. Liposome preparation Since lipids stick to plasticware, glassware has to be used until liposomes are formed. Furthermore, chloroform can extract components from plasticware, reinforcing the need to use glassware. Glass tubes as well as Hamilton syringes are used for the lipid film preparation. To reduce unwanted evaporation of chloroform during pipetting, lipid stocks are handled on ice. Note: Chloroform is a hazardous solvent. Conduct all work in a fume hood while wearing proper protective clothing. Lipids are received in chloroform packaged in sealed glass ampoules; store at -20 °C until use. Note: For long-term storage, evaporate the solvent and store the lipids at -80 °C to avoid oxidation of unsaturated lipids. To prepare lipid stocks, transfer 10 mg aliquots from the Avanti glass ampoule into glass screw neck vials. Evaporate the solvent from the glass vials in a desiccator at 250 mbar for 3 h with an additional incubation for 1 h at 30 mbar. Close the vials with screw caps and store at -80 °C until further use. Remove desired lipid stocks from freezer, place on ice, and dissolve in methanol:chloroform (1:1, v/v) to a final lipid concentration of 10 mg/mL. Note: Lipids other than POPC and POPG may have limited or very poor solubility in chloroform:methanol and require a mixture of chloroform/methanol/water. For a ratio of 9:1, transfer 450 µL of 10 mg/mL POPC and 50 µL of 10 mg/mL POPG into a grounded round bottom glass tube for a final lipid film of 5 mg. Dry lipid film in a rotary evaporator at 150 mbar until no residual solvent is visible. Go down to the lowest possible pressure (here 30 mbar) and dry lipid film for an additional 1 h. Rehydrate the lipid film (5 mg, greasy film in the bottom of the tube, appearance ranges from clear to whitish depending on lipid composition) with 334 μL of reconstitution buffer (see Recipe 1) to yield a final lipid concentration of 15 mg/mL (approximately 20 mM total phospholipid). Complete rehydration is achieved by addition of a glass bead and vortexing until no lipid film is visible on the tube bottom anymore, but for a minimum of 5 min. Note: The mixing of the lipid film with aqueous solution results in spontaneous formation of liposomes. Subject the lipid solution to five freeze-thawing cycles by placing the tube alternating into liquid nitrogen for 30 s for freezing and in a water bath at 50 °C for 90 s for thawing. Note: Wear face shield and insulating gloves when handling liquid nitrogen. Use glass tubes with high thermal shock resistance. Assemble the Avanti mini extruder according to the manual with two filter supporters on each side and two 200 nm pore sized polycarbonate membranes in between. Check tightness by flushing with 1 mL of reconstitution buffer (see Recipe 1), i.e., pass back and forth between syringes three to four times. Only continue if the buffer volume stays the same for each passing. Note: In the first passage, buffer volume can be reduced due to the void volume of the extruder. Fill one Hamilton glass syringe with approximately 334 µL of liposome solution and place the filled syringe into one end of the mini extruder. Carefully place an empty syringe into the opposite end of the mini extruder. The plunger of the empty syringe should be moved completely into the syringe barrel. Perform manual extrusion through assembled Avanti mini extruder with 21 passages. Note: Number of passages needs to be uneven to finish in the other syringe, as residual suspension in the starting syringe might have never passed the extruder. Final liposomes can be stored in 1.5 mL microcentrifuge tubes in the fridge for up to two weeks. Reconstitution Note: Reconstitution is performed here by using detergent-mediated vesicle destabilization (Rigaud et al., 1988). The method was optimized for the needs of the used protein. For detailed reviews on this topic see Amati et al. (2020). Dilute 66 μL liposome suspension (4–5 mM lipid concentration) with 124 μL of reconstitution buffer (see Recipe 1) in a 1.5 mL microcentrifuge tube. Destabilize liposomes with 9 μL of detergent (45 mM final concentration, OG stock, see Recipe 2) with 5 min head-over-head rotation. Note: Addition of the appropriate amount of detergent can be monitored by measuring light scattering of the liposome-containing solution at 600 nm using a spectrofluorometer. With increasing concentrations of detergent added to the vesicles, the absorbance starts to decrease (point of detergent saturation) until complete solubilization of the vesicles. The best stage for protein insertion for AHA2 was on the turning point between detergent saturation and total solubilization of the liposomes. Add 25 μg of purified AHA2 (in storage buffer, see Recipe 3) to the liposome–detergent mixture and incubate with head-over-head rotation for another 5 min. Meanwhile, prepare the G50 filtration column: cut two filter supporters from the side to the middle and place in a disposable 2 mL syringe without the plunger to create a stopper. Place syringe into a disposable 15 mL reaction tube, add 3 mL Sephadex G50 fine slurry (see Recipe 5), centrifuge at 180 × g for 5 min, and transfer column to a new 15 mL reaction tube (see Figure 2). Figure 2. Preparation of G50 column. (1) Two filter papers are cut from the side up to the middle. (2) After wetting the papers with the reconstitution buffer, they are inserted into a plastic syringe without the plunger. (3) The pre-soaked G50 slurry is applied to the syringe, which is placed in a 15 mL tube. (4) After centrifugation (180 × g for 5 min) the G50 column is transferred to a fresh 15 mL tube and is ready to use. Add lipid–protein–detergent suspension on top of the column and incubate for 5 min. Centrifuge at 180 × g for 8 min. Note: This size-exclusion column is trapping free detergent, i.e., OG. Transfer the flow-through to a 2 mL microcentrifuge tube. Note: The wider bottom of the 2 mL compared to the 1.5 mL tube is important for better rotation and mixing of the liquid in the next step. Add 50 mg of pre-washed BioBeads (see Recipe 6) to the sample and incubate for 60 min, with head-over-head rotation. Note: BioBeads are hydrophobic and extract the leftover detergent from the liposomes by adsorption. Punch a small hole in the bottom of the 2 mL microcentrifuge tube with a 26-gauge needle and place it in a 15 mL reaction tube. Note: The plastic is thickest at the center of the bottom; punch next to this for easier handling. Punch a hole in the lid of the 2 mL microcentrifuge tube for pressure equilibrium and centrifuge at 180 × g for 30 s to separate liposome sample from pre-washed BioBeads. Move the flow-through to a fresh 1.5 mL microcentrifuge tube. Store in the fridge for up to four days, use as soon as possible. Asymmetric protein labeling and analysis Liposomes containing the reconstituted SNAP-tagged membrane protein are labeled separately or sequentially with membrane-impermeable and permeable SNAP dyes as outlined in Figure 3. Figure 3. Double labeling assay workflow. The proteoliposome sample is split into conditions I, II, and III. Sample I is only labeled with the membrane-impermeable SNAP-Alexa488 and serves later as bleed-through correction. Sample II is labeled with the membrane-permeable SNAP-647-SIR for the 100% labeling signal. Sample III is labeled with the membrane-impermeable SNAP-Alexa488 first, to block all the outward-facing proteins, and in a second step with the cell-permeable SNAP-647-SIR to obtain a signal corresponding to the inward-facing proteins. Samples are prepared in duplicates and loaded on an SDS-PAGE. Gel is sequentially imaged for in-gel fluorescence in green and red channels and subsequently stained for total protein amount via colloidal Coomassie staining. Dilute SNAP dye stock 1:100 with reconstitution buffer supplemented with 1 mM DTT (added from a 0.5 M DTT stock, see Recipe 4) to yield a 10 µM diluted stock. Pipette 10 µL of liposome suspension (approximately 62.5–125 µg protein, 5–10 pmol) in six 1.5 mL microcentrifuge tubes. This corresponds to the three different sample types (I, II, III) in duplicates. Add the impermeable SNAP-dye in a 1:2 (mol protein:mol dye) excess (1–2 µL from 10 µM SNAP-Surface Alexa488 diluted stock) to sample I and II. Incubate for 2 h at 25 °C. Note: To achieve maximum labeling of the protein, use an excess of the dyes (at least 1:2, mol protein:mol dye) and test different incubation times until the labeling intensity does not further increase. We have experienced slower labeling with the red SNAP-Cell 647-SIR compared to the green dye SNAP-Surface Alexa488. Add membrane-permeable dye (1–2 µL from 10 µM SNAP-Cell 647-SiR diluted stock) to sample II and III. Incubate for 2 h at 25 °C. Note: Consider testing different times to reach maximum labeling. Stop reaction by adding 5 µL of Laemmli buffer (see Recipe 7). Load samples directly on 10% 1.5 mm SDS gel (see Recipe 10). Note: Membrane protein samples are not boiled before loading to prevent aggregation. Use marker for the molecular weight (e.g., 3 µL of PageRulerTM Plus Prestained Protein Ladder, 10–250 kDa). Run SDS-PAGE for approximately 2 h at 100 V, until running front is 0.25 cm from the end of the gel. Take the gel out of the cassette and image with Chemidoc imager with pre-programmed option for Alexa488 (460–490 nm, 530/28 nm filter) and Alexa647 (625–675 nm, 695/50 nm filter). Note: Adjust exposure times so that the signal is not saturated, since we want to quantify it. Due to the weak signal of some SNAP dyes exposure, times up to 10 s might be used. First, take an image of the entire gel to see the free dye; most of the actual protein–dye conjugate is not yet visible. Afterwards, cut the lower part containing the free dye. Take a second image with higher exposure time to properly visualize the protein-dye bands. Save the images as ImageLab inbuilt data format “.scn.” Exemplary results from dual color labeling experiment are shown in Figure 5. Colloidal Coomassie staining, according to Dyballa and Metzger (2009) Transfer the gel into a bowl with ddH2O and shake twice for 10 min to get rid of residual SDS. Stain shaking in Colloidal Coomassie solution (see Recipe 13) for up to 4 h. Destain for several hours with destaining solution (see Recipe 14). Use pre-programmed option for Coomassie stained gels (white trans illumination) in Chemidoc. Save the images as ImageLab inbuilt data format “.scn.” Data analysis Extract signal intensities with ImageLab software Open the gel images with the red dyes, green dyes, and the Coomassie image as “.scn” with ImageLab Version 5.2.1. From each image, extract the band intensities: Click on “Lane and bands” menu. Click on the tab “Lanes” and use the lane finder (type number of lanes in the “manual” field) to detect lanes. Adjust lane positions, fitting to the actual lane position. Note: All lanes should have an equal width, so that errors in background correction are minimized. Click on the tab “Bands” and use “Band Finder” by clicking on “detect bands” (default settings: band sensitivity – low; lanes – all). Thereby, all prominent bands are detected. Add additional bands via “Bands” – “add.” Check your result by using the “Lane Profiler”: the intensity of the bands over the lane is displayed as intensity function. Do quality control: the entire area under the band peak has to be covered by the band borders. Adjust by moving the band boundaries (see Figure 4). Note: It is important to treat all bands equally, thus the same area fraction should be chosen. To avoid mistakes by manual selection of band borders, include the entire area. Adjust the background correction by going back to the “Lanes” tab and changing “Background Subtraction.” Specify the disk size, as background correction is done by rolling ball method (see Figure 4). Do not tick the “apply to selected lane” as all lanes should be treated equal. Note: Too high disk size leads to non-separated peaks or too long tails increasing the area too much. Too low disk size might lead to cropping of the area under the peak as partly considered as background. Figure 4. Gel bands quantification in ImageLab. ImageLab graphical user interface showing the data analysis. The boundaries of the selected gel lane were adjusted so that the entire peak is covered between the boundaries. In addition, the background subtraction was done by a rolling ball correction (upper panel, blue line), optimizing the disk size to make it fit to the red line (actual signal). After iterating steps A2e and A2f until the areas under the peak are marked properly, export the peak data to an Excel file. Combine the exported Excel sheets and use the extracted volume intensities. Normalize the intensities of the SNAP dyes signal (volume intensities) by dividing by the Coomassie signal (volume intensities). Note: In more current versions of the software, the background corrected data needs to be used. In the version used here, only the background corrected value is given as Volume (Int). The quotient of inside-oriented protein is obtained by division of the signals from the membrane-permeable SNAP-Cell 647-SIR dye derived from sample III (only inside labeling) by sample II (all protein labeled). Fluorescence values of the red channel are used here after normalization to the signal of a Coomassie staining. FIII: fluorescence of the inward-labeled protein (average); FII: fluorescence of total labeled protein (average); FI: bleed-through, fluorescence of the lane without red dye (average). Note: As control for bleed-through, determine also the quotient of the signals from the membrane-permeable SNAP-Cell 647-SIR dye derived from sample I (no label) by sample II (all protein labeled). For a robust assay, bleed-through of less than 1% is recommended to ensure minimal interference with image analysis. Figure 5. Exemplary results from dual color labeling experiment. Proteoliposomes are separated in samples I, II, and III, and, after SNAP-labeling, in-gel fluorescence is measured in both channels. Subsequently, colloidal Coomassie staining is performed for normalization on total protein amount. All samples are prepared and loaded in duplicates. Sample I is labeled with the membrane-impermeable SNAP-Cell Alexa488. Sample II is labeled with the membrane-permeable SNAP-Cell 647-SIR and served as 100% protein signal. Sample III is blocked on the outside with membrane-impermeable SNAP-Surface Alexa488 first, followed by the labeling of inward-facing proteins only with the membrane-permeable SNAP-Cell 647-SIR. All fluorescence intensities are normalized on corresponding Coomassie signal. The normalized signal intensities in the SNAP-Cell 647-SIR channel are used to determine the percentage of inside-oriented protein by division of the signals derived from sample III (only inside labeling) by sample II (all protein labeled), showing here 25%. Bleed-through in the SNAP-Cell 647-SIR channel can be determined by division of the signals derived from sample I (no label) by sample II (all protein labeled), showing here less than 1%. Recipes Reconstitution buffer (1 L) 20 mM MOPS-KOH (4.185 g), pH 7, 50 mM K2SO4 (8.713 g) 1 M OG stock Dissolve 4.386 g OG to a final volume of 15 mL in ddH2O. Freeze aliquots at -20 °C. Protein storage buffer (1 L) 50 mM MES-KOH (9.76 g; pH 7), 50 mM KCl (3.728 g), 20% (v/v) glycerol, 1 mM EDTA (0.292 g), and 1 mM DTT (2 mL from 0.5 M stock) supplemented with 0.04% (w/v) DDM. 0.5 M DTT stock Dissolve 1 g of DTT in 42.81 µL of 3 M Na-acetate (pH 5.2), and add 12.93 mL of ddH2O. Freeze aliquots at -20 °C. Note: DTT is volatile; thaw and freeze directly before and after use. Sephadex G50 fine slurry Dissolve 2 g of Sephadex G50 to a final volume of 50 mL of reconstitution buffer, soak for 3 h at room temperature or overnight in the fridge. Pre-washed BioBeads Weight approximately 1 g of BioBeads in a 50 mL reaction tube. Wash twice for 10 min in methanol, twice for 10 min in ddH2O, and once for 10 min in reconstitution buffer at room temperature. BioBeads can be stored in reconstitution buffer for up to one week in the fridge. For usage, the buffer is removed to have moist BioBeads. Laemmli buffer (100 mL) 60 mM Tris (0.727 g), 0.75% (v/v) SDS (3.75 mL from 20% stock), 10 mM DTT (2 mL from 0.5 M stock), 2.6% (w/v) sucrose (2.6 g), 0.005% (w/v) bromophenol blue (0.005 g), and 2 mM EDTA (0.058 g), pH 6.8 Note: DTT is volatile; thaw and freeze directly before and after use. 20% SDS stock (100 mL) Dissolve 20 g of SDS to a total volume of 100 mL in ddH2O. SDS-PAGE running buffer (1 L) 25 mM Tris (3.029 g), 192 mM Glycine (14.413 g), and 1% SDS (50 mL from 20% stock), pH 8.6 SDS gel 10% 1.5 mm SDS-polyacrylamide separation gel (50 mL) 10% (v/v) acrylamide (5 mL), 375 mM Tris, pH 8.8 (18.75 mL of 1 M stock), 0.1% (v/v) SDS (250 µL of 20% stock), 0.1% (v/v) APS (500 µL of 10% stock), and 0.01% (v/v) TEMED (5 µL) 5% stacking gel (10 mL) 5 % (v/v) acrylamide (2.5 mL), 125 mM Tris, pH 6.8 (6.25 mL of 1 M stock), 0.1 % (v/v) SDS (250 µL of 20 % stock), 0.1 % (v/v) APS (100 µL of 10 % stock), and 0.01 % (v/v) TEMED (5 µL) 10% APS stock Dissolve 1 g to a total volume of 10 mL in ddH2O. 1 M Tris (100 mL) Dissolve 12.114 g Tris to a total volume of 100 mL in ddH2O, adjust the pH to either 8.8 or 6.8, respectively. Colloidal Coomassie staining solution (1 L) 0.02% (w/v) Coomassie brilliant blue G-250 (0.2 g), 0.02% (w/v) aluminum sulfate hydrate (0.2 g), 10% (v/v) ethanol (100 mL), and 2% (v/v) 85% orthophosphoric acid (20 mL) Colloidal Coomassie destaining solution (1 L) 10% (v/v) ethanol (100 mL), 2% (v/v) 85% orthophosphoric acid (20 mL) Acknowledgments This protocol was adapted from our previous work (Veit et al., 2022). The work was funded by grants from the German Research 545 Foundation (GU 1133/11-1) and DAAD (57386621) to TGP, LCP and SV acknowledge funding by the Studienstiftung des deutschen Volkes. We thank Huriye Deniz Uzun and Bo Højen Justesen for the generation of the AHA2-SNAP construct. All figures are prepared using Biorender.com. Competing interests The authors declare that no competing interests exist. References Amati, A. M., Graf, S., Deutschmann, S., Dolder, N. and von Ballmoos, C. (2020). Current problems and future avenues in proteoliposome research. Biochem Soc Trans 48(4): 1473-1492. Cid, A., Perona, R. and Serrano, R. (1987). Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences. Curr Genet 12(2): 105-110. Cole, N. B. (2013). Site-specific protein labeling with SNAP-tags. Curr Protoc Protein Sci 73: 30 31 31-30 31 16. Deutschmann, S., Rimle, L. and von Ballmoos, C. (2022). Rapid Estimation of Membrane Protein Orientation in Liposomes. Chembiochem 23(2): e202100543. Dyballa, N. and Metzger, S. (2009). Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp (30): 1431. Eisinger, M. L., Nie, L., Dorrbaum, A. R., Langer, J. D. and Michel, H. (2018). The Xenobiotic Extrusion Mechanism of the MATE Transporter NorM_PS from Pseudomonas stutzeri. J Mol Biol 430(9): 1311-1323. Islam, S. T., Eckford, P. D., Jones, M. L., Nugent, T., Bear, C. E., Vogel, C. and Lam, J. S. (2013). Proton-dependent gating and proton uptake by Wzx support O-antigen-subunit antiport across the bacterial inner membrane. mBio 4(5): e00678-00613. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. and Johnsson, K. (2004). Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101(27): 9955-9959. Lanfermeijer, F. C., Venema, K. and Palmgren, M. G. (1998). Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast. Protein Expr Purif 12(1): 29-37. Lukinavičius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V., Reymond, L., Correa, I. R., Jr., Luo, Z. G., et al. (2013). A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem 5(2): 132-139. Marek, M., Milles, S., Schreiber, G., Daleke, D. L., Dittmar, G., Herrmann, A., Muller, P. and Pomorski, T. G. (2011). The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity. J Biol Chem 286(24): 21835-21843. Murray, D. T., Griffin, J. and Cross, T. A. (2014). Detergent optimized membrane protein reconstitution in liposomes for solid state NMR. Biochemistry 53(15): 2454-2463. Rigaud, J. L. and Lévy, D. (2003). Reconstitution of membrane proteins into liposomes. Methods Enzymol 372: 65-86. Rigaud, J. L., Paternostre, M. T. and Bluzat, A. (1988). Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin. Biochemistry 27(8): 2677-2688. Schuette, C. G., Hatsuzawa, K., Margittai, M., Stein, A., Riedel, D., Kuster, P., Konig, M., Seidel, C. and Jahn, R. (2004). Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc Natl Acad Sci U S A 101(9): 2858-2863. Serek, J., Bauer-Manz, G., Struhalla, G., van den Berg, L., Kiefer, D., Dalbey, R. and Kuhn, A. (2004). Escherichia coli YidC is a membrane insertase for Sec-independent proteins. EMBO J 23(2): 294-301. Tunuguntla, R., Bangar, M., Kim, K., Stroeve, P., Ajo-Franklin, C. M. and Noy, A. (2013). Lipid bilayer composition can influence the orientation of proteorhodopsin in artificial membranes. Biophys J 105(6): 1388-1396. Veit, S., Paweletz, L. C., Bohr, S. S., Menon, A. K., Hatzakis, N. S. and Pomorski, T. G. (2022). Single Vesicle Fluorescence-Bleaching Assay for Multi-Parameter Analysis of Proteoliposomes by Total Internal Reflection Fluorescence Microscopy. ACS Appl Mater Interfaces 14(26): 29659-29667. Wilhelm, J., Kuhn, S., Tarnawski, M., Gotthard, G., Tunnermann, J., Tanzer, T., Karpenko, J., Mertes, N., Xue, L., Uhrig, U., et al. (2021). Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag. Biochemistry 60(33): 2560-2575. Zhang, W., Bogdanov, M., Pi, J., Pittard, A. J. and Dowhan, W. (2003). Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition. J Biol Chem 278(50): 50128-50135. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biological Engineering > Synthetic biology Biochemistry > Protein > Fluorescence Biophysics Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana Yen-Ning Chen and Cheng-Hsun Ho Aug 20, 2023 931 Views An in vitro Assay to Probe the Formation of Biomolecular Condensates Yu Zhang and Shen Lisha Sep 5, 2023 1442 Views Utilizing FRET-based Biosensors to Measure Cellular Phosphate Levels in Mycorrhizal Roots of Brachypodium distachyon Shiqi Zhang [...] Maria J. Harrison Jan 20, 2025 398 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,543	https://bio-protocol.org/en/bpdetail?id=4543&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products SN Salete M. Newton PK Phillip E. Klebba Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4543 Views: 1369 Reviewed by: Chiara Ambrogio Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Mar 2022 Abstract Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores, the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms. Graphical abstract: Keywords: Fluorescent sensor Site-directed mutagenesis Cys scanning mutagenesis Covalent modification Background The covalent modification of proteins to investigate their biochemical properties (Means and Feeney, 1971) was initially limited by the often uncertain location of amino acid side chains in protein tertiary structure, and the variable reactivity of different side chains with extrinsic chemical probes. However, the combination of protein sequence information, crystallographic depictions of protein folding, site-directed mutagenesis, and specific alkylation chemistry allowed high yield, often stoichiometric labeling of genetically engineered cysteine (Cys) sulfhydryls at strategic sites in proteins. Cys sulfhydryls are selectively modified by reagents like methanthiosulfonates, maleimides, and iodoacetamides (Means and Feeney, 1971). When combined with site-directed spin labeling experiments and electron paramagnetic resonance (EPR) spectroscopy, this approach provided information about the structure and function of membrane proteins (Altenbach et al., 1989, 1990). In addition, while exploring the properties of the major facilitator lactose permease, LacY, Kaback and colleagues adapted the method to site-directed fluorescent labeling (Nie et al., 2007; Smirnova et al., 2008). However, LacY is a Gram-negative bacterial inner membrane (IM) protein that is shielded by the barrier properties of the outer membrane [OM; Nikaido, 2003; Nikaido and Vaara, 1985] and hence recalcitrant to labeling in vivo. Our protocol focuses on fluorescent modification of engineered Cys residues on the external loops of Gram-negative bacterial OM proteins (OMPs), to transform them into sensors. This methodology enables labeling of living cells, which has many advantages, including facile preparation, high sensitivity, biochemical durability, multifunctionality, and convenient storage (Nairn et al., 2017; Chakravorty et al., 2019; Kumar et al., 2022). We also genetically engineered, purified, and fluorescently labeled binding proteins [e.g., human siderocalin (HsaSCN)] for use as soluble sensors. These manipulations transform both types of proteins into sensitive, quantitative biosensors that detect the presence of biochemicals, metabolites, natural products, enzymes, and other molecules. Materials and Reagents Appropriate growth media [e.g., MOPS minimal media (Neidhardt et al., 1974)] 50 mM NaHPO4, pH 6.7 Phosphate-buffered saline (PBS) Talon Superflow metal affinity resin (Takara, catalog number: 635670; for purification of soluble proteins) A stock solution of ~1 mM fluorescein 5' maleimide (FM; AnaSpec, catalog number: AS-81405) in anhydrous dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) 10 mM Tris-Cl, pH 8 β-mercaptoethanol (Fisher, catalog number: O3446I-100) Sensor storage: solutions containing 10% glycerol, either labeled bacterial cells in media, or purified protein buffer, respectively. Store samples frozen at -70 °C. Iron-deficient MOPS Minimal Medium (Neidhardt et al., 1974) (see Recipes) Iron-free glassware (see Recipes) 50 mM sodium phosphate, pH 6.7 (see Recipes) Phosphate-buffered saline (see Recipes) Equipment High-speed centrifuge (Beckman Coulter, model: Avanti J25) Spectrophotometer (Beckman Coulter, model: DU800) Fluorometer (OLIS Clarity; model: OLIS-SLM-Aminco 8100) and quartz fluorescence cuvettes (Fisher 14-958-128) Typhoon Biomolecular Imager (GE/Amersham, model: 8600) Procedure Overall design and engineering of site-directed fluorescent sensors This methodology is predicated on locating sites in the primary structure of OM or soluble binding proteins that tolerate Cys substitution and chemical modification with extrinsic fluorophores. Using either crystallographic data or structural models [e.g., from the MODELLER algorithm of CHIMERA (Pettersen et al., 2004)], we predict accessible sites, genetically engineer single Cys residues at those locations, and subject the resulting Cys mutant proteins to labeling with fluorophores. Regardless of the basis of such predictions, the extent of labeling and biochemical functionality of each construct must be experimentally verified. Consequently, for each protein of interest we select 4–6 candidate target residues, mutagenize them to substitute Cys, evaluate their expression, their susceptibility to alkylation with different maleimide fluorophores, and their overall sensitivity to interactions with ligands. Selection of target residues for Cys substitution mutagenesis For a protein whose tertiary or quaternary structure is fully delineated, we pick candidate residues with side chains that project near its ligand binding site, such that adsorption of a ligand may result in the quenching of an attached fluorophore. For example, Escherichia coli FepA (EcoFepA) binds the siderophore ferric enterobactin (FeEnt) in the loops of its surface vestibule (Smallwood et al., 2014). Its crystal structure (Buchanan et al., 1999) correctly predicted that Cys mutant proteins EcoFepA_S271C and EcoFepA_A698C are accessible to fluoresceination, and then sensitive to FeEnt binding (Smallwood et al., 2014). For Klebsiella pneumoniae IroN (KpnIroN), which is not yet structurally solved but has 82% identity to EcoFepA, we relied on the guideline that >25% sequence identity predicts an identical overall protein fold (Ginalski, 2006; Stokes-Rees and Sliz, 2010). To determine labeling targets in KpnIroN we used CLUSTALW to align it with EcoFepA [PDB sequence 1FEP (Buchanan et al., 1999)], and then employed the MODELLER of CHIMERA (UCSF) to predict KpnIroN tertiary structure, including surface loops. This led to the selection and engineering of mutant KpnIroN_T210C, located in L2, that is quantitatively modified by FM (Chakravorty et al., 2019). Cys mutant proteins As a general approach, we use PCR to clone a binding protein of interest from the chromosomes of a particular bacterial species. When the sensors originate from E. coli OMPs, we clone the nucleotide sequences encoding their mature proteins and insert them downstream from the native promoter of EcofepA in pITS23 (Scott et al., 2001), which is a derivative of the low-copy pHSG575 (Takeshita et al., 1987). One may generate Cys substitution mutants in proteins by a variety of methods; we use QuikChange mutagenesis (Agilent) of the wild-type genes on pITS23 (Ma et al., 2007), with complementary oligonucleotides flanking the mutation, following the manufacturer’s instructions. After confirming the mutations by sequencing (Genewiz) of purified plasmids, express and fluorescently label the sensor proteins in intact cells, that may be stored frozen at -70 °C. If the desired sensor derives from a soluble binding protein, then clone the relevant structural genes in plasmid pET28a, that adds a 6-histidine (6H)- tag at either the N- or C-termini of the mature protein, and purify it by metal ion affinity chromatography (Talon Superflow, Takara Bio Inc.). Cys mutant protein expression and analysis of fluorescence labeling Although other plasmid systems are likely also acceptable, our vector for OMP production, pITS23 (Scott, 2000), carries wild-type EcofepA under control of its native, Fur-regulated promoter. For expression of other E. coli OMP sensors, we precisely replace EcofepA in pITS23 with the alternate OMP structural gene (with its own signal sequence), such that the iron-regulated EcofepA promoter controls biosynthesis of the OM protein. For OM proteins of other bacterial species, we replace the sequence encoding mature EcoFepA with the sequence encoding the foreign, mature OM protein, downstream from the EcoFepA signal sequence, and regulated by the EcofepA promoter. This approach allows for the EcoFepA signal sequence to direct secretion and assembly of the foreign OMP in the E. coli OM. Next, choose appropriate conditions for high-level expression of the OM sensor proteins of interest. We utilize the E. coli host OKN1359, because its inability to make enterobactin (entA) or to transport iron (∆tonB) leads to overexpression of iron-regulated sensor proteins during growth in iron-deficient MOPS media (Chakravorty et al., 2019; Kumar et al., 2022). Additionally, OKN1359 is devoid of several OM proteins (∆fiu, ∆fepA, ∆cir), which facilitates expression of foreign OM proteins. Expose the Cys mutant constructs in situ, in living cells, to FM or other fluorophore maleimides (e.g., coumarin maleimides and Alexa Fluor maleimides; see below). Evaluate the expression and labeling of each cloned OMP by growing bacteria harboring the appropriate plasmid construct in MOPS medium to late log phase (A600nm = 2.5–3.5), and analyze SDS-PAGE resolved samples, or perform immunoblots of bacterial OM or soluble purified protein fractions, to visualize the production and extent of modification of the sensor proteins. For soluble binding proteins, purify a 6H-tagged Cys mutant protein from cell lysate by metal affinity chromatography (e.g., Talon Superflow, Takara Bio Inc), modify it with FM or other fluorophore maleimides, and re-purify the fluorescently labeled sensor by acetone precipitation or gel filtration chromatography. Fluorescence labeling For modifications with fluorophore maleimides, inoculate bacteria harboring plasmids that encode a Cys mutant OMP from frozen stocks into LB, grow the strain with shaking (200 rpm) at 37 °C overnight, and sub-culture at 1% into MOPS minimal media with shaking (200 rpm) at 37 °C for 10–12 h, until late exponential phase. Collect the cells by centrifugation at 7,500 × g for 15 min; wash with and resuspend in 50 mM NaHPO4, pH 6.7. Use a micropipettor tip to transfer a small amount of maleimide fluorophore (e.g., FM) powder to 0.5 mL of anhydrous DMF or DMSO, and determine the concentration of this stock solution from its absorbance at 488 nm in 10 mM Tris-Cl, pH 8 (ϵmM = 80). The stock solution may be stored at -20 °C. Calculate the appropriate dilution factor of the stock, and then label the bacterial cells or purified proteins in 50 mM NaHPO4, pH 6.7, by adding FM to a final concentration of 5 μM, at 37 °C for 15 min. Terminate the labeling reaction by adding β-mercaptoethanol to 140 μM, which instantaneously reacts with excess fluorophore maleimide. After collecting the fluoresceinated cells by centrifugation at 7,500 × g for 15 min, wash with phosphate-buffered saline (PBS), and resuspend the labeled bacteria in PBS. Immediately use the labeled cells in spectroscopic experiments or rest them on ice (up to 24 h), or add glycerol to 15% and store them (indefinitely) as 1-mL aliquots at -70 °C. In the latter case, after thawing the labeled cells, pellet them by centrifugation in a microfuge, wash them once, and resuspend them in PBS. For evaluation of protein expression or the extent of FM-labeling, solubilize aliquots of the cell suspensions with sample buffer, and subject them to SDS-PAGE (Figure 1). After electrophoresis, first visualize the extent of labeling with a fluorescence imager (Typhoon 8600GE/Amersham), and then stain the gels with Coomassie blue R (Ames, 1974). Fluorescence spectroscopic binding determinations We observe fluorophore-labeled cells in an OLIS-SLM AMINCO 8100 fluorescence spectrometer, upgraded with an OLIS operating system and software (OLIS SpectralWorks, OLIS Inc., Bogart, GA), to control its shutters, polarizers, and data collection. We also utilize an OLIS Clarity fluorescence spectrometer, with the same operating software, for fluorescence assays. For binding determinations, deposit 2.5 × 107 labeled cells in a quartz cuvette (final volume: 2 mL) with stirring at 37 °C, measure the initial fluorescence (F0), and then add increasing concentrations of a ligand, while monitoring the quenching of fluorescence emissions (F) at 520 nm. Step-by-step protocol Grow sensor strains to maximize Cys-mutant OMP expression, or purify soluble Cys-mutant binding proteins. For bacteria, determine cell density from absorbance at 600 nm; for purified proteins, determine the concentration from absorbance at 280 nm. Pellet bacterial cells by centrifugation at 7,500 × g for 15 min and resuspend in the same volume of 50 mM NaHPO4, pH 6.7. Repeat. For soluble binding proteins, dialyze the solution of purified protein (1–5 mg/mL) overnight, using appropriate molecular weight–cutoff tubing or membrane, against 50 mM NaHPO4, pH 6.7. Dissolve ~1 mg of FM in 0.5 mL of anhydrous DMF or DMSO; determine [FM] by measuring the absorbance of a 1/100 dilution at 488 nm in 10 mM Tris-Cl, pH 8 (ϵmM = 8.1). Expose bacterial cells or purified proteins to 5 μM FM (or other maleimide fluorophore) in 50 mM NaHPO4, pH 6.7, at 37 °C for 15 min, and quench the reaction with 140 μM β-mercaptoethanol. Pellet bacterial cells by centrifugation at 7,500 × g for 15 min and resuspend in the same volume of PBS. Repeat the centrifugation and resuspend the cells in PBS at ~109/mL; dialyze fluoresceinated soluble binding proteins against PBS overnight, to remove excess fluorophore. Assess the efficacy of covalent modification by diluting aliquots of fluoresceinated cells or proteins in PBS in a fluorometer; analyze with excitation at 488 nm and emission at 520 nm. Calculate the specific fluorescence of the samples (fluorescence intensity/109 cells or /mg protein). Measure the extent of fluorescence quenching by natural ligands. Use, store on ice (up to 24 h), or preserve at -70 °C in 10% glycerol. Data analysis For fluorescence spectroscopic measurements of sensor-ligand quenching, perform each measurement in triplicate, and calculate the mean value of F/F0 at each ligand concentration (Figure 1). For analysis and curve-fitting of fluorescence quenching data, plot 1-F/F0 vs. [ligand] and analyze the data by a 1-site binding model, using Grafit 6.0.12 (Erithacus Ltd. West Sussex, UK), or Enzfitter (Biosoft, Cambridge, UK), that fit data to a single site saturation curve, where the amount of ligand bound is plotted as a function of the amount free: . These plots yield KD values of for each receptor-ligand interactions, with associated standard errors. Figure 1. Representative data. A. Fluorescent labeling of Cys substitutions in E. coli FepA. Wild-type (++) EcoFepA and its seven Cys mutants were grown in MOPS media and labeled with 5 µM FM in 50 mM NaHPO4, pH 6.7, at 37 °C for 15 min. Cell lysates were resolved by SDS-PAGE (Ames, 1974), and the gel was scanned for fluorescence at 520 nm on a Typhoon imager. FM did not react with wild-type FepA, but it labeled the Cys mutants to different extents. The gel proteins were transferred to nitrocellulose and probed with α-FepA mAb 45 (Murphy et al., 1990)/[125I]-protein A to monitor expression. Certain mutants (T216C, S271C, A698C) were stoichiometrically labeled by FM. B. Analysis of fluorescence quenching during FeEnt binding to EcoFepA-A698C-FM. Cells [OKN3 (∆fepA) (Ma et al., 2007)] producing EcoFepA or its mutant A698C were exposed to FM, washed, and assayed in an OLIS/SLM-Aminco 8100 fluorometer, with excitation at 488 nm and emission at 520 nm. EcoFepA_A698C-FM showed intense fluorescence, which was quenched when 10 nM FeEnt was added at 100 s. Data points are the means of triplicate measurements; grey error bars represent the associated standard deviations of means. Notes Host bacteria OM proteins from many Gram-negative bacteria, including E. coli, K. pneumoniae (Kumar et al., 2022), Acinetobacter baumannii (Nairn et al., 2017), Caulobacteri crescentus (Balhesteros et al., 2017) and more, are susceptible to FM labeling in situ, in the cell envelopes of those living cells. However, the cell surface of laboratory E. coli is a preferred environment for expression and labeling of both native and foreign OM proteins, because it is not obscured by an LPS O-antigen nor capsular polysaccharide. Hence, we perform most labeling reactions in laboratory E. coli host strains. Protein expression It is advisable to adopt a cloning/expression strategy that maximizes production of the target OM protein. We use iron-regulated Fur promoters in conjunction with iron-deficient MOPS media, to maximize the expression of foreign proteins in E. coli. However, other promoters (e.g., lac or tac) are equally effective. Media for bacterial growth and target protein expression As a result of the iron-regulated promoters in our experiments, we utilize MOPS minimal media, that is a complete minimal media for Enterobacteriaceae (Neidhardt et al., 1974), and readily rendered iron-deficient by excluding FeSO4 and tricine from its formulation. We have no experience with the chemical modification of cells grown in other defined media or broths, but we anticipate that regardless of growth media, preliminary washing with 50 mM NaHPO4, pH 6.7, will lead to efficacious maleimide labeling reactions. Recipes Iron-deficient MOPS Minimal Medium (Neidhardt et al., 1974) 10× MOPS concentrate The following recipe is for 1 L of 10× concentrate: MOPS 83.7 g NH4Cl 5.1 g K2SO4 0.48 g CaCl2 0.56 mg MgCl2·6H2O 1.07 g NaCl 29.25 g Add MOPS to about 800 mL of double distilled H2O. Adjust pH to 7.4 with KOH pellets (~14 g), add the other components, bring the volume to 1 L, and pass through a 0.2 μm filter. DO NOT AUTOCLAVE. 1,000× Micronutrient solution The amounts are for 100 mL of 1,000× micronutrient solution. (NH4)6Mo7O24·4H2O 37 mg CoCl2·6H2O 71 mg HBO3 250 mg CuSO4·5H2O 25 mg MnCl2 160 mg ZnCl2 13.6 mg Pass through a 0.2 μm filter. DO NOT AUTOCLAVE. Dilute 1:1,000 into final media. 1,000× KH2PO4 solution 30 g in 100 mL. Dilute 1:1,000 into the final media. To prepare the final media Add 1 mL of KH2PO4 solution to 900 mL of double-distilled H2O in an iron-free flask, and autoclave. Add 100 mL of filter-sterilized 10× MOPS concentrate and 1 mL of filter-sterilized 1,000× micronutrient solution. Before inoculation, add a carbon source (e.g., 0.4% glucose), amino acids, and vitamins for auxotrophic markers, and appropriate antibiotics. Iron-free glassware To remove adventitious iron from glassware, fill the flasks or bottles with 0.1 N HCl, soak overnight at room temperature, and thoroughly rinse with distilled water. 50 mM sodium phosphate, pH 6.7 6.9 g Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) in 1 L of distilled water. Adjust pH to 6.7 with 1 M NaOH. Phosphate-buffered saline Dissolve the following in 1 L distilled water: NaCl 8 g KCl 0.2 g Na2HPO4 1.44 g KH2PO4 0.24 g Acknowledgments This research was supported by National Institutes of Health grants GM53836 and R21AI115187, and National Science Foundation grant MCB0952299 to P.E. Klebba and S.M.C. Newton. The authors thank the many undergraduate students, graduate students, and postdoctoral researchers who successfully applied these procedures in their experiments. The original research articles from which these methodologies derived are: Cao et al. (2003), Chakravorty et al. (2019), Hanson et al. (2016), Kumar et al. (2022), Ma et al. (2007), Nairn et al. (2017), Payne et al. (1997), and Smallwood et al. (2009, 2014). Competing interests The authors declare no competing financial interests. These procedures are included in part or whole in U.S. Patent No.: US 10,604,782 B2, and US Patent Application No.: US 2021/0263014 A1. Ethics The procedures reported herein do not involve human or animal subjects. References Altenbach, C., Flitsch, S. L., Khorana, H. G. and Hubbell, W. L. (1989). Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines. Biochemistry 28(19): 7806-7812. Altenbach, C., Marti, T., Khorana, H. G. and Hubbell, W. L. (1990). Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. Science 248(4959): 1088-1092. Ames, G. F. (1974). Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem 249(2): 634-644. Balhesteros, H., Shipelskiy, Y., Long, N. J., Majumdar, A., Katz, B. B., Santos, N. M., Leaden, L., Newton, S. M., Marques, M. V. and Klebba, P. E. (2017). TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA. J Bacteriol 199(6): e00723-16. Buchanan, S. K., Smith, B. S., Venkatramani, L., Xia, D., Esser, L., Palnitkar, M., Chakraborty, R., van der Helm, D. and Deisenhofer, J. (1999). Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat Struct Biol 6(1): 56-63. Cao, Z., Warfel, P., Newton, S. M. and Klebba, P. E. (2003). Spectroscopic observations of ferric enterobactin transport. J Biol Chem 278(2): 1022-1028. Chakravorty, S., Shipelskiy, Y., Kumar, A., Majumdar, A., Yang, T., Nairn, B. L., Newton, S. M. and Klebba, P. E. (2019). Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens. J Biol Chem 294(12): 4682-4692. Ginalski, K. (2006). Comparative modeling for protein structure prediction. Curr Opin Struct Biol 16(2): 172-177. Hanson, M., Jordan, L. D., Shipelskiy, Y., Newton, S. M. and Klebba, P. E. (2016). High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport. J Biomol Screen 21(3): 316-322. Kumar, A., Yang, T., Chakravorty, S., Majumdar, A., Nairn, B. L., Six, D. A., Marcondes Dos Santos, N., Price, S. L., Lawrenz, M. B., Actis, L. A., et al. (2022). Fluorescent sensors of siderophores produced by bacterial pathogens. J Biol Chem 298(3): 101651. Ma, L., Kaserer, W., Annamalai, R., Scott, D. C., Jin, B., Jiang, X., Xiao, Q., Maymani, H., Massis, L. M., Ferreira, L. C., et al. (2007). Evidence of ball-and-chain transport of ferric enterobactin through FepA. J Biol Chem 282(1): 397-406. Means, G. E. and Feeney, R. E. (1971). Chemical Modification of Proteins. Holden-Day, Inc., San Francisco, CA. Murphy, C. K., Kalve, V. I. and Klebba, P. E. (1990). Surface topology of the Escherichia coli K-12 ferric enterobactin receptor. J Bacteriol 172(5): 2736-2746. Nairn, B. L., Eliasson, O. S., Hyder, D. R., Long, N. J., Majumdar, A., Chakravorty, S., McDonald, P., Roy, A., Newton, S. M. and Klebba, P. E. (2017). Fluorescence High-Throughput Screening for Inhibitors of TonB Action. J Bacteriol 199(10): e00889-16. Neidhardt, F. C., Bloch, P. L. and Smith, D. F. (1974). Culture medium for enterobacteria. J Bacteriol 119(3): 736-747. Nie, Y., Ermolova, N. and Kaback, H. R. (2007). Site-directed alkylation of LacY: effect of the proton electrochemical gradient. J Mol Biol 374(2): 356-364. Nikaido, H. (2003). Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67(4): 593-656. Nikaido, H. and Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49(1): 1-32. Payne, M. A., Igo, J. D., Cao, Z., Foster, S. B., Newton, S. M. and Klebba, P. E. (1997). Biphasic binding kinetics between FepA and its ligands. J Biol Chem 272: 21950-21955. Scott, D. C. (2000). Mechanism of ferric enterobactin transport through Escherichia coli FepA: evolution of a bacterial venus fly trap, Department of Chemistry & Biochemistry. University of Oklahoma, Norman. Scott, D. C., Cao, Z., Qi, Z., Bauler, M., Igo, J. D., Newton, S. M. and Klebba, P. E. (2001). Exchangeability of N termini in the ligand-gated porins of Escherichia coli. J Biol Chem 276(16): 13025-13033. Smallwood, C. R., Jordan, L., Trinh, V., Schuerch, D. W., Gala, A., Hanson, M., Shipelskiy, Y., Majumdar, A., Newton, S. M. and Klebba, P. E. (2014). Concerted loop motion triggers induced fit of FepA to ferric enterobactin. J Gen Physiol 144(1): 71-80. Smallwood, C. R., Marco, A. G., Xiao, Q., Trinh, V., Newton, S. M. and Klebba, P. E. (2009). Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB. Mol Microbiol 72(5): 1171-1180. Smirnova, I. N., Kasho, V. and Kaback, H. R. (2008). Protonation and sugar binding to LacY. Proc Natl Acad Sci U S A 105(26): 8896-8901. Stokes-Rees, I. and Sliz, P. (2010). Protein structure determination by exhaustive search of Protein Data Bank derived databases. Proc Natl Acad Sci U S A 107(50): 21476-21481. Takeshita, S., Sato, M., Toba, M., Masahashi, W. and Hashimoto-Gotoh, T. (1987). High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene 61(1): 63-74. Neidhardt, F. C., Bloch, P. L. and Smith, D. F. (1974). Culture medium for enterobacteria. J Bacteriol 119(3): 736-747. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and Ferrin, T. E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25(13): 1605-1612. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > Pathogen detection > Biosensor Molecular Biology > Protein > Activity Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method Elena Maklashina and Gary Cecchini Apr 5, 2020 3682 Views In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes Hanako Nishikawa [...] Ken-ichi Nishiyama May 20, 2020 4317 Views Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE Joel Weinberger II and Christopher W. Lennon Aug 20, 2021 3134 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,544	https://bio-protocol.org/en/bpdetail?id=4544&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method OK Oleg Klykov * DB Daija Bobe * MP Mohammadreza Paraan JJ Jake D. Johnston CP Clinton S. Potter BC Bridget Carragher MK Mykhailo Kopylov AN Alex J. Noble (*contributed equally to this work) Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4544 Views: 3535 Reviewed by: Manjula MummadisettiVignesh Kasinath Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Communications Apr 2022 Abstract Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the “Waffle Method” protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100–200 nm and dimensions of approximately 12 μm width and 15–20 μm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h. Graphical abstract: Keywords: FIB-SEM HPF Waffle Method cryo-ET Sample preparation In situ AutoTEM Cryo Background Studying biological systems in close-to-native or in situ states opens wide frontiers towards precise contextual understandings of the mechanistic behaviors of molecular machines. In cryo-electron tomography (cryo-ET) pipelines, the sample is vitrified, which 1) captures and preserves biological processes in their close-to-native state, 2) maintains sample hydration, and 3) protects it from the vacuum of the electron microscope (Saibil, 2022). Vitrification is commonly followed by sample thinning with cryo-focused ion beam (cryo-FIB) milling (Marko et al., 2007) before cryo-ET data acquisition, providing the highest resolution in situ three dimensional (3D) views into the structures of individual protein complexes and cell parts in native contexts (Wang et al., 2022). Cryo-ET also allows for the assessment of the overall morphology of more complex biological systems, including cellular organelles, whole cells, intact organisms, and tissues (Bauerlein and Baumeister, 2021). However, vitrification with common plunge-freezing approaches is limited to samples up to ~5–10 μm thick. For thicker samples, a high-pressure freezer (HPF) must be used, which vitrifies specimens up to at least 60 μm thick (Harapin et al., 2015). HPF vitrification has been successfully applied to multicellular biological samples (Harapin et al., 2015), and its applicability has been extended with the waffle method (Kelley et al., 2022). The waffle method allows for 1) vitrification of samples with thicknesses of tens of microns, 2) elimination of preferred orientation issues, 3) production of large (between 10 μm × 10 μm and 70 μm × 25 μm in x,y) and specimen-dense cryo-lamellae, and 4) an increase in cryo-FIB lamellae production throughput. Specimens vitrified with an HPF cannot be directly imaged by cryo-ET and need to be thinned down to <300 nm to be transparent enough to the electron beam. During the last decade, cryo-FIB has gained substantial traction for thinning vitrified biological specimens, overtaking the promising but technologically challenging method of cryo-ultramicrotomy (McDowall et al., 1983; Marko et al., 2007). Until several years ago, HPF vitrification of large (10+ μm thick) specimens followed by cryo-FIB/SEM and cryo-ET has typically been demonstrated on multicellular C. elegans organisms (Harapin et al., 2015; Mahamid et al., 2015; Schaffer et al., 2019). Direct cryo-FIB milling of these samples requires a significant amount of continuous milling (>1 day) and has a low success rate. To increase the throughput, Mahamid et al. (2015) and Schaffer et al. (2019) developed a cryo-liftout (cryo-LO) approach, where a section of the specimen is extracted with a micro-manipulator and transferred to a special half-moon grid, for further thinning. Cryo-LO allows for region of interest (ROI) extraction, and decreases milling times per single lamella of ~150 nm thickness and a suitable imaging area of ~8 μm × 7 μm to ~10 h (Schaffer et al., 2019); however, it requires specialized milling equipment and expertise. To address these issues, we developed the waffle method, as described in Kelley et al. (2022). Here, we present a detailed protocol to support the waffle method, including sample vitrification (Procedure A), and waffle sample milling (Procedure B), that is applicable to a variety of biological samples. Samples prepared with Procedure A are generally tens of microns thick. Milling of these waffle samples does not require specialized instrumentation and can be performed manually or semi-automatically. Procedure B describes semi-automated milling, where milling takes ~2 h per lamellae site, with target thickness of 150–200 nm (as defined by the AutoTEM Cryo software), and approximate dimensions of 12 μm in width × 15–20 μm in length. Procedure B utilizes the MAPS software for defining ROIs and lamellae sites, then TFS AutoTEM Cryo automates the majority of the waffle milling process similarly to freely-available milling software packages (Buckley et al., 2020; Klumpe et al., 2021). Waffle lamellae are typically larger in cross-sectional area than lamellae generated with conventional plunge-frozen cryo-FIB-milling, and may be produced at a higher rate (~2,800 μm2 per 24 h for waffle lamellae versus ~600 μm2 per 24 h for conventional lamellae; see Notes for calculations). To increase the quality and efficiency of the protocol, and to ensure that the resulting cryo-ET tomograms contain objects of interest, ROI localization is optionally done by correlating fluorescent images of the sample before or after freezing. Cryo-fluorescent light microscopy (cryo-FLM) is used to illuminate fluorescently-tagged objects in the vitrified sample and may be performed with a stand-alone cryo-FLM, or with an FLM integrated inside the milling instrument (iFLM, Gorelick et al., 2019). Cryo-FLM can be done before (as screening), during (for FIB-milling navigation), and after FIB-milling (for guiding data collection) (Watanabe et al., 2020; Gupta et al., 2021). However, it is important to account for autofluorescence for certain samples (Carter et al., 2018). In addition to fluorescence-based approaches, two other methods for localizing or obtaining statistical information of objects of interest in situ exist, but fall outside the scope of this protocol: samples with EM-tags for localization in cryo-ET data (Silvester et al., 2021; Tan et al., 2021), and structural mass spectrometry (Klykov et al., 2022). Materials and Reagents Figure 1 shows several of the required items listed below together with common laboratory equipment. 200-mesh cryo-EM grids (e.g., SPI Supplies, catalog number: 4220C-CF) Note: Grids need to be coated with an extra ~20 nm thick carbon layer. While most commonly used grid material can be utilized, we typically use 200-mesh copper grids. Larger and smaller mesh sizes will decrease and increase lamellae dimensions, respectively. Grid foil type is not important, as it will be milled through. Gridboxes (e.g., SubAngstrom, catalog number: GBV01) Freezer planchettes (hats) type “B” (e.g., Ted Pella, catalog number: 39201) SEM pin stub (e.g., Ted Pella, catalog number: 16261) Abrasive sandpaper with grits 1,200, 7,000, 15,000, or similar (e.g., ADVcer, ADV_Sandpaper_Sheets_S01_5_10BUGY) HPF holder (e.g., Technotrade International, catalog number: 290-1) POL Metallpflege metal-polish (discontinued, substituted by, e.g., Ted Pella, catalog number: 892) Filter papers (e.g., Whatman, catalog number: 1004090) Kimwipe tissues (e.g., Kimtech, catalog number: 34155) C-clip ring (Thermo Fisher Scientific, catalog number: 1036173) Cryo-FIB AutoGrid (Thermo Fisher Scientific, catalog number: 1205101) Yeast, bacteria, mammalian cells, proteins, or protein complexes Concentrating supplies For cells or organisms, Conical Sterile Polypropylene Centrifuge Tubes (e.g., Thermo Fisher Scientific, catalog number: 339652) For purified proteins and protein complexes, protein concentrators with MWCO based on the protein size (e.g., Thermo Fisher Scientific, catalog number: 88513) Respective cell culture or yeast media Industrial grade dry nitrogen tank Industrial grade liquid nitrogen tank 1-Hexadecene (e.g., Millipore Sigma, catalog number: 822064) Figure 1. Waffle preparation instrumentation. The items from the Materials and Reagents section are shown with their corresponding numbering. For the waffle assembly, please refer to the original waffle paper by Kelley et al. (2022). Other common cryo-EM laboratory equipment included for completeness (black box on the bottom-right): P10 pipette with corresponding tips, fine-point tweezers, and AutoGrid box opening tool. Equipment Personal protective equipment for work with liquid nitrogen (e.g., gloves, eye protection, oxygen monitors) Stereoscope (e.g., Zeiss, catalog number: 455053-0000-000) Clipping station and clipping tools (e.g., Thermo Fisher Scientific, catalog number: 1000068) Pipettors and tips (10 μL, 200 μL, 1,000 μL) Centrifuge (e.g., Thermo Fisher Scientific, catalog number: 75004240) Tweezers (e.g., Ted Pella, catalog number: 5220) AutoGrid tweezers (e.g., Ted Pella, catalog number: 47000-600) Grid transfer dewar (e.g., Thomas Scientific, catalog number: 5028M40) Liquid Nitrogen dewar with 4 L capacity Liquid Nitrogen dewar or puck system for grid storage (e.g., SubAngstrom, catalog number: GSS02-E) Note: In our case, storing lamellae separated from other samples reduces contamination. CCU-010 HV high vacuum coater (e.g., Safematic, catalog number: 100001) CT-010 carbon fibre evaporation head module (e.g., Safematic, catalog number: 100003) Plasma Cleaner (e.g., Gatan Inc., Gatan Solarus II Model 955) Wohlwend Compact 01 HPF or comparable (M. Wohlwend GmbH) Cryo-FIB/SEM dual beam microscope, including relevant grid shuttles (Thermo Fisher Scientific, Aquilos 2) Note: The standard Aquilos 2 shuttle has a 45° pretilt. We found that the 27° pretilt shuttle supplied with the TFS iFLM module is more convenient for performing precleans (Procedure B, Step 30) because the pretilt allows for higher milling angles without lowering the stage. Anti-contamination cryo-shield/cryo-shutter system (e.g., Delmic CERES Ice Shield, catalog number: DM-2707-999-0003-1) Note: Such a low-contamination setup is optional. We found it useful in terms of reducing lamellae contamination during automated milling and allowing for automated overnight milling. Software FIB-milling software: Microscope operating platform. Instruments from TFS are typically operated with xT. Here, we used xT v. 20.1.1. Software for defining ROIs and aligning fluorescent signals, when applicable. Here we used MAPS v. 3.16-3.17. Software for milling automation. Here we used AutoTEM Cryo v. 2.2. Procedure Note: Working with liquid nitrogen is potentially dangerous and should be done with caution and proper protection (i.e., cryo-gloves, protective glasses, oxygen monitors, etc.). Sample Vitrification with High-Pressure Freezing (HPF) Note: For additional instructions and demonstrations of Procedure A, refer to Video 1. Video 1. Sample preparation with the waffle method. HPF sample preparation with the waffle method, including HPF planchette polishing (0:22) followed by application of 1-Hexadecene (2:25), waffle assembly in the HPF tip holder (2:50), and high pressure freezing of the waffle grid (4:10; Procedure A). For more information, including intermediate states of the planchettes, please refer to Supplementary Figure 1 in the original waffle paper (Kelley et al., 2022). Rigidify grids with an extra layer of carbon: Place grids on a glass slide, grid bar side down. Place the glass slide on the stage of a carbon evaporator, and evaporate 20 nm of carbon onto the film side of the grids. Sand and polish planchettes: Sand the flat side of Type B planchettes on 1,200, 7,000, and 15,000 grit sandpaper to get rid of the concentric rings present from manufacturing. Dip the flat side of the planchettes in POL metallpflege metal polish, and rub it by hand in circular motions on a piece of filter paper. Get rid of any excess polish with a Kimwipe tissue, then rub the planchettes on a piece of filter paper again to ensure all of the polish is gone. Coat planchettes in 1-Hexadecene: Place all planchettes flat side up on a piece of filter paper. Apply a droplet of 1-Hexadecene (4–6 μL) to the top of the planchette, then drag the pipette tip so the drop spreads onto the filter paper. Apply another droplet of 1-Hexadecene on top of the planchette. Keep the planchettes coated in 1-Hexadecene for at least 15 min before waffle assembly. Prepare a concentrated sample. Note: The sample should typically be as concentrated as possible while remaining pipette-able. However, the sample does not necessarily need to be viscous. Plasma clean carbon-rigidified grids: Plasma clean grids with the grid bar side up (extra carbon side down) using a recipe of 80% O2 gas and 20% H2 gas for 30 s, with a forward radio frequency (RF) target of 50 W. Waffle assembly: Blot away 1-Hexadecene from a planchette hat, and place it on the HPF holder tip, flat side up. Place the grid on the planchette hat, grid bar side up. Apply 5 µL of the sample to the grid. Blot away 1-Hexadecene from a second planchette hat, and place it on top of the sample, flat side down. If necessary, lightly blot away excess sample that has squeezed out of the planchette hats. Close the HPF tip. Tighten the assembly with tweezers. HPF: Note: Typically, with our setup (Wohlwend Compact 01 HPF), the HPF pressure is 2,100 bar. Transfer the assembled waffle sample to the HPF chamber. Vitrify the sample. Transfer the HPF holder into the liquid nitrogen chamber. Loosen the HPF holder tip, and remove the waffle assembly. Note: Ideally, the planchettes will separate fully from the grid. If the assembly is stuck together, use tweezers to press between the two planchettes; another pair of tweezers may be used to hold the assembly in place. If only one planchette hat separates, grab the grid by the outer rim and carefully lift it off of the other planchette hat. Clipping waffle grids Note: Waffle clipping is done in the same setup as clipping of grids of conventional samples prior to cryo-FIB/SEM thinning with the addition of a stereoscope. Mark the notch of the AutoGrid with a marker (Figure 2A). This helps with grid bar alignment when clipping. It is also helpful for orienting the lamellae with respect to the TEM tilt axis during loading. Under a stereoscope and before clipping with a c-clip ring, rotate the grid with tweezers until the grid bars are square to the notch of the AutoGrid (Figure 2B, C). It is possible that the grid moves slightly while clipping, which is not detrimental to the method, as the grid bars are aligned primarily to maximize the area within a square where a lamella can be placed. Figure 2. Waffle grid clipping after HPF. Properly orientating the grid bars helps to maximize the area within a square in which you can place a lamella. (A) Example of a cryo-FIB AutoGrid with the notch marked. The opposite side of the AutoGrid is shown with a marking that corresponds to the notch, which is visible during the clipping process. (B) Alignment of the grid bars as seen in a stereoscope without sample and (C) with sample in the grid clipping chamber. Sample Thinning using cryo-FIB Milling Notes: Steps B1–B6, B8–B12, B14–B23, B25, and B29–B30 are done in xT software, B7, B13, and B23–B24 in MAPS, and B25–B28 and B31–B32 in AutoTEM. For additional instructions and a demonstration of how to prepare lamellae sites before automated milling, refer to Video 2. The process of automated milling is demonstrated in Video 3. Electron beam (EB) settings of 2 kV and 13 pA are kept throughout the whole protocol. Ion beam (IB) starting settings are 30 kV and 10 pA. While the IB voltage is kept the same throughout the protocol, currents are changed according to the text. For imaging, we typically use an ETD detector. Video 2. Setting up milling sites. Two-screen recording of xT, MAPS, and AutoTEM windows for the site preparation before automated milling of waffle lamellae (Procedure B). After creating a new project in MAPS, a SEM grid overview is recorded (0:08), followed by platinum sputtering and gas injection system (GIS) coating inside the instrument chamber, then defining of preliminary lamellae sites (1:09). Next, the grid is positioned orthogonally to the IB, and precuts are milled for several lamellae sites (1:24). After finishing the precuts, the grid is placed back to the mapping position, and a new grid overview is recorded (3:12) and used to define final lamellae sites (3:33). Then, an AutoTEM project is created, and eucentric positions, milling angle, and preliminary lamellae positions for each site are defined (3:35). Precleans and notch milling are shown starting at 5:25. Final lamellae placement is shown at 8:40. Video 3. AutoTEM milling. Recording of automated milling in AutoTEM (Step B32d). First, Rough Milling, Medium Milling, Fine Milling, and Finer Milling are performed for each lamella (0:01). Then, Polishing 1 and Polishing 2 are performed for each lamella (0:45). Parameters are listed in Table 3. Before loading the specimen, purge the GIS lines. This step can be done while the FIB/SEM system is cooling down. No sample should be present during GIS purging. Wake up the system and switch on both beams, then make sure that the “touch alarm” is enabled. Apply a scan rotation of 180° to the EB and IB. Select the grid, then go to the predefined “Mapping” position. Lower the magnification, center on the grid, and focus the EB on the grid. Increase the magnification until one square is visible, focus the EB, and then link the Z height (press “link Z to FWD”). Create a new project and acquire an overview image of the grid in the EB: Create and name a new Layer. Create and name a new Tileset. Define Tileset parameters (Table 1). Table 1. Parameters for the acquisition of EB grid overviews in MAPS Name [Name of your Tileset] Acquisition Type Electron Tiles X,Y 5,7 Tile HFW 600 µm Total HFW 2.76 mm Resolution 1,536 × 1,024 Pixel Size 390.625 nm Dwell 1 µs Frames Check, 1 Reduced Area Uncheck Click “Prepare for sputtering” and run conductive platinum deposition with parameters from Table 2. Table 2. Platinum sputter coating parameters Current 30.0 mA Pressure 0.10 mbar Voltage Not changeable; values are 1 kV (while EB is 2 kV) Run Time 15 s Click “Recover from Sputtering” when finished to shut down the sputtering functionality (the “Run” button should be active again). Go to the predefined “Deposition” position. Add a layer of organometallic platinum (also known as GIS platinum): in the cryo GIS Deposition tab, select a grid and define the timing. With our milling settings, specifically the milling angle, 2 min of GIS works well for lamellae being milled to 200 nm thickness, or 2 min 15 s for 150 nm thickness. Go back to the “Mapping” position. Define the ROIs and preliminarily lamellae sites. Notes: For fluorescently labeled targets, ROIs and lamellae sites are defined based on the presence of fluorescence signals. This can be done either with a grid overview acquired in advance with an external cryo-FLM system, or with an internal FLM module. In both cases, MAPS allows for fluorescent image export, which is aligned with an EB grid overview. The general workflow for using fluorescence in FIB-SEM is described elsewhere, e.g., Gorelick et al. (2019). In most cases, the grid will have distinct features, which can be used for alignment (e.g., broken squares or the grid center mark). We typically perform three-point alignment. First, rough alignment is performed using three points; then, MAPS allows for fine adjustments. Alignments are usually performed after platinum sputtering and GIS coating. From the “Mapping” position, place the grid orthogonal to the IB: Notes: Make sure that “touch alarm” is enabled during all steps. Additionally, one can lower the stage, then rotate it, tilt, and move the stage back up to move to the orthogonal position. Values are indicated for the standard TFS Aquilos 2 shuttle with 45° pretilt. For systems equipped with an iFLM, the shuttle pretilt is 27°, which requires tilting the stage to 25° in Step B14a. Rotate the stage to the actual value of 108.1°, or relative 180° from the Mapping position, then tilt the stage to 7°, so the IB angle is 90° to the sample. Save the shuttle position and use this saved position for future navigation. Wake up the system, which automatically shuts down after sputtering. With the IB at 10 pA current, navigate to one of the preliminary lamellae sites defined in Step B13. Adjust the contrast so that any features on the surface of the lamella site are clearly visible, then acquire an image, and focus the IB while the image is being acquired. This will minimize charging. Check the surface quality. Ideally, the surface should be “flat” and without contamination. Locate the grid bars. If they are not visible, mill small windows into the surface at 15 nA where you expect the grid bars to be (Figure 3). If you hit two or more orthogonal grid bars, draw the corresponding lines, extending to all the squares with preliminary lamellae sites (Kelley et al., 2022). Precuts: For 200-mesh grids where the grid bars are aligned during clipping, define rectangular milling patterns in {x,y} as {22 µm, 37 µm} (top) and {20 µm, 17 µm} (bottom), with the distance between the boxes set to 25 µm. Precut patterns for xT are available as Supplementary File 1. Note: We recommend using these extensively-tested pattern dimensions while initially implementing the waffle method. Mill the patterns at 15 nA (milled precuts are shown in Figure 3D). Figure 3. Milling precuts with the corresponding timing as in Video 2. Initial milling patterns (Supplementary File 1) are placed at the region of interest (A). Prior to milling, an additional smaller pattern (yellow pattern) is used to localize the grid bar location (B). The precut patterns are then adjusted to avoid the grid bars (C). The finished precuts are shown in panel (D). Change back to 10 pA, zoom out, navigate to the next preliminary lamella site, and repeat Steps B17–B20. Go back to the “Mapping” position. Acquire a new Tileset with the same parameters as in Table 1. Delete old preliminary lamellae sites and define new lamellae sites on the new grid overview according to the precut positions. Open AutoTEM and select the project with the same Project Name created in MAPS. Choose the corresponding template for milling, and apply it to all lamellae sites (Table 3). Table 3. Milling parameters in AutoTEM Cryo Lamella thickness (defined as “Pattern Offset”) (µm) Front Width Overlap (µm) Rear Width Overlap (µm) Milling Current (nA) Pattern Type DCM Rescan Interval (s) Rough Milling 1.0 1.5 1.0 1.0 Rectangle 120 Medium Milling 0.8 0.65 0.5 1.0 CCS 90 Fine Milling 0.6 0.35 0.1 0.5 CCS 60 Finer Milling 0.4 0.05 0.05 0.3 CCS 30 Polishing 1 0.15 N/A N/A 0.03 CCS 30 Polishing 2 0 N/A N/A 0.01 CCS 10 The template can be found as Supplementary File 3. Initially, we recommend setting the Depth Correction to 100% for 10 μm lamellae length (see Notes for an additional discussion). Run “Preparation” for all lamellae sites in the “Guided” mode. Follow the prompts for eucentric tilt and milling angle (20°). Position the lamellae to guide the notch placement in Step B31. Prepare “Precleans” (Figure 4): Note: Confirm with EB images that material below the lamellae sites are milled away completely (Supplementary Figure 1). Notes: Steps B30b, d assume that apertures are aligned well, so there is no shift between milling patterns after changing the current. If there is a significant shift of milling windows between 10 pA and 7 nA or 3 nA, take a quick snapshot at the milling current in question to accurately position the rectangular milling pattern. Tilt the shuttle to a high milling angle, so the bottom of any material below each lamella is visible. Note: The standard Aquilos 2 shuttle has a 45° pretilt, and the maximum angle typically is around 25–30°. To reach the bottom of the material below lamellae, a standard shuttle might require moving the stage down in z. The iFLM shuttle has a 27° pretilt, and tilting the stage to achieve a high milling angle does not require moving the stage down. For the iFLM shuttle, a milling angle of 45° can be used as a starting point, followed by milling at 30°, and finally, at the milling angle in Step B30c. Define the milling rectangular pattern at 10 pA, and then mill at 7 nA until the bottom of the bulk material is gone (Figure 4A, B). Tilt to the final milling angle (20° in this protocol). Define the milling rectangular patterns at 10 pA and position the pattern a few microns away from the edge of the slab. Mill at 3 nA until the excess material is gone (Figure 4C). Figure 4. Precleans. Milling precleans with the corresponding timing, as in Video 2. (A, B) Milling at 7 nA is performed at high milling angles. (C) The stage is tilted to the milling angle (20° in this protocol), and a further preclean is performed at 3 nA current. In all cases, it is important to place milling patterns slightly below the edge of the slab (orange arrow), and to make sure that excess material below the potential lamella is milled away completely. Notch milling Align the notch patterns in xT with the initial lamella position in AutoTEM (Figure 5A). This step does not have to be accurate since the lamella position will be redefined later. The total size of the notch pattern is 3.5 µm in x, 8.1 µm in y, and 3 µm in z. The size of the vertical patterns, except for the box on the side, is 3.5 µm in x and 0.2 µm in y, and vice versa for horizontal patterns. The overlap between patterns is 0.1 µm. The side pattern is 0.2 µm in x and 1.3 µm in y. The spacing between top and bottom parts is 1.1 µm (Figure 5B). The pattern can be found as Supplementary File 1. Mill at 0.3 nA. With the z depth defined in Step B31b, milling should take ~2 min 15 s in total (Figure 5C). Figure 5. Milling notch with the corresponding timing as in Video 2. (A) The notch pattern (Supplementary File 2) is placed according to the pre-defined lamella site as in Step B29 and after precleans are done. (B) After digitally zooming on the pattern, the position of the Notch pattern with the defined dimensions is adjusted to be close to the edge of the slab. (C) Example of the milled notch before complete detachment. Final lamellae positioning: Rerun “Image Acquisition” and “Lamella placement.” Place the lamella ~1 µm away from the notch, with the top and bottom mill boxes overlapping with the notch, as shown in Figure 6. Typical lamellae dimensions are 12 µm × 15–20 µm × 150–200 nm. Run in step-wise mode based on the AutoTEM template in Table 3 and Supplementary File 3. Note: Step-wise mode is used to minimize contamination growth on lamellae after milling. Figure 6. Final lamella placement during the Preparation step in AutoTEM with the corresponding timing as in Video 2. Typical lamella dimensions are 12 μm in width, 10 µm length and 150 nm thick. Overtilts or Final Polishing: Note: This step is optional and performed depending on the final lamellae thickness and lamellae quality. If the lamella or GIS layer is damaged, do not perform automated post-milling operations. Otherwise, define a positive 0.5° overtilt (milling angle goes to 20.5°) in the Polishing 2 step, and run automated milling only for that step. Alternatively, manually place a CCS milling pattern on top of the lamella. In both cases, milling for 1–2 min is sufficient. Data analysis Figure 7 shows an example of a final cryo-FIB-prepared waffle method lamella of yeast cells. Generated lamellae are further subjected to imaging with cryo-ET. Examples of the procedure, as well as more examples of milled lamellae, can be found in Kelley et al. (2022). Figure 7. Example of waffle lamellae of yeast cells with 150 nm target thickness prepared with this protocol. (A) Electron beam view with ETD detector, and (B) Ion beam view with ETD detector. Notes The protocol is highly reproducible in our hands, with a success rate close to 100% for most of the samples. The most often occurring issue is poor vitrification of the samples. Poor vitrification becomes apparent during TEM data acquisition, not during cryo-FIB/SEM or cryo-FLM. While we are using relatively high milling currents during precleans, the milling windows are placed away from the lamellae sites, and, therefore, we consider that poorly vitrified areas are from insufficient vitrification in the HPF. To reduce poor vitrification issues, we recommend adding cryo-protectant to the sample before the HPF; typically we add 5%–10% glycerol (Bauerlein et al., 2021). If poor vitrification still occurs, we suggest increasing the sample concentration. Initially, we recommend using the settings as in Supplementary File 3. However, depending on the sample type, milling time can be reduced. To speed up the milling process, the Depth Correction parameter can be adjusted. Depth Correction should be decreased when material behind the lamella is milled during automated milling (see overmill in Supplementary Figure 1). Other potential issues which may arise are summarized in Supplementary Figure 1. Avoiding overmilling (Supplementary Figure 1A, B) reduces milling times and gallium implantation of the lamellae. Ensuring that the bulk layer of material is removed during precleans (Supplementary Figure 1A, B) is critical. We notice that lamellae deformations, such as sagging (Supplementary Figure 1B) and buckling (Supplementary Figure 1C), often occur due to incomplete notch milling. While we do not currently have a solution for sagging, buckling may be avoided by ensuring that the notch is milled through completely. Double lamellae (Supplementary Figure 1D, E) are often a consequence of displacement of the milling patterns by AutoTEM. While we cannot highlight precise reasons for such a displacement, it is likely caused by errors in cross-correlation during re-navigation to the milling site, and by improper drift correction. We have found that replacing the TFS pole piece shutter assembly with a Delmic CERES Ice Shield on our TFS Aquilos 2 reduced the lamellae contamination rate by a factor of approximately 3. Prior to this change, we were unable to perform automated milling overnight without accruing considerable contamination on lamellae (presumably from the electron beam pole piece). Our current contamination rate after this upgrade is ~1.5 nm/h, previously ~4.4 nm/h, which allows for up to 16 waffle lamellae to be milled automatically overnight with acceptably low contamination, as opposed to our previous limit of 2–4 waffle lamellae milled during an 8-h workday. As each cryo-FIB/SEM instrument setup is different, we recommend testing contamination during overnight waffle milling. Based on contamination rates, the cryo-FIB/SEM operator may either restrict automated waffle milling to a normal 8-h workday, or find a way to lower the contamination rate in the cryo-FIB/SEM chamber to allow for significantly higher automated overnight waffle milling throughput. Additionally, we did not experience any clogs in the cooling lines during up to 72 h of continuous operation at cryo-temperatures. Automated cryo-FIB/SEM workflows in the literature (i.e., Tacke et al., 2021) claim approximately 24 lamellae in 24 h, where each lamellae has an imageable area of ~5 μm × 5 μm, resulting in a total imageable area of ~600 μm2. The low-contamination waffle protocol we describe here produces 16 lamellae in 24 h, where each lamella has an imageable area of ~11 μm × 16 μm, resulting in a total imageable area of ~2,800 μm2. Acknowledgments This work was performed at the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NIH NIGMS (GM103310), and NIH (U24GM139171). Competing interests The authors declare no competing interests. References Buckley, G., Gervinskas, G., Taveneau, C., Venugopal, H., Whisstock, J. C. and de Marco, A. (2020). Automated cryo-lamella preparation for high-throughput in-situ structural biology. J Struct Biol 210(2): 107488. Bauerlein, F. J. B., Pastor-Pareja J. C. and Fernández-Busnadiego R. (2021). Cryo-Electron Tomography of Native Drosophila Tissues Vitrified by Plunge Freezing. BioRxiv. doi: https://doi.org/10.1101/2021.04.14.437159 Bauerlein, F. J. B. and Baumeister, W. (2021). Towards Visual Proteomics at High Resolution. J Mol Biol 443(20): 167-187. Carter, S. D., Mageswaran, S. K., Farino, Z. J., Mamede, J. I., Oikonomou, C. M., Hope, T. J., Freyberg, Z. and Jensen, G. J. (2018). Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells. J Struct Biol 201(1): 15-25. Gorelick, S., Buckley, G., Gervinskas, G., Johnson, T. K., Handley, A., Caggiano, M. P., Whisstock, J. C., Pocock, R. and de Marco, A. (2019). PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. Elife 8: e45919. Gupta, T. K., Klumpe, S., Gries, K., Heinz, S., Wietrzynski, W., Ohnishi, N., Niemeyer, J., Spaniol, B., Schaffer, M., Rast, A., et al. (2021). Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity. Cell 184(14): 3643-3659 e3623. Harapin, J., Bormel, M., Sapra, K. T., Brunner, D., Kaech, A. and Medalia, O. (2015). Structural analysis of multicellular organisms with cryo-electron tomography. Nat Methods 12(7): 634-636. Kelley, K., Raczkowski, A. M., Klykov, O., Jaroenlak, P., Bobe, D., Kopylov, M., Eng, E. T., Bhabha, G., Potter, C. S., Carragher, B. and Noble, A. J. (2022). Waffle Method: A general and flexible approach for improving throughput in FIB-milling. Nat Commun 13(1): 1857. Klumpe, S., Fung, H. K., Goetz, S. K., Zagoriy, I., Hampoelz, B., Zhang, X., Erdmann, P. S., Baumbach, J., Muller, C. W., Beck, M., et al. (2021). A modular platform for automated cryo-FIB workflows. Elife 10: e70506. Klykov, O., Kopylov, M., Carragher, B., Heck, A. J. R., Noble, A. J. and Scheltema, R. A. (2022). Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology. Mol Cell 82(2): 285-303. Mahamid, J., Schampers, R., Persoon, H., Hyman, A. A., Baumeister, W. and Plitzko, J. M. (2015). A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms. J Struct Biol 192(2): 262-269. Marko, M., Hsieh, C., Schalek, R., Frank, J. and Mannella, C. (2007). Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat Methods 4(3): 215-217. McDowall, A. W., Chang, J. J., Freeman, R., Lepault, J., Walter, C. A. and Dubochet, J. (1983). Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples. J Microsc 131(Pt 1): 1-9. Saibil, H. R. (2022). Cryo-EM in molecular and cellular biology. Mol Cell 82(2): 274-284. Schaffer, M., Pfeffer, S., Mahamid, J., Kleindiek, S., Laugks, T., Albert, S., Engel, B. D., Rummel, A., Smith, A. J., Baumeister, W. (2019). A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.Nat Methods 16(8): 757-762. Silvester, E., Vollmer, B., Prazak, V., Vasishtan, D., Machala, E. A., Whittle, C., Black, S., Bath, J., Turberfield, A. J., Grunewald, K., et al. (2021). DNA origami signposts for identifying proteins on cell membranes by electron cryotomography. Cell 184(4): 1110-1121 e1116. Tacke, S., Erdmann, P., Wang, Z., Klumpe, S., Grange, M., Plitzko, J. and Raunser, S. (2021). A streamlined workflow for automated cryo focused ion beam milling. J Struct Biol 213(3): 107743. Tan, Z. Y., Cai, S., Noble, A. J., Chen, J. K., Shi, J., and Gan, L. (2021). Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ. BioRxiv. doi: https://doi.org/10.1101/2021.04.04.438362 Watanabe, R., Buschauer, R., Böhning, J., Audagnotto, M., Lasker, K., Lu, T. W., Boassa, D., Taylor, S., Villa, E. (2020). The in-situ structure of Parkinson’s disease-linked LRRK2. Cell 182(6): 1508-1518.e16. Wang, Z., Grange, M., Pospich, S., Wagner, T., Kho A.L., Gautel, M., Raunser S. (2022). Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin. Science 6582(375): eabn1934. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biophysics > Electron cryotomography Cell Biology > Cell structure Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,545	https://bio-protocol.org/en/bpdetail?id=4545&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed An Unbiased CRISPR-Cas9 Screening Method for the Identification of Positive and Negative Regulatory Proteins of Cell Adhesion YT Yvonne J. Thus * MR Martin F. M. de Rooij * RB Roderick L. Beijersbergen MS Marcel Spaargaren (contributed equally to this work) Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4545 Views: 1115 Reviewed by: Giusy TornilloAlfano Luigi Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Communications Apr 2022 Abstract Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor–controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion–regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types. Graphical abstract: Keywords: CRISPR-Cas9 Adhesion Kinases Screening B-cell receptor signaling Integrins Background In mature B-cell lymphomas, the malignant B cells are highly dependent on their protective lymphoid organ microenvironment; the stromal cells, T cells, and macrophages in the lymph node and bone marrow secrete a variety of cytokines and chemokines and express several adhesion molecules, which stimulate survival and proliferation of the malignant B cells (Burger and Wiestner, 2018). The retention of the malignant cells in these protective niches is mediated by cell adhesion to extracellular matrix components (e.g., fibronectin) and cellular ligands (e.g., VCAM-1, ICAM-1, and cadherins). In many mature B-cell malignancies, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM), the activation of integrins is controlled by the B-cell receptor (BCR) signaling (Figure 1), among others (de Rooij et al., 2012; Chang et al., 2013; de Rooij et al., 2016). Targeting the BCR signalosome [e.g., by the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib or the PI3Kδ inhibitor idelalisib] has demonstrated an unprecedented clinical efficacy in these malignancies, with objective response rates of 70%–90% (Burger and Wiestner, 2018). We and others have shown that these drugs do not act by directly killing the lymphoma cells, but rather by mobilizing the cells from their protective niches into the peripheral blood, where they are deprived from survival and growth signals, eventually dying (de Rooij et al., 2012, 2016; Byrd et al., 2013; Chang et al., 2013; Wang et al., 2013; Brown et al., 2014; Treon et al., 2015). Notwithstanding the high clinical efficacy of these drugs, a subset of patients does not respond at all or develops resistance upon prolonged treatment (Stephens and Byrd, 2021). Considering the microenvironment dependence as the Achilles’ heel of many B-cell malignancies, we aimed to identify novel signaling molecules involved in the regulation of B-cell homing and retention. Over the last decade, pooled genetic screening platforms, either using shRNAs or CRISPR-Cas9 libraries, have been widely used not only in the context of (synthetic) lethality and drug resistance, but also in more functional assays, such as FACS-based reporter or signaling screens (Przybyla and Gilbert, 2022). Here, we describe the detailed method of a pooled genetic CRISPR-Cas9-mediated knockout screening method to identify genes involved in integrin-mediated adhesion: a loss-of-adhesion screen. This screen is suitable for studying a variety of different cell types, stimuli (chemokines or cytokines), cell adhesion molecules (integrins, cadherins, and selectins), and their ligands [(extra)cellular matrix components]. Figure 1. The B-cell receptor signaling pathway. Antigen (Ag) binding to the B-cell receptor (BCR) induces a signaling cascade resulting in the activation of integrins. In black, all kinase(-related) proteins are depicted. Adapted from De Rooij et al. (2022). Ag: antigen (αIgM); BCR: B-cell receptor; PIP2: phosphatidylinositol-4,5-diphosphate; PIP3: phosphatidylinositol-3,4,5-triphosphate; DAG: diacylglycerol; IP3: inositol-1,4,5-triphosphate; Ca2+: cytosolic calcium ions; PMA: phorbol-12-myristate-13-acetate; GMP/GDP/GTP: guanosine-mono/di/triphosphate. Materials and Reagents Plasmids and vectors LentiCas9-Blast (Addgene, catalog number: 52962) Cas9-reporter [pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W] (Addgene, catalog number: 67980) psPAX2 (Addgene, catalog number: 12260) pMD2.G (Addgene, catalog number: 12259) pEGFP-N3 (Clontech, catalog number: 6080-1) LentiGuide-Puro Brunello kinome library (Addgene, catalog number: 1000000082) Cell culture, transfection, and transduction 6-well plate (Greiner, catalog number: 657160) T175 flasks (Greiner, catalog number: 658170) 15 mL tubes (Falcon, catalog number: 734-0452) 0.45 µm filter (Millipore, catalog number: SLHA033SS) Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, GibcoTM, catalog number: 41966-052) Iscove’s modified Dulbecco’s medium (IMDM, Thermo Fisher Scientific, GibcoTM, catalog number: 12440-061) Polyethylenimine, linear (PEI, Polysciences, catalog number: 24313-2) HEK293T-17 (ATCC, catalog number: CRL11268) Namalwa (DSMZ, catalog number: ACC24) Cryovials (Costar, catalog number: 430488) Puromycin (Invivogen, catalog number: ant-pr-1) Blasticidin S hydrochloride (Thermo Fisher Scientific, GibcoTM, catalog number: R210-01) Supplements for cell culture medium (added before use; see Recipes) Fetal bovine serum (FBS) (HyClone, catalog number: SH30071.03) L-glutamine (Sigma-Aldrich, catalog number: 59202C) Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333) Library amplification 94 × 16 mm plates (Greiner, catalog number: 633181; other sizes can be used if desired) EnduraTM electrocompetent cells (Lucigen, catalog number: 60242-1) Agar (BD, catalog number: 214010) Ampicillin (Sigma, catalog number: A5354) NucleoBond Xtra midi prep kit (Macherey-Nagel, catalog number: 740420.50) LB (BD, catalog number: 240230) LB medium containing antibiotics (see Recipes) SOC medium (see Recipes) D-glucose (Merck Millipore, catalog number: 1083371000) Agar plates containing antibiotics (see Recipes) Adhesion 96-well high binding, flat bottom plates (Greiner, catalog number: 655061) Human plasma fibronectin (FN) (Sigma-Aldrich, catalog number: F2006) Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P6282) Bovine serum albumin fraction V (BSA) (Roche, catalog number: 10735094001) Ibrutinib (SelleckChem, catalog number: S2680) Goat F(ab’)2 anti-human IgM (Southern Biotech, catalog number: 2022-14) Phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139) Multichannel pipette reservoir (Corning, catalog number: 4870) 25% glutaraldehyde (Sigma-Aldrich, catalog number: 354400) Crystal violet (Sigma-Aldrich, catalog number: C0775) Ethanol (Merck Millipore, catalog number: 1009831000) Phosphate buffered saline (PBS) (Fresenius Kabi, catalog number: M090001/13) Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884) 0.5% crystal violet in 20% ethanol (see Recipes) 0.5 M EDTA, pH 8.0 (see Recipes) Genomic DNA isolation Quick DNA mini prep kit (Zymo Research, catalog number: D3024) Qubit broad range kit (Thermo Fisher Scientific, catalog number: Q32850) PCR and next-generation sequencing PhusionTM high-fidelity DNA polymerase (contains 5× HF buffer) (Thermo Fisher Scientific, catalog number: F530S) 10 mM dNTP mix (Sigma-Aldrich, 71004-3) 10 µM PCR1 barcoded forward primer (Sigma-Aldrich, Tables 1, 2) 10 µM PCR1 reverse primer (Sigma-Aldrich, Table 1) 10 µM PCR2 forward primer (Sigma-Aldrich, Table 1) 10 µM PCR2 indexed reverse primer (Sigma-Aldrich, Tables 1, 2) Agarose (Meridian Biosciences, catalog number: BIO-41025) Ethidium bromide (EtBr) (Sigma-Aldrich, catalog number: E1385) NucleoSpin gel and PCR clean-up kit (Macherey-Nagel, catalog number: 740609.250) Qubit high sensitivity kit (Thermo Fisher Scientific, catalog number: Q32851) Tris base (Sigma-Aldrich, catalog number: T1503) Acetic acid (Sigma-Aldrich, catalog number: A6283) PhiX Control v3 (Illumina, catalog number: FC-110-3001) TAE buffer (see Recipes) Agarose gel (see Recipes) Table 1. Primers PCR1 and PCR2 Primer Sequence PCR 1 barcoded forward ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGGCTTTATATATCTTGTGGAAAGGACG PCR 1 reverse GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTGACGGGCACCGGAGCCAATTCC PCR 2 forward AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT PCR 2 indexed reverse CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT The NNNNNN in the PCR1 forward and PCR2 reverse primers are replaced by the desired barcodes/ indexes listed in Table 2. Table 2. Barcodes/indexes for PCR1 and PCR2 primers Catalog number Barcode PCR1 Index PCR2 1 CGTGAT ATCACG 2 ACATCG CGATGT 3 GCCTAA TTAGGC 4 TGGTCA TGACCA 5 CACTGT ACAGTG 6 ATTGGC GCCAAT 7 GATCTG CAGATC 8 TCAAGT ACTTGA 9 CTGATC GATCAG 10 AAGCTA TAGCTT 11 GTAGCC GGCTAC 12 TACAAG CTTGTA Equipment 12-well multichannel pipette (Eppendorf Research, catalog number: 4926000069) An electronic multichannel, which releases the fluid at a given speed, might be of interest, especially when a large number of 96-well plates are used for the screen. We used the Picus 1200 (Sartorius, catalog number: 735491) Spectrophotometer (CLARIOstar Plus, BMG Labtech) Fluorescence microscope (Olympus Lifesciences) FACSCanto II (BD Biosciences) 37 °C, 5% CO2 incubators (cell culture) Electroporation system (Bio-Rad, Gene Pulser) 37 °C incubator (bacteria) 100 mL erlenmeyers Microcentrifuge (Hettich, MIKRO 220R) Centrifuge (Hettich, ROTINA 420) Vortex (Heidolph, Reax Top) Qubit (ThermoFisher) Thermal cycler (Westburg, Biometra TAdvanced) Gel electrophoresis system (LKB Power supply; CBS scientific) Sequencer (Illumina, HiSeq2000) -80 °C freezer -20 °C freezer Software FlowJo (BD Biosciences; https://www.flowjo.com/) Perl (https://www.perl.org/) or Python (Anaconda) (https://www.anaconda.com/) R (https://www.r-project.org/) RStudio (https://www.rstudio.com/products/rstudio/) DESeq2 R-package (http://www.bioconductor.org/packages/release/bioc/htmL/DESeq2.htmL) (Love et al., 2014) and/or MAGeCK (only in Linux) (https://sourceforge.net/p/mageck/wiki/Home/) (Li et al., 2014) Procedure Setting up the adhesion assay Note: For choosing a suitable cell line and optimal conditions for the screen, several small-scale adhesion assays should be performed to determine which cell line shows the best adhesive capacity (either unstimulated or after stimulation, depending on the aim of the screen), which adhesion molecules and corresponding ligands are suitable for the screening analysis, which stimuli are suitable for the screen, what concentrations and incubation times should be used, etc. Example: BCR-controlled integrin-mediated adhesion of Namalwa cells to fibronectin Coat 96-well high binding plates with 100 µL of PBS containing 2.5 µg/mL fibronectin at 4 °C overnight or with 100 µL of PBS containing 1 mg/mL PLL for 15 min at 37 °C. The PLL should be coated on a separate plate. See Figure 2 for a layout example of an adhesion experiment. To optimize the concentration for the coating (adhesion substrate) and, if applicable, the stimulus, perform a titration experiment with both stimulated and unstimulated cells. Using too high concentrations could result in high basal/unstimulated adhesion (Figure 3A, U2932) and/or such strong stimulated adhesion that it is difficult to inhibit (Figure 3C); using a too low concentration of coating or stimulus results in such weak adhesion that it decreases the sensitivity of the assay. If available, take along an inhibitor that targets adhesion as a positive control. In our case, ibrutinib-treated cells were taken along to establish the concentration of fibronectin and αIgM at which unstimulated (no αIgM) and untreated (no ibrutinib) cells hardly or not adhered, whereas stimulated (with αIgM) and untreated (no ibrutinib) cells showed strong adhesion that could be efficiently inhibited by ibrutinib (Figure 3C). For Namalwa, the optimal concentrations were 2.5 µg/mL fibronectin and 100 ng/mL αIgM. Apart from fibronectin, other cell adhesion molecules and their ligands can also be used as coating (e.g., VCAM-1, ICAM-1, selectins, cadherins, laminin, or collagen) (Figure 3D). We used fibronectin since we obtained strong adhesion with this molecule and it binds to several distinct integrins, whereas for example VCAM-1 mainly binds to integrin α4β1 (Takada et al., 2007). Wash the wells twice with 100 µL of PBS. Block the wells with 100 µL of 4% BSA in serum-free medium for at least 1 h at 37 °C. For Namalwa cells, we used IMDM both as culture medium and to perform the adhesion assay. Incubate the cells in 1% BSA in serum-free medium for 1 h at 37 °C at a cell density of 1.5 × 106 per mL. If using inhibitors, add your inhibitor of interest to this medium. Adjust the time period of pre-incubation with inhibitor as desired. Add the desired stimulus to your cells and plate 100 µL cells per well (= 1.5 × 105 cells/well). Incubate for 30 min at 37 °C (Figure 2). Adjust the time period of incubation with the stimulus as desired. For Namalwa cells, we used 100 ng/mL αIgM or 50 ng/mL PMA as stimuli. As with fibronectin, titrate these concentrations down to prevent too strong or too weak adhesion. If possible, we recommend taking along a stimulus relatively distal in the pathway of interest. Since the viability/fitness of the cells will also affect their adhesive capacity, the knock out of a gene that impairs cell fitness will also reduce cell adhesion, irrespective of its role in the regulation of adhesion. The downstream stimulus functions as a control for the fitness (and adhesive capacity) of the cell. Here, we used PMA as distal stimulus, which is an activator of PKC and CalDAG-GEFs (Figure 1). The incubation time can differ per stimulus. For example, for chemokine-induced adhesion, the optimal incubation time is 2 or 5 min. Some stimuli, such as chemokines, do not maximally induce adhesion when added soluble. These molecules need to be co-coated with the adhesion molecule in step A1. Wash the fibronectin-coated plate with 100 µL of warm 1% BSA in serum-free medium by smashing the fluid out of the plate, dapping it dry on a tissue, and gently adding new medium to remove non-adherent cells. Repeat step A6 until (1) the uncoated wells are empty, (2) the effect of the stimulation is maximal (i.e., with a maximal number of stimulated cells and a minimal number of unstimulated cells remaining adherent), and (3) the effect of the inhibitor is maximal. Write down the number of washes necessary to reach this point, as this is an indication for the number of washes during the screening procedure. Remove the 1% BSA in serum-free medium and fix the cells by adding 100 µL of 10% glutaraldehyde in PBS. For the PLL plate, without washing or removing cells, add 67 µL of 25% glutaraldehyde in PBS. Incubate for 10 min at room temperature. Wash twice with PBS. Add 100 µL of 0.5% crystal violet in 20% ethanol. Incubate for 45 min at room temperature. Wash the plate extensively with H2O to remove the overload crystal violet. Add 100 µL of ethanol to elute the crystal violet. Incubate for 30 min at room temperature. Measure absorbance at 570 nm on a spectrophotometer. Determine adhesion rate by first subtracting absorbance due to nonspecific adhesion, as determined in the uncoated, 4% BSA-blocked wells, followed by dividing it by the maximal absorbance, as determined in the PLL-coated wells. Figure 2. Plate layout adhesion assay. An example of the design of an adhesion assay (in triplicate) evaluating the effect of an inhibitor on adhesion. The PLL-coating should be on a separate plate. Figure 3. Titration experiments adhesion. Namalwa and U2932 cells were allowed to adhere to a coated plate as indicated. Cells were stimulated with the indicated concentration stimuli and/or pretreated with 100 nM Ibrutinib. (A) Namalwa and U2932 cells were allowed to adhere to a plate coated with different concentrations of FN. Whereas high concentrations did not affect unstimulated adhesion of Namalwa cells, U2932 cells showed increased unstimulated adhesion. Therefore, for U2932 cells the optimal concentration of FN would be 0.25 µg/mL. (B) Namalwa cells were allowed to adhere to a plate coated with 2.5 µg/mL of FN for different time frames. For αIgM and PMA, 30 min adhesion showed the strongest adhesion. (C) Ibrutinib pre-treatment prevented adhesion induced by low concentrations of αIgM and adhesion to low concentrations of VCAM-1, whereas this effect was largely gone when higher concentrations of αIgM or VCAM-1 were used. Optimal concentrations would be 100 ng/mL of αIgM and 600 ng/mL of VCAM-1, although higher concentrations resulted in an increased fold-change of stimulated adhesion over unstimulated adhesion. (D) For CXCL12α-induced adhesion to VCAM-1, an adhesion optimum was reached with 25 ng/mL of CXCL12α, reflecting the typical bell-shaped curve for stimulation of chemokine receptors due to receptor desensitization/downregulation at higher chemokine concentrations. For αIgM we did not observe a bell-shaped curve when increasing the concentration. P < 0.05; P < 0.01; *P < 0.001; ns: not significant; two-way ANOVA with Tukey’s multiple comparison test of unstimulated versus stimulated condition. Choosing the cell line of interest To select a suitable cell line, the following aspects should be considered: The cell line should be transducible with the sgRNA library. If using poorly transducible cell lines, we recommend using a dual-vector system with Cas9 and the sgRNA in two separate constructs, instead of a one-vector system with Cas9 and the sgRNA in the same construct. The vector size of the dual-vector sgRNA construct (approximately 10 kb) is significantly smaller than the one-vector Cas9/gRNA construct (approximately 15 kb), allowing for higher transduction efficacy of the library. The cells should adhere sufficiently (see Section A) to reach a certain power of the screen: If the goal is to identify genes involved in basal/unstimulated adhesion, we suggest using a cell line for which the basal adhesion is as high as possible, even after more than five washing steps. The disadvantage of using a poorly adhering cell line is that in order to maintain the complexity of the sgRNA library post-adhesion, the quantity of cells employed for the screen and the number of adhesion assays that need to be performed is significantly higher. The disadvantage of using a cell line that detaches after a low number (e.g., three) of washing steps is that the sensitivity of the screen is low: only all-or-nothing genes will be identified, not the intermediate adhesion inducers. For example, if the knock out of a certain gene will cause a cell line that normally detaches after ten washing steps to detach after eight washing steps, this gene will not be identified in another cell line that detaches after three washing steps; the sensitivity of the assay with this cell line is too low. Since most signaling pathways may display (partial) redundancy, a minimum of five washing steps will increase the chance of identifying genes. If interested in stimulated adhesion, we suggest using a cell line that shows minimal basal adhesion (close to zero) and at least a 2-fold increase in adhesion after stimulation. In an ideal situation, as with identifying genes involved in basal adhesion, the fraction of cells that is adherent after stimulation is as high as possible, even after more than five washing steps. We typically observe a distribution of the sgRNAs of 1.5-fold from the mean, so if the basal adhesion is 30% and the stimulated adhesion is 2-fold higher (60%), even a perfect dropout could only slightly drop out from the cloud in an MA-plot. Since the chance of having a perfect dropout is small, with most signaling pathways displaying (partial) redundancy, a minimum of 2-fold increase in adhesion after stimulation will increase the chance of identifying genes with the screen. Obtaining a plasmid pool of the sgRNA library of interest Note: Either design (and construct) the sgRNA library yourself or obtain it from a distributor (e.g., Addgene). Nowadays, many companies provide custom-designed oligo libraries, which you can clone into a vector yourself, or completely cloned customized libraries. If designing the library yourself, include control sgRNAs targeting essential and non-essential/non-expressed genes. Furthermore, make sure that known positive and negative regulators of adhesion are included in the library. For our BCR-controlled adhesion screen, we made sure that the established adhesion-regulators BTK, SYK, and PI3K (PIK3R1) (Figure 1) were present in our selected library, the Brunello kinome library. Generating a Cas9-expressing cell line Note: If using a one-vector library or if a stable Cas9-expressing cell line is already available, proceed directly to Section E. Obtain a Cas9 expression vector of your interest. We used lentiCas9-Blast, since our sgRNA library contained puromycin as selection marker. This is preferred as it allows selection of transduced cells within a limited time (approximately three days). Produce lentivirus containing the Cas9 expression vector. Plate 1 × 106 HEK293T-17 cells in 2 mL in a 6-well plate and incubate for 24 h. After 24 h, the cells should be adherent and at 80% confluency. Use DMEM as culture medium for HEK293T-17. Mix 2 µg of LentiCas9-Blast, 1 µg of psPAX2, 0.5 µg of pMD2.G, and 0.1 µg of pEGFP-N3 with 8 µg of PEI in 400 µL of serum free DMEM. Note: The use of pEGFP-N3 is optional; we only added it to check the transfection efficiency of the HEK cells. Incubate for 15 min at room temperature. Carefully add the transfection mixture to HEK293T-17 cells and incubate for 16 h at 37 °C. Check for GFP expression in HEK cells using a fluorescence microscope. If >50% of HEK cells are GFP-positive, replace medium with fresh medium and incubate for 24 h at 37 °C. Take viral supernatant from HEK cells and filter with a 0.45 µm filter to remove cell debris. Use the virus directly (step D3) or store aliquots at -80 °C. Transduce a cell line with the Cas9 expression vector by adding the virus to your cells. Make sure that your cells of interest are at a density compatible with their exponential growth. If your transduction efficiency is low, you might consider adding polybrene during the viral transduction. However, in our cells polybrene was slightly toxic, so first check the effect of polybrene itself and perform a titration experiment for viability. We added our virus in a 1:2 dilution to obtain the highest multiplicity of infection (MOI) possible. The higher the number of infections that take place during this transduction, the higher the Cas9 expression and, thus, the Cas9 activity will probably be, and the more efficient your cell line will generate your desired knockouts. If a cell line is used for which such a high concentration of virus is toxic, or if your quantity of virus is scarce, lower dilutions can also be used. Select transduced cells by culturing the cells in the presence of 10 µg/mL blasticidin for seven days. Use untransduced cells as control. Check Cas9 efficacy with a Cas9-reporter. We used the pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W lentiviral construct, which results in the expression of BFP, GFP, and a sgRNA targeting GFP. Produce Cas9-reporter-lentivirus as in step D2. Transduce the Cas9-expressing cells and, as control, the parental cells with the virus as in step D3. As in step D3c, we added the virus in a 1:2 dilution to obtain the highest MOI possible, and thus the strongest effect of the reporter. Lower dilutions can be used when your cells have a high transduction efficiency, if the quantity of virus is limited, or if a high concentration of virus is toxic for your cells. After six days, validate Cas9 efficacy by checking for GFP and BFP expression by flow cytometry. These six days are necessary to allow the cells to induce the GFP knockout. If transduction with reporter was successful, the parental cells are GFP- and BFP-positive (Figure 4; green). If all Cas9-expressing cells have active Cas9, those cells will only be BFP-positive since the sgRNA in the reporter construct results in defective GFP (Figure 4; blue). If less than 90% of the Cas9-expressing cells show active Cas9 (e.g., as for Granta-519 in Figure 4), you might consider either increasing the blasticidin concentration to improve the selection pressure or transducing the cells again with Cas9-lentivirus. Figure 4. Cas9-reporter assay. An example of the Cas9-reporter assay with the parental and Cas9-expressing cells without Cas9-reporter in red and yellow, being both GFP- and BFP-negative. In green, parental cells transduced with the Cas9-reporter are depicted, being both GFP- and BFP-positive. In blue, the Cas9 expressing cells transduced with the Cas9-reporter are depicted, being largely only BFP-positive, indicating efficient Cas9/gRNA-mediated GFP knockout. The percentages depicted in the quadrants describe the percentage of the Cas9 cell line with the Cas9-reporter (blue) per quadrant. In Namalwa, Cas9 is active in 62/(62+1) = 98% of cells, whereas in Granta-519 only 48/(48+20) = 70% of cells contain active Cas9. Lentiviral sgRNA library production Note: A major determinant of successful screening is the maintenance of the complexity of the library throughout the screening process. For a dropout screen, this means that during every step of the screen (e.g., after amplifying the library, after puromycin selection, after adhesion, after DNA isolation, etc.) all sgRNAs should be represented >1,000-fold on average in your sample, to warrant sufficient (statistical) power of the screen. In the case of the Brunello kinome library, which contains 6,204 unique sgRNAs, this means that during every step of the screen at least 6.2 × 106 colonies/cells should be present. Amplify the sgRNA library. Electroporate 0.1–1 µg plasmid library in one vial EnduraTM electrocompetent cells at 1.8 kV. Enable the EnduraTM cells to recover in 1 mL SOC medium for 1 h at 37 °C. Take 1 µL from EnduraTM cells and perform four consecutive 10× dilutions in 100 µL of LB (1:103, 1:104, 1:105, and 1:106). Plate these four dilutions on agar plates with 50 µg/mL of ampicillin and incubate plates upside down overnight at 37 °C. Note: Agar plates are incubated upside down to reduce evaporation of moisture from the agar during overnight incubations. Dilute the remaining 999 µL of EnduraTM cells in 400 mL of LB with 50 µg/mL of ampicillin and incubate overnight at 37 °C. Count the number of colonies on the agar plates. If enough colonies are present on the agar plates (at least 100 but preferably 1,000 times the number of unique sgRNAs present in the library), perform plasmid DNA isolation of the 400 mL of LB according to manufacturer’s protocol. Use multiple columns depending on the optical density of the bacterial culture. In our case, we typically used four columns of the NucleoBond Xtra midi prep kit. We suggest storing a master stock of your library at -80 °C for future library amplifications, instead of amplification of consecutively produced stocks. This prevents accumulating random changes in sgRNA composition by amplification errors. Produce lentivirus containing the library as in step D2, but with HEK293T-17 cells grown in five T175 culture flasks instead of a 6-well plate. The packaging mixture should be scaled up relative to the surface area of the culture flasks (6-well plate = 9.6 cm2). After harvesting viral supernatant, aliquot the virus in cryovials (for titration) and 15 mL tubes (screening aliquots) and store at -80 °C. As the quality of your virus decreases with every freeze-thaw cycle, do not immediately start with the virus titration (Section F) but first freeze down the virus. This way, virus used for the screen will have the same number of freeze-thaw cycles and thus probably the same quality as the virus used for titration. Lentiviral sgRNA library titration Note: To maximize the number of cells with a single integration and to limit the number of cells needed for viral transduction, cells should be transduced at a MOI of 0.3. To determine the efficacy of your virus batch and the quantity of virus to be added to the cells, first perform titration assays. Plate 1 mL of your Cas9-expressing cell line in a 6-well plate at a 2-fold higher density than the density at which the cells would not become overgrown by the end of the assay in the absence of (puromycin) selection. Plate enough wells to allow testing of all desired conditions (Figure 5). Thaw an aliquot of the virus and prepare the desired pre-dilutions of the virus. In our case, we made pre-dilutions of 1:32, 1:16, 1:8, 1:4, and 1:2 virus and added 1 mL per well to the cells, testing a final concentration of 1:64 until 1:4. If your cell line has a higher transduction efficiency, you might consider testing for example 1:200 until 1:12.5 virus dilutions. Incubate for 24 h. Note: Usually, 24 h is sufficient to allow cells to become resistant to puromycin. However, in some cases, cells may need up to 48 h to become resistant. Add an appropriate concentration of puromycin to the second half of the plate to allow selection for transduced cells. Incubate for 48 h. If the appropriate puromycin concentration is unknown, a puromycin titration experiment needs to be performed in advance. Measure cell density and viability by flow cytometry and calculate at which concentration of virus 30% of the cells remain viable in the puromycin-condition versus the non-puromycin-condition. This will be the desired concentration of virus to be used during the screen (i.e., a MOI of 0.3). Figure 5. Plate layout virus titration. An example of the design of a virus titration assay in 6-well plates. The depicted layout is performed in duplicate. Viral transduction of the library for the screen Expand your cells to warrant obtaining sufficient numbers of cells for the infection at a MOI of approximately 0.3, to maintain the complexity of the library. We suggest performing three independent transductions, which you keep as separate replicate samples throughout the whole screening procedure, to increase the statistical power of your screen and thereby the chance of identifying relevant proteins. We often observe a lower transduction efficiency in a T175 compared to a 6-well plate in which the virus titration is performed. To warrant maintenance of the desired complexity of the library (1,000× coverage), we suggest transducing more cells than calculated, if possible, to compensate for the anticipated reduced transduction efficiency. Since we required at least 6.2 × 106 transduced cells after transduction at a MOI of 0.3, we transduced 6.2 × 106/0.3 + a bit extra = 24 × 106 cells. The size of a bit extra depends on the standard deviation of your transduction efficiency. To be sure to maintain the complexity of the library, we transduced at least 10% more cells than required based upon the MOI calculation. Divide your cells over T175 flasks, so that your cells are at a density compatible with their exponential growth. Prepare an extra flask for an untransduced, puromycin selection control. Thaw and pool sufficient virus aliquots to transduce all flasks. Add virus to each flask, except for the puromycin selection control, and incubate for 24 h. Add the appropriate concentration of puromycin to achieve complete cell death of the untransduced cells and incubate for 48 h. Allow cells to recover from the puromycin selection until cells are growing with an acceptable viability (less than 10% below standard culture viability) and proliferation rate (both close to the parental cells), and until the number of viable cells is sufficient to set up all conditions. Allow recovery for a minimum of six days to allow efficient gene knockouts. Adhesion screen Pool all transduced cells of each replicate. For each replicate, take a cell aliquot for the pre-adhesion reference sample. This aliquot should contain at least a 1,000-fold representation of the unique sgRNAs in your library. If possible, we recommend increasing this number of cells to maintain the complexity of the library even after genomic DNA (gDNA) extraction, since the efficiency of gDNA extraction is often 50%–70%. Pellet the aliquot of the reference sample by centrifugation (e.g., 600 × g for 3 min), aspirate supernatant, and resuspend in 200 µL of PBS per sample. Store these samples at -20 °C, until all selected samples have been collected. Use the remaining cells for the adhesion assay as in steps A1–A6. Coat all wells of an appropriate number of 96-well plates with fibronectin (Figure 6), but add unstimulated cells or cells pretreated with an inhibitor in the corner-wells, as a control for the number of washes. Despite the large number of used plates and the time-inefficient workload, we did not scale up the adhesion assay to, for example, 6-well plates (or Petri dishes), since high-binding 6-well plates are not available and since washing with an equal force (stringency) over the entire surface is feasible with a 96-well plate, but not with a 6-well plate. Use separate plates for different stimuli to be able to collect the cells separately. After extensive washing (step A6), add 100 µL of 5 mM EDTA/PBS to detach all adherent cells and incubate for 10 min. For some cell types, trypsin might be beneficial over EDTA. Collect the adherent cells. Wash the plate once with 100 µL of PBS and add this to the 5 mM EDTA/PBS suspension with adherent cells. Pellet the samples as in step H3. Figure 6. Plate layout adhesion assay screen. Use the wells in the corners as control wells to establish the number of washes needed during the adhesion assay. These control wells either contain unstimulated cells or cells pre-incubated with an inhibitor with an established effect on adhesion. Genomic DNA isolation Extract genomic DNA from cell pellets, for example, using the Quick DNA mini prep kit according to manufacturer’s protocol. Measure DNA concentration per sample, for example using the Qubit broad range kit and a Qubit fluorometer according to manufacturer’s protocol. PCR and next-generation sequencing of sgRNA inserts For the first round of PCR amplification reaction (Figure 7) for each sample and for a water control (no DNA), prepare the following: 1 µg of gDNA, 10 µL of 5× HF buffer, 1 µL of 10 mM dNTPs, 1 µL of 10 µM PCR1 barcoded forward primer, 1 µL of 10 µM PCR1 reverse primer, 0.5 µL of Phusion polymerase, and H2O to 50 µL. Use a distinct barcoded forward primer for every sample. This can be scaled up for any screening library size used. A typical human diploid cell contains 6 pg of DNA and, therefore, we used 40 reactions/40 µg DNA per sample, which is representative for 6.7 × 106 cells, maintaining the coverage of the sgRNA library (>6.2 × 106). If more than 12 samples are being processed, barcoding can also be applied in the second round of PCR. We suggest sequencing the sgRNA library as well, to assess the starting distribution of the library. To do so, use 1 ng library plasmid in one PCR reaction (50 µL). The primers suitable for the lentiGuide-Puro and lenti-CRISPRv2 backbone are listed in Table 1 and 2 (Evers et al., 2016). Run the following protocol: (1) 98 °C for 30 s; (2) 21 cycles of 98 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; (3) 72 °C for 5 min; (4) hold at 4 °C. Pool the PCR reactions. Use the product of PCR1 to set up the second round of PCR reactions (Figure 7) using distinct indexed reverse primers if processing more than 12 samples: 2 µL of pooled PCR1 product, 10 µL of 5× HF buffer, 1 µL of 10 mM dNTPs, 1 µL of 10 µM PCR2 forward primer, 1 µL of 10 µM PCR2 indexed reverse primer, 0.5 µL of Phusion polymerase, and 34.5 µL of H2O (final volume is 50 µL). Run the following protocol: (1) 98 °C for 30 s; (2) 15 cycles of 98 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; (3) 72 °C for 5 min; (4) hold at 4 °C. Apply 4 µL of each PCR2 product, including the H2O control, to a 2% agarose gel to inspect the presence of an appropriately sized product (589 bp) across the samples and the absence of any product in the H2O (no DNA) control (see Figure 8 for an example). Purify the PCR product using the PCR purification kit according to the manufacturer’s protocol. In case of a prominent primer dimer band, first apply the full PCR product to a 2% agarose gel and isolate the proper band from the gel. Measure the DNA concentration per sample using the Qubit high sensitivity kit and a Qubit fluorometer according to the manufacturer’s protocol. Pool equimolar quantities of all samples. Run samples on an Illumina next-generation sequencer, according to the manufacturer’s protocol. For a screen with nine conditions (samples of pre-adhesion and two stimuli post-adhesion, all in triplicate) generated with a library containing 6,000 sgRNA constructs, we would require 5.4 × 107 reads. This can easily be achieved on a single HiSeq X lane (expected number of reads being approximately 3 × 109). Addition of 30% PhiX is recommended, because of the limited variation of initial nucleotides. The barcoded/indexed primers can be adjusted to the favorite design of your sequence facility, like the TruSeq design, and can be run together with RNAseq samples. Figure 7. Schematic overview of PCR reactions for sequencing library preparation. (A) The first round of amplification is carried out with primers flanking the sgRNA sequence. The PCR1 forward primer adds a barcode and the Illumina sequencing primer 1, while the reverse primer adds the Illumina indexing read primer sequence. (B) The second round of PCR uses the product of PCR1 as template to add the P5 and P7 adapters, as well as a further optional index in the reverse primer. (C) The final product is a 589 bp fragment containing all the elements required for annealing and sequencing on Illumina high throughput sequencers. Adapted from Jastrzebski et al. (2016). Figure 8. Agarose gel with PCR2 product. The PCR2 product of 589 bp is present in most samples and not in the H2O controls of PCR1 and PCR2. The third and ninth samples failed and were repeated. Data analysis Note: All used scripts for R, Perl, and Python are available on the public GitHub repository https://github.com/MFMdeRooij/CRISPRscreen/https://doi.org/10.5281/zenodo.6342853. In these scripts, the analysis is more extensively explained, while here we only provide a brief description. The raw data of the Namalwa adhesion screen are available in the NCBI SRA database under the accession number SRR16971271. Generate a count table from the fastq file as described on the GitHub repository (/CRISPRScreenAnalysis/FastqToCountTable.pl). In brief, this script: Sorts the reads on barcode (i.e., per sample). Determines the guide sequence and counts unique guides. Aligns the count table to the reference table of the library. For barcode determination one mismatch was allowed; for guide sequence determination no mismatches were allowed but a shift of maximum three nucleotides was allowed. These shifts are caused by indels in the synthetic PCR oligos. Optionally, you can order HPLC-purified PCR primers, which have fewer mutations. Perform DESeq2 analysis (Love et al., 2014) and αRRA from the MAGeCK pipeline (Li et al., 2014) as described on the GitHub repository (/CRISPRScreenAnalysis/CRISPRScreen Analysis.R) to obtain statistics at guide and gene level. This analysis can include shrinkage of non-informative fold changes, which makes it different from standard MAGeCK analysis. The script generates guide and gene output tables, and for example replicate, dispersion, and MA plots (see Figure 9 for an example). The median fold changes of hits in a (synthetic) lethality screen are generally higher than in a loss-of-adhesion screen, since in a lethality screen samples are typically compared after 10 cell divisions. If the event (apoptosis/cell cycle arrest) occurs after two cell divisions, this effect is amplified for eight more cell divisions (so times 28 = 256-fold depletion). In the loss-of-adhesion screen, an event is not amplified by cell divisions. Nonetheless, the loss-of-adhesion screen provides sufficient power to identify proteins involved in adhesion (de Rooij et al., 2022). To determine whether cell viability affects your assay, either perform a lethality screen in parallel or define (strong) viability genes by comparing read counts of the pre-adhesion samples with the distribution of the library (significant: FDR < 0.1 DESeq2 followed by αRRA). If present in your library, the essential genes of Hart et al. (2014) can also be used. Generate volcano plots by plotting the median fold change of the guides per gene against the αRRA depletion and enrichment scores, as described on the GitHub repository (/CRISPRScreenPlots/VolcanoSplit_GenesOfInterest.py) or fold change plots (/CRISPRScreenPlots/GuideFCPlot.py), which plotted the fold changes of all the guides against the genes of interest. Optionally, check the sequence quality as described on the GitHub repository using Pearl and R (/CRISPRScreenAnalysis/Quality.pl followed by /CRISPRScreenAnalysis/Quality.R) or using Python (/CRISPRScreenAnalysis/FastqToCountTable.py). With these scripts you can determine the number of CRISPR reads per barcode, the number of reads perfectly mapped to the library, the number of primer dimer reads, and the most abundant read. If this most abundant read is not present in your library, this indicates a PCR contamination. Figure 9. Example of DESeq2 analysis of the loss-of-adhesion screen of Namalwa. (A) The replicate plots of guide distribution in the three different replicates of the Namalwa screen. nrc: normalized read counts; black line y=x; purple line: linear fit. (B) The dispersion is plotted versus the mean of normalized counts per guide. (C) The log10-count of the guides in the αIgM-stimulated post-adhesion sample is plotted (horizontally) against the log2-fold change of the guides in the αIgM-stimulated post-adhesion sample versus the PMA-stimulated post-adhesion sample (vertically). In black, the guides targeting BTK are depicted. Recipes Supplemented culture medium Reagent Final concentration Amount DMEM/IMDM n/a 500 mL FCS 10% 50 mL L-glutamine (200 mM) 2 mM 5 mL Penicillin-streptomycin 1% 5 mL Total n/a 560 mL 0.5% crystal violet in 20% ethanol Reagent Final concentration Amount Crystal violet 5% 25 g Ethanol (absolute) 20% 100 mL H2O n/a 400 mL Total n/a 500 mL Agar plates containing antibiotics Reagent Final concentration Amount LB 20 g/L 15 g Agar 15 g/L 11.25 g H2O n/a 750 mL Total n/a 750 mL Autoclave this solution in a 1 L bottle. Cool down to approximately 55 °C. Add the appropriate antibiotic to the plate, depending upon which antibiotic resistance gene the plasmid carries. We used 750 µL of 50 mg/mL ampicillin (final concentration is 100 µg/mL). Pour approximately 10 mL per plate. Leave your plates out on the bench to solidify. LB medium containing antibiotics Reagent Final concentration Quantity LB 20 g/L 15 g H2O n/a 750 mL Total n/a 750 mL Autoclave this solution in a 1 L bottle. Cool down to approximately 55 °C. Add the appropriate antibiotic, depending upon which antibiotic resistance gene the plasmid carries. We used 750 µL 50 mg/mL ampicillin (final concentration is 100 µg/mL). SOC medium Reagent Final concentration Quantity LB 20 g/L 5 g D-glucose 20 mM 900.8 mg H2O n/a 250 mL Total n/a 250 mL Produce as LB but with D-glucose added. 0.5 M EDTA, pH 8.0 Reagent Final concentration Quantity EDTA 0.5 M 186.1 g H2O n/a 1,000 mL Total n/a 1,000 mL Stir 186.1 g of EDTA into 800 mL of distilled water. Add NaOH solution to adjust the pH to 8.0. Dilute the solution to 1 L with distilled water. TAE buffer (10×) Reagent Final concentration Quantity Tris-base 400 mM 48.4 g Acetic acid (100%) 200 mM 11.4 ml EDTA (0.5 M, pH 8.0) 10 mM 20 mL H2O n/a 970 mL Total n/a 1,000 mL Dissolve 48.4 g of Tris-base in approximately 700 mL of deionized H2O. Carefully add 11.4 mL of 100% acetic acid and 20 mL of 0.5 M EDTA. Adjust the solution to a final volume of 1 L. For the preparation of the 1× TAE working solution, dilute 100 ml of 10× TAE buffer in 900 mL H2O. Agarose gel Reagent Final concentration Quantity Agarose 2% 3 g TAE buffer n/a 150 mL Total n/a 150 mL Heat the agarose/TAE buffer mixture in a microwave. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved. Add 0.5 μg/mL ethidium bromide. Pour gel in casting apparatus. Allow the agarose to set at room temperature. Acknowledgments This research was supported by grants from the Dutch Cancer Society (KWF) to MS (catalog number: 7873, catalog number: 102750) and to RLB (catalog number: 12539), and by grants from Lymph&Co and the International Waldenstrom’s Macroglobulinemia Foundation (IWMF)/Leukemia & Lymphoma Society (LLS) to MS. This protocol was adapted from de Rooij et al. (2022). Competing interests The authors have no competing interests to declare. Ethics The study did not involve human subjects or animal work. References Brown, J. R., Byrd, J. C., Coutre, S. E., Benson, D. M., Flinn, I. W., Wagner-Johnston, N. D., Spurgeon, S. E., Kahl, B. S., Bello, C., Webb, H. K., et al. (2014). Idelalisib, an inhibitor of phosphatidylinositol 3-kinase p110δ, for relapsed/refractory chronic lymphocytic leukemia. Blood 123(22): 3390-3397. Burger, J. A. and Wiestner, A. (2018). Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nature Reviews Cancer 18(3): 148-167. Byrd, J. C., Furman, R. R., Coutre, S. E., Flinn, I. W., Burger, J. A., Blum, K. A., Grant, B., Sharman, J. P., Coleman, M., Wierda, W. G., et al. (2013). Targeting BTK with Ibrutinib in Relapsed Chronic Lymphocytic Leukemia. New Engl J Med 369(1): 32-42. Chang, B. Y., Francesco, M., De Rooij, M. F. M., Magadala, P., Steggerda, S. M., Huang, M. M., Kuil, A., Herman, S. E. M., Chang, S., Pals, S. T., et al. (2013). Egress of CD19+CD5+ cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients. Blood 122(14): 2412-2424. de Rooij, M. F. M., Kuil, A., Geest, C. R., Eldering, E., Chang, B. Y., Buggy, J. J., Pals, S. T. and Spaargaren, M. (2012). The clinically active BTK inhibitor PCI-32765 targets B-cell receptor– and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 119(11): 2590-2594. de Rooij, M. F. M., Kuil, A., Kraan, W., Kersten, M. J., Treon, S. P., Pals, S. T. and Spaargaren, M. (2016). Ibrutinib and idelalisib target B cell receptor- but not CXCL12/CXCR4-controlled integrin-mediated adhesion in Waldenström macroglobulinemia. Haematologica 101(3): e111-e115. de Rooij, M. F. M., Thus, Y. J., Swier, N., Beijersbergen, R. L., Pals, S. T. and Spaargaren, M. (2022). A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways. Nat Commun 13(1): 2136. Evers, B., Jastrzebski, K., Heijmans, J. P. M., Grernrum, W., Beijersbergen, R. L. and Bernards, R. (2016). CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat Biotechnol 34(6): 631-633. Hart, T., Brown, K. R., Sircoulomb, F., Rottapel, R. and Moffat, J. (2014). Measuring error rates in genomic perturbation screens: gold standards for human functional genomics. Mol Syst Biol 10(7): 733. Jastrzebski, K., Evers, B. and Beijersbergen, R. L. (2016). Pooled shRNA Screening in Mammalian Cells as a Functional Genomic Discovery Platform. In: Azorsa, D. O. and Arora, S. (Eds.) High-Throughput RNAi Screening: Methods and Protocols. Springer New York, 49-73. Li, W., Xu, H., Xiao, T., Cong, L., Love, M. I., Zhang, F., Irizarry, R. A., Liu, J. S., Brown, M. and Liu, X. S. (2014). MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15(12): 554. Love, M. I., Huber, W. and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12): 550. Przybyla, L. and Gilbert, L. A. (2022). A new era in functional genomics screens. Nat Rev Genet 23(2): 89-103. Stephens, D. M. and Byrd, J. C. (2021). Resistance to Bruton tyrosine kinase inhibitors: the Achilles heel of their success story in lymphoid malignancies. Blood 138(13): 1099-1109. Takada, Y., Ye, X. and Simon, S. (2007). The integrins. Genome Biology 8(5): 215. Treon, S. P., Tripsas, C. K., Meid, K., Warren, D., Varma, G., Green, R., Argyropoulos, K. V., Yang, G., Cao, Y., Xu, L., et al. (2015). Ibrutinib in Previously Treated Waldenström’s Macroglobulinemia. New Engl J Med 372(15): 1430-1440. Wang, M. L., Rule, S., Martin, P., Goy, A., Auer, R., Kahl, B. S., Jurczak, W., Advani, R. H., Romaguera, J. E., Williams, M. E., et al. (2013). Targeting BTK with Ibrutinib in Relapsed or Refractory Mantle-Cell Lymphoma. New Engl J Med 369(6): 507-516. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > Invasion & metastasis > Drug discovery and analysis Drug Discovery > Drug Screening Cell Biology > Cell movement > Cell adhesion Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells Seymen Avci [...] Tsvee Lapidot Feb 20, 2017 10721 Views Analysis of B Cell Migration by Intravital Microscopy Michael Schnoor [...] Eduardo Vadillo Dec 5, 2020 3068 Views Static Adhesion Assay for Human Peripheral Blood Mononuclear Cells Giulia Vanoni [...] Sara Trabanelli Jan 5, 2022 2428 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,546	https://bio-protocol.org/en/bpdetail?id=4546&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed X-ray Crystallography: Seeding Technique with Cytochrome P450 Reductase BZ Bixia Zhang JL Jacob A. Lewis RH Rishi Hazra CK ChulHee Kang Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4546 Views: 1068 Reviewed by: Joana Alexandra Costa ReisLaura Alvigini Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Apr 2022 Abstract Cytochrome P450 reductase (CPR) is a multi-domain protein that acts as a redox partner of cytochrome P450s. The CPR contains a flavin adenine dinucleotide (FAD)–binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting domain. To achieve catalytic events, the FMN-binding domain needs to move relative to the FAD-binding domain, and this high flexibility complicates structural determination in high-resolution by X-ray crystallography. Here, we demonstrate a seeding technique of sorghum CPR crystals for resolution improvement, which can be applied to other poorly diffracting protein crystals. Protein expression is completed using an E. coli cell line with a high protein yield and purified using chromatography techniques. Crystals are screened using an automated 96-well plating robot. Poorly diffracting crystals are originally grown using a hanging drop method from successful trials observed in sitting drops. A macro seeding technique is applied by transferring crystal clusters to fresh conditions without nucleation to increase crystal size. Prior to diffraction, a dehydration technique is applied by serial transfer to higher precipitant concentrations. Thus, an increase in resolution by 7 Å is achieved by limiting the inopportune effects of the flexibility inherent to the domains of CPR, and secondary structures of SbCPR2c are observed. Graphical abstract: Keywords: Cytochrome P450 reductase X-ray crystallography Protein crystal seeding Crystal dehydration Structure disorder Resolution improvement Background Cytochrome P450 reductase (CPR, EC 1.6.2.4) is located at the cytosol side of the endoplasmic reticulum membrane in eukaryotic cells, acting as the electron donor to cytochrome P450s and other heme proteins (Phillips and Langdon, 1962). It has been known that there are one to three CPR genes in most vascular plants (Jensen and Møller, 2010). The CPR contains a flavin adenine dinucleotide (FAD)/NADPH-binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting (or linker) domain with a flexible hinge region that links these two domains (Munro et al., 2001; Wang et al., 1997). We have reported on the structural flexibility of sorghum CPR isoforms (Zhang et al., 2022b) and the rapid interdomain opening and closing motion that resulted in fast interdomain electron transfer in SbCPR (Zhang et al., 2022a). Especially in the SbCPR2c isoform, which was crystallized in full-length, the FMN-binding domain was not resolved due to the aforementioned flexibility. Such flexibility causes uncertainty of the residue atomic coordinates and dihedral angles of the protein crystal, which leads to local disordered structure and overall low-resolution diffraction. Crystallization of diffraction quality crystals is the largest bottleneck in protein structure determination (Holcomb et al., 2017). These difficulties have been overcome through the use of various techniques. Seeding uses previously nucleated crystals to initiate the growth of larger crystals in a fresh drop where protein concentration has not yet been depleted by the many nucleation sites of the small crystals (Rhodes, 2006). Micro streaking operates by running a fiber over an existing crystal—often a whisker from an animal—through a fresh drop to increase growth of the nucleated particles in a supersaturated drop (Stura and Wilson, 1991). In contrast, a macro seeding technique utilizes larger crystals, and thus requires more precision as a chosen crystal is washed and transferred from its original drop to a new mother liquor. This method tends to be more meticulous and can fracture the original crystal, causing smaller fragments to be placed into the new drop, which induces micro seeding (Zhu et al., 2005). Seeding techniques have allowed for growth of larger crystals and increased resolution of diffraction; cellobiohydrolase II (CBHII) crystallization was first achieved by using seeding to avoid formation of microcrystals and diffraction greater than 2.0 Å (Bergfors et al., 1989). High mosaicity and cracking/sliding of the lattice caused by impurities have been overcome by using seeding to reduce crystal deficits and improve the resolution of crystals (Caylor et al., 1999). Another method for improving crystal lattice packing is the dehydration of the crystalline to reduce water contents and to consequently confer tighter packing (Timasheff, 1995). Dehydration can be accomplished via exposure to the atmosphere or via serial dilution into higher cryoprotectant-containing solutions (Heras and Martin, 2005). Crystal dehydration has been one of the most successful post crystallization treatments in increasing resolution. Notably, the resolution of disulfide bond isomerase was improved from 7 to 2.6 Å and from 12 to 2.6 Å in E. coli YbgL (Abergel, 2004; Haebel et al., 2001). We incorporated a macromolecule seeding technique—additive solution optimization—together with dehydration to improve the resolution of the SbCPR2c crystal structure from approximately 12 to 4.5 Å. This enabled the determination of the secondary structure for SbCPR2c. It is worth noting, however, that crystal growth is a very tedious process with repeatability differing in trials due to miniscule changes of parameters and the complexity of the system. Despite the aforementioned difficulties, our protocol of seeding and dehydration could be widely applied to resolve the resolution problems to a variety of crystals hindered in resolution by their dynamic disorder. Materials and Reagents MRC 2-well crystallization plate (Swissci, Hampton Research, catalog number: HR3-082) 24-well XRL plate (Molecular Dimensions, catalog number: MD3-11) Additive screen (Hampton Research, catalog number: HR2-428) Microscope cover glass (Fisher Scientific, Fisherbrand, catalog number: 19803) Glass capillary (Charles Super Company, catalog number: 02-SG) Amicon 8050 ultrafiltration cell (Amicon, catalog number: UFSC05001) 30 kDa ultrafiltration discs (EMD Millipore Corporation, Biomax, catalog number: PBTK04310) Escherichia coli Rosetta 2 (DE3) competent cells (Sigma-Aldrich, Millipore Sigma, catalog number: 71402) pET-30a(+) vector (Sigma-Aldrich, Millipore Sigma, catalog number: 69909) Sorghum bicolor CPR2c gene (Sobic.006G245400) (synthesized by Genscript) Kanamycin (IBI Scientific, catalog number: IB02120) IPTG (GoldBio, catalog number:I2481C100) NADP+ (Alfa Aesar, catalog number: T23B015) PEG 3350 (Sigma-Aldrich, catalog number: P4338) Nickel-NTA resin (G-biosciences, catalog number: 786-940) Index crystal screen kit (Hampton Research, catalog number: HR2-144) Luria-Bertani (LB) medium (see Recipes) Lysis/wash buffer (see Recipes) Elution buffer (see Recipes) Hydroxyapatite column buffer A (see Recipes) Hydroxyapatite column buffer B (see Recipes) Final buffer (see Recipes) Crystallization buffer (crystal screen F12) (see Recipes) Equipment Crystal Phoenix (Art Robbins Instruments) ÄKTA pure (GE Healthcare, ÄKTA pure, catalog number: 29014834) Model 450 sonicator (Branson Ultrasonics, catalog number: 22-309783) Software HKL2000 (https://hkl-xray.com/hkl-2000) Phenix (https://phenix-online.org/) Coot (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/) Procedure Gene cloning Clone the Sorghum bicolor CPR2c gene (Sobic.006G245400) with a N-terminal 6×His tag and without the native N-terminal hydrophobic membrane–anchoring region (Δ1–50) into vector pET-30a(+) for overexpression. Cell transformation Put E. coli Rosetta 2 (DE3) competent cells on ice. Add 1 μL of plasmid (20 ng/μL) into 20 μL of competent cells. Incubate on ice for 5 min. Heat shock for 30 s. Incubate on ice for 2 min. Add 80 μL of LB medium to competent cells. Shake at 37 °C at 220 rpm for 1 h. Plate all the cells on an LB agar plate with 50 ng/μL of kanamycin and incubate overnight. Protein expression Pick a single colony from the LB agar plate and grow in 5 mL of LB medium supplemented with 50 ng/μL kanamycin overnight at 37 °C. Grow 20 mL of starting culture from the 5 mL culture with 50 ng/μL kanamycin overnight at 37 °C. Transfer 20 mL of starting culture to 3 L of LB medium with 50 ng/μL kanamycin. Shake at 220 rpm and 37 °C until reaching a OD600 of 0.4–0.6. Cool down the medium to 25 °C and add 0.5 mM of IPTG to induce. Induce the cells at 25 °C for 16 h. Harvest the cells at 8,000 × g and 4 °C for 10 min. Protein purification Resuspend the cells with lysis buffer. Set the output control at 9 and duty cycle at 90%. Sonicate the cells for 30 s followed by resting on ice for 5 min until homogenous. Centrifuge at 34,000 × g and 4 °C for 1 h to remove the cell debris. Load the clear lysate on a gravity column containing 20 mL of nickel-NTA resin pre-equilibrated with lysis buffer. Wash with 200 mL of the same buffer. Elute the protein with 100 mL of elution buffer. Concentrate and buffer exchange against hydroxyapatite column buffer A using an Amicon 8050 ultrafiltration cell with a 30 kDa cutoff membrane. Load the buffer-exchanged protein on the hydroxyapatite column. Wash with 100 mL of 5% hydroxyapatite column buffer B. Elute protein with 10% hydroxyapatite column buffer B. Concentrate and buffer exchange against final buffer using an Amicon 8050 ultrafiltration cell with a 30 kDa cutoff membrane. Initial crystallization setup Concentrate the purified protein to 30 mg/mL, determined by the molar extinction coefficient of CPR (21.4 mm−1 cm−1). Add 1.2 mM of NADP+ prior to crystallization. Run Crystal Phoenix with an MRC 2-well crystallization plate with the index crystal screen kit and set up at room temperature (22 °C). The drop contains 0.2 μL of protein solution and 0.2 μL of screening solution. Small crystals show up in condition F12 (see crystallization buffer in Recipes) in two days (Figure 1A). Reproduce and optimize the crystal by mixing 1.6 μL of crystallization buffer, 2 μL of protein solution, and 0.4 μL of additive screen on a cover glass. Set up the hanging drop vapor diffusion in 24-well plates with 0.5 mL of crystallization buffer in reservoir. Needle-shaped crystals show up with additive screen condition 31 (1,5-diaminopentane dihydrochloride) in 3–5 days (Figure 1B). Crystal seeding Mix 2.4 μL of crystallization buffer, 3 μL of protein solution, and 0.6 μL of additive screen. Suck one needle crystal from the previous drop, place into the new 6 μL crystallization drop, and set up the hanging drop. Large crystals show up in one week (Figure 1C). Transfer the cover glass to the reservoir filled with 0.5 mL of 30% (v/w), 40% (v/w), and 50% (v/w) PEG 3350 every third day for sequential dehydration. Fast freeze the dehydrated crystals in liquid nitrogen for X-ray diffraction after equilibrating in 50% (v/w) PEG 3350 for two days. Figure 1. Optimization of SbCPR2c crystals. (A) Crystals obtained from the hit [index condition F12 (see crystallization buffer in Recipes)] of screening. (B) Crystals obtained from hanging drop with additive solution optimization. (C) Crystals obtained from seeding technique. Data collection and structure determination Diffract the crystals at the beamline by using beam size 100 × 100 μm, 10 s, and 1 degree per frame and 180 degrees in total. Index, integrate, and scale the data using HKL2000. Do molecular replacement with the proper model [in our case, SbCPR2b (PDBID: 7SUX)] with PHENIX, run AutoBuild, and refine the structure with PHENIX and Coot. Figure 2. The resolution of the crystal has been improved from 11.7 Å (from non-seeding hanging drop crystals) to 4.5 Å (seeding crystals) Data analysis The diffraction data was indexed, integrated, and scaled with HKL2000. The molecular replacement was performed with the SbCPR2b structure (PDBID: 7SUX) and with Phaser-MR (full-featured) in the PHENIX software. The number of molecules in an asymmetric unit was calculated with Matthews coefficients (Matthews, 1975). AutoBuild was then run for model building with the SbCPR2c sequence. A rigid body refinement was completed before full refinement of XYZ (reciprocal-space), XYZ (real-space), and individual B-factors with phenix.refine. The individual residues were refined in Coot. Notes The crystallization and additive solutions are specific for SbCPR2c. It is necessary to initially screen for the optimal condition for the protein of interest. If crystals are of poor shape, using additive screen solution can rectify poor crystal edges. Using additive screen solution may cause precipitation. When placing the crystal seed, you should avoid disruption to the drop, which can mix the solutions and will impede crystal growth. Recipes Luria-Bertani (LB) medium Reagent Final concentration Amount Tryptone 10 g/L 10 g Yeast extract 5 g/L 5 g NaCl 10 g/L 10 g dH2O n/a n/a Total n/a 1,000 mL Lysis/wash buffer Reagent Final concentration Amount NaCl 300 mM 17.532 g Tris 50 mM 6.057 g Imidazole 20 mM 1.36 g dH2O n/a n/a HCl (12 M) n/a As needed to adjust pH to 8 Total n/a 1,000 mL Elution buffer Reagent Final concentration Amount NaCl 300 mM 17.532 g Tris 50 mM 6.057 g Imidazole 250 mM 17.02 g dH2O n/a n/a HCl (12 M) n/a As needed to adjust pH to 8 Total n/a 1,000 mL Hydroxyapatite column buffer A Reagent Final concentration Amount KH2PO4 5 mM 0.68 g NaOH n/a As needed to adjust pH to 6.8 dH2O n/a n/a Total n/a 1,000 mL Hydroxyapatite column buffer B Reagent Final concentration Amount KH2PO4 500 mM 68 g NaOH n/a As needed to adjust pH to 6.8 dH2O n/a n/a Total n/a 1,000 ml Final buffer Reagent Final concentration Amount Hydroxyapatite column buffer B 50 mM 100 mL dH2O n/a 900 mL Total n/a 1,000 mL Crystallization buffer (crystal screen F12) Reagent Final concentration Amount NaCl (2 M) 200 mM 5 mL HEPES (1 M, pH 7.5) 100 mM 5 mL dH2O n/a n/a PEG 3350 (50%, m/v) 25% (w/v) 25 mL Total n/a 50 mL Acknowledgments Original research was supported by grant from NSF (CHE-1804699, MCB-2043248) and Murdock Charitable Trust (CHK). This research used resources of the Advanced Light Source (beamline 5.0.2 and beamline 5.0.3), which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. The protocol was derived from original research paper (Zhang et al., 2022b). Competing interests There is no competing interest. References Abergel, C. (2004). Spectacular improvement of X-ray diffraction through fast desiccation of protein crystals. Acta Crystallogr D Biol Crystallogr 60(Pt 8): 1413-1416. Bergfors, T., Rouvinen, J., Lehtovaara, P., Caldentey, X., Tomme, P., Claeyssens, M., Pettersson, G., Teeri, T., Knowles, J. and Jones, T. A. (1989). Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei. J Mol Biol 209(1): 167-169. Caylor, C. L., Dobrianov, I., Lemay, S. G., Kimmer, C., Kriminski, S., Finkelstein, K. D., Zipfel, W., Webb, W. W., Thomas, B. R., Chernov, A. A., et al. (1999). Macromolecular impurities and disorder in protein crystals. Proteins 36(3): 270-281. Jensen, K. and Møller, B. L. (2010). Plant NADPH-cytochrome P450 oxidoreductases. Phytochemistry 71: 132-141. Haebel, P. W., Wichman, S., Goldstone, D. and Metcalf, P. (2001). Crystallization and initial crystallographic analysis of the disulfide bond isomerase DsbC in complex with the alpha domain of the electron transporter DsbD. J Struct Biol 136(2): 162-166. Heras, B. and Martin, J. L. (2005). Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr D Biol Crystallogr 61(Pt 9): 1173-1180. Holcomb, J., Spellmon, N., Zhang, Y., Doughan, M., Li, C. and Yang, Z. (2017). Protein crystallization: Eluding the bottleneck of X-ray crystallography. AIMS Biophys 4(4): 557-575. Matthews, B. W. (1975). Comparison of the predicted and observed secondary structure of T4 phage lysozyme. Biochim Biophys Acta 405(2): 442-451. Munro, A. W., Noble, M. A., Robledo, L., Daff, S. N. and Chapman, S. K. (2001). Determination of the redox properties of human NADPH-cytochrome P450 reductase. Biochemistry 40(7): 1956-1963. Phillips, A. H. and Langdon, R. G. (1962). Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J Biol Chem 237: 2652-2660. Rhodes, G. (2006). Chapter 3 - Protein Crystals. In: Rhodes, G. (Ed.) Crystallography Made Crystal Clear (Third Edition). Academic Press, 31-47. Stura, E. A. and Wilson, I. A. (1991). Applications of the streak seeding technique in protein crystallization. J Cryst Growth 110(1): 270-282. Timasheff, S. N. (1995). Solvent stabilization of protein structure. Methods Mol Biol 40: 253-269. Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S. S. and Kim, J.-J. P. (1997). Three-dimensional structure of NADPH–cytochrome P450 reductase: prototype for FMN-and FAD-containing enzymes. Proc Natl Acad Scie U S A 94(16): 8411-8416. Zhang, B., Kang, C. and Davydov, D. R. (2022a). Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy. Biology (Basel) 11(4). Zhang, B., Munske, G. R., Timokhin, V. I., Ralph, J., Davydov, D. R., Vermerris, W., Sattler, S. E. and Kang, C. (2022b). Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition. J Biol Chem 298(4): 101761. Zhu, D. Y., Zhu, Y. Q., Xiang, Y. and Wang, D. C. (2005). Optimizing protein crystal growth through dynamic seeding. Acta Crystallogr D Biol Crystallogr 61(Pt 6): 772-775. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant biochemistry > Protein Biophysics > X-ray crystallography Biochemistry > Protein > Structure Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Localization and Topology of Thylakoid Membrane Proteins in Land Plants Salar Torabi [...] Jörg Meurer Dec 20, 2014 12380 Views Heterologous Expression and Purification of Catalytic Domain of CESA1 from Arabidopsis thaliana Venu Gopal Vandavasi and Hugh O’ Neill Oct 20, 2016 6695 Views Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1) Joana Rocha [...] Christelle Breton Dec 20, 2016 7876 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,547	https://bio-protocol.org/en/bpdetail?id=4547&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay RB Rebecca J. Burge KJ Katie H. Jameson AW Anthony J. Wilkinson JM Jeremy C. Mottram Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4547 Views: 854 Reviewed by: Alexandros AlexandratosAndrew MacLean Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in PLOS Pathogens Oct 2020 Abstract Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues. Keywords: Ubiquitin Ubiquitination E2 conjugating E3 ligase Chain formation Background Ubiquitination is a three-step process involving the sequential action of E1, E2, and E3 enzymes. In the first step, catalyzed by an E1 ubiquitin-activating enzyme, ubiquitin-adenylate is formed followed by ubiquitin transfer to a conserved cysteine on the enzyme. The second step involves a trans-thioesterification, in which the ubiquitin is transferred to a cysteine on the E2. In the third step, the E2, known as a ubiquitin-conjugating enzyme, cooperates with an E3 ubiquitin ligase to transfer ubiquitin to the target substrate, typically through the formation of an isopeptide bond to a lysine side chain (Zheng and Shabek, 2017). In many cases, target substrates can become poly-ubiquitinated through the attachment of further ubiquitin molecules. This attachment can take place on any of ubiquitin’s seven lysine side chains or on its amino terminal methionine, resulting in many permutations of ubiquitin conjugation (Zheng and Shabek, 2017). Specific ubiquitin linkages are associated with different cellular outcomes, although the understanding of this is incomplete (Komander and Rape, 2012). In an important “off-pathway” reaction, some E2s are also able to catalyze the formation of free di-ubiquitin, alone or with the assistance of a ubiquitin E2 variant (Wu et al., 2003; Wang et al., 2016; Burge et al., 2020). The parasite Leishmania mexicana assumes different morphologies depending on its host: in the sand fly, it takes the motile promastigote form, whereas in a mammalian host, it takes the immotile amastigote form. Key to mammalian infection is the ability to undergo promastigote to amastigote differentiation. In L. mexicana, the E2 UBC2 and the ubiquitin E2 variant UEV1, which form a heterodimeric complex, are required for this process (Burge et al., 2020). Prior studies of the Saccharomyces cerevisiae UBC2 and UEV1 homologs UBC13 and MMS2, respectively, demonstrated that they could form K63-linked ubiquitin chains in the absence of an E3 (Hofmann and Pickart, 1999; McKenna et al., 2001; Wu et al., 2003; Andersen et al., 2005; Pastushok et al., 2005). By adapting methods used in these studies, the in vitro ubiquitin conjugation assays described here were established to delineate the ubiquitination activities of UBC2 and UEV1: firstly, to assess the cooperation of UBC2 and UEV1 in di-ubiquitin formation and, secondly, to assess the cooperation of UBC2 and UEV1 with E3 ubiquitin ligases (Figure 1). The method described here allows for the assessment of di-ubiquitin formation by an E2 and E2 variant pair and their collective cooperation with an E3 ligase. The assay uses western blotting specifically to detect ubiquitin conjugates. Blotting with an antibody specific to K63-conjugation also enables the assessment of whether the ubiquitin-conjugate products are linked at K63. Although we have not tested other antibodies, we anticipate that the method could easily be adapted to assay antibodies specific to other ubiquitin linkages. Figure 1. Ubiquitination outcomes mediated by UBC2 and UEV1. In the presence of ATP, ubiquitin (Ub) is activated and covalently attached in step 1 to the E1 UBA1a. In the presence of UEV1 and UBC2, the ubiquitin is transferred in step 2 to the UBC2 component of the UBC2–UEV1 heterodimer, and subsequently in step 3 onto a second ubiquitin molecule to form a K63-linked ubiquitin dimer, as described in Assay A. In the absence of UEV1, a UBC2–Ub complex is formed in step 4, which in the presence of an E3 such as RNF8 or BIRC2 forms polyubiquitin chains that may be unanchored or anchored to an unidentified substrate protein as described in Assay B. The linkage(s) in these chains are not known but are not K63-based. Materials and Reagents 1.5 mL reaction tubes (Sarstedt, catalog number: 72.690.001) 100 mm square Petri dishes (Thermo Scientific, catalog number: 11349273) HEPES (Fisher Bioreagents, catalog number: BP310-1) NaCl (Fisher Chemicals, catalog number: S/3161/65) MgCl2 (Sigma, catalog number: 63064) DTT (Melford Laboratories, catalog number: D11000) ATP (Sigma-Aldrich, catalog number: A2383) Ubiquitin (R&D Systems, catalog number: U-100H-10M) NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, mini protein gels (Invitrogen, catalog number: NP0322BOX) NuPAGE MES-SDS running buffer (20×) (Invitrogen, catalog number: NP0002) NuPAGE LDS sample buffer (4×) (Invitrogen, catalog number: NP0007) β-Mercaptoethanol (PanReac AppliChem, catalog number: A1108) SeeBlue Plus2 pre-stained protein standard (Invitrogen, catalog number: LC5925) PierceTM 20× TBS buffer (ThermoFisher, catalog number: 28358) Tween-20 (Sigma, catalog number: P1379) iBlot 2 nitrocellulose transfer stacks (Invitrogen, catalog number: IB23001) Ethanol (VWR chemicals, catalog number: MFCD00003568) Bovine serum albumin (BSA) (Sigma, catalog number: A3294) Milk powder (Marvel) Mono and polyubiquitylated conjugates, mAb (FK2) antibody (Ubiquigent, catalog number: 68-0121-500) Ub-K63 mouse anti-human, clone: HWA4C4 (Affymetrix eBioscienceTM, catalog number: 14-6077-82) HRP-conjugated anti-mouse antibody (Promega, catalog number: W4021) Clarity Western ECL substrate (Bio-Rad, catalog number: 1705060) Clarity Max Western ECL substrate (Bio-Rad, catalog number: 1705062) Purified recombinant E1 (UBA1a, prepared under reducing conditions, storage: -70 °C) (Burge et al., 2020) Purified recombinant E2 (UBC2, prepared under reducing conditions, storage: -70 °C) (Burge et al., 2020) Purified recombinant E2 variant (UEV1, storage: -70 °C) (Burge et al., 2020) Purified recombinant E3 (GST-BIRC2 or GST-RNF8, storage: -70 °C) (Ubiquigent, catalog number: 63-0015-025 or 63-0021-025) di-ubiquitin, K63-linked (storage: -70 °C) (Ubiquigent, catalog number: 60-0107-010) Reaction buffer (10×) (see Recipes) SDS-PAGE sample buffer (4×) (see Recipes) 1× TBSt (see Recipes) Equipment Dry block heater (Grant QBD1) Protein electrophoresis equipment (XCell SureLock Mini-Cell, EI0001) Protein transfer system (iBlot 2 Dry Blotting System, Thermo Fisher Scientific) Gel rocker Microplate shaker (Grant, PMS-1000i) Chemi-blot imager (ChemiDoc Imaging System, Bio-Rad) Procedure Di-ubiquitin formation assay Prepare three reaction mixes in 1.5 mL tubes on ice containing the following reagents: 100 nM E1, 2.5 µM E2, 2.5 µM E2 variant, and 100 µM ubiquitin, in a buffer of 50 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2, 2 mM DTT, and 5 mM ATP in a total volume of 40 µL. Typical reagent concentrations and volumes are given in Table 1. If a controlled reaction start is desired, the E1 or ATP can be omitted from the reaction mix during setup and added to initiate the reaction (mix by gently flicking the tube or stirring with a pipette tip during the final reagent addition). Three control reactions should also be performed, omitting either the E2, the E2 variant, or both the E2 and E2 variant, demonstrating that di-ubiquitin formation is not a consequence of activity from either the E2 or E2 variant alone, or from activity of the E1. Table 1. Reaction composition Component Working concentration Volume per reaction Final concentration E1 4 µM 1 µL 100 nM E2 25 µM 4 µL 2.5 µM E2 variant* 25 µM 4 µL 2.5 µM E3** 5 µM 8 µL 1.0 µM Ubiquitin 1167 µM 3.4 µL 100 µM Reaction buffer 500 mM HEPES pH 7.5 1 M NaCl 100 mM MgCl2 20 mM DTT 4 µL 50 mM HEPES pH 7.5 100 mM NaCl 10 mM MgCl2 2 mM DTT ATP 100 mM 2 µL 5 mM dH2O Up to 40 µL Up to 40 µL - Can be excluded in the E3 cooperation assay *Include only in the E3 cooperation assay Incubate the reaction mixes in a dry block heater at 37 °C for 0 min, 30 min, and 90 min. Stop the reactions by adding 1× SDS-PAGE sample buffer (13.3 µL) (see Recipes). Heat the samples at 70 °C for 5 min using a dry block heater. Run 20 µL of each sample on a NuPAGE mini protein gel in 1× NuPAGE MES-SDS running buffer using an electrophoresis system. Load 5 µL of SeeBlue Plus2 pre-stained protein standard to a single lane of the gel to enable molecular weight comparison of bands detected later during the western blot. Run the gel at 200 V until the lowest molecular weight marker is approximately 0.25 cm from the bottom of the gel (approximately 35 min). If analysis of both ubiquitin conjugates and K63-linked ubiquitin conjugates is desired, run two gels with 20 µL samples from each reaction on each gel. When assessing the presence of K63-linked ubiquitin, a sample of 100 ng K63-linked di-ubiquitin should be included on the gel, which will act as a positive control when blotting with the Ub-K63 antibody. Incubate the gel in 20% ethanol in a square Petri dish for 10 min on a gel rocker at room temperature. Transfer the proteins to a nitrocellulose membrane using the iBlot 2 Dry Blotting System and an iBlot 2 nitrocellulose transfer stack according to the manufacturer’s instructions. Perform the transfer at 20 V for 1 min, then 23 V for 4 min, and finally 25 V for 7 min. Assessing the formation of ubiquitin conjugates using the FK2-antibody (Ubiquigent) Block the membrane by covering it with 5% BSA dissolved in 1× TBSt (see Recipes) in a square Petri dish on a microplate shaker at 4 °C overnight. Apply the mono and polyubiquitylated conjugates, mAb (FK2) antibody at a 1:1,000 dilution in 10 mL of 1% BSA in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker for 1 h. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker. Apply the HRP-conjugated anti-mouse secondary antibody at 1:10,000 dilution in 10 mL of 5% BSA in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker for 1 h. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker operating at speed 4. Apply clarity Western ECL substrate according to manufacturer’s instructions. Measure chemiluminescence using a suitable imaging system (e.g., ChemiDoc imaging system, Bio-Rad). Assessing the formation of K63-linked ubiquitin conjugates using the Ub-K63 antibody (ThermoFisher) Block the membrane by covering it with 5% (w/v) milk dissolved in 1× TBSt. Incubate in a square Petri dish on a microplate shaker at 4 °C for 1 h. Apply the Ub-K63 mouse antibody at 1:250 dilution in 5 ml of 5% milk in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker overnight. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker. Apply the HRP-conjugated anti-mouse secondary antibody at 1:10,000 dilution in 10 mL of 5% (w/v) milk in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker overnight. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker. Apply clarity Western ECL substrate according to manufacturer’s instructions. Measure chemiluminescence using a suitable imaging system. E3 cooperation assay Prepare reaction mixes in 1.5 mL reaction tubes on ice containing the following reagents: 100 nM E1, 2.5 µM E2, 1 µM E3, and 100 µM ubiquitin in a buffer of 50 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2, 2 mM DTT, and 5 mM ATP in a total volume of 40 µL. Where relevant, 2.5 µM of an E2 variant can also be included (e.g., when involved in di-ubiquitin formation in conjunction with an E2). Typical reagent concentrations and volumes are given in Table 1. If a controlled reaction start is desired, the E1 or ATP can be omitted from the reaction mix during setup and added to initiate the reaction (mix by gently flicking the tube or stirring with a pipette tip during the final reagent addition). To determine whether the observed activity is due to the E3, four control reactions should be carried out, omitting either the E3, the E2, the E2 variant, or both the E2 and E2 variant. This allows for the assessment of any activity that may be a consequence of the E1 and E2s in the absence of the E3, and also whether the E3 works in cooperation with the E2 or E2 variant, alone or in combination. Incubate reactions in a dry block heater at 30 °C for 60 min or other times as required. Carry out steps A3–A21. Data analysis The FK2 antibody detects all ubiquitin conjugates, which are observed as chemiluminescent bands on the western blot. Comparison of these bands with the pre-stained ladder allows confirmation of di-ubiquitin formation. This is seen in Figure 2A, where incubation of Leishmania UBC2 and UEV1 for 30 or 90 min resulted in the formation of a clear band for di-ubiquitin (minor bands were also observed for UBA1a-Ub and UBC2-Ub complexes because these also contain a conjugated ubiquitin). The FK2 antibody can also be used to detect substrate and/or poly-ubiquitination, as desired in the E3 cooperation assay (Figure 3A). In this example, multiple ubiquitin conjugate bands were seen when Leishmania UBC2 was incubated with the human E3s BIRC2 or RNF8. Blotting with the Ub-K63 antibody allows the specific assessment of the formation of K63-linked ubiquitin conjugates (Figure 2B and Figure 3B). Thus, assessment of the same reaction products using both the FK2 and Ub-K63 antibodies enables the analysis of whether conjugates detected by the FK2 antibody are K63-linked. For example, in Figure 2, the di-ubiquitin band that was observed when blotting with the FK2 antibody (Figure 2A) was also observed when blotting with the Ub-K63 antibody (Figure 2B), indicating that the di-ubiquitin formed by UBC2 and UEV1 is K63-linked. However, in the E3 cooperation assay (Figure 3), conjugates produced by incubation of UBC2 with BIRC2 or RNF8 that were detected by the FK2 antibody (Figure 3A) were not detected by the Ub-K63 antibody (Figure 3B), suggesting that these conjugates are not K63-linked. Figure 2. Di-ubiquitin formation assay. A) Blot using FK2 ubiquitin conjugate antibody. B) Blot using K63-linked ubiquitin antibody. Reproduced from Burge et al. (2020). Figure 3. E3 Cooperation assay. A) Blot using FK2 ubiquitin conjugate antibody. B) Blot using K63-linked ubiquitin antibody. Reproduced from Burge et al. (2020). Notes UBA1a and UBC2 need to be purified under reducing conditions to maintain catalytic activity: 5 mM β-mercaptoethanol was included in the purification buffers for nickel affinity purification and 2 mM DTT or 1 mM TCEP for size exclusion chromatography. Proteins were stored at -70 °C in buffers containing 2 mM DTT or 1 mM TCEP (Note: UBA1a and UBC2 purified in buffers without reducing agents were not catalytically active). Avoid freeze-thaw cycles to increase the storage lifetime of the proteins. Successful assays were achieved with the following changes in gel-running and transfer conditions: firstly, running samples on 4%–20% Mini-Protean TGX gels (Bio-Rad) in a running buffer of 25 mM Tris and 192 mM glycine pH 8.3, at 200 V for 35 min; secondly, incubating the gel, a nitrocellulose membrane, and filter paper in a transfer buffer of 25 mM Tris, 192 mM glycine, and 10% v/v methanol for 30 min before transfer using a Trans-Blot SD semi-dry transfer cell (Bio-Rad) at 25 V for 25 min. If low activity is seen in the E3 cooperation assay, try increasing the incubation temperature to 37 °C. Recipes Reaction buffer (10×) 500 mM HEPES pH 7.5 1 M NaCl 100 mM MgCl2 20 mM DTT SDS-PAGE sample buffer (4×) 95% NuPAGE LDS sample buffer (4×) 5% β-mercaptoethanol 1× TBSt 5% (v/v) PierceTM 20× TBS buffer 0.05% (v/v) Tween-20 94.95% (v/v) deionized H2O Acknowledgments This protocol was adapted from the research published in Burge et al. (2020). We would like to thank Ubiquigent for providing a 3 month internship for RB, during which this protocol was developed. This work was supported by a Medical Research Council Studentship to RB (MRC MR/N018230/1) and the Wellcome Trust to JCM (200807/Z/16/Z). Competing interests The authors declare no competing interests References Andersen, P. L., Zhou, H., Pastushok, L., Moraes, T., McKenna, S., Ziola, B., Ellison, M. J., Dixit, V. M. and Xiao, W. (2005). Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J Cell Biol 170(5): 745-755. Burge, R. J., Damianou, A., Wilkinson, A. J., Rodenko, B. and Mottram, J. C. (2020). Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex. PLoS Pathog 16(10): e1008784. Hofmann, R. M. and Pickart, C. M. (1999). Non-canonical MMS2-Encoded Ubiquitin-Conjugating Enzyme Functions in Assembly of Novel Polyubiquitin Chains for DNA Repair. Cell 96: 645-53. Komander, D. and Rape, M. (2012). The ubiquitin code. Annu Rev Biochem 81: 203-229. McKenna, S., Spyracopoulos, L., Moraes, T., Pastushok, L., Ptak, C., Xiao, W. and Ellison, M. J. (2001). Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J Biol Chem 276(43): 40120-6. Pastushok, L., Moraes, T. F., Ellison, M. J. and Xiao, W. (2005). A single Mms2 "key" residue insertion into a Ubc13 pocket determines the interface specificity of a human Lys63 ubiquitin conjugation complex. J Biol Chem 280(18): 17891-17900. Wang, S., Cao, L. and Wang, H. (2016). Arabidopsis ubiquitin-conjugating enzyme UBC22 is required for female gametophyte development and likely involved in Lys11-linked ubiquitination. J Exp Bot 67(11): 3277-3288. Wu, P. Y., Hanlon, M., Eddins, M., Tsui, C., Rogers, R. S., Jensen, J. P., Matunis, M. J., Weissman, A. M., Wolberger, C. and Pickart, C. M. (2003). A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J 22(19): 5241-5250. Zheng, N. and Shabek, N. (2017). Ubiquitin Ligases: Structure, Function, and Regulation. Annu Rev Biochem 86: 129-157. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > Microbial biochemistry > Protein Biochemistry > Protein > Activity Biological Sciences > Biological techniques > Microbiology techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Liposome Disruption Assay to Examine Lytic Properties of Biomolecules John R. Jimah [...] Niraj H. Tolia Aug 5, 2017 12791 Views Activity-based Crosslinking to Identify Substrates of Thioredoxin-domain Proteins in Malaria Parasites David W. Cobb [...] Vasant Muralidharan Feb 20, 2022 1791 Views H2 Production from Methyl Viologen–Dependent Hydrogenase Activity Monitored by Gas Chromatography Nuttavut Kosem Dec 5, 2023 561 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,548	https://bio-protocol.org/en/bpdetail?id=4548&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Binding Affinity Measurements Between DNA Aptamers and their Virus Targets Using ELONA and MST GP Gregory T. Pawel YM Yuan Ma YW Yuting Wu YL Yi Lu AP Ana Sol Peinetti Published: Vol 12, Iss 21, Nov 5, 2022 DOI: 10.21769/BioProtoc.4548 Views: 1394 Reviewed by: Alka MehraAbhilash Padavannil Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Sep 2021 Abstract Aptamers have been selected with strong affinity and high selectivity for a wide range of targets, as recently highlighted by the development of aptamer-based sensors that can differentiate infectious from non-infectious viruses, including human adenovirus and SARS-CoV-2. Accurate determination of the binding affinity between the DNA aptamers and their viral targets is the first step to understanding the molecular recognition of viral particles and the potential uses of aptamers in various diagnostics and therapeutic applications. Here, we describe protocols to obtain the binding curve of the DNA aptamers to SARS-CoV-2 using Enzyme-Linked Oligonucleotide Assay (ELONA) and MicroScale Thermophoresis (MST). These methods allow for the determination of the binding affinity of the aptamer to the infectious SARS-CoV-2 and the selectivity of this aptamer against the same SARS-CoV-2 that has been rendered non-infectious by UV inactivation, and other viruses. Compared to other techniques like Electrophoretic Mobility Shift Assay (EMSA), Surface Plasmon Resonance (SPR), and Isothermal Titration Calorimetry (ITC), these methods have advantages for working with larger particles like viruses and with samples that require biosafety level 2 facilities. Keywords: Aptamer SARS-CoV-2 Microscale Thermophoresis Binding affinity ELONA Virus detection Background Infectious diseases remain one of the biggest challenges for human health, with the current COVID-19 pandemic as a primary example. Accurate and early detection of viruses is crucial for transmission control through clinical diagnosis and therapy. To achieve this goal, DNA aptamers have emerged as a promising approach to develop sensors for on-site and real-time detection and quantitation. DNA aptamers are short single-strand DNA molecules with a specific sequence that allow them to form a specific three-dimensional conformation to recognize a certain target with high affinity and selectivity (Huizenga 1997; Cho et al., 2009; Liu et al., 2009; Xiang and Lu 2014; Xing et al., 2014; Li et al., 2019; Ma et al., 2021; Xie et al., 2021). They have been shown to be highly specific for binding viruses (Balogh et al., 2010; González et al., 2016; Zou et al., 2019; Kukushkin et al., 2019; Peinetti et al., 2021), rivaling antibodies. Furthermore, DNA aptamers have gained considerable attention because they are cost-effective, have easy chemical modifications, and have high chemical stability, binding affinity, repeatability, and reusability (Liu et al., 2009; Fang and Tan 2010; Dunn et al., 2017; Hong et al., 2020). All these features make them ideal candidates for developing affordable, rapid, sensitive methods able to identify new or emerging viruses, including virus variants (Xing et al., 2014; Zou et al., 2019; Li et al., 2021; Peinetti et al., 2021; Chakraborty et al., 2022). Aptamers are obtained from a random DNA library by an iterative procedure called Systematic Evolution of Ligands by EXponential enrichment (SELEX), which is carried out in test tubes with large sampling libraries (~1015) and is performed in a much shorter time with less cost than the generation of antibodies, peptides, and small molecule agents (Ellington and Szostak 1990; Tuerk and Gold 1990). More importantly, counter selection during SELEX to remove the DNA molecules that bind similar competing species can enhance the selectivity of aptamers and give them unique properties (Bruesehoff et al., 2002; Shen et al., 2016), such as differentiating an intact infectious virus from its non-infectious version (Peinetti et al., 2021). Given the success of SELEX in obtaining aptamers for a wide variety of targets, including infectious viruses, it is important to measure their binding affinity to the targets and to similar competing species to understand the interactions of the aptamer with their targets (Sun et al., 2021; Zhang et al., 2021). In addition to validating the capability of the aptamers to bind their intended targets but not other targets, the results provide quantitative measures on how strong and how selective the binding is and thus play a vital role in optimizing their performance as sensors, imaging tools, or therapeutic agents. Viruses generally present several proteins in high copy number on their surfaces that are potential targets for aptamer recognition (Kwon et al., 2020; Li et al., 2022). Considering the high copy number and possibility of higher-ordered structures and arrangements of the proteins on the virus surface, it is more reasonable to determine the binding affinity of aptamers using the whole virus, rather than using the viral protein or a domain of the protein in solution. There are several sources of discrepancies between using the whole virus or just the purified protein. One of the most critical differences is that surface proteins exist in their native state on the surface of the virus. For example, the spike protein of SARS-CoV-2 is in trimeric form on the virus surface, and thus it is very challenging to purify the protein in its native-like environments that preserve their conformation (Cai et al., 2020; Tai et al., 2020; Juraszek et al., 2021). Moreover, as there are multiple copies of the same protein in each virion, the avidity effect jointly with multivalent effects may significantly affect the binding capability (Kwon et al., 2020; Kim et al., 2022). An aptamer that would bind to a single protein may have a significantly different binding affinity or may not even bind at all to the protein when expressed on the surface of a virion, and vice versa. Therefore, to allow direct detection of the virus, it is best to determine the binding affinity between the aptamer and the intact virus. To perform binding assays between aptamers and intact viruses, there are some aspects and challenges that need to be considered. First, the DNA aptamer structures depend on the experiment conditions more than other recognition macromolecules such as antibodies or other folded proteins (Liu et al., 2009; Xia et al., 2013). Therefore, determining the affinity constant of an aptamer requires rigorous control of the experimental conditions to obtain reproducible results. Second, the available techniques for binding assays that can be performed with the intact virus are limited compared to proteins. For instance, viruses are larger than proteins, and a common binding assay like Electrophoretic Mobility Shift Assay (EMSA) cannot be performed. Furthermore, handling the whole intact virus or intact pseudoviruses (when working with BSL3 viruses such as SARS-CoV-2) could require a biosafety level 2 (BSL2) facility or even more stringent safety considerations. As a result, the equipment for the measurement needs to be housed in a BSL2 lab and requires regular decontamination with solutions like bleach that can be damaging to instruments if there are not available disposable options to introduce the viral sample. Because of these requirements, it is difficult to use some techniques such as Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC) that have been successfully applied to characterize other aptamer-target bindings (Ramakrishnan et al., 2012; Chen et al., 2015; Harazi et al., 2017). Here, we describe two different techniques that can be used to obtain binding affinity of aptamers to viruses. First, Enzyme-Linked Oligonucleotide Assay (ELONA), which has been more extensively used for studying the binding between aptamers and viruses (Drolet et al., 1996; Balogh et al., 2010; Bai et al., 2018). This method immobilizes the virus on a surface and uses biotin-labeled aptamers and streptavidin-labeled horseradish peroxidase (HRP) to carry out colorimetric assays through oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to 3,3',5,5'-tetramethylbenzidine diamine. By changing the concentration of the aptamer relative to the virus and measuring the amount of bound aptamer, it is possible to determine the binding affinity as a dissociation constant (KD). Second, a more recent method, MicroScale Thermophoresis (MST), which monitors thermophoretic mobility change in a fluorescent DNA aptamer as a function of the concentration of virus to determine the binding affinity (Duhr et al., 2004; Plach et al., 2017). MST is a simpler assay to set up and offers the possibility to determine binding affinity, binding stoichiometry, and interaction thermodynamics (Baaske et al., 2010; Jerabek-Willemsen et al., 2011). In this work, we used both protocols to measure the binding of DNA aptamers and SARS-CoV-2 pseudoviruses (Peinetti et al., 2021). It is important to mention that pseudoviruses are used for SARS-CoV-2 to reduce the biosafety level required for SARS-CoV-2 viruses from BSL3 to BSL2. However, these methods can be used for other viruses, including different types of viruses, from enveloped RNA viruses like the one shown here, to non-enveloped DNA viruses, as demonstrated in our recent paper (Peinetti et al., 2021). Materials and Reagents Materials PCR tubes (USA Scientific, catalog number: AB1183) Eppendorf tubes (1.5 mL) (Sigma-Aldrich, catalog number: T9661) Pipette tips (10 μL, 200 μL, 1,000 μL) (Thomas Scientific, catalog numbers: 1158U43, 1159M44, 1158U40) NanoTemper capillary tubes (NanoTemper, catalog number: MO-K022) Corning® 96-Well Half-Area Clear Flat Bottom Polystyrene High Bind Microplate, without lid, nonsterile (Sigma-Aldrich, catalog number: CLS3690) Paraffin wax (CAS number: 8002-74-2) (Sigma-Aldrich, catalog number: 327204) Reagents PBS 1× without calcium & magnesium (Corning catalog number: 21-040-CV) Tris(hydroxymethyl)aminomethane (Tris, CAS number: 77-86-1) (Sigma-Aldrich, catalog number: T1503) Sodium chloride (NaCl, CAS number: 7647-14-5) (Sigma-Aldrich, catalog number: S7653) Magnesium chloride (MgCl2, CAS number: 7786-30-3) (Sigma-Aldrich, catalog number: M8266) Calcium chloride (CaCl2, CAS number: 10043-52-4) (Sigma-Aldrich, catalog number: C4901) Tween® 20 (CAS number: 9005-64-5) (Sigma-Aldrich, catalog number: P1379) Bovine Serum Albumin (BSA, CAS number: 9048-46-8) (Sigma-Aldrich, catalog number: A7030) Sodium acetate (CH3COONa, CAS number: 127-09-3) (Sigma-Aldrich, catalog number: S2889) 3,3′,5,5′-Tetramethylbenzidine (TMB, CAS number: 54827-17-7) (Sigma-Aldrich, catalog number: 860336) 100% Ethanol (CH3CH2OH, CAS number: 64-17-5) (Sigma-Aldrich, catalog number: E7023) 30% Hydrogen peroxide (H2O2, CAS number: 7722-84-1) (Sigma-Aldrich, catalog number: 216763) Concentrated sulfuric acid (H2SO4, CAS number: 7664-93-9) (Sigma-Aldrich, catalog number: 339741) Horseradish Peroxidase-Streptavidin (HRP-S) (Sigma-Aldrich, catalog number: OR03L) SARS-CoV-2, SARS-CoV-1, and H1N5 pseudoviruses were provided by Prof. Rong’s lab (UIC) in PBS buffer (Guo et al., 2009; Wang et al., 2014; Peinetti et al., 2021). The titer of the stock solution was 1 × 1011 copies/mL. The stock solution was aliquoted in tubes containing 20 μL each and kept at -80 °C. Before each experiment, a new aliquot was thawed. SARS2-AR10 aptamer DNA (Integrated DNA technologies) FAM-labeled: 5’/56-FAM/CCCGACCAGCCACCATCAGCAACTCTTCCGCGTCCATCCCTGCTG’3 Biotinylated: 5’/5Biosg/CCCGACCAGCCACCATCAGCAACTCTTCCGCGTCCATCCCTGCTG’3 SARS-CoV-2 Pseudoviruses (see Recipes) SELEX buffer (see Recipes) PBS-T (see Recipes) PBS-T-BSA (see Recipes) Acetate buffer (see Recipes) Stop solution (see Recipes) Equipment Biosafety Cabinet (Labconco Purifier Logic+ Class II, Type A2) Pipettes (Eppendorf Reference® 20, 100, 200, 1,000) (Eppendorf, catalog numbers: 4920000032, 4920000059, 4920000067, 4920000083) BioTek H1 Synergy Hybrid Reader (Winooski, VT) NanoTemper Monolith NT.115 (NanoTemper Technologies, Munich, Germany) Procedure Enzyme-linked oligonucleotide assay (ELONA) Working principles. ELONA works by measuring the activity of an enzyme that is present only upon aptamer’s binding to the target. First, the target virus is incubated in a 96-well microplate with high-binding polymer wells, which allow the virus to stick to the well through non-specific binding. Biotinylated DNA aptamer is added to each well, which binds to the target virus but not other viruses. Overdosed or unbound DNA is washed away. A reporter enzyme, labeled with a streptavidin, is then added to bind to the biotin end of the aptamer. Then, the enzyme catalyzes a colorimetric reaction. The amount of color product will be proportional to the amount of enzyme that is correlated to aptamer binding events. The enzyme used here is Horseradish Peroxidase (HRP). In the presence of H2O2, HRP oxidizes TMB, which produces a blue color. After quenching the reaction with H2SO4, the blue color turns yellow, and the measured absorbance of the yellow color correlates to the amount of HRP. In a biosafety cabinet (all procedures from here need to be inside of the biosafety cabinet), dilute the pseudovirus in SELEX buffer to 5 × 108 copies/mL. Mix by flipping the tube. Do NOT vortex. Incubate 50 μL of pseudovirus in each well of a 96-well microplate (Figure 1.1A) at room temperature for 2 h. During this time, the virus will bind to the walls of the well (Figure 1.1B). The plate needs to be covered by parafilm during all the incubation steps. Note: The pseudovirus can be destroyed by intense mechanical forces. Vortexing is likely to destroy the structure and render it ineffective for this experiment. Similarly, excess pipetting to mix could also be problematic, but it is difficult to say how much is too much pipetting for stability. Instead, we recommend using a gentler method of mixing whenever possible. Wash each well three times with PBS-T buffer. Flip the plate and drain the virus solution into a prepared container with bleach (10% v/v) inside. Try to remove the liquid as much as possible by tapping. Note: There are many ways to remove the liquid from solution. Flipping over a bucket of 10% bleach is our recommendation due to its speed and simplicity of decontaminating. For example, the 96-well plate could also be tapped on an absorbing sheet, but then the sheet must be decontaminated and treated as hazardous waste. Any similar method, including pipetting, would be acceptable; however, flipping and tapping the solution into a bleached waste container is safe, quick, easy, and effective. Add 100 μL of buffer to each well. Invert over waste bucket, tapping forcefully if necessary to remove all solution. Add 50 μL of PBS-T-BSA buffer to each well and incubate at room temperature for 1 h in order to avoid non-specific binding to reduce the background signal (Figure 1.1C). Wash three times with PBS-T as before. Prepare array of 11 different biotinylated-ssDNA aptamer concentrations. Serial dilute to prepare the ssDNA aptamer at the concentration of 1 μM, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, 10 nM, 2.5 nM, 1 nM, 0.25 nM, 0.1 nM, 0 nM in SELEX buffer. The total volume of each solution should be sufficient to add 40 μL for each replicate, plus a little to account for losses during pipetting. In this case, use three replicates of two different virus conditions (intact and UV-inactivated pseudovirus), for a total of six. To do this effectively, make at least 240 μL of each DNA solution. Add 40 μL of ssDNA aptamer at different concentrations to each well of the corresponding column (Figure 1.1D), and just SELEX buffer to the first column as a blank (consider loading the plate as shown Figure 1.2). Incubate at room temperature for 1 h. Wash three times with PBS-T as before. Add 40 μL of freshly made 1:500 dilution of HRP-S in PBS-T buffer to each well. Incubate for 1 h (Figure 1.1E). Prepare TMB solutions. TMB stock solution (should always be made fresh during experiment) Dissolve 5 mg of TMB in 5 mL of 100% ethanol. TMB working solution (Should always be made fresh during experiment) Add 1 mL of TMB stock solution to 4 mL of acetate buffer. Add 3 μL of 30% hydrogen peroxide. Wash three times with PBS-T as before. Add 50 μL of TMB working solution to each well (Figure 1.1F). Incubate in the dark until the light blue color builds up for high concentration groups (approximately 20–30 min). Quench the reaction by adding 10 μL of stop solution. After quenching, the blue product turns yellow in the acid solution (Figure 1.1G). Measure the yellow product, which has an absorption peak at 450 nm (OD450), using the BioTek microplate reader (Figure 1.3). Figure 1. Scheme and example of sample loading of the Enzyme-Linked Oligonucleotide Assay (ELONA). 1A) Empty well of 96-well microplate. 1B) Add pseudovirus (step A2). 1C) Add BSA to block non-specific binding (step A4). 1D) Add biotinylated aptamer according to the concentrations in Figure 1.2 (steps A6–7). 1E) Add Avidin-HRP (step A9). 1F) Add TMB (step A12). 1G) Add stop solution (step A14). 2) A plate layout of the 96-well plate. All the wells contain the same amount of virus, either SARS-CoV-2 or UV-inactivated SARS-CoV-2, as indicated in the layout. Concentrations in the scheme correspond to biotinylated aptamer concentrations. 3) A picture of the instrument used. Microscale Thermophoresis (MST) Working principles The physical phenomenon ‘‘thermophoresis” describes molecules’ movement in temperature gradients dependent on their size, charge, and hydration shell. MST works by heating a small spot in the middle of a capillary such that the molecules inside move faster. Smaller molecules move faster than larger ones. Fluorescence is measured in the heated area. The fluorescent DNA aptamer is a relatively small molecule, which becomes much larger and slower when binding to the virus. After aptamers bind to the virus, the fluorescent molecule will leave the tracked area slower, thereby producing a change in the fluorescence intensity. Preparation In a biosafety cabinet, create a 1:2 serial dilution of 16 pseudovirus solutions in SELEX buffer from 3.9 × 108 copies/mL maximum to 1.2 × 105 copies/mL (Figure 2). Start by making 20 μL of the maximum concentration (3.9 × 108 copies/mL) in SELEX buffer in a PCR tube (tube 1). Arrange 15 PCR tubes in a line and add 10 μL of SELEX buffer to each (tubes 2–16). Remove 10 μL from tube 1 and mix thoroughly with tube 2 by pipetting up and down. Remove 10 μL from tube 2 and mix thoroughly with tube 3 by pipetting up and down. Repeat to end of the line, remove 10 μL from tube 16 and discard. To cover a wider concentration range, use a 1:3 serial dilution. Prepare aptamer solution. Dissolve FAM-functionalized DNA aptamer in SELEX buffer to a final concentration of 500 nM. Heat the aptamer solution at 95 °C for 5 min, then cool on ice for 3 min. Add 10 μL of 500 nM FAM-functionalized DNA aptamer to each tube from step B2a, pipette up and down, and incubate for 10 min at room temperature. Heat wax until melted (Figure 2.5.). Insert a MO-K022capillary tube into each PCR tube and tap gently to load solution into tube. The liquid should be loaded approximately two thirds full. The measurement takes place in the middle of the length of the capillary, so the solution must be safely past the midpoint. Dip each end of the tube in melted wax and allow to dry to seal tube (Figure 2.5.). Note: This is a safety recommendation for removing the pseudovirus samples from the biosafety cabinet. Moreover, sealing the tube can prevent solution evaporation during an MST experiment. The wax layer should be thick enough to be robust but not so thick to create bulbous ends that raise the capillary out of the instrument tray. Do not touch the central testing region of the capillary tube, as this may significantly affect the result. Always grab capillary tubes at the ends. Figure 2. Schematic of the MicroScale Thermophoresis (MST) experiment. 1 to 4) Scheme of MST sample preparation and capillary tube loading. 5) Sealing the capillary in wax. First, dip the end of the capillary in the melted wax. Then, allow to cool and solidify. Repeat for both ends. 6) Layout of samples loaded to the MST tray with components labeled A) the tray, B) magnetic bars to clamp down capillaries, and C) white bar illustrating where capillaries are placed. Measurement Arrange the tubes 1–16 in the NanoTemper Monolith NT.115 tray and clamp down using the magnetic bars (Figure 2.6.). Start the instrument, insert the tray, and close the instrument door. Set the Instrument control temperature to 25 °C. Select the LED channel. For fluorescein-modified DNA, select the blue channel. Select the option to perform an MST experiment. Set the LED power to 20%. Set MST power to Medium. This setting controls the power of the IR laser, which heats the solution during the measurement. If the signal change is too large or too small, it can be adjusted down to lower or to higher settings, respectively. Set the time sequence to 5 s cold, 30 s hot (on), followed by 5 s cold. This time sequence corresponds to the time for each phase of measurement. The initial “cold” time establishes a baseline equilibrium fluorescence. The 30 s “hot” period is the time in which the IR laser is heating the solution, causing a temperature gradient, and making the molecules move faster. The hot period may or may not establish a new equilibrium, but the rate at which the measured fluorescence changes will vary related to the amount of virus in the tube. At some time during the hot period, there will be a region that presents a binding curve in the final data. The final 5 s “cold” time is to return towards the initial equilibrium. Enter the maximum concentration (3.9 × 108 copies/mL) and then select 1:2 serial dilution. The instrument will autofill the concentrations of tubes 2–16 according to this dilution factor. Note: The instrument will expect a nanomolar amount as a concentration. It is not advised to try to convert the concentration of the pseudovirus from particles/mL to molarity, but to keep track of the units manually. We recommend just inputting “3.9 × 108 nM” in the unit of nM instead of the correct unit of copies/mL and then correcting the unit back to copies/mL at the end of the analysis. The reason is that converting to 3.9 × 108 copies/mL to its actual nM would result in extremely low concentrations (6.5 × 10-7–2.0 × 10-11 nM), which can cause computational round-off errors in the equipment software when fitting a curve. Make sure all fields are properly filled in, including the concentration of the fluorescent DNA ligand. Provide a filename for the .ntp file that will be created and can be opened with the analysis software of the equipment. An ntp-file can contain several MST experiments that are listed accordingly to their name and the date & time when they were recorded. An MST experiment can contain several runs that are listed according to the “Table of Runs” that you defined for your assay (e.g., different MST powers). Start the measurement. Repeat two more times from step B2a to obtain all data in triplicate. Data analysis ELONA The BioTek plate reader data will be in the form of a grid with one value for each well in the 96-well grid. Each column representing one concentration of DNA. Average the values in each column and calculate the standard deviation. This will create a 1×12 grid. Subtract the absorbance at 450 nm of the blank (A0, column 12) from each value in the grid (denoted as A). Normalize the data for the entire plate by then dividing by A0. Plot (A-A0)/A0 against the concentration of aptamer used in each column. To determine the KD fit the data to Equation A, which is derived from Jaroskaite et al. (2020), where PF is the measured value of free DNA (fit min), PB is the measured value of bound DNA (fit max), LT is the concentration of pseudovirus (known constant), KD is the binding coefficient (fit), and c is the concentration of DNA ligand (known variable). PF and PB are included to set a numeric range for the fitting equation. Use any software that can plot (A-A0)/A0 vs. the concentration of aptamer, as well as calculate statistical parameters and fit nonlinear curves. The following steps are further explained assuming Origin Inc is used. In a new sheet, add one column containing the concentration of aptamer, and in the following columns, the (A-A0)/A0 values calculated for each replicate, typically triplicates. Select the three columns containing the values for the triplicates and click on Origin: Statistics → Descriptive statistics → Statistics on rows, to obtain the mean and standard deviation for each value corresponding to each concentration. Plot the concentration of aptamer against the mean of (A-A0)/A0. Set the standard deviation as the error in Y. Select the following fitting option in Origin: Analysis → Fitting → Nonlinear fitting. Choose the KD function if you already have the function available or create the KD function (eq. A) with the Fitting Function Builder. Note: If in the experimental conditions the target concentration is in excess related to the aptamer concentration, it is possible to simplify the eq. A to eq. B: In this case, choose the Hyperbola function already available in the function options of Origin. The Hyperbola function has the same expression as equation B, where P2 is the KD, and P1 is PB. Example data of this experiment from our publication is shown below in Figure 3. The SARS2-AR10 aptamer is shown to bind to the SARS-CoV-2 pseudovirus with a KD of 79 ± 28 nM (red). The same aptamer shows no significant binding to the same pseudovirus, which has been rendered non-infectious by UV light (blue). Figure 3. Binding curves obtained from the ELONA assay after the immobilization of 5 × 108 copies/mL pseudotyped SARS-CoV-2 on a 96-wells plate. The dissociation constant ( Kd) of the SARS2-AR10 sequence for the infectious pseudotyped SARS-CoV-2 is 79 nM, while no changes in the absorbance at 450 nm are observed for the non-infectious pseudotyped SARS-CoV-2. n = 3 technical replicates (mean ± SD). This figure has been reprinted with permission from Peinetti et al. (2021). MST Open the NanoTemper MST analysis software MO.Affinity Analysis (Supplementary Material). A User Manual is available for the instrument online. Load the dataset(s) from the .ntp file saved by the instrument. Choose the MST analysis type. Combine any replicate datasets to be analyzed together by using the + icon. By clicking next, the software will automatically analyze the data. The raw data is in the form of data traces comparing the fluorescence (Y-axis) to time (x-axis). Figure 4 includes 42 data traces (14 concentrations × 3 replicates each). Each data trace will begin with an equilibrium “cold” region of 5 s followed by a gentle arc to approach a new apparent equilibrium during the 30 s “hot” region while the IR laser is on. In the standard evaluation mode of the instrument, it will generate the Fcold and FHot (The fluorescence with the IR laser off and on, respectively) automatically, but it is possible to manually adjust them to find better signal-to-noise and an improved binding curve. All datasets being compared should be treated uniformly. Note: The data is converted by calculating the average value within each region. Fcold is the average value in the blue region of Figure 4. Fhot is the average value in the red region of Figure 4. The second cold region is not labeled but is shown as the downturn in the data at the end of the plot. These calculations are made by the Analysis software and reported. Because each capillary tube may have slightly different overall fluorescence, Fcold is normalized to be 1. The amount that the fluorescence has changed from Fcold to Fhot (denoted as Fnorm) is recorded at the same time point for each capillary tube and plotted against the concentrations provided during setup. The Fnorm is calculated for each concentration as an average and standard deviation of each replicate. Note: Fnorm, as well as (A-A0)/A values in ELONA assay, are two different variables that represent the fraction of the aptamer that binds to the virus. At this point, there are two available options currently implemented in the MO.Affinity Analysis software for models to fit to the data: KD model and Hill model. The SARS-CoV-2 pseudovirus contains multiple copies of the spike protein on the surface. As the DNA aptamer binds to this protein, multiples aptamers can bind to each virus. Select the KD model. The KD model corresponds to a single site model, which can be adapted also to multiple identical and independent sites, meaning there are many binding sites that have the same binding affinity and are unaffected by cooperativity. It is defined as follows, where PF is the measured value of free DNA (fit min), PB is the measured value of bound DNA (fit max), LT is the concentration of DNA (known constant), KD is the apparent binding constant (fit), and c is the concentration of pseudovirus (known variable). A derivation of the equation with stated assumptions can be found elsewhere (Jarmoskaite et al., 2020). The equation is modified for our terms. Note: Sometimes the model will fail to fit the data. This happens when either there is no trend, indicating no specific interaction, or if the binding curve is incomplete and does not reach maximum/minimum at either end of the data. If this happens, adjust the maximum/minimum concentrations used and try the whole experiment again, or export the values and do the fitting manually according to the fitting model in any data processing software like MS Excel or Origin. Example data of this experiment from our paper is shown below: This experiment was carried out with four different pseudotyped viruses and 229E coronavirus in order to show aptamer selectivity. All pseudoviruses (pSARS-CoV-2, UV-inactivated pSARS-CoV-2, pSARS-CoV-1, and pH5N1) and 229E coronavirus were obtained from Prof. Rong lab (University of Illinois Chicago). Figure 5a shows the average Fnorm and standard deviation (error bar in Y axis) of each replicate. At low concentrations of virus a low Fnorm is observed, and a nice curve to higher Fnorm at higher concentrations. The Fnorm observed with the lower and higher concentration of virus in Figure 5a are significantly different with a 99% confidence. The KD model could be accurately fitted and resulted in KD = 4.8 × 105 copies/mL. Note: The KD is reported in different units in the MST experiment compared with the ELONA experiment. This is because in the ELONA experiment, the DNA concentration (denoted in nM) is varied, but in the MST the DNA concentration is constant, and the virus concentration (denoted in copies/mL) is varied. The sensitivity of this assay would vary for each biochemical system being measured and for this specific assay in the above figure, the LOD = 2.7 × 105 copies/mL. The specificity of this assay is shown in Figure 5b–c. Figure 5b and 5c show MST datasets where no binding is observed, which shows selectivity of the aptamer for the pSARS-CoV-2. Figure 4. MST raw data trace for infectious SARS-CoV-2. Each color data trace represents one replicate data series. The subset of the hot region used for analysis is highlighted in red, and the cold region is highlighted in blue. Figure 5. MST results for infectious SARS-CoV-2 (A), UV-inactivated pseudotyped SARS-CoV-2 (B), and other viruses (C). 229E coronavirus, pseudotyped SARS-CoV-1, and pseudotyped H5N1. SARS2-AR10 was labeled with FAM at the 5’ end, and its concentration was fixed at 250nM. These results confirmed the binding of the aptamer to infectious pseudotyped SARS-CoV-2 and its selectivity. n = 3 technical replicates (mean ± SD). This figure has been reprinted with permission from Peinetti et al. (2021). Notes Some of the materials described in this paper are biosafety level 2 (BSL2), and that requires extra care to handle safely and appropriately. The most basic engineering control for handling biohazardous materials is to do so in a biosafety cabinet. All steps of these procedures that include pseudovirus must be performed in a biosafety cabinet or the virus solution must be in a sealed container (e.g., wax sealed capillary tubes or sealed 96-well plate). Recipes SARS-CoV-2 pseudoviruses SARS-CoV-2 pseudoviruses were provided by Prof. Rong lab (University of Illinois Chicago) in PBS buffer (Guo et al., 2009; Wang et al., 2014). The titer of the stock solution was 1 × 1011 copies/mL. The stock solution was aliquoted in tubes containing 20 μL each and kept at -80 °C. Before each experiment, a new aliquot was thawed. Once thawed, it should not be refrozen but can be stored at 4 °C for one week. Please note that the contents should not be used more than one week after thawing. SELEX buffer 1× PBS, 2.5 mM MgCl2, and 0.5 mM CaCl2, pH = 7.4 Prepare 25 mM MgCl2 solution. Weight out 238 mg MgCl2 and dissolve in 100 mL of molecular grade water. Prepare 5 mM CaCl2 solution. Weight out 55 mg CaCl2 and dissolve in 100 mL of molecular grade water. Mix 20 mL of 10× PBS, 20 mL of 25 mM MgCl2 solution and 20 mL of 5 mM CaCl2 solution. Dilute with 140 mL of molecular grade water. It can be stored at room temperature for several months. If the salts crystalize and are visually apparent, discard and make fresh buffer. Higher concentrated stock solutions of SELEX buffer begin to have solubility issues and are not advised. Individual components can be stored as up to 10× stock solutions. PBS-T Add Tween 20 to PBS to 10% v/v (e.g., 10 mL of Tween 20 + 90 mL of PBS) and mix thoroughly Further dilute the solution to a final concentration of 0.05% v/v by adding 0.5 mL from step 3a to 99.5 mL of PBS. Note: The Tween 20 is very viscous and makes pipetting small volumes accurately difficult. The stepwise dilution improves the accuracy of volumes dispensed. The 10% Tween stock solution can be stored at 4 °C for up to one month for future usage. PBS-T-BSA Dissolve 0.3 g BSA in 6 mL of PBS-T to prepare a 5% w/v BSA solution. Acetate buffer Dissolve sodium acetate in ultrapure water to a concentration of 0.1 M. Adjust pH to 6.5 by adding glacial acetic acid or sodium hydroxide. If pH > 6.5, the reaction will not work. Prepare 80 mL of distilled water in a suitable container. Add 772.142 mg of sodium acetate to the solution. Adjust solution to a final pH of 6.5 by adding acetic acid or sodium hydroxide. Add distilled water until the volume is 100 mL. Stop solution SLOWLY add 1 mL of concentrated sulfuric acid to 9 mL of ultrapure water inside of a fume hood. Because concentrated sulfuric acid is volatile, it can drop from pipettes. Take care in transferring to open tubes. Acknowledgments The authors thank Ms. Laura M. Cooper and Dr. Lijun Rong for providing the pseudovirus of SARS-Cov-2 used in this protocol and Dr. Daiana Capdevila for her insightful discussions. This work was supported by a RAPID grant from the National Science Foundation (CBET 20-29215) and a seed grant from the Institute for Sustainability, Energy, and Environment at University of Illinois at Urbana-Champaign and Illinois-JITRI Institute (JITRI 23965). A.S.P. thanks the PEW Latin American Fellowship for financial support. We also thank the Robert A. Welch Foundation (Grant F-0020) for support of the Lu group research program at the University of Texas at Austin. The protocols described here are adapted from our previous work (Peinetti et al., 2021). Competing interests The authors have applied for a patent (WO2021236828A1) based on the contents of the original paper from which these protocols are derived (Peinetti et al., 2021). References Baaske, P., Wienken, C. J., Reineck, P., Duhr, S. and Braun, D. (2010). Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew Chem Int Ed Engl 49(12): 2238-2241. Bai, C., Lu, Z., Jiang, H., Yang, Z., Liu, X., Ding, H., Li, H., Dong, J., Huang, A., Fang, T., et al. (2018). Aptamer selection and application in multivalent binding-based electrical impedance detection of inactivated H1N1 virus. Biosens Bioelectron 110: 162-167. Balogh, Z., Lautner, G., Bardoczy, V., Komorowska, B., Gyurcsanyi, R. E. and Meszaros, T. (2010). Selection and versatile application of virus-specific aptamers. FASEB J 24(11): 4187-4195. Cai, Y., Zhang, J., Xiao, T., Peng, H., Sterling, S. M., Walsh, R. M., Jr., Rawson, S., Rits-Volloch, S. and Chen, B. (2020). Distinct conformational states of SARS-CoV-2 spike protein. Science 369(6511): 1586-1592. Chakraborty, B., Das, S., Gupta, A., Xiong, Y., Vysnavi, T-V., Kizer, M. E., Duan, J., Chandrasekaran, A. R. and Wang, X. (2022). Aptamers for Viral Detection and Inhibition. ACS Infect Dis 8(4): 667-692. Chen, M., Hu, D., Li, X., Yang, S., Zhang, W., Li, P. and Song, B. (2015). Antiviral activity and interaction mechanisms study of novel glucopyranoside derivatives. Bioorg Med Chem Lett 25(18): 3840-3844. Cho, E. J., Lee, J. W. and Ellington, A. D. (2009). Applications of aptamers as sensors. Annu Rev Anal Chem (Palo Alto Calif) 2: 241-264. Drolet, D. W., Moon-McDermott, L. and Romig, T. S. (1996). An enzyme-linked oligonucleotide assay. Nat Biotechnol 14(8): 1021-1025. Duhr, S., Arduini, S. and Braun, D. (2004). Thermophoresis of DNA determined by microfluidic fluorescence. Eur Phys J E Soft Matter 15(3): 277-286. Dunn, M. R., Jimenez, R. M., and Chaput, J. C. (2017). Analysis of aptamer discovery and technology. Nat Rev Chem 1 (10):1-16. Ellington, A. D. and Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346(6287): 818-822. Fang, X. and Tan, W. (2010). Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach. Acc Chem Res 43(1): 48-57. González, V. M., Martin, M. E., Fernandez, G. and Garcia-Sacristan, A. (2016). Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses. Pharmaceuticals (Basel) 9(4): 78. Guo, Y., Tisoncik, J., McReynolds, S., Farzan, M., Prabhakar, B. S., Gallagher, T., Rong, L. and Caffrey, M. (2009). Identification of a new region of SARS-CoV S protein critical for viral entry. J Mol Biol 394(4): 600-605. Harazi, A., Becker-Cohen, M., Zer, H., Moshel, O., Hinderlich, S. and Mitrani-Rosenbaum, S. (2017). The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant. Mol Neurobiol 54(4): 2928-2938. Hong, S., Zhang, X., Lake, R. J., Pawel, G. T., Guo, Z., Pei, R. and Lu, Y. (2019). A photo-regulated aptamer sensor for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells. Chem Sci 11(3): 713-720. Huizenga, D. E. (1997). Isolation and analysis of novel functional single-stranded DNAs (aptamer, ribozyme, deoxyribozyme). Jarmoskaite, I., AlSadhan, I., Vaidyanathan, P. P., Herschlag, D. (2020). How to measure and evaluate binding affinities. eLife 9: e57264. Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P. and Duhr, S. (2011). Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 9(4): 342-353. Juraszek, J., Rutten, L., Blokland, S., Bouchier, P., Voorzaat, R., Ritschel, T., Bakkers, M. J. G., Renault, L. L. R. and Langedijk, J. P. M. (2021). Stabilizing the closed SARS-CoV-2 spike trimer. Nat Commun 12(1): 244. Kim, H., Choi, H., Heo, Y., Kim, C., Kim, M., and Kim, K.T. (2022). Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds. Appl Sci 12 (3):1717. Kukushkin, V. I., Ivanov, N. M., Novoseltseva, A. A., Gambaryan, A. S., Yaminsky, I. V., Kopylov, A. M. and Zavyalova, E. G. (2019). Highly sensitive detection of influenza virus with SERS aptasensor. PLoS One 14(4): e0216247. Kwon, P. S., Ren, S., Kwon, S. J., Kizer, M. E., Kuo, L., Xie, M., Zhu, D., Zhou, F., Zhang, F., Kim, D., et al. (2020). Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibition. Nat Chem 12(1): 26-35. Li, L., Xing, H., Zhang, J. and Lu, Y. (2019). Functional DNA Molecules Enable Selective and Stimuli-Responsive Nanoparticles for Biomedical Applications. Acc Chem Res 52(9): 2415-2426. Li, L., Xu, S., Yan, H., Li, X., Yazd, H. S., Li, X., Huang, T., Cui, C., Jiang, J. and Tan, W. (2021). Nucleic Acid Aptamers for Molecular Diagnostics and Therapeutics: Advances and Perspectives. Angew Chem Int Ed Engl 60(5): 2221-2231. Liu, J., Cao, Z. and Lu, Y. (2009). Functional nucleic acid sensors. Chem Rev 109(5): 1948-1998. Ma, Y., Mou, Q., Yan, P., Yang, Z., Xiong, Y., Yan, D., Zhang, C., Zhu, X. and Lu, Y. (2021). A highly sensitive and selective fluoride sensor based on a riboswitch-regulated transcription coupled with CRISPR-Cas13a tandem reaction. Chem Sci 12(35): 11740-11747. Peinetti, A. S., Lake, R. J., Cong, W., Cooper, L., Wu, Y., Ma, Y., Pawel, G. T., Toimil-Molares, M. E., Trautmann, C., Rong, L., et al. (2021). Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors. Sci Adv 7(39): eabh2848. Bruesehoff, P. J., Li, J., Augustine, A. J. 3rd and Lu, Y. (2002). Improving Metal Ion Specificity During In Vitro Selection of Catalytic DNA. CCHTS 5 (4): 327-335. Plach, M.G., Grasser, K., and Schubert, T. (2017). MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes. Bio-protocol 7 (23): e2632. Ramakrishnan, M., Alves De Melo, F., Kinsey, B. M., Ladbury, J. E., Kosten, T. R. and Orson, F. M. (2012). Probing cocaine-antibody interactions in buffer and human serum. PLoS One 7(7): e40518. Shen, Z., Wu, Z., Chang, D., Zhang, W., Tram, K., Lee, C., Kim, P., Salena, B. J. and Li, Y. (2016). A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen. Angew Chem Int Ed Engl 55(7): 2431-2434. Sun, M., Liu, S., Wei, X., Wan, S., Huang, M., Song, T., Lu, Y., Weng, X., Lin, Z., Chen, H., et al. (2021). Aptamer Blocking Strategy Inhibits SARS-CoV-2 Virus Infection. Angew Chem Int Ed Engl 60(18): 10266-10272. Tai, W., He, L., Zhang, X., Pu, J., Voronin, D., Jiang, S., Zhou, Y. and Du, L. (2020). Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol 17(6): 613-620. Tuerk, C. and Gold, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249(4968): 505-510. Wang, J., Cheng, H., Ratia, K., Varhegyi, E., Hendrickson, W. G., Li, J. and Rong, L. (2014). A comparative high-throughput screening protocol to identify entry inhibitors of enveloped viruses. J Biomol Screen 19(1): 100-107. Xia, T., Yuan, J. and Fang, X. (2013). Conformational dynamics of an ATP-binding DNA aptamer: a single-molecule study. J Phys Chem B 117(48): 14994-15003. Xiang, Y. and Lu, Y. (2014). DNA as sensors and imaging agents for metal ions. Inorg Chem 53(4): 1925-1942. Xie, S., Ai, L., Cui, C., Fu, T., Cheng, X., Qu, F. and Tan, W. (2021). Functional Aptamer-Embedded Nanomaterials for Diagnostics and Therapeutics. ACS Appl Mater Interfaces 13(8): 9542-9560. Xing, H., Hwang, K., Li, J., Torabi, S. F. and Lu, Y. (2014). DNA Aptamer Technology for Personalized Medicine. Curr Opin Chem Eng 4: 79-87. Zhang, Z., Pandey, R., Li, J., Gu, J., White, D., Stacey, H. D., Ang, J. C., Steinberg, C. J., Capretta, A., Filipe, C. D. M., et al. (2021). High-Affinity Dimeric Aptamers Enable the Rapid Electrochemical Detection of Wild-Type and B.1.1.7 SARS-CoV-2 in Unprocessed Saliva. Angew Chem Int Ed Engl 60(45): 24266-24274. Zou, X., Wu, J., Gu, J., Shen, L., and Mao, L. (2019). Application of Aptamers in Virus Detection and Antiviral Therapy. Front Microbiol 10: 1462. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biophysics > Bioengineering Biochemistry > DNA Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Lentivirus-mediated Conditional Gene Expression Leah Rommereim [...] Naeha Subramanian Nov 5, 2021 4505 Views Generation, Maintenance and HBV Infection of Human Liver Organoids Shahla Romal [...] Tokameh Mahmoudi Mar 20, 2022 2831 Views Visualization, Quantification, and Modeling of Endogenous RNA Polymerase II Phosphorylation at a Single-copy Gene in Living Cells Linda S. Forero-Quintero [...] Timothy J. Stasevich Aug 5, 2022 1371 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,549	https://bio-protocol.org/en/bpdetail?id=4549&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Babesia duncani in Culture and in Mouse (ICIM) Model for the Advancement of Babesia Biology, Pathogenesis and Therapy VK Vandana Kumari AP Anasuya C. Pal PS Pallavi Singh CM Choukri Ben Mamoun Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4549 Views: 723 Reviewed by: Kristin L. ShinglerWenn-Chyau LeeLucy Xie Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Infectious Diseases Sep 2022 Abstract Babesiosis is a tick-borne disease caused by pathogens belonging to the genus Babesia. In humans, the disease presents as a malaria-like illness and can be fatal in immunocompromised and elderly people. In the past few years, human babesiosis has been a rising concern worldwide. The disease is transmitted through tick bite, blood transfusion, and transplacentally in rare cases, with several species of Babesia causing human infection. Babesia microti, Babesia duncani, and Babesia divergens are of particular interest because of their important health impact and amenability to research inquiries. B. microti, the most commonly reported Babesia pathogen infecting humans, can be propagated in immunocompetent and immunocompromised mice but so far has not been successfully continuously propagated in vitro in human red blood cells (hRBCs). Conversely, B. divergens can be propagated in vitro in hRBCs but lacks a mouse model to study its virulence. Recent studies have highlighted the uniqueness of B. duncani as an ideal model organism to study intraerythrocytic parasitism in vitro and in vivo. An optimized B. duncani in culture and in mouse (ICIM) model has recently been described, combining long-term continuous in vitro culture of the parasite in human red blood cells with an animal model of parasitemia (P) and lethal infection in C3H/HeJ mice. Here, we provide a detailed protocol for the use of the B. duncani ICIM model in research. This model provides a unique and sound foundation to gain further insights into the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites, and has been validated as an efficient system to evaluate novel strategies for the treatment of human babesiosis and possibly other parasitic diseases. Graphical abstract: ICIM model [Adapted and modified from Pal et al. (2022)] Keywords: Human babesiosis Babesia duncani Infection Parasite Erythrocyte Virulence Smear Mice Background Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia. Like the other apicomplexan parasite Plasmodium falciparum, the causative agent of human malaria, Babesia parasites also invade human red blood cells (hRBCs) to cause the pathological symptoms associated with human babesiosis, with clinical outcomes ranging from mild to severe and, in some cases, leading to death. More than 100 species of Babesia are known to cause infection in a wide range of mammalian hosts, including livestock, with significant health and economic impacts (Renard and Ben Mamoun, 2021). Human babesiosis is an emerging worldwide concern; although humans are not the natural host of Babesia. Over the past 50 years there has been a rapid increase in cases of human babesiosis caused by different Babesia species (Renard and Ben Mamoun, 2021). While B. microti is the most common species causing human babesiosis, other species such as B. divergens and B. duncani have also been shown to lead to severe and sometimes lethal clinical outcomes (Persing et al., 1995; Rożej-Bielicka et al., 2015; Vannier et al., 2015; Kumar et al., 2021; Renard and Ben Mamoun, 2021). The first case of human babesiosis was identified in a splenectomized patient in Europe, but most cases of babesiosis found in northeastern and midwestern United States had no history of immune impairment (Skrabalo and Deanovic, 1957; Hunfeld et al., 2008; Vannier et al., 2015). Although humans are a dead-end host of Babesia parasites, cases of accidental transmission through blood transfusion from Babesia-infected individuals have been reported, and some rare cases of transplacental transmission have also been documented (Fox et al., 2006, Walker et al., 2022). The first case of transfusion-transmitted babesiosis (TTB) was reported in 1979, ten years after the first reported clinical case of B. microti human babesiosis in the United States (Scholtens et al., 1968, Jacoby et al., 1980). Although B. microti is the most common agent of TTB, cases caused by B. duncani have also been reported (Kjemtrup and Conrad, 2000; Kjemtrup et al., 2002). As the recipients are often immunocompromised, TTB could be fatal (Herwaldt et al., 1997; Claycomb et al., 1998; Conrad, 2000; Leiby, 2011; Renard and Ben Mamoun, 2021). The blood donors carrying Babesia parasites are often asymptomatic, which highlights the necessity for generating tools for efficient diagnosis of parasite infection and for the development of a vaccine and new therapies for the prevention and treatment of human babesiosis. For a long time, the life cycle and biology of Babesia parasites have been poorly elucidated, mainly due to the lack of suitable tools for continuous in vitro propagation of the parasites and/or lack of animal models to study their pathogenesis and virulence. The in culture and in mouse (ICIM) model for Babesia infection described herein will assist in answering some key questions about Babesia biology, pathogenesis, and survival in human red blood cells, namely how these parasites interact with the host and modulate its immune response. The ICIM model has also been important in the discovery and development of novel antibabesial drugs (Lawres et al., 2016; Chiu et al., 2021; Pal et al., 2022). In previous studies (Abraham et al., 2018), the continuous in vitro culture of B. duncani in hRBCs was reported in commercially available Claycomb (Sigma) and HL1 (Lonza) media. The HL1 medium has been discontinued since March 2021; the Claycomb medium often suffers supply shortages, is relatively expensive, and contains several mammalian proteins (bovine albumin, fetuin, transferrin, human insulin, long R3IGF-1, and long EGF) that, while important for HL-1 cardiomyocytes, are of no importance to Babesia intraerythrocytic development (Claycomb et al., 1998; White et al., 2004; Singh et al., 2022). We have recently reported that the DMEM/F-12 is an alternative growth medium for continuous in vitro culture of B. duncani in hRBCs (Singh et al., 2022). It differs from a standard DMEM medium, which does not support the growth of the parasite, by the presence of six amino acids (alanine, asparagine, aspartic acid, cysteine, glutamic acid, and proline), two vitamins (biotin and cobalamin), and four inorganic salts (cupric sulfate, ferric sulfate, magnesium chloride, and zinc sulfate) as well as hypoxanthine, thymidine, linoleic acid, lipoic acid, and putrescine (Singh et al., 2022). Furthermore, an optimized animal model of B. duncani infection that allows a consistent and reproducible evaluation of parasite development and virulence in mice has been recently described (Pal et al., 2022). The ICIM model, which combines the in vitro propagation of the parasite in human red blood cells and parasite virulence in mice, will usher a new era of advanced research on Babesia by facilitating the use of genetic tools and resources to conduct large scale functional analysis to link gene expression and function to disease progression and parasite virulence. This model will greatly enhance our understanding of this disease, as well as help in developing novel therapeutic strategies with improved efficacy. Materials and Reagents For in vitro culture 6-well plate (Corning, catalog number: 353046) 1.7 mL microcentrifuge tubes (Thomas Scientific, catalog number: 1149K01) Centrifuge tubes (Corning, Falcon tubes, catalog numbers: 430829 [50 mL], 352096 [15 mL]) Pipette tips [USA Scientific, catalog numbers: 1121-3810 (10 μL), 1120-8810 (200 μL), 1111-2721 (1,000 μL)] Plugged serological sterile pipettes [Corning, Falcon pipettes, catalog numbers: 357543 (5 mL), 357551 (10 mL), 357525 (25 mL)] Millex filter units [Millipore, catalog numbers: SLGSR33SS (Syringe driven), S2GPU10RE (vacuum driven)] Pipettes [Eppendorf, catalog numbers: K24694J (1,000 μL), J46084J (200 μL), H16607J (20 μL), I54818J (10 μL)] Pipette AID (Drummond Scientific) Aspiration pipettes (Santa Cruz, catalog number: 357781) DMEM/F-12 (Lonza, catalog number: BE04-687/U1, Basel, Switzerland; or Thermo Fisher Scientific, catalog number: 21331020) DMEM (Thermo Fisher Scientific, Gibco, catalog number: 11-966-025) RPMI (Thermo Fisher Scientific, Gibco, catalog number: 11-875-093) FBS (Thermo Fisher Scientific, Gibco, catalog number: 10438-026, Waltham, MA, USA) 50× HT media supplement Hybrid-MaxTM (Sigma, catalog number: H0137) L-glutamine (Thermo Fisher Scientific, Gibco, catalog number: 25030-081) Antimycotic (antibiotic) (Thermo Fisher Scientific, Gibco, catalog number: 15240-062) Gentamicin reagent solution (Thermo Fisher Scientific, Gibco, catalog number: 15710-072) A+ human RBCs (American Red Cross or Interstate Blood Bank, Inc.) B. duncani WA1 strain (BEI Resources, NR-12311) DMEM/F-12 complete media (250 mL) (see Recipes) For cryopreservation Glycerolyte 57 solution (Baxter Healthcare corporation, Deerfield, IL, USA, catalog number: 4A7831) Cryotube vials (Thermo Scientific, catalog number: 363401) For slide preparation Premium microscope slides (Fisherfinest, catalog number: 22038-103) Hemacolor solution I, fixative (Sigma-Aldrich, catalog number: 65044A-85) Hemacolor solution II (Sigma-Aldrich, catalog number: 65044B-85) Hemacolor solution III (Sigma-Aldrich, catalog number: 65044C) Immersion oil (Cargille, catalog number: 16482) For mouse infection BD PrecisionGlideTM 27G needle (BD, catalog number: 305109) Lithium heparin–coated blood collection tube (McKesson 574507, Greiner Bio-One, MiniCollect, catalog number: 450477) Heparinized capillary tubes (Fisher Scientific, catalog number: 22-260-950) Isoflurane (Covetrus, catalog number: 11695-6777-2) PEG 400 (Thermo Fisher Scientific, Avantor J.T. Baker U216-07, catalog number: 02-003-646) 1× PBS diluted from 10× PBS pH 7.4 (Gibco, catalog number: 70011-044) Mouse strains: C3H/HeJ (from Jackson Laboratories, Bar Harbor, ME) Note: Our studies have shown that C3H/HeJ mice are susceptible to B. duncani infection following IV inoculation with doses of infected RBCs (iRBCs) ranging between 102 and 107. We found that 107 and 106 iRBC doses elicit an acute increase in parasitemia (P) (up to 35%) within a short span of time [3–5 days post infection (DPI)], whereas mice inoculated with doses of 102–105 iRBCs show a delayed onset of infection with a lower peak P (Pal et al., 2022). Parasitemia levels in C3H/HeJ mice were always higher in females compared to males at the same infection dose (Aguilar-Delfin et al., 2001, Pal et al., 2022). In contrast, C57BL/6J mice showed 100% survival with no detectable P following inoculation with 104 B. duncani–iRBC. Infection of Balb/cJ with 104 B. duncani–iRBC resulted in increased P over time in all mice; however, approximately 50% of the mice cleared the infection and survived, whereas the other half continued to show detectable P and succumbed to infection (Pal et al., 2022). 30% isoflurane (50 mL) (inhalation anesthetic; see Recipes) Equipment Sorvall legend XTR centrifuge (Thermo Scientific, catalog number: 75004521) Microscope (Nikon Eclipse 5Oi) SterilGARD III advance biological safety cabinet (The Baker Company, catalog number: SG603) Water bath (Fisher brand, model: FSGPD15D) Tri gas incubator (Thermo Scientific, model: HERACELL VIOS 160i) Eppendorf MiniSpin (Millipore-Sigma, catalog number: EP022620100) Software GraphPad Prism version 9.4.1 Procedure In vitro culture of B. duncani WA1 isolate in hRBCs Prepare media by adding all the components (see Figure 1) in sterile conditions inside a biosafety cabinet (Figure 2). Note: In specific circumstances when DMEM/F-12 is not available from the different mentioned sources, DMEM base medium supplemented with missing components (those present in DMEM/F-12) could be successfully used for parasite culture (see Table 1 provided below). Figure 1. Representative image showing media components and materials required for B. duncani in vitro culture Figure 2. Representative image showing biosafety cabinet BSL-2 grade required for handling and culturing of B. duncani parasite Table 1. Supplement required to complete DMEM base medium to be used as a replacement of DMEM/F-12 Catalog number DMEMb Catalog number: 11965092 (Thermo Fisher Scientific) Conc. (mg/L) DMEM/F-12 Catalog number: BE04-687F/U1 (LONZA) Conc. (mg/L) Amino Acids L-Alanine A7219, Sigma - 4.45 L-Asparagine-H2O A7094, Sigma - 7.5 L-Aspartic acid A7219, Sigma - 6.65 L-Cysteine hydrochloride-H2O C6852, Sigma - 17.56 L-Glutamic Acid G8415, Sigma - 7.35 L-Proline P5067, Sigma - 17.27 Vitamins Biotin B4639, Sigma - 0.004 Vitamin B12 V6629, Sigma - 0.68 Inorganic Salts Cupric sulfate (CuSO4·5H2O) C8027, Sigma - 0.0012 Ferric sulfate (FeSO4·7H2O) F8633, Sigma - 0.42 Magnesium Chloride (MgCl2) anhydrous M8266, Sigma - 28.57 Zinc sulfate (ZnSO4·7H2O) Z0251, Sigma - 0.43 Lipids Linoleic Acid L1012, Sigma - 0.044 Lipoic Acid T1395, Sigma - 0.013 Other components Putrescine 2HCl P5780, Sigma - 0.081 Hypoxanthine and Thymidine mix H0137-10VL, Sigma - 0.0112 - 0.001468 Wash hRBCs as follows: After receiving blood (bag containing approximately 500 mL of total blood) from a donation center, mix gently by inverting the bag 2–3 times. Aliquot blood samples into 50 mL centrifuge tubes and store at 4 °C until washing. Unwashed blood can be stored at 4 °C for up to one month. To wash an aliquot of blood, use incomplete DMEM or RPMI media. Use roughly four volumes of medium per one volume of packed RBCs. Mix the suspension gently to resuspend the RBCs and centrifuge at 1,800 rpm (757 × g) for 10 min at room temperature (RT). Note: For washing RBCs, DMEM/RPMI from any source can be used. Following centrifugation, gently aspirate the medium and carefully remove the buffy coat containing the white blood cells from the top RBC layer. Repeat the washing steps twice. After the final wash, add an equal volume of incomplete DMEM or RPMI media to make 50% hematocrit (HC). The washed RBCs (50% HC) can be immediately used for in vitro culture or stored at 4 °C for further use. Washed RBCs can be stored at 4 °C and used for up to two weeks in cell culture. Thawing cryo-preserved B. duncani–infected erythrocytes: Take a cryovial of frozen parasite from the liquid nitrogen and immediately put in a 37 °C water bath for a few seconds until the contents of the vial turn into liquid. Transfer the contents of the thawed vial containing the B. duncani–infected hRBCs to a 50 mL centrifuge tube inside the biosafety cabinet. Add 200 μL of 12% (w/v) NaCl (0.2× original cryovial volume) dropwise while gently shaking the tube. Incubate for 5 min at RT. Add 9 mL of 1.6% (w/v) NaCl dropwise while gently shaking the tube. Centrifuge at 1,500 rpm (526 × g) for 5 min at RT. Remove the supernatant without disturbing the pellet. Add 25 mL of the incomplete DMEM/F-12 medium to wash the parasite pellet. Centrifuge at 1,500 rpm (526 × g) for 5 min at RT. Repeat the above step one more time and resuspend the pellet in 3.6 mL of complete DMEM/F-12 media (see Recipes). Add 400 μL of washed 50% HC A+ RBCs (step 2) to maintain culture at 5% HC. Seed the culture into a well of 6-well plate and place it in the incubator at 2% O2, 5% CO2, and 93% N2 atmosphere with 95% humidity setting (see Figure 3). Figure 3. Representative image of Tri gas incubator used for B. duncani in vitro culture Maintain the culture as follows: Replace culture medium every day by aspirating old medium with aspirating pipette and replace with complete DMEM/F-12 medium prewarmed to 37 °C. Note: Take extra precaution while aspirating the medium to not disturb the bottom of the plate containing the iRBCs. Monitor the culture and P level by smear preparation as follows: Aspirate medium as described above and resuspend culture in fresh medium by gently pipetting a few times. Take 50 μL culture in a microcentrifuge tube and spin at 2,500 rpm (700 × g) for 2 min at RT. Carefully remove supernatant with a 200 μL pipette tip (set to 40 μL), leaving behind 10 μL of medium. Resuspend RBCs pellet by gently pipetting a few times. Place a drop of the resuspended RBCs on a slide and make a smear by sliding another slide at an angle of 25°–45° as shown in Figure 4. Fix the blood smear for 10 s in the fixative solution I followed by 10 s in solution II. Stain with the solution III for 25 s. Rinse the stained slide in water, air dry, and visualize the slide under the light microscope at 100× (representative image of blood smear with 10% P shown in Figure 5). Estimate P as follows: Make a smear from in vitro culture and perform Giemsa staining as described above in steps i–v. Air dry for 3–4 min. Put a drop of immersion oil and visualize under microscope. Count iRBCs and total RBCs (tRBCs) (tRBCs = iRBCs + uninfected RBCs) in the field. Repeat step 3 for different fields until the tRBCs count from adding all the fields is 1,000–2,000 RBCs (for more accuracy). Calculate percentage of iRBCs as follows: Field 1=(9 iRBCs)/(80 tRBCs) Field 2=(10 iRBCs)/(110 tRBCs) Field 3=(11 iRBCs)/(120 tRBCs) Field 5=(8 iRBCs)/(70 tRBCs) Field 6=(12 iRBCs)/(130 tRBCs) (9+10+11+8+12)/(80+110+120+70+130)= 50/510×100=9.8% Figure 4. Representative image demonstrating angular position of slide and RBC pellet (50 μL of in vitro culture after centrifugation) for smear preparation Figure 5. Representative image depicting 10% P of B. duncani–infected human erythrocytes Note: Increasing the number of fields for calculating the proportion of iRBCs and tRBCs and percentage of P levels allows a more accurate determination of P. Maintain the parasite culture by inoculating 0.5%–1% P of original culture once the P level reaches 10%–15%. Example: For a starter culture of 5 mL with 0.5% P and 5% HC, take 250 μL of the stock culture (10% P and 5% HC) and add 475 μL of 50% HC washed hRBCs and 4.275 mL of complete DMEM/F-12 medium. For specific assays that require high P, culture the parasites continuously with media replacement after every 24 h until it reaches the desired P. B. duncani in vitro cultures can reach up to 20%–25% P. Cryopreservation of in vitro cultured B. duncani–iRBCs Estimate P levels by light microscopy following Giemsa staining of blood smears prepared from in vitro cultured parasites as described in step A4a if P is >5%, then proceed with the following steps of cryopreservation. Pellet the culture (5 mL) by centrifugation at 1,800 rpm for 5 min at RT. Aspirate the supernatant leaving behind the RBC pellet. Add 250 μL of filter-sterilized glycerolyte dropwise while tapping the bottom of the tube to mix. Gently mix the suspension and put in a cryovial. Label the cryovial describing the parasite strain and % P of the culture on the day of cryopreservation. Store the cryovial at -80 °C. For long-term storage (more than six months), take out the cryovial from -80 °C and place in liquid nitrogen. Purification of B. duncani merozoites Initiate and maintain B. duncani in vitro culture in hRBCs in DMEM/F-12 medium until P reaches 18%–20% and free merozoite can be observed in blood smears (Figure 6). Figure 6. Representative image depicting 18%–20% P with free merozoites indicated by arrows To isolate the free merozoites, centrifuge the parasite culture at 1,800 rpm (757 × g) and 37 °C for 5 min. Use the pellet containing the B. duncani iRBCs for initiating fresh starter cultures or for other assays. The supernatant containing the free merozoites is collected using a pipette in a fresh centrifuge tube. The supernatant is centrifuged at 4,000 rpm (1,932 × g) and 37 °C for 10 min. The resulting pellet contains the free merozoites. Resuspend the merozoite pellet in warm DMEM/F-12 in 1:5 ratio (v/v). For example, for a packed pellet of 10 μL, add 50 μL of culture medium. Estimate the purity of the merozoites by Giemsa staining. The purity of a merozoite preparation is determined by the amount of free merozoites with little to no intact uninfected or infected RBCs. Figure 7 shows an example of a merozoite preparation with no RBCs detected. The merozoite preparations that are more than 95% pure are considered suitable for subsequent cell biological and molecular analyses. Figure 7. Representative image showing free merozoites (black arrows). Pink structures in the image represent RBC membranes resulting from lysis of infected erythrocytes. Mouse infection Use 5–6 weeks old female C3H/HeJ mice after one week acclimatization post procurement. To infect mouse, use either B. duncani in vitro cultured purified merozoites (2 × 107) or human iRBCs (8.5 × 105) and inject by retro-orbital intravenous (IV) route. Note: Higher doses of parasite infection will expedite the establishment of infection in mouse. However, results may vary depending on the success of infection and handling. Calculate the amount of ihRBCs containing 8.5 × 105 parasites as follows: Calculation of iRBCs in the in vitro culture: 1 mL of human blood = 5 × 109 RBCs (100% HC) 1 mL of 5% HC human blood = 2.5 × 108 RBCs If the P estimated by Giemsa stain is 10% 1 mL of 5% HC with 10% P = 10/100 × 2.5 × 108 = 2.5 × 107 iRBCs Take the desired amount of parasite (8.5 × 105) by serial dilution as follows: Take 100 μL of above culture (step D3a) containing 2.5 × 106 iRBCs and dilute 1:10 by adding 900 μL of 1× PBS to attain a concentration of 2.5 × 103 parasites per μL. For 8.5 × 105 parasites from above diluted culture, take 340 μL (8.5 × 105 ÷ 2.5 × 105 = 340 μL) and pellet down by centrifugation at 1,800 rpm (757 × g) for 5 min at RT. Carefully aspirate the supernatant leaving behind 100 μL of supernatant. Resuspend by gentle tapping and inject as described below in step D5. Note: A serial dilution to take the desired amount of parasite is important, rather than taking a very little amount (e.g., 34 μL) directly from original culture (to avoid pipetting error). Perform IV infection as follows: Use a jar with cotton soaked in 30% isoflurane (inhalation anesthetic) diluted in PEG 400 placed at the bottom and covered with a mesh. Place a single mouse inside the sealed chamber containing the above-mentioned inhalation anesthetic (see Figure 8). Figure 8. Representative image showing reagents and materials required for mouse infection Inject 100 μL of parasite (in PBS) diluted as mentioned above in step D3b and inject via IV route. Monitor P by microscopy of thin blood smears prepared from blood collected from the tail vein on a regular interval of 24 or 48 h. Plot the P and survival curve using GraphPad Prism. Blood from an infected mouse can be used to infect another uninfected mouse to maintain the parasite stock. Parasitemia in the stock mouse can be monitored by microscopic examination of Giemsa-stained blood smears. Calculate the amount of blood required for mouse-to-mouse infection: Calculation of iRBCs 1 mL of mouse blood = 10 × 109 RBCs If P (estimated by Giemsa stain) is 10% 1 mL of 10% P = 10/100 × 10 × 109 = 109 iRBCs Dilution of iRBCs If the desired dose of infection is 104 parasites, collect 30 μL of blood from the mouse by retroorbital bleeding in a heparinized blood collection tube and dilute as follows: Take 10 μL of blood (1.0 × 107 iRBCs) and add 990 μL of 1× PBS to make 1 × 107 iRBC/mL. Dilute above diluted iRBCs to 1:10 for two more times by taking 100 μL each time from previous dilution and adding 900 μL of 1× PBS (serial dilution). This will result in 1 mL containing 1 ×105 iRBCs (102 iRBCs/μL) at final step. Use 100 μL (1 × 104 iRBC) of this dilution to inject each mouse. Notes: 1. Calculate the dose for N+2 mice to account for any volume loss. Infection from mouse to mouse is established faster than from in vitro culture to mouse (see Pal et al., 2022). 2. A dose response infection (ranging from 102–107 iRBCs) was tested in mouse-to-mouse transmission of B. duncani and successful infection was achieved at all doses (Pal et al., 2022). Compared to higher doses, subsequent lower doses led to delays in the establishment of infection. However, results may vary depending on the handling. Any animal showing signs of distress should be humanely euthanized per approved IACUC protocol by CO2 asphyxiation followed by cervical dislocation. Data analysis For in vitro studies (Figure 9), percent P on the indicated days (days 0, 3, 6, 9, 12, and 15) is determined by counting the number of iRBCs out of approximately 3,000 tRBCs per blood smear prepared from cultures grown in different media, as described in step A4a. The values are then plotted using GraphPad prism. Statistical significance (P-value) is calculated using two-way ANOVA. For in vivo studies (Figure 10), P is estimated as described in step A4.a.vi from thin blood smears prepared from mouse blood at the indicated time points (DPI 1, 3, 4, 6, 7, 8, 9, 10, and 11). The number of iRBCs in a total of approximately 2,000 tRBCs is determined for each mouse. At least three mice are used per group. Data are then plotted using GraphPad prism 9.4.1 software. Kaplan-Meier survival curves are generated using GraphPad prism 9.4.1. Figure 9. In vitro propagation of B. duncani WA1 in different growth media. (A) Continuous in vitro growth of B. duncani WA1 in different media for a period of 15 days in hRBCs. The cultures were diluted (D) on days 3, 6, and 9 (indicated by arrows). (B) Representative images of Giemsa-stained smears of in vitro cultured B. duncani WA1 parasites showing different stages, including rings (R), double rings (DR), filamentous forms (FF), and tetrads (T). (C) Graph showing percentage of different developmental stages of B. duncani WA1 in DMEM/F-12 and Claycomb media. Data presented are mean ± SD of two independent experiments performed in biological duplicates. No significant differences (p > 0.99, two-way ANOVA) were found between the different stages in the two media [adapted and modified from Singh et al. (2022)]. Figure 10. Lethal B. duncani infection in immunocompetent mice. (A) Parasitemia profile over time in female C3H/HeJ mice (n = 3/group) following infection with B. duncani parasites from in vitro culture in hRBCs (purple; 8.5 × 105 iRBC/mouse), or B. duncani parasitized mouse RBCs (mRBC) collected from infected mice (green; 1 × 104 iRBC/mouse). Uninfected mice profile depicted in red. E: euthanized. (B) Kaplan-Meier plot of percent survival of uninfected mice (red), mice infected with in vitro cultured B. duncani parasites (purple), or mice infected with B. duncani parasitized mouse blood (green) at indicated doses [adapted and modified from Pal et al. (2022)]. Recipes DMEM/F-12 complete media (250 mL) DMEM/F-12 188.5 mL FBS 50 mL HT media supplement (50×) hybrid max 5 mL L-Glutamine 2.5 mL Antimycotic 2.5 mL Gentamycin 2.5 mL 30% isoflurane (50 mL) Isoflurane 15 mL PEG 400 35 mL Acknowledgments Funding: The published protocols by Pal and colleagues (Pal et al., 2022) and Singh and colleagues (Singh et al., 2022) were supported by the National Institutes of Health grants (AI123321, AI138139, AI152220, and AI136118), the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and the Global Lyme Alliance to CBM. Competing interests None of the named authors have any conflict of interest, financial or otherwise. Ethics All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) at Yale University (Protocol #2020-07689). Animals were acclimatized for one week after arrival before the start of an experiment. Animals that showed signs of distress or appeared moribund were humanly euthanized using approved protocols. Institutional Review Board Statement: All animal studies conducted were approved by the Institutional Animal Care and Use Committees (IACUC) at Yale University (Protocol #2020-07689). Institutional Biosafety Statement: All studies involving the use of human blood and Babesia parasites in culture were approved by the Institutional BioSafety Committee at Yale University. References Abraham, A., Brasov, I., Thekkiniath, J., Kilian, N., Lawres, L., Gao, R., DeBus, K., He, L., Yu, X., Zhu, G., Graham, M. M., Liu, X., Molestina, R. and Ben Mamoun, C. (2018). Establishment of a continuous in vitro culture of Babesia duncani in human erythrocytes reveals unusually high tolerance to recommended therapies. J Biol Chem 293(52): 19974-19981. Aguilar-Delfin, I., Homer, M. J., Wettstein, P. J. and Persing, D. H. (2001). Innate resistance to Babesia infection is influenced by genetic background and gender. Infect Immun 69(12): 7955-7958. Chiu, J. E., Renard, I., Pal, A. C., Singh, P., Vydyam, P., Thekkiniath, J., Kumar, M., Gihaz, S., Pou, S., Winter, R. W., et al. (2021). Effective Therapy Targeting Cytochrome bc1 Prevents Babesia Erythrocytic Development and Protects from Lethal Infection. Antimicrob Agents Chemother 65(9): e0066221. Claycomb, W. C., Lanson, N. A., Jr., Stallworth, B. S., Egeland, D. B., Delcarpio, J. B., Bahinski, A. and Izzo, N. J., Jr. (1998). HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A 95(6): 2979-2984. Fox, L. M., Wingerter, S., Ahmed, A., Arnold, A., Chou, J., Rhein, L. and Levy, O. (2006). Neonatal babesiosis: case report and review of the literature. Pediatr Infect Dis J 25(2): 169-173. Herwaldt, B. L., Kjemtrup, A. M., Conrad, P. A., Barnes, R. C., Wilson, M., McCarthy, M. G., Sayers, M. H. and Eberhard, M. L. (1997). Transfusion-transmitted babesiosis in Washington State: first reported case caused by a WA1-type parasite. J Infect Dis 175(5): 1259-1262. Hunfeld, K. P., Hildebrandt, A. and Gray, J. S. (2008). Babesiosis: recent insights into an ancient disease. Int J Parasitol 38(11):1219-37. Jacoby, G. A., Hunt, J. V., Kosinski, K. S., Demirjian, Z. N., Huggins, C., Etkind, P., Marcus, L. C. and Spielman, A. (1980). Treatment of transfusion-transmitted babesiosis by exchange transfusion. N Engl J Med 303(19): 1098-1100. Kjemtrup, A. M. and Conrad, P. A. (2000). Human babesiosis: an emerging tick-borne disease. Int J Parasitol 30(12-13): 1323-1337. Kjemtrup, A. M., Lee, B., Fritz, C. L., Evans, C., Chervenak, M. and Conrad, P. A. (2002). Investigation of transfusion transmission of a WA1-type babesial parasite to a premature infant in California. Transfusion 42(11): 1482-1487. Kumar, A., O'Bryan, J. and Krause, P. J. (2021). The Global Emergence of Human Babesiosis. Pathogens 10(11): 1447. Lawres, L. A., Garg, A., Kumar, V., Bruzual, I., Forquer, I. P., Renard, I., Virji, A. Z., Boulard, P., Rodriguez, E. X., Allen, A. J., et al. (2016). Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone. J Exp Med 213(7): 1307-1318. Leiby, D. A. (2011). Transfusion-transmitted Babesia spp.: bull's-eye on Babesia microti. Clin Microbiol Rev 24(1): 14-28. Pal, A. C., Renard, I., Singh, P., Vydyam, P., Chiu, J. E., Pou, S., Winter, R. W., Dodean, R., Frueh, L., Nilsen, A. C., et al. (2022). Babesia duncani as a Model Organism to Study the Development, Virulence, and Drug Susceptibility of Intraerythrocytic Parasites In Vitro and In Vivo. J Infect Dis 226(7):1267-1275. Persing, D. H., Herwaldt, B. L., Glaser, C., Lane, R. S., Thomford, J. W., Mathiesen, D., Krause, P. J., Phillip, D. F. and Conrad, P. A. (1995). Infection with a Babesia-like organism in northern California. N Engl J Med 332(5): 298-303. Renard, I. and Ben Mamoun, C. (2021). Treatment of Human Babesiosis: Then and Now. Pathogens 10(9): 1120. Rożej-Bielicka, W., Stypułkowska-Misiurewicz, H. and Gołąb, E. (2015). Human babesiosis. Przegl Epidemiol 69(3): 489-94, 605-8. Scholtens, R. G., Braff, E. H., Healey, G. A. and Gleason, N. (1968). A case of babesiosis in man in the United States. Am J Trop Med Hyg 17(6): 810-3. Singh, P., Pal, A. C. and Mamoun, C. B. (2022). An Alternative Culture Medium for Continuous In Vitro Propagation of the Human Pathogen Babesia duncani in Human Erythrocytes. Pathogens 11(5): 599. Skrabalo, Z. and Deanovic, Z. (1957). Piroplasmosis in man; report of a case. Doc Med Geogr Trop 9(1): 11-16. Vannier, E. G., Diuk-Wasser, M. A., Ben Mamoun, C. and Krause, P. J. (2015). Babesiosis. Infect Dis Clin North Am 29(2): 357-70. Walker, S., Coray, E., Ginsberg-Peltz, J. and Smith, L. (2022). A Five-Week-Old Twin With Profound Anemia: A Case Report of Asymmetric Congenital Babesiosis. Cureus 14(3): e22774. White, S. M., Constantin, P. E. and Claycomb, W. C. (2004). Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol 286(3): H823-829. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > Microbial cell biology > Cell isolation and culture Microbiology > in vivo model > Protozoan Cell Biology > Cell isolation and culture Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Isolation of GFP-expressing Malarial Hypnozoites by Flow Cytometry Cell Sorting Annemarie Voorberg-van der Wel [...] Clemens H. M. Kocken May 5, 2021 3326 Views Sex-specific Separation of Plasmodium falciparum Gametocyte Populations Melanie C. Ridgway [...] Alexander G. Maier Jun 5, 2021 2603 Views Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement Jessica Jie Ying Ong [...] Jin-Hee Han Sep 5, 2021 2205 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
455	https://bio-protocol.org/en/bpdetail?id=455&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei Laia Armengot Jordi Moreno-Romero Published: Vol 3, Iss 7, Apr 5, 2013 DOI: 10.21769/BioProtoc.455 Views: 23549 Download PDF Ask a question How to cite Favorite Cited by Original Research Article: The authors used this protocol in The Plant Journal Aug 2012 Abstract Micrococcal nuclease (MNase) is able to produce double-strand breaks within nucleosome linker regions. The efficiency of MNase digestion depends on the degree of chromatin compaction, being more easily digested the regions of less compacted chromatin. The MNase protocol described here can be used to asses changes in the chromatin structure of nuclei extracted from Arabidopsis seedlings. Keywords: Arabidopsis thaliana Nuclei extraction Microccocal nuclease digestion Chromatin density Materials and Reagents Micrococcal Nuclease (MNase) (Roche Applied Science, catalog number: 10107921001 , 15,000 U) Proteinase K (Roche Applied Science, catalog number: 03115836001 ) PIPES (Sigma-Aldrich, catalog number: P-9291 ) Liquid nitrogen Sucrose KCl MgCl2 CaCl2 Triton X-100 1 mM PMSF (freshly added) Tris/HCl (pH 7.8) EDTA SDS Ethidium bromide Nuclei extraction buffer A, B, C (see Recipes) MNase buffer (see Recipes) 2x stop buffer (see Recipes) 10x Proteinase K buffer (see Recipes) Equipment Pestle and mortar Centrifuges 37 °C oven 70 μm Nylon mesh 50 ml Falcon tubes Centrifuge tubes 2 ml microtubes Nylon mesh Procedure Grind frozen Arabidopsis plantlets (2 g) with a pestle and a mortar (previously cooled with liquid nitrogen) under liquid nitrogen. Note: Perform the following steps on ice. Add the homogenized plant material to 10 ml of nuclei extraction buffer A in a 50 ml Falcon tube. Mix it well by vortexing. Filter twice the plant homogenate obtained in step 2, using a 70 μm nylon mesh placed in a funnel. Note: perform the second filtering step in the centrifuge tubes needed for step 4. Centrifuge at 10,000 x g for 20 min at 4 °C. Discard the supernatant by decantation. Note: Discard all the supernatant by pipetting. Resuspend the pellet in 200-500 μl of nuclei extraction buffer B (this volume can be adjusted depending on the size of the pellet). Pipet 200-500 μl of nuclei extraction buffer C (it must be the same volume as the one used in step 6) and place it into an empty 2 ml Eppendorf tube. Note: Nuclei extraction buffer C is viscous because of its high sucrose content. You should perform this step slowly in order to avoid the formation of bubbles. Add the resuspended pellet from step 6 onto the layer of buffer C obtained in step 7. Note: Pay attention in not disturbing the layer of Buffer C when you add the resuspended pellet. Centrifuge at 12,000 x g for 1 h at 4 °C. Discard all the supernatant by pipetting. Resuspend the pellet in 250 μl of MNase buffer (this volume can be adjusted depending on the size of the pellet). Note: At this point you can add 35% glycerol to the nuclei samples, submerge them into liquid nitrogen, and keep frozen at -80 °C until MNase digestion is going to be performed. At that moment, nuclei are defrozen and centrifuged at 16,000 x g for 20 min at 4 °C in order to remove the glycerol. Finally, the nuclei are resuspended in MNase buffer. Quantify the DNA concentration by measuring the absorbance at 260 nm. Usually a dilution 1:20 should be used. Optional: Analyze the DNA integrity before performing the MNase digestion. For this purpose, mix equal volumes of nuclei and 2x stop buffer, and centrifuge at 16,000 x g for 10 min at 4 °C. Analyze the supernatant on a 1.2% agarose gel stained with ethidium bromide. Figure 1. Analysis of DNA integrity before performing the MNase digestion. Several procedures were used for this purpose: (A) mixing equal volums of nuclei and milliQ water; (B) mixing equal volumes of nuclei and 2x stop buffer and, (C) mixing equal volumes of nuclei and 2x stop buffer, and centrifugation as described in the protocol. The procedure in (C) gave the best results and was used routinely. Perform this step if you want to compare different samples, if not, you can directly proceed to MNase digestion (step 14). Dilute the sample(s) with MNase buffer in order to obtain the same DNA concentration in all of them (usually a range of concentrations between 300 ng/μl and 600 ng/μl). Note: The final volume for all the samples must be the same and it must be adjusted depending on the number of MNase digestions you want to perform. As a rule, use between 20 μl and 40 μl per reaction. To study chromatin sensitivity to MNase, two complementary protocols can be used: Digestion with different concentrations (Units) of MNase (step 14) and/or digestion with a desired MNase concentration during different incubation times (step 15). Incubate the suspension of nuclei from step 13 with different Units of MNase, at 37 °C for 15 min. For instance, you can use 1, 2.5, 5, 10, 20, 40 U/ml or higher concentrations, until you observe total degradation of the high molecular weight DNA band (Figure 2). For this purpose, prepare the MNase solutions by making serial dilutions from the enzyme stock (for example 10,000 U/ml), in order to add the same volume of MNase to each individual reaction. For example, aliquot 30 μl of nuclei and add 10 μl of MNase at the desired concentration. To stop the reaction, add 40 μl of 2x stop buffer. Finally, add Proteinase K 1x buffer and 1 μl of Proteinase K (stock 10 mg/ml), and incubate overnight at 37 °C. Incubate the diluted nuclei with the MNase at 4 °C () for different periods of time, in order to perform time-course studies at a desired concentration of MNase. At each time point, transfer 20 μl of the reaction to an eppendorf tube containing 20 μl of stop buffer 2x and mix. Add Proteinase K 1x buffer and 1 μl of Proteinase K (stock 10 mg/ml), and incubate overnight at 37 °C. () The optimal digestion temperature of MNase is 37 °C. At 4 °C MNase digestion is slower than at 37 °C, so you can use longer incubation time of digestion at 4 °C than at 37 °C. Note: RNase treatment can be performed at the end of step 14 and step 15 if you observe RNA contamination in your samples. Add DNA loading buffer to the sample obtained in step 14 and step 15 and visualize the results by electrophoresis on 1.2% agarose gels stained with ethidium bromide. Figure 2. Example of MNase digestion using diferent concentrations of MNase. Same amounts of nuclei were digested with increasing concentrations of MNase at 37 ºC for 15 min and analysed in an 1.2% agarose gel. Recipes Nuclei extraction buffer A 0.25 M sucrose 60 mM KCl 15 mM MgCl2 1 mM CaCl2 15 mM PIPES (pH 6.8) 0.8% Triton X-100 1 mM PMSF (freshly added) Nuclei extraction buffer B 0.25 M sucrose 10 mM Tris/HCl (pH 8.0) 10 mM MgCl2 1% v/v Triton X-100 5 mM β-mercaptoethanol (freshly added) 1 mM PMSF (freshly added) Nuclei extraction buffer C 1.7 M sucrose 10 mM Tris/HCl (pH 8.0) 10 mM MgCl2 0.5% Triton X-100 5 mM β -mercaptoethanol 1 mM PMSF MNase buffer 0.3 M sucrose 20 mM Tris/HCl (pH 7.5) 3 mM CaCl2 2x stop buffer 50 mM EDTA 1% SDS 10x Proteinase K buffer 100 mM Tris/HCl (pH 7.8) 50 mM EDTA 5% SDS Acknowledgments This protocol was developed for the work previously published in Plant Journal (Moreno-Romero et al., 2012). This work was supported by grants BFU2007-60569, BFU2010-15090 and Consolider Ingenio 2010 CSD2007-00036 from the Ministerio de Educación y Ciencia, (Spain) and grants 2005SGR-00112 and 2009SGR-795 from the Generalitat de Catalunya, Catalunya (Spain). L.A. and J.M.-R. were recipients of fellowships from the Ministerio de Educación y Ciencia (Spain) and the Universitat Autònoma de Barcelona respectively. References Moreno-Romero, J., Armengot, L., Mar Marques-Bueno, M., Britt, A. and Carmen Martinez, M. (2012). CK2-defective Arabidopsis plants exhibit enhanced double-strand break repair rates and reduced survival after exposure to ionizing radiation. Plant J 71(4): 627-638. Article Information Copyright © 2013 The Authors; exclusive licensee Bio-protocol LLC. How to cite Armengot, L. and Moreno-Romero, J. (2013). Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei. Bio-protocol 3(7): e455. DOI: 10.21769/BioProtoc.455. Download Citation in RIS Format Category Cell Biology > Organelle isolation > Nuclei Molecular Biology > DNA > DNA structure Plant Science > Plant molecular biology > DNA > DNA structure Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells Xingwang Li and Changyin Wu Jul 20, 2014 10830 Views Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants Nathaniel D. Graham [...] James A. Birchler Sep 20, 2015 8284 Views Isolation of Plant Nuclei Compatible with Microfluidic Single-nucleus ATAC-sequencing Sandra B. Thibivilliers [...] Marc Y. Libault Dec 5, 2021 3276 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,550	https://bio-protocol.org/en/bpdetail?id=4550&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Fast and Efficient Decellularization Method for Tissue Slices MN Maria Narciso AU Anna Ulldemolins CJ Constança Júnior JO Jorge Otero DN Daniel Navajas RF Ramon Farré NG Núria Gavara IA Isaac Almendros Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4550 Views: 1516 Reviewed by: Alessandro DidonnaEmilie ViennoisRAMESH KUDIRA Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Frontiers in Bioengineering and Biotechnology Mar 2022 Abstract The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue’s vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2–3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies. Keywords: Decellularization Extracellular matrix Tissue slices Glass slide Mechanobiology Sodium deoxycholate Background The extracellular matrix (ECM) is composed of more than 300 core structural components (Burgstaller et al., 2017), whose physical and chemical features regulate crucial cellular mechanisms (Gattazzo et al., 2014) including differentiation, migration, and proliferation. Thus, the study of the ECM is essential for understanding some pathological conditions and diseases including cancer and fibrosis (Elowsson Rendin et al., 2019, Wishart et al., 2020, Júnior et al., 2021). Also, decellularized extracellular matrix (dECM) scaffolds have many potential applications in tissue engineering and regenerative medicine. In fact, dECM has been used for the generation of ECM hydrogels (Marhuenda et al., 2022) and the recellularization of whole previously decellularized organs (Ohata and Ott, 2020), as well as several applications in the regeneration of tissues (Zhu et al., 2019). Therefore, it is not surprising the increased interest in physiomimetic tissue scaffolds by producing decellularized tissue samples (Mendibil et al., 2020). The elimination of cells from tissue to obtain the ECM is possible by using physical, chemical, enzymatic, or a combination of these approaches (Mendibil et al., 2020). Physical strategies include freeze/thawing cycles, which induce ice crystals in the matrix, disrupting the cell membrane. Chemical strategies include detergents that solubilize the cell membrane and hypertonic or hypotonic solutions causing cell disruption by osmotic shock. Finally, enzymatic approaches can target the cell’s nuclear material, such as with deoxyribonuclease (DNase), or the cell-ECM adhesion, such as with trypsin. However, the available decellularization protocols have important limitations. Many of them can take up to several days (Wishart et al., 2020, Wüthrich et al., 2020) and are still not particularly flexible or accessible since they are designed for the decellularization of full organs or thick sections (tissue blocks). This type of protocol would not suit the decellularization of many tissue samples, as is the case for clinical biopsies. In fact, clinical biopsies are scarce and cannot be decellularized by accessing the tissue’s vasculature. Since no current protocols have explored the decellularization of glass-attached tissue sections, a method that allows for the study of the exact location before and after decellularization is needed to fill this gap. The method presented here is significantly faster and less wasteful (i.e., can produce a single acellular tissue slice, instead of requiring a large sample portion) than other available methods, while maintaining the sample’s mechanical properties and being suited for cell culture applications (Narciso et al., 2022). Additionally, it provides the option for studying the same tissue section before and after decellularization, which is invaluable for studies of certain pathologies and of scarce or valuable clinical samples. Patient biopsies, for example, are tested for several markers and histopathological features; hence, the entire sample cannot be decellularized. This method allows for studies of both native tissue and dECM to be carried out in the same sample. For early-stage tumors especially, this is of paramount importance as cancer cells are removed during decellularization and their location cannot be pinpointed. Furthermore, the tissue's architecture is preserved throughout decellularization by pre-attaching the samples to a glass slide. For tissues like bladder and lung, where organ inflation is required to emulate different conditions, this inflation can be performed on the native tissue, guaranteeing a more physiological result. Materials and Reagents Nail polish Coverslips (Labbox, catalog number: COVN-050-100) Blades (Ted Pella, Inc. St/Steel, Single Edge, 38 mm, catalog number: 121.4) Slide tray (Histoline, Tray Slide Staining System, catalog number: M920-1) 250 mL glass beaker (VWR, catalog number: 213-1124) Pasteur pipettes 3 mL (Deltalab, catalog number: 200006.C.) Hydrophobic pen (Sigma-Aldrich, catalog number: Z377821-1EA), storage: room temperature (RT) SuperFrost Plus glass slides (ThermoFisher, EprediaTM SuperFrost PlusTM Adhesion slides, catalog number: 10149870), storage: RT SuperFrost Gold glass slides (ThermoFisher, EprediaTM SuperFrost Ultra PlusTM GOLD Adhesion Slides, catalog number: 11976299), storage: RT Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25-1G), storage: -20 °C MgCl2 (Sigma-Aldrich, catalog number: M8266-1KG), storage: RT CaCl2 (Sigma-Aldrich, catalog number: C1016-500G), storage: RT 1 M Tris-HCl, pH 7.5 (ThermoFisher, Invitrogen, catalog number: 15567027), storage: 2–8 °C Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750-500G), storage: RT Ultrapure water/Milli-Q water (obtained via Equipment #6), storage: RT PBS 10× (ThermoFisher, catalog number: 70011-036), storage: RT Optimum cutting temperature (OCT) compound (Sakura, Tissue-Tek®, catalog number: 4583), storage: RT Cryomolds (Sakura, Tissue-Tek® Cryomold® Standard 25 × 20 × 5 mm, catalog number: 4557) Paraformaldehyde, 4% in PBS (ThermoFisher, catalog number: J61899.AK), storage: 2–8 °C Corning® 50 mL centrifuge tubes (Sigma-Aldrich, catalog number: CLS430290-500EA), storage: RT Hoechst 33342 staining (ThermoFisher, Invitrogen, NucblueTM Live Cell Stain ReadyProbesTM reagent, catalog number: R37605), storage: 2–30 °C Fluoromount (Southern Biotech, catalog number: 0100-01), storage: RT Lint-free paper (KIMTECH Science Precision, catalog number: 7551), storage: RT Fresh tissue sample (protocol tested on heart, lungs, bladder, and kidneys. Origins tested: murine and porcine) DNase solution (see Recipes) SD 2% solution (see Recipes) Equipment Tweezers (rubisTech, catalog number: 1-SA) Scale (Sartorius Lab Instruments, ENTRIS124l-1S, catalog number: 31603742) Cryostat (Leica, model: CM3050S) Vortex (Scientific Industries Inc., Vortex Genie 2, model: G-560E) Orbital shaker (IKA, Model: KS 130 basic, catalog number: 0002980000) Milli-Q Gradient (Millipore, catalog number: ZMQ55V001) Inverted microscope (Leica, SP5) equipped with a CCD camera (Hamamatsu Photonics C9100) and using a 10× Plan Fluor objective (Nikon, Tokyo, Japan) Incubator (Nuaire, model: NU-4750) Software ImageJ (National Institutes of Health, LOCI, University of Wisconsin, https://imagej.nih.gov/ij/) Procedure Sample preparation and OCT embedding Tip: Work near a -80 °C freezer. After tissue retrieval from the animal/patient, place the sample in an appropriate container and keep refrigerated on ice for up to 2 h. For longer periods of time, freeze the sample at -80 °C and remove it from the freezer before OCT embedding until the sample is completely thawed. Choose the appropriate size of cryomold for your sample so that the sample has at least 0.5 cm between the sample and the walls of the cryomold. Place a single drop of OCT compound in the center of the cryomold. With the help of tweezers, position your fresh tissue sample in the center. Note: For some applications, the tissue can also be positioned in a specific orientation in relation to the blade. Cover the whole tissue sample with OCT, without overflowing the cryomold. Tip: Pour the OCT compound without squeezing the tube to avoid air bubbles, which can interfere with cryosectioning. Make sure there is a thick layer of OCT on top of the sample in a way that the surface of the OCT is as smooth as possible. If any bubbles have formed, gently remove them by slowly aspirating with a pipette. Place the cryomold with the sample in the freezer on a flat surface. Tip: Depending on the type of tissue used, the sample will tend to float on the OCT, so it is important to freeze as soon as possible. Note: Other freezing procedures, such as snap freezing with liquid nitrogen, dry ice, liquid nitrogen vapors, or freezing at -20 °C, are also compatible with this protocol. Let the samples freeze overnight. Tissue sectioning Set the appropriate cryostat temperature for the tissue sample to be sliced. Note: The cryostat temperature will have to be optimized depending on the device used and the tissue to be sliced. In the setup described, the temperature that produces the best tissue sections for lungs and heart is -18 °C to -20 °C, for bladder is -20 °C to -22 °C, and for liver is -15 °C to -18 °C. Place the blade inside the cryostat at least 20 min before use. Withdraw the sample from the freezer and place it inside the cryostat for 30 min before sectioning. Trim the beginning of the cryoblock until reaching the sample. Select the desired thickness. Note: For mechanical testing we suggest 20–30 µm sections, while for tissue staining and imaging we suggest 10 µm. Section the samples. Carefully make contact between the positive area of the glass slide and the tissue section (depending on the size of your sample, you can place 1–3 consecutive tissue sections in one glass slide). Note: For thicker sections (50 µm and higher) consider using a more adhesive glass slide, like Superfrost Gold. Allow the samples to dry at RT for 10–20 min. Store the samples at -80 °C. Tissue section decellularization (10–70 μm sample thickness) Withdraw samples from the freezer and place them on the slide tray (sample side up). Allow samples to thaw at room temperature for 20 min. Trace the edges of the sample with a hydrophobic pen and allow the ink to completely dry for 1 min. Using a Pasteur pipette, cover the sample in PBS 1× for 20 min (RT) to remove the OCT compound. Please note that depending on the tissue size, the volume required to cover the sample could vary from 100 µL to up to 1 mL. Tip: When pouring liquid during decellularization, never pour it directly on top of the sample to avoid damaging or detaching the tissue. Also avoid adding the liquid as droplets, as that might also create too much turbulence. Instead, approach the tip of the pipette to a nearby bare glass region of the sample and slowly deposit the liquid in a continuous flow. Remove the PBS (and all other incubations throughout the decellularization) by inverting the glass slide over the glass beaker. Warning: Be quick in between washes so as to never let the sample completely dry. Cover the sample in Milli-Q water for 10 min to provoke cell lysis. Remove the Milli-Q water by inversion and repeat step C6. Remove the Milli-Q water by inversion. Cover the sample in SD 2% solution for 15 min. Remove the SD 2% solution by inversion. Cover the sample again in SD 2% solution for 15 min. Remove the SD 2% solution by inversion. Tip: In the following steps, be careful when inverting the glass slide over the glass beaker, as the SD solution with cell remnants is extremely viscous and could damage the sample if done too quickly. Incubate with PBS 1× for 5 min and remove it by inverting the sample. Repeat step C13 three times. With the Pasteur pipette, aspirate and release the solution of DNase to ensure it is homogeneous (and that the DNase does not sink to the bottom). Cover the sample in DNase solution and incubate at RT for 20 min. Remove the DNase by inversion and repeat step C13 three times. Leave the sample in PBS until further staining or testing. Do not allow to dry. Tissue section decellularization (70–100 μm sample thickness) For the decellularization of thicker tissue sections, follow section C with these alterations to the following steps: (Instead of step C2)—Allow samples to thaw at RT for 40 min. (Instead of step C4)—To remove the OCT compound from the sample, cover the sample in PBS 1× and incubate for 30 min. (Instead of step C16)—Cover the sample in the DNase solution and incubate at 37 °C for 40 min. For short-term storage (<24 h), non-fixed decellularized samples can be left in PBS at 4 °C. For long-term storage (1–10 days), we advise fixing samples in PFA for 15 min, removing the PFA with PBS, and leaving the samples in 50 mL PBS-filled tubes at 4 °C. Do not freeze thin decellularized samples to avoid ice crystal formation and structural damage to the samples. Nuclear staining and mounting Note: Any nuclear stain like DAPI or Hoechst 33342 would work for decellularization quantification. Thaw consecutive tissue sections for 20 min at RT. Trace the edges of the sample with a hydrophobic pen and allow the ink to completely dry for 1 min. Remove the OCT compound from the native sample by covering it in PBS 1× for 20 min. Prepare the Nucblue Hoechst 33342 staining solution according to the manufacturer’s instructions (2 drops per mL of PBS). Cover both the native and decellularized sample in Nucblue solution and protect the slide tray from light. Incubate for 20 min at 80 rpm using an orbital shaker at RT. Remove the Nucblue solution by inversion and wash the samples three times with PBS for 5 min each wash. Note: Keep your samples protected from light as much as possible in between washes. If no opaque slide tray lid is available, aluminum foil paper will suffice. Remove the excess PBS from the glass slide by inversion. Add a drop of fluoromount from the container and place it on top of your samples so as not to touch it directly. Tip: Do this step by using a yellow pipette tip to avoid bubble formation. With the tweezers, remove the coverslip from the container and very carefully place one edge of the coverslip on one of the sides of the sample. Slowly lower the other edge until it covers the sample. Avoid dropping the coverslip on top of the sample as this will create bubbles on the sample. With nail polish, trace the edges of your coverslip and secure the coverslip to the glass slide. Wait for it to dry and store at 4 °C protected from light. Image acquisition and decellularization quantification Tip: Use a low magnification objective to image as much tissue area as possible (5×–20×). Place the samples at room temperature 30 min before imaging. Clean the glass slides with lint-free paper and ethanol 70%. Image the native tissue sample and set the UV (420 nm) exposure as high as possible without causing pixel saturation (Figure 1B). Acquire 5–10 image sets of phase contrast and UV fluorescence (Figure 1A and 1B). Image decellularized samples using the same exposure settings (Figure 1C and 1D). Figure 1. Representative images of the decellularization of mice bladder (20 µm). Phase contrast images of a native (A) and decellularized (C) 20 µm mice bladder tissue section and corresponding UV images (B and D, respectively). Contrast was increased in phase contrast images for the sake of readability. Data analysis Open ImageJ. Drag the phase contrast image and its corresponding UV image to ImageJ. Using the Freehand selections tool, trace the edges of the sample in the phase-contrast photo. Edit > Selections > Create mask, to obtain Figure 2B. Edit > Selections > Create selection. On the UV image: Shift+E to apply the selection (Figure 2C). Figure 2. Decellularization quantification. A. Phase contrast image of a 20 µm mice bladder tissue section with the freehand outline in yellow. B. Mask of the outline of A. C. Area of interest selection of the corresponding UV image. Scale bar = 200 µm. Analyze > Set Measurements > Mean gray value > OK. Analyze > Measure. Repeat this procedure for all native and decellularized images to obtain the mean signal intensity of the nuclear staining for both groups (Figure 3). Tip: The approach described in this protocol is manual. For an automatic approach, you can use the previously described algorithm for decellularization quantification (Narciso et al., 2021). Figure 3. Representative signal quantification results of bladder decellularization. The left bar represents the mean UV intensity signal (± SD) for native bladder samples stained with Hoechst 33342 that was normalized to 100%, while the right bar represents the mean intensity signal of the decellularized bladder sections. Recipes DNase solution Reagent Final concentration Amount Deoxyribonuclease I from bovine pancreas 0.3 mg/mL 3 mg Tris-HCl (1 M, pH 8.0) 10% 100 µL MgCl2 10 mM 9.5 mg CaCl2 10 mM 11 mg H2O (ultrapure) n/a 9.9 mL Total n/a 10 mL SD 2% solution Reagent Final concentration Amount Sodium deoxycholate 2% 0.2g H2O (Ultrapure) n/a (until reaching a total of 10 mL) Total n/a 10 mL Acknowledgments This protocol was derived from the previously published work “Novel Decellularization Method for Tissue Slices” in Frontiers in Bioengineering and Biotechnology in March 2022 (Narciso et al., 2022). The authors acknowledge the funding received: IA is funded by Ministerio de Ciencia e Innovación (PID2019-108958RB-I00 / AEI/ 10.13039/501100011033) and SEPAR (900-2019). MN and CJ were funded by the H2020 European Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement “Phys2BioMed” contract no. 812772. The Spanish Ministry of Sciences funded RF, JO, and NG, Innovation and Universities, PID2020-113910RB-I00-AEI/10.13039/501100011033, PGC2018-097323-A-I00, and PID2020-116808RB-I00 AEI-Retos, respectively. Competing interests There are no conflicts of interest or competing interests. Ethics All animal procedures were approved by the Institutional Committee of Universitat de Barcelona and the Animal Experimentation Committee of regional authorities (Generalitat de Catalunya, OB 168/19 and 10972). References Burgstaller, G., Oehrle, B., Gerckens, M., White, E. S., Schiller, H. B. and Eickelberg, O. (2017). The instructive extracellular matrix of the lung: basic composition and alterations in chronic lung disease. Eur Respir J 50(1): 1601805. Elowsson Rendin, L., Lofdahl, A., Ahrman, E., Muller, C., Notermans, T., Michalikova, B., Rosmark, O., Zhou, X. H., Dellgren, G., Silverborn, M., et al. (2019). Matrisome Properties of Scaffolds Direct Fibroblasts in Idiopathic Pulmonary Fibrosis. Int J Mol Sci 20(16): 4013. Gattazzo, F., Urciuolo, A. and Bonaldo, P. (2014). Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta 1840(8): 2506-2519. Junior, C., Narciso, M., Marhuenda, E., Almendros, I., Farre, R., Navajas, D., Otero, J. and Gavara, N. (2021). Baseline Stiffness Modulates the Non-Linear Response to Stretch of the Extracellular Matrix in Pulmonary Fibrosis. Int J Mol Sci 22(23): 12928. Marhuenda, E., Villarino, A., Narciso, M. L., Camprubi-Rimblas, M., Farre, R., Gavara, N., Artigas, A., Almendros, I. and Otero, J. (2022). Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells. Int J Mol Sci 23(9): 4888. Mendibil, U., Ruiz-Hernandez, R., Retegi-Carrion, S., Garcia-Urquia, N., Olalde-Graells, B. and Abarrategi, A. (2020). Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds. Int J Mol Sci 21(15): 5447. Narciso, M., Otero, J., Navajas, D., Farre, R., Almendros, I. and Gavara, N. (2021). Image-Based Method to Quantify Decellularization of Tissue Sections. Int J Mol Sci 22(16): 8399. Narciso, M., Ulldemolins, A., Junior, C., Otero, J., Navajas, D., Farre, R., Gavara, N. and Almendros, I. (2022). Novel Decellularization Method for Tissue Slices. Front Bioeng Biotechnol 10: 832178. Ohata, K. and Ott, H. C. (2020). Human-scale lung regeneration based on decellularized matrix scaffolds as a biologic platform. Surg Today 50(7): 633-643. Wishart, A. L., Conner, S. J., Guarin, J. R., Fatherree, J. P., Peng, Y., McGinn, R. A., Crews, R., Naber, S. P., Hunter, M., Greenberg, A. S., et al. (2020). Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis. Sci Adv 6(43): eabc3175. Wüthrich, T., Lese, I., Haberthur, D., Zubler, C., Hlushchuk, R., Hewer, E., Maistriaux, L., Gianello, P., Lengele, B., Rieben, R., et al. (2020). Development of vascularized nerve scaffold using perfusion-decellularization and recellularization. Mater Sci Eng C Mater Biol Appl 117: 111311. Zhu, M., Li, W., Dong, X., Yuan, X., Midgley, A. C., Chang, H., Wang, Y., Wang, H., Wang, K., Ma, P. X., et al. (2019). In vivo engineered extracellular matrix scaffolds with instructive niches for oriented tissue regeneration. Nat Commun 10(1): 4620. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biological Engineering > Biomedical engineering Biophysics > Bioengineering > Medical biomaterials Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Generation of Human Blood Vessel and Vascularized Cerebral Organoids Xin-Yao Sun [...] Zhen-Ge Luo Nov 5, 2023 1659 Views Iterative Immunostaining and NEDD Denoising for Improved Signal-To-Noise Ratio in ExM-LSCM Lucio Azzari [...] Elina Mäntylä Sep 20, 2024 570 Views An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene Srisathya Srinivasan and Hsin-Yi Henry Ho Jan 20, 2025 980 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,551	https://bio-protocol.org/en/bpdetail?id=4551&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Fluorescence Screens for Identifying Central Nervous System–Acting Drug–Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics ZB Zoe G. Beatty AM Anand K. Muthusamy EU Elizabeth K. Unger DD Dennis A. Dougherty LT Lin Tian LL Loren L. Looger AS Amol V. Shivange KB Kallol Bera HL Henry A. Lester AN Aaron L. Nichols Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4551 Views: 1376 Reviewed by: Geoffrey C. Y. LauSrinidhi Rao Sripathy RaoAndrew L. EagleZheng Zachory Wei Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Jan 2022 Abstract Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)–acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13–18 candidate biosensors and 44–84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. Graphical abstract: Keywords: Biosensor Fluorescence screening Pharmacokinetics Directed evolution iDrugSnFR CNS-acting drugs Background Low-molecular-weight central nervous system (CNS)–acting drugs typically bind to receptors, transporters, and ion channels (both ligand- and neurotransmitter-gated). Such CNS-acting drugs have therapeutic uses, but some are also abused (Lester et al., 2012; Henderson and Lester, 2015). Sources for new drug compounds typically include nature (mostly plants) or medicinal chemistry. The protein targets of CNS-acting drugs are synthesized, assembled, and processed within intracellular exocytotic pathways before eventually reaching the plasma membrane. In some cases, membrane-permeant drugs also interact with their targets in intracellular compartments. For this reason, many papers report on pharmacokinetic characteristics of CNS-acting drugs at the subcellular scale: dynamics and intracellular concentrations. Optical methods provide appropriate time and distance scales, and the genetically encoded fluorescent biosensors we have developed bear strong resemblance to those developed for individual neurotransmitters (Marvin et al., 2018; Unger et al., 2020). The reversibility and linearity of such sensors also render them useful for the more conventional application of monitoring within extracellular biofluids (Muthusamy et al., 2022). A general term we use to describe our biosensors is iDrugSnFR (intensity-based drug-sensing fluorescent reporter), following the lead of iGluSnFR (an early sensor for the neurotransmitter glutamate) (Marvin et al., 2018). All the iDrugSnFRs described here consist of circularly permuted green fluorescent proteins (cpGFP) inserted into a suitably mutated OpuBC periplasmic choline binding protein (PBP) from Thermoanaerobacter sp513. Upon the development of the first iDrugSnFR (iNicSnFR3a for nicotine), we conducted several screens with the goal of developing iDrugSnFRs for other drugs (Bera et al., 2019; Shivange et al., 2019; Nichols et al., 2022; Muthusamy et al., 2022). The present report shows the protocol for such screens. In most cases, screening is required to develop a novel biosensor because, at present, we cannot predict atomic-scale details of the interaction between a drug of interest and the PBP site. Some pharmacological structure–activity relations (such as cation-π boxes) that govern the interaction between a CNS-acting drug and its putative binding partner are recapitulated in the drug-iDrugSnFR binding site, while others are not (Bera et al., 2019; Nichols et al., 2022; Muthusamy et al., 2022) (Protein Data Bank files 7S7T, 7S7U, 7S7X, and 7S7Z). Screens reported here have two classes of molecular input: purified biosensor candidate proteins (13–18 per screen) and drugs of interest (DOIs, 44–84 drugs per screen). The DOIs we have chosen to study typically contain nitrogen (i.e., are alkaloids), have molecular weights (MW) below 500, and are weak bases (6 < pKa < 10). The weakly basic nature of these molecules allows passive diffusion through membranes into cells and organelles, enabling us to study their subcellular pharmacokinetics. We define a hit pair as a drug–biosensor pair that displays an acceptable fluorescence signal (ΔF/F0 > 1). This definition of a hit arises from our experience that ΔF/F0 > 1 is required for subsequent directed evolution of an optimal biosensor variant. This report describes screens with single DOI concentrations and validations with full dose-response relations (several concentrations of the DOI) (Bera et al., 2019). In general, the biosensor of the hit pair becomes the starting construct for directed evolution of an optimized variant that senses the DOI. Other papers describe the directed evolution of optimized variants (Bera et al., 2019; Shivange et al., 2019; Nichols et al., 2022; Muthusamy et al., 2022). In all cases, the directed evolution also includes optimizing the selectivity against other drugs. This workflow has resulted in iDrugSnFRs for cholinergic compounds, opioids, and the rapidly acting antidepressant S-ketamine (Shivange et al., 2019; Nichols et al., 2022; Muthusamy et al., 2022). Reports on additional iDrugSnFRs are in preparation. Materials and Reagents Disposable borosilicate glass culture tubes (VWR, catalog number: 47729-578) 10 mL syringes (Becton Dickinson, catalog number: 309605) Sterile syringe filters with 0.2 µm cellulose acetate membrane (VWR, catalog number: 28145-477) PrecisionGlideTM 18G 11/2 needles (Becton Dickinson, catalog number: 305196) 360 µL 96-well plates (Corning, catalog number: 3915) 2.2 mL 96-square-well storage plates (Thermo Scientific, catalog number: AB0932) 15 mL conical centrifuge tubes (Falcon, catalog number: 14-959-53A) 50 mL Amicon Ultra centrifugal filter tubes (30 kDa cutoff) (Sigma-Aldrich, catalog number: UFC903096) 50 mL conical centrifuge tubes (Falcon, catalog number: 14-432-22) 1.5 mL Premium microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129) 0.5 mL tubes (Fisher Scientific, catalog number: AB0350) Sterile film (AeraSeal) (Sigma-Aldrich, catalog number: A9224) Sterile glass beads Ice/ice bucket Dewar flask Dimethyl sulfoxide (DMSO, C2H6OS) (Fisher Scientific, catalog number: BP231100) Methanol (CH3OH) (EMD Millipore, catalog number: 179337) 0.5 M sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465) Ethylenediaminetetraacetic acid (EDTA) (C10H16N2O8) (Sigma-Aldrich, catalog number: E9884) Sodium dodecyl sulfate (SDS) [CH3(CH2)11SO4)] (Sigma-Aldrich, catalog number: 436143) Ethanol (EtOH, C2H5OH) (DeConLabs, catalog number: V1016) Nickel sulfate (NiSO4(H2O)6) (Fisher Scientific, catalog number: AC415611000) Buffer A (1× PBS, pH 7.4, 10 mM imidazole) [made from 10× PBS, pH 7.4 (Fisher Scientific, catalog number: 10-010-023)] Buffer B (1× PBS, pH 7.4, 200 mM imidazole) [made from 10× PBS, pH 7.4 (Fisher Scientific, catalog number: 10-010-023)] 1× PBS, pH 7.0 [made from 10× PBS, pH 7.4 (Fisher Scientific, catalog number: 10-010-023)] 3× PBS, pH 7.0 [made from 10× PBS, pH 7.4 (Fisher Scientific, catalog number: 10-010-023)] 1 M MgSO4 (Sigma-Aldrich, catalog number: 230391) Ampicillin 1,000× stock (Sigma-Aldrich, catalog number: A9518) SOC media (MP Biomedicals, catalog number: 3031-012) Difco LB media (Fisher Scientific, catalog number: DF0402) BL21(DE3) chemically competent cells (Thermo Fisher Scientific, catalog number: C600003) ddH2O LB agar plates (ampicillin, 100 mg/mL) Liquid nitrogen (N2) 2 mM drug stock solutions Biosensor plasmid DNA 4× Laemmli sample buffer (Bio-Rad, catalog number: 161-0747) 2-Mercaptoethannol (β-ME) (Bio-Rad, catalog number: 161-0710) HiTrapTM 5ml IMAC FF Nickel NTA column (Cytiva, catalog number: 17-0921-04) Mini-PROTEAN TGXTM precast protein gels, 10-well, 30 μL (Bio-Rad, catalog number: 456-1093) Tris-glycine buffer 10× concentrate (Sigma-Aldrich, catalog number: T4904) Mini-PROTEAN Gel Box (Bio-Rad, catalog number: 1658005) Precision Plus protein dual color standards ladder (Bio-Rad, catalog number: 161-0374) QC Colloidal Coomassie stain (Bio-Rad, catalog number: 161-0803) 50× M (see Recipes) 50× 5052 (see Recipes) Equipment Heat block (VWR Scientific, catalog number: 13259-005) Precision GP 05 water bath (Thermo Fisher Scientific, catalog number: TSGP02) Excella E25 shaking incubator (New Brunswick Scientific, catalog number: M1353-0000) G-25 incubator shaker (New Brunswick Scientific, catalog number: 76901-1) Mini centrifuge (Thermo Fisher Scientific, catalog number: 05-090-100) Fluorospectrometer (Thermo Scientific, catalog number: ND-3300) ÄKTA Start protein purification system with Frac30 fraction collector (Cytiva, catalog number: 29023051) XPR204 scale (Mettler Toledo, catalog number: 30355419) Vortex mixer (Baxter Scientific, catalog number: S8223-1) epMotion 5075t liquid handling robot and appropriate tips (Eppendorf, catalog number: 5075006022) Spark 10M multimode plate reader (Tecan, catalog number: 30086376) Agilent ELx50 microplate strip washer (BioTek) Sonicator [Sonifier SFX550 control box (Branson, catalog number: 101-063-969), 20 kHz 102-C converter (Branson, catalog number:101-135-066R), tapered 1/8 inch microtip (Branson, catalog number: 101-148-062)] Allegra 25R centrifuge (Beckman Coulter, catalog number: 369434) Nanodrop 1000 (Thermo Scientific) Orion VersaStar Pro pH meter (Thermo Scientific, catalog number: VSTAR90) Covered container for gel staining/destaining Rotary shaker (VWR, catalog number: 89032) Xplorer variable volume 12 channel motorized pipette (50–1,200 µL) (Eppendorf, catalog number: 4861000830) PowerPacTM HC high-current power supply (Bio-Rad, catalog number: 1645052) Software Tecan v3.1 software Eppendorf EpMotion software version 40.4.0.38 Microsoft Excel version 16.55 Origin Pro version 9.1 64-bit Procedure Expression of biosensor proteins Chemical transformation of plasmid into BL21(DE3) chemically competent cells Determine biosensor plasmid to express. Coding regions of each candidate biosensor are given in the iSnFRbase database (a repository for tracking biosensor mutagenesis and drug affinities), which will be deposited on GitHub. Figures 4, 5, and 6 give the iSnFRbase identifiers under nicknames. Add 300 ng of biosensor of chosen plasmid DNA to 100 µL of BL21(DE3) chemically competent cells on ice for 10–30 min. After incubation, heat-shock the sample on a 42 °C heat block for 30 s and place back on ice for 2 min. Add 700 µL of SOC media to the sample, resuspend solution with a pipette, and place the sample in a 37 °C shaker for 1 h at 225 rpm. Spin the sample in a microcentrifuge for 2 min. Remove 500 µL of supernatant and resuspend the pellet in the remaining supernatant. Add 60 µL of resuspended sample to an LB agar plate containing 100 mg/mL ampicillin (or appropriate antibiotic) and spread media on plate using 5–10 sterile glass beads. Keep the plate in a 37 °C incubator for 16–20 h. Note: The volume of BL21(DE3) chemo-competent cells plated can be reduced if colony density on LB agar plates is too high after 37 °C incubation. Inoculation Pick a single colony from the previously grown LB agar plate and add it to a mixture of 192 mL autoclaved LB (made from Difco LB media) and auto-induction additives (4 mL of 50× M, 4 mL of 50× 5052, 200 µL of ampicillin 1,000× stock, and 40 µL of 1 M MgSO4) (Studier, 2005). Swirl the flask and place in a 30 °C shaking incubator for 24–30 h at 240 rpm. Transfer the culture into four 50 mL Falcon tubes and centrifuge for 15 min at 5,300 × g and 4 °C. Decant samples, gently wash each pellet once with 5 mL of 1× PBS, pH 7.0, and store the tubes at -80 °C for later purification (storage at temperatures above -80 °C may result in degraded, non-functional protein). Note: Initial auto-induction volume can be increased or decreased depending on protein yield as calculated in step A6h. Preparation for purification Thaw 50 mL Falcon tubes containing pelleted biosensor culture on ice. Add 5 mL 1× PBS, pH 7.0, to each tube and completely resuspend pellet while on ice. Pool resuspensions into a single 50 mL Falcon tube and place it on ice. Sonicate the resuspended biosensor at 13% amplitude, 0.7 s time-on, and 0.2 s time-off for 30 s. Repeat sonication three to six times, with 3 min between each sonication to allow for cooling. CRITICAL STEP Effective sonication is required for maximum protein yield. Increase the number of sonication cycles or sonication amplitude if the yield calculated in Step A6h is low. Centrifuge the sample for 15 min at 5,300 × g and 4 °C. Collect the supernatant while being careful to avoid collecting any pellet and transfer it to a new 50 mL Falcon tube. CRITICAL STEP If some pellet is gathered with the supernatant, this will markedly lengthen the filtration step in Step A3h. Note: An additional 15 min centrifugation and transfer to a new 50 mL Falcon tube may facilitate filtration in later steps. Attach a 0.2 µm filter to a 10 mL syringe and place over a new 50 mL Falcon tube. Remove syringe plunger and add the previously collected supernatant to the 10 mL syringe, replace syringe plunger, and depress plunger to filter. Note: Multiple 0.2 µm filters may be required to filter one sample; these may be replaced on the 10 mL syringe. Ensure that filtered and unfiltered supernatant are not mixed during 0.2 µm filter replacement. CRITICAL STEP Unfiltered supernatant can result in bacterial growth in fast protein liquid chromatography (FPLC) systems. Repeat filtration step with remaining supernatant until the entire sample is filtered. Note: Multiple 0.2 µm filtrations may be performed in parallel. Pool 0.2 µm filtered supernatant into one 50 mL Falcon tube (as needed). FPLC purification Equilibrate Ni-NTA column according to manufacturer’s guidelines. Note: For general FPLC setup, see Figure 1. Equilibrate appropriate FPLC lines with Buffer A and Buffer B. Run an appropriate protein purification method (example program: seven column volume (CV) applications of 100% Buffer A, sample application 10–30 mL, seven CV washouts of unbound protein with 100% Buffer A, 0%–100% Buffer B gradient elution over 10 CV, five CV applications of 100% Buffer B, and collect 4 mL fractions starting at Buffer B gradient elution step). When prompted to load the sample, load the filtered biosensor solution using either the 1) sample loop or 2) sample valve as appropriate for the supernatant volume and FPLC model. When purification program is complete, remove fractions from the FPLC carousel and store at 4 °C. CRITICAL STEP To ensure minimal cross-contamination of biosensors, column stripping and regeneration is recommended when purifying multiple constructs in succession. Run an appropriate program to strip the column using 50 mM EDTA and 0.05% SDS, followed by 0.5 M NaOH (example program: five CV applications of EDTA 0.05% SDS, two CV applications of H2O, and five CV applications of 0.5 M NaOH). Note: If changing bottles between programs, wash the lines with water before equilibrating with the new solution. CRITICAL STEP Direct mixing of buffers without a water wash may result in precipitation of nickel in FPLC lines, affecting FPLC function. TROUBLESHOOTING If precipitation is seen, wash lines extensively with water to remove precipitate. Run an appropriate program to regenerate the column using 0.2 M NiSO4, followed by storage in 20% EtOH (example program: five CV applications of nickel sulfate, three CV applications of H2O, and five CV applications of 20% EtOH). Ensure that all lines are stored in 20% EtOH to reduce bacterial growth. Figure 1. Fast protein liquid chromatography (FPLC) setup. The FPLC machine used for protein purification is shown with lines, waste, and fraction collector. Protein gel electrophoresis Add 20 µL of 2-mercaptoethanol (β-ME) and 180 µL of 4× Laemmli sample buffer to a 1.5 mL microcentrifuge tube and mix by a brief microcentrifuge spin. Identify protein fractions to sample from the FPLC purification. Note: A general rule is to sample the range that covers the fractions of detectable protein elution, with a buffer of several fractions on either end of this range. If needed, sample every third or fourth fraction. Add 20 µL of each chosen protein fraction from purification to 1.5 mL microcentrifuge tubes. Add 10 µL of the mixture of β-ME and Laemmli sample buffer made in StepA5a a to each protein fraction sample. Mix tube contents by a brief microcentrifuge spin. Place samples in a 95 °C heat block for 10 min. Add 30 µL 10-well Mini-PROTEAN TGXTM precast protein gel to a gel box and fill the box with Tris-glycine buffer (diluted from 10× concentrate). After 95 °C incubation is completed, briefly spin down 1.5 mL microcentrifuge tubes and fill lanes 2–10 of the gel with 20 μL of the FPLC-purified fraction solution. Add 1.5 µL of Bio-Rad Precision Plus protein dual color standards to lane 1. Run the gel for 1 h and 10 min at 110 V (or for the time recommended by manufacturer). Remove the gel from its casting by crimping, place in a coverable container, add Coomassie stain, and place on a rotary shaker on a light setting overnight. The next morning, decant the Coomassie stain (which can be saved for future gels) and rinse the gel with water. Incubate the gel with water on the rotary shaker until it becomes clear (the protein bands should be blue). Note: To hasten the destaining process, replace the water every 10–15 min. Once the gel is clear, examine the protein bands in lanes 2–10 and compare these to the protein ladder (lane 1) to determine whether the protein bands match the molecular weight of the desired protein. Note: Bands of lower or higher molecular weight may be visible along with the desired protein and indicate unwanted proteins. CRITICAL STEP Care should be taken to concentrate biosensor protein fractions with minimal contamination from these other proteins. If desired, size-exclusion chromatography can be performed to further increase protein purity. Concentration of purified protein Transfer 8 mL of 1× PBS, pH 7.0, to a 50 mL centrifugal filter (30 kDa cutoff). Centrifuge the tube for 9 min at 3,400 × g and 4 °C to wash the filter. Add 11 mL of the chosen FPLC purified protein fractions to the 50 mL centrifugal filter (30 kDa cutoff) and centrifuge using the settings in Step A6b. Note: Single-chain biosensors that merge OpuBC, cpGFP, and two 4-residue linkers have a molecular weight of approximately 62 kDa. The 30 kDa cutoff is probably appropriate for concentrating all known biosensors that merge a PBP moiety with a fluorescent protein moiety. Repeat Step A6c until all desired fractions are added to the filter and centrifuged. Add 8 mL of 1× PBS, pH 7.0, to the centrifugal filter and centrifuge using the settings in Step A6b. Repeat Step A6e six more times to buffer exchange the biosensor solution and remove all residual imidazole. When the final centrifugation is reached, determine the volume of the biosensor in the centrifugal filter. If it is 500–1,000 µL, continue to Step A6h. If it is >1,000 μL, centrifuge the biosensor solution at 3,400 × g in 3 min increments until the volume is 500–1,000 µL. Note: A volume of 500–1,000 µL generally provides a protein concentration of 50–200 μM. 250 μL of biosensor protein at 50 μM is generally sufficient to run several screens and perform the multiple concentration drug–biosensor fluorescence validations. Measure the A280 of the protein on Nanodrop 1000 (or equivalent UV-Vis spectrophotometer) using the manufacturer’s instructions. After obtaining the A280, determine the concentration using Beer’s Law. The appropriate extinction coefficient for each biosensor being purified is calculated from the amino acid composition. Note: Purification of protein from 200 mL of autoinduction media generally provides a final yield of 0.2–2.0 mg. Aliquot the biosensor solution into 0.5 mL tubes and flash freeze in liquid nitrogen. CRITICAL STEP Multiple freeze-thaw cycles are not recommended, so storage in 20–50 µL aliquots is recommended. Store the biosensor solutions at -80 °C. TROUBLESHOOTING If biosensor yield is low (less than 250 μL at 50 μM), the following suggestions are recommended: increase autoinduction time to the 30 h maximum, remake auto-induction solutions and repeat autoinduction, increase auto-induction volume, increase number of sonication rounds, and increase sonication amplitude. Although GFP-based biosensor protein production in E. coli is generally robust, some variability remains. Creation of drug plate Assembly of drug library Choose drugs based on criteria relevant to the project of interest (i.e., controlled substance scheduling, absence of existing biosensors, and clinical importance). Collect information on each drug and compile in Microsoft Excel for reference and comparison; this also allows tracking of delivery status and storage. Note: In our case, information was collected on each drug’s commercial availability, pharmacological or therapeutic class, trade name, rationale for study, IUPAC name, stereochemistry, chemical formula, formula weight, CAS number, formulation, structure, additional pharmacological properties, EC50, activity (agonist/antagonist), solubility, pKa, logP (octanol-water partition coefficient) or logDpH7.4 (logP corrected for fraction of uncharged (deprotonated) molecules at pH 7.4), controlled substance scheduling, handling instructions, vendor, price, and purity. Drug solvation Choose a solvation method for each drug based on solubility in water and organic solvents. CRITICAL STEP If using PBP-based biosensors, avoid DMSO when possible (interactions between DMSO and PBP-based biosensors can result in high baseline fluorescence). Use 1× PBS (pH 7.0) as the solvent for water-soluble drugs. TROUBLESHOOTING It is suggested to perform small-scale tests of drug solubility in various solvent systems prior to drug plate formation to ensure complete dissolution. Water, 3× PBS (pH 7.0), ethanol, or methanol are recommended solvation systems. Calculate desired amount of drug to result in a drug solution at 2 mM, 2–8 mL. For drugs in powder form, use the following steps: Measure on a Mettler Toledo XPR204 scale and add the powder to a 15 mL Falcon tube. Add approximately 80% of the calculated appropriate solvent required by serological pipette and mix by vortexing. Measure the pH of the resulting solution and adjust to pH 7.0. CRITICAL STEP The pH of a solution affects the baseline fluorescence of iDrugSnFRs (Shivange et al., 2019). This variation amounts to 10-fold per pH unit, or 26% per 0.1 pH unit. Consistency of pH across drug solutions will reduce variability in screen results. Add the remaining volume required to obtain a 2 mM concentration. Note: For solutions that do not dissolve fully by vortexing, place in a 42 °C heat bath until solvation is complete. CRITICAL STEP Ensure that the drug is completely dissolved before continuing to drug plate formation. For drugs in liquid form, use the following steps: Dilute drug directly into 80% of the calculated appropriate solvent. Mix drug by vortexing. Measure the pH of the resulting solution and adjust to pH 7.0. CRITICAL STEP The pH of a solution affects the baseline fluorescence of iDrugSnFRs (Shivange et al., 2019). This variation amounts to 10-fold per pH unit, or 26% per 0.1 pH unit. Consistency of pH across drug solutions will reduce variability. Add the remaining volume required to obtain a 2 mM concentration. CRITICAL STEP Ensure that the drug is completely dissolved before continuing to drug plate formation. Drug plate formatting and compilation Design a 2.2 mL (deep-well) 96-well drug plate with drugs of interest (Figure 2). Add three control wells for each solvation condition and intersperse these across the plate to monitor potential cross contamination due to pipetting. Add 1 mL of the appropriate drug solution or solvent to each well. Store the remaining drug solution as described in MSDS for future screening plates. Figure 2. Drug plate design example of a drug–biosensor fluorescence screen. The drug plate design used for a single concentration drug–biosensor fluorescence screen is shown, with drugs organized by class and control wells mixed throughout the plate. Single concentration drug–biosensor fluorescence screen Biosensor dilution Thaw a biosensor protein aliquot on ice. Dilute the biosensor in 3× PBS, pH 7.0, so that the diluted solution has a concentration of 111 nM and volume of 30–50 mL. Transfer the solution to a 50 mL Falcon tube and mix by inversion. Mixing of drug–biosensor solution using epMotion liquid handling robot Create a program in the Eppendorf ePBlue software for biosensor screening. Note: This program needs a location for a 50 µL Eppendorf pipette tip box (for distributing drug solution), 300 µL Eppendorf pipette tip box (for distributing biosensor solution), the 2.2 mL 96-well drug plate containing 1 mL drug solutions created in Step B3, three 360 µL 96-well plates (for mixed drug–biosensor solution), and a 50 mL tube containing biosensor solution (Figure 3). The program must allow for 100 µL biosensor solution to be added from the 50 mL tube to each well of the three 360 µL 96-well plates (using 300 µL tips), 11 µL of drug solution to be added from the 2.2 mL 96-well drug plate containing 1 mL drug solution to corresponding wells of the three 360 µL 96-well plates (using 50 µL tips), and a step for the mixing of drug and biosensor solutions. The data from drug-containing wells on these plates will serve as the Drug–Biosensor Fluorescence values for ΔF/F0 calculations in Equation 1 (Data analysis). The data from the solvent control wells from this plate (i.e., 25% DMSO, 3× PBS, pH 7.0) will serve as Baseline Biosensor Fluorescence for ΔF/F0 calculations in Equation 1 (Data analysis). Run the program outlined in Step C2a. Note: This step can also be performed manually if a liquid handling robot is not available. Run the program outlined in Steps C2a-b with no biosensor present (i.e., 3× PBS, pH 7.0 only). Note: The data from these plates will serve as Baseline Drug Fluorescence for ΔF/F0 calculations in Equation 1 (Data analysis). Figure 3. Eppendorf epMotion setup for single concentration drug–biosensor fluorescence screen. The setup to mix drug and biosensor solutions is shown on the ePMotion 5075 liquid handling robot. This shows a 2.2 mL 96-well plate with 1 mL of drug/solvent solutions in position B1, p50 tips in B2, p300 tips in B3, 360 µL 96-well plates in C2–C4, and containers of biosensor solution (2× 50 mL tubes) in C5, as desired. Fluorescence measurements using Spark 10M Remove 360 µL 96-well plates one at a time from the Eppendorf machine and place into the plate holder of the Spark 10M Fluorescence Reader. Design a program to measure fluorescence of the drug–biosensor solution. Our program contains the following elements: it shakes the plate for 10 s at an amplitude of 1.5 mm to allow for additional mixing and then measures fluorescence with an excitation wavelength of 480 nm and emission wavelength of 535 nm. Note: An excitation wavelength of 480 nm and emission wavelength of 535 nm is appropriate for all GFP-based biosensors. If a different fluorescent protein is being used, the excitation and emission wavelengths should be adjusted to match its fluorophore. Example results: 2017 single concentration drug–biosensor fluorescence screen In 2017, a single concentration drug–biosensor fluorescence screen was conducted with a primary focus on drugs used to treat psychiatric disorders (Figure 4). Drugs were chosen both based on clinical importance and to encompass a range of pharmacological or therapeutic classes; classes present included opioids, anticholinergics, antipsychotics, antidepressants, benzodiazepines, anti-seizure medications, and sedatives. In total, 84 drugs and 13 biosensors were screened. We also grouped results into general categories based on the most common treatment use: schizophrenia, major depressive disorder, anxiety disorders, epilepsy, and other. As noted, a hit pair comprises a pair of molecules: drug and biosensor. In total, 190 drug–biosensor hit pairs were identified, with ΔF/F0 ranging from 1.0 to 18.5. From these, 108 hit pairs had 1 < ΔF/F0 < 3 and thus could likely be used to generate optimized biosensor variants through directed evolution. The remaining 82 hits had ΔF/F0 > 3 and thus could likely be used for cellular imaging without further mutations. Hit pairs were concentrated in three clinical use categories (schizophrenia, major depressive disorder, and other), with no hit pairs identified for anxiety and epilepsy drugs. The v7.1.2 biosensor participated in the largest number of hit pairs; these also had the highest ΔF/F0 values (Figure 4). Figure 4. Results map from the 2017 single concentration drug–biosensor fluorescence screen. Fluorescence response (as represented by ΔF/F0) for drug–biosensor pairs included in the 2017 screen. Fluorescent response shading from green (ΔF/F0 ≥ 3) to white (ΔF/F0 ≤ 0), in which stronger hits are represented by darker green, weaker hits by lighter green, and non-hits by white. Drug–biosensor pairs that were not screened are in black. This screen allowed for the identification of drug–biosensor pairs, from which the biosensor could potentially be evolved for engineering of novel variant biosensors for various neuropsychiatric medications. Biosensors for two nicotinic agonists, cytisine and dianicline, were engineered based on hit pairs obtained from this screen. The directed evolution of these sensors, as well as associated experiments on the subcellular pharmacokinetics of these drugs, are detailed in Nichols et al. (2022). The hit pair involving methadone became the basis for a selective variant, iS-methadoneSnFR (Muthusamy et al., 2022). Because many of the drugs included in this screen are sparingly water-soluble, DMSO was often used for dissolution. However, this screen showed that DMSO interacts with several biosensor candidates, even in the absence of a DOI. This phenomenon resulted in the presence of many negative ΔF/F0s, as the baseline biosensor fluorescence was higher than would be reasonable if the biosensor and solvent were not interacting. This showed that additional normalizations and corrections would be required when DMSO was used as a solvent with iDrugSnFRs (see Notes). We have not systematically studied the residues responsible for DMSO sensitivity. The 2017 screen was also informative in showing a failure: we found no hit pairs involving S-ketamine, its enantiomers, or its metabolites. Faute de mieux, we performed site-saturated mutagenesis on a residue, Tyr357, that makes a key cation-π interaction with nicotinic drugs (Shivange et al., 2019; Unger et al., 2020). This resulted in the S-ketamine responsive Tyr357Gly construct termed AK1, and we evolved AK1 into variants that satisfactorily sense S-ketamine. We included these variants in subsequent screens described below. 2018 single concentration drug–biosensor fluorescence screen The 2018 screen was conducted to identify biosensor–drug hit pairs for opioids of several categories (Figure 5). Drugs included in the screen were chosen based on clinical importance, as well as presence in the existing literature. Most opioids included activate the μ opioid receptor, though several drugs target κ and 𝛿 opioid receptors. An Other category was composed of opioid inhibitors. In total, 48 drugs and 16 biosensors were included. From these pairs, 205 total drug–biosensor hits were identified. Of these, 128 hits had 1 < ΔF/F0 < 3, while 76 had ΔF/F0 > 3. Thirty of the 48 drugs tested participated in at least one hit pair, with most having hit pairs with several biosensors (one drug had a single hit pair). Additionally, each biosensor participated in a hit pair with at least one drug. The proportion of hit pairs varied by drug category. No hit pairs were found for µ opioids ligands, while the largest proportion of hit pairs were found for µ opioid ligands. Hit pairs also varied in fluorescence response, with the weakest pair having a ΔF/F0 of 1.0 (our at least definition for a hit pair) and the strongest pair having a ΔF/F0 of 19.0 (tapentadol × biosensor v7). Generally, the v7 biosensor series (including v7, v7.1, v7.1.2, v8, and v9) had strong responses with μ opioids (Figure 5). This screen allowed for the identification of drug–biosensor pairs that could lead to the engineering of novel opioid biosensor variants. Furthermore, this screen showed many strong hit pairs involving clinically relevant μ opioid ligands, which could allow for direct cellular imaging or efficient generation of novel biosensors for this drug class. Figure 5. Results map from the 2018 single concentration drug–biosensor fluorescence screen. Fluorescence response (as represented by ΔF/F0) for drug–biosensor pairs included in the 2018 single concentration drug–biosensor fluorescence screen. Drugs are grouped by opioid category. Fluorescent response shading from green (ΔF/F0 ≥ 3) to white (ΔF/F0 = 0), in which stronger hits are represented by darker green, weaker hits by lighter green, and non-hits by white. 2019 single concentration drug–biosensor fluorescence screen The 2019 screen was completed to identify hit pairs involving previously unscreened classes of CNS-acting drugs (Figure 6). Several drugs were included from each of five classes: 5-HT3 antagonists, anticholinergics, CB1/CB2 ligands, opioids, and neonicotinoids. Individual drugs of interest from a range of classes were also included and designated by an Other category. In total, 44 drugs were included and tested against 18 biosensor proteins. A total of 106 drug–biosensor hit pairs were identified, with 81 having 1 < ΔF/F0 < 3 and 24 having ΔF/F0 > 3. Twenty-one of the 44 drugs chosen participated in a hit pair with at least one biosensor. Additionally, each biosensor had a hit pair with at least one drug. Certain biosensors showed strong responses with several drugs in the same class. For example, L194D1 [a precursor to the biosensor iSeroSnFR, which detects serotonin (Unger et al., 2020)] displayed hit pairs for six CB1/CB2 ligands, with responses ranging from ΔF/F0 of 3.5–5.0. Several other biosensors (v4.6, v.4.8.1.2, v7 436A, and Scop4) also participated in hit pairs with several CB1/CB2 ligands, albeit less strongly (Figure 6). This screen expanded the number of drug–biosensor hit pairs identified that could be used for engineering of novel variants, as well as the range of drug classes. 5-HT3 antagonists participated in many hit pairs and also showed promising results in validation through dose-response measurements (below). Figure 6. Results map from the 2019 single concentration drug–biosensor fluorescence screen results map. Fluorescence response (as represented by ΔF/F0) for drug–biosensor pairs included in the 2019 single concentration drug–biosensor fluorescence screen. Drugs are grouped by class. Fluorescent response shading from green (ΔF/F0 ≥ 3) to white (ΔF/F0 = 0), in which stronger hits are represented by darker green, weaker hits by lighter green, and non-hits by white. Drug–biosensor fluorescence validation of hit pairs through multiple concentration response experiments Creation of drug dilution plate Choose a drug-biosensor hit pair for a multiple concentration response experiment. Create a drug dilution plate that allows for a triplicate dose-response relation measurement of the desired drug. CRITICAL STEP Accurate drug concentrations in the drug dilution plate are necessary for accurate calculations of the drug–biosensor interaction. Pipette 1 mL of 2 mM drug solution into wells A1–C1 of a 2.2 mL 96-well plate. Perform a √1 0 dilution eight times. Transfer 316 µL of solution from A1–C1 wells to A2–C2. Add 684 µL of 3× PBS, pH 7.0, to wells A2–C2 and mix by pipette. Transfer 316 µL of solution from wells A2–C2 to wells A3–C3, add 684 µL of 3× PBS, pH 7.0, and mix. Repeat above steps until wells A9–C9 are filled. Add 684 µL of 3× PBS, pH 7.0, to wells A10–C10 to serve as a blank. Biosensor dilution Thaw a biosensor protein aliquot from Step A6j on ice. Dilute the biosensor in 3× PBS, pH 7.0, so that the diluted solution has a concentration of 111 nM and volume of 30–50 mL. Transfer the solution to a 50 mL Falcon tube and mix by inversion. Mixing of drug–biosensor solution using epMotion liquid handling robot Create a program in the Eppendorf ePBlue software for validating biosensor hit pairs. Note: This program needs a location for a 50 µL Eppendorf pipette tip box (for distributing drug solution), 300 µL Eppendorf pipette tip box (for distributing biosensor solution), the 2.2 mL 96-well drug plate containing 1 mL drug solutions created in Step D1, one 360 µL 96-well plate (for mixed drug–biosensor solution), and a 50 mL Falcon tube containing biosensor solution. The program must allow for 11 µL of drug solution to be added from the 2.2 mL 96-well drug plate containing 1 mL drug solutions to corresponding wells of the 360 µL 96-well plate, 100 µL of biosensor solution to be added from the 50 mL Falcon tube to the same wells of the 360 µL 96-well plate, and for these solutions to be mixed (there will only be drug and biosensor solution in wells A–C 1–9 of the plate if the 2.2 mL 96-well drug plate containing 1 mL drug solutions is set up as instructed in step D1). The data from columns 1–9 will serve as the Drug–Biosensor Fluorescence values for ΔF/F0 calculations in Equation 1 (Data analysis). The data from column 10 will serve as Baseline Biosensor Fluorescence for ΔF/F0 calculations in Equation 1 (Data analysis). Run the program outlined in Step C2a. Notes: This step can also be performed manually if a liquid handling robot is not available. The range of drug concentration sampled can be increased or decreased as needed to sample a full dose-response relation. Fluorescence measurements using Spark 10M Once the program on the epMotion liquid handling robot is complete, remove the 360 µL 96-well plate from the Eppendorf machine and place it into the plate holder of the Tecan Spark 10M Fluorescence Reader. Utilize the same program as in step C3b. Repeat steps D3–4 with no biosensor present (i.e., 3× PBS, pH 7.0 only) Note: The data from these plates will serve as Baseline Drug Fluorescence for ΔF/F0 calculations in Equation 1 (Data analysis). Example results for future studies: 5-HT3 receptor Antagonists Four 5-HT3 antagonists were included in the 2019 screen: ondansetron, tropisetron, palonosetron, and alosetron. In this class, 23 hit pairs involved drugs, including several pairs found for ondansetron, tropisetron, and palonosetron. No hit pairs were found for alosetron. Top hit pairs for each drug were validated through multiple concentration drug–biosensor fluorescence dose-response studies as described in step D above (Figure 7). Additionally, these validations allowed for the calculation of EC50, Hill coefficient (nH, a measure of apparent cooperativity among binding sites), and S-slope (as defined in Data analysis) of the drug–biosensor hit pairs. Validation screens were conducted for the four strongest hit pairs for each DOI. Thus, ondansetron was studied against the biosensors AK1, cc70, v7.1.2, and Scop4; tropisetron was studied against the biosensors AK1, v7.1, v7.1.2, and v8; and palonosetron was studied against the biosensors AK1, v7.1, v7.1.2, and Scop4. From these validation screens, the drug–biosensor pairs with the most favorable profile were chosen (Figure 7). This was determined based on the S-slope, EC50, and Fmax of the drug–biosensor pairs, with a higher Fmax, higher S-slope, and lower EC50 being most desirable. Several 5-HT3 antagonist × biosensor hit pairs were validated through these dose-response relations. Two strong hit pairs were validated for palonosetron. Palonosetron × v7.1.2 displayed an Fmax of 5.4, and palonosetron × v7.1 displayed an Fmax of 3.2. We expect that both hit pairs have Fmax values large enough (i.e., > 3) to be used directly for cellular imaging. For ondansetron and tropisetron, a single hit pair was identified, with an Fmax of 2 for ondansetron × cc70 and an Fmax of 2.2 for tropisetron × v7.1.2. Because the Fmax values for these pairs are < 3, further directed evolution will be required to optimize a variant for successful use for imaging. For all hit pairs shown, nH is near 1, suggesting that the biosensors would have desired binding properties with these drugs. Figure 7. Multiple concentration drug–biosensor fluorescence validations for 5HT3 antagonists. Dose-response relations (A) and analysis [Fmax, EC50, Hill coefficient (nH), and S-slope] (B) for 5-HT3 antagonists. Only the most promising dose-response relation(s) for each drug of interest are shown. Biosensors included are cc70, v7.1.2, and v7.1. CB1/CB2 Ligands The 2019 screen included nine CB1/CB2 ligands: SLV-319 (+/-), AM 6545, PSNCBAM-1, O-2050, WIN-55 212,22, CP 94545, NIDA 41020, rimonabant, and leelamine. In total, 34 hit pairs were found for drugs in this class, with several hit pairs being found for each of six drugs [SLV-319 (+/-), AM 6545, PSNCBAM-1, WIN-55 212,22, NIDA 41020, and rimonabant]. We found no hit pairs including the remaining three drugs (O-2050, CP 94545, and leelamine). The top four hit pairs for each drug were validated through multiple concentration drug–biosensor fluorescence dose-response measurements. Thus, SLV-319 (+/-) was measured against the biosensors v4.6, v7A, and L194D1; AM 6545, rimonabant, and PSNCBAM-1 were measured against the biosensors AK1, v4.6, v7A, and L194D1; WIN-55 212,22 was measured against the biosensors v4.6, v4.8.1.2, v7A, and L194D1; and NIDA 41020 was measured against the biosensors v4.6, v4.8.1.2, Scop4, and L194D1. From these screens, the drug–biosensor pairs with the most favorable profile were chosen (Figure 8). This was determined based on the S-slope, EC50, and Fmax of the drug–biosensor pairs, with a higher Fmax, higher S-slope, and lower EC50 being most desirable. The multiple concentration drug–biosensor validations showed promising data for CB1/CB2 ligands: when calculated as S-slopes ((ΔF/F0)/[Drug]) at 0.6 µM, the sensitivities plotted in Figure 8 range from 1.6 to 6 µM-1. Our limited supplies of the test drugs vitiated full dose-relations at saturated concentrations and Hill equation fits; but these data already suggest that it will be possible to conduct cellular experiments with these ligands at 1 < µM. Further optimization may present the challenge of directed evolution in non-aqueous solvents such as DMSO or methanol. Figure 8. Multiple concentration drug–biosensor fluorescence validations for CB1/CB2 ligands. Dose-response relations for CB1/CB2 ligands. Only the most promising dose-response relation(s) for each drug of interest are shown. Data analysis Single concentration drug–biosensor fluorescence screen Data analysis for our single concentration drug–biosensor fluorescence screens is relatively straightforward. When fluorescence measurements are obtained using the Tecan fluorescence reader, measurements are obtained for the biosensor alone, the drug of interest alone, and the biosensor when the drug of interest is added. These measurements are automatically output into an Excel file through the Tecan program and can be used to calculate ΔF/F0 for each drug–biosensor pair. This calculation allows for a direct comparison of fluorescence between drug–biosensor pairs with varying drug and biosensor baseline fluorescence. Additionally, it allows for determination of the strength of a hit regardless of whether the involved biosensor has high or low baseline fluorescence. The calculation used for ΔF/F0 is given in Equation 1. ΔF/F0=[(Drug-Biosensor Fluorescence)-(Baseline Biosensor Fluorescence)-(Baseline Drug Fluorescence)]/(Baseline Biosensor Fluorescence) EQUATION 1. Relative fluorescence values (ΔF/F0) Equation used to calculate relative fluorescence values (ΔF/F0) for each drug–biosensor combination in single concentration screens and dose-response relations. Multiple concentration drug–biosensor fluorescence validation For our multiple concentration drug–biosensor fluorescence validations, Tecan measurements include biosensor baseline fluorescence, fluorescence of biosensor when the DOI is added at eight concentrations (0.02–2 µM final drug concentration), and drug baseline fluorescence. These measurements yield ΔF/F0 for each drug concentration using Equation 1. A well-behaved dose-response relation suggests that more complex interactions do not occur. Therefore, we wish to obtain values for ΔFmax/F0, EC50, and nH. ΔFmax/F0 represents the estimated maximum fluorescence value as drug concentration approaches infinity. EC50 represents the drug concentration at which ΔF/F0 increases to ΔFmax/2F0. The Hill coefficient is nH. These classical values can be obtained via curve-fitting through Lineweaver-Burke plots or Hill plots; we use the Origin Pro 9.1 program. One goal of many drug development efforts is to find drugs with high potency, i.e., the lowest possible EC50 and IC50. Because we aim for biosensor variants that respond at pharmacologically relevant drug concentrations, we often work near the linear start of the drug–biosensor dose-response relation. To evaluate sensitivity, we define the S-slope metric as a linear fit to (∆F/F0 )/[ligand] at the beginning of the dose-response relation, with units of (μM)-1. Such measurements do not require a measurement of the maximal response, ΔFmax/F0, and are therefore appropriate for dose-response relations that do not extend to saturating levels (i.e., Figure 8). When a fully characterized dose-response is available (nH is always near 1.0), we also characterize S-slope by combining the values for ΔFmax/F0 and EC50 (Equation 2). Note that S-slope can be increased either by increasing ΔFmax/F0 or by decreasing EC50. S-slope=(ΔFmax/F0)/EC50 EQUATION 2. S-slope Equation used to calculate S-slope dose-response relations from maximum fluorescence increase (ΔFmax/F0) and half-maximal concentration (EC50). These values are determined via curve fitting to complete dose-response relations, for instance during multiple concentration drug–biosensor fluorescence validation. Database Availability The iSnFRbase numbers in Figures 4, 5, and 6 refer to the posted file, https://github.com/lesterha/lesterlab_caltech/blob/main/iSnFRBase0711%20as%20of%207-15-22_for_%E2%80%8BBeatty_et_al_2022b.xlsx Notes Through our single concentration screens, over 500 drug–biosensor hit pairs were identified that show promise for creation of novel biosensors for CNS-acting drugs. While most hit pairs had 1 < ΔF/F0 < 3 (approximately 65%) and thus will likely require further directed evolution before they can be used for imaging with their paired drug, a substantial portion (approximately 35%) had ΔF/F0 > 3 and thus could likely be directly used for cellular imaging. However, one must note that the screened biosensors were engineered to bind another drug, so directed evolution will still need to be conducted to establish selectivity for the new drug of interest. Once hit pairs have been successfully identified, they can be further assessed by multiple concentration validations. This is particularly useful in cases where a single drug participates in multiple hit pairs, and a single sensor needs to be chosen to begin directed evolution. Several idiosyncrasies underlie the described protocols. First, many iDrugSnFRs of the OpuBC family bind and respond to amine-containing buffers at the tens of mM concentrations used in typical biological and molecular experiments. This is likely due to the amine-binding OpuBC parentage with the cation-π box at the binding site (Bera et al., 2019; Nichols et al., 2022; Muthusamy et al., 2022) (pdb files 7S7T, 7S7U, 7S7X, 7S7Z). Thus, we use only phosphate- and/or bicarbonate-based buffers. Second, most cpGFP-based biosensors are sensitive to pH, which is likely due to the proton candle snuffer mechanism (Barnett et al., 2017; Muthusamy et al., 2022; Nichols et al., 2022). Thus, one is limited to imaging in nearly pH-neutral organelles such as the cytoplasm, endoplasmic reticulum, and cis-Golgi apparatus. Third, many iDrugSnFRs are activated by DMSO alone, which implies that one cannot use commercially available screening libraries formatted in DMSO and must be cautious when using DMSO for water-insoluble drugs. Lastly, one must consider the fact that some drugs may have detectable fluorescence independent of biosensor interaction and require correction for this fluorescence in ΔF/F0 calculations (as outlined in Equation 1 above). Additionally, the protocol has been revised with each screen to increase efficiency. Given the wide range of drugs for which hit pairs were identified in the screens shown, our fluorescence screening protocol is probably generalizable for drug classes other than those we have studied. There are several rubrics for describing drug classes. One definition of drug class invokes chemistry. We do not know the upper limit for MW of ligands that could participate in hit pairs; we have not screened drugs with MW > 500. Wild-type OpuBC and its orthologs bind permanently charged quaternary amines such as choline, betaine, and proline betaine. Only a few clinically useful drugs (for instance tiotropium) are quaternary amines. Our screens have concentrated on primary, secondary, and tertiary amines that are partially protonated at neutral pH, i.e., they are weakly basic. We assume that only the protonated form of the drug makes a cation-π interaction with the iDrugSnFR (Bera et al., 2019; Nichols et al., 2022; Muthusamy et al., 2022) (Protein Data Bank files 7S7T, 7S7U, 7S7X, and 7S7Z). In many alkaloids, the nitrogen remains unprotonated at neutral pH. For instance, the iDrugSnFRs that bind nicotinic drugs do not sense cotinine, an amide, and we doubt that OpuBC-based iDrugSnFRs would detect amides. In unpublished work related to the 2018 screen (Figure 5), we found no hit pairs involving opioid peptides (the peptide bond is an amide bind). Another rubric for drug class invokes target pharmacology (receptor, channel, transporter, or enzyme). As noted in the Introduction, the screens we describe are necessary because little correlation exists between the structure–activity relations that govern, on one hand, target binding and, on the other hand, binding to OpuBC variants, other than the presence of a weakly basic amine. Other rubrics for drug class invoke the disorder being treated or the type of drug abuse. Within the former rubric, the figures show hit pairs for classical antidepressants, rapidly acting antidepressants, smoking cessation therapeutics, analgesics, antiemetics, antipsychotics, and appetite suppressants. Our unpublished data show hit pairs including the antidementia drug tacrine. Within the latter rubric, we found hit pairs including nicotine dependence, opioid dependence, and dissociative effects. We have not screened US Drug Enforcement Administration Schedule 1 drugs; among these, it is likely that hit pairs could be identified for some psychedelics (especially psilocin and analogs), cocaine, and MDMA. Hit pairs for other drug classes (however one defines the classes) should probably be approached by merging circularly permuted fluorescent protein GFP with variants of a different PBP (Scheepers et al., 2016) Hit pairs for amino acid drugs might be generated by mutating the binding site in the glutamate-sensing iGluSnFR variants (Marvin et al., 2018). Many drugs that inhibit intracellular enzymes are amides; we have not considered the likeliest PBPs for binding amides. Finally, several scientists have proposed the intriguing challenge of generating PBP-based sensors for peptides. Recipes 50× M Reagent Final concentration Amount Na2HPO4 1.25 M 17.75 g KH2PO4 1.25 M 17.0 g NH4Cl 2.5 M 13.4 g Na2SO4 H2O Total 0.25 M n/a n/a 3.55 g remainder 100 mL 50× 5052 Reagent Final % Amount Glycerol 25% 25 mL Glucose 2.5% 2.5 g Alpha-lactose 10% 10 g H2O Total n/a n/a remainder ~100 mL Acknowledgments We have been supported by The Brain and Behavior Research Foundation (NARSAD Award 2014-23069), California Tobacco-Related Disease Research Program (Aaron L. Nichols, 27FT-0022) (Dennis A. Dougherty, 27IP-0057), The Della Martin Foundation (Kallol Bera), Howard Hughes Medical Institute (Jonathan S Marvin and Loren L Looger), National Institute on Drug Abuse (Henry A Lester, DA043829) (Henry A Lester and Anand K Muthusamy, DA049140), National Institute of General Medical Sciences (Henry A Lester, GM-12358) (Anand K Muthusamy T32-GM7616), National Institute of Mental Health (Henry A Lester, 213MH120823), National Institute of Neurological Disorders and Stroke fellowship (Anand K Muthusamy T32NS105595), the Caltech CI2 program, and Caltech SURF donors Samuel P. and Frances Krown (Zoe G Beatty). This protocol was derived from the original research paper “Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands” (Nichols et al., 2022). Competing interests Anand Muthusamy, Henry Lester, Loren Looger, and Jonathan Marvin have filed a patent application that includes opioid biosensors. Lin Tian is a co-founder of Seven Biosciences. Ethics There are no ethical concerns relevant to this paper. References Barnett, L. M., Hughes, T. E. and Drobizhev, M. (2017). Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence. PLoS One 12(2): e0170934. Bera, K., Kamajaya, A., Shivange, A. V., Muthusamy, A. K., Nichols, A. L., Borden, P. M., Grant, S., Jeon, J., Lin, E., Bishara, I., et al. (2019). Biosensors Show the Pharmacokinetics of S-Ketamine in the Endoplasmic Reticulum. Front Cell Neurosci 13: 499. Henderson, B. J. and Lester, H. A. (2015). Inside-out neuropharmacology of nicotinic drugs. Neuropharmacology 96(Pt B): 178-193. Lester, H. A., Miwa, J. M. and Srinivasan, R. (2012). Psychiatric drugs bind to classical targets within early exocytotic pathways: therapeutic effects. Biol Psychiatry 72(11): 907-915. Marvin, J. S., Scholl, B., Wilson, D. E., Podgorski, K., Kazemipour, A., Muller, J. A., Schoch, S., Quiroz, F. J. U., Rebola, N., Bao, H., et al. (2018). Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat Methods 15(11): 936-939. Muthusamy, A. K., Kim, C. H., Virgil, S. C., Knox, H. J., Marvin, J. S., Nichols, A. L., Cohen, B. N., Dougherty, D. A., Looger, L. L. and Lester, H. A. (2022). Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids. J Am Chem Soc 144(19): 8480-8486. Nichols, A. L., Blumenfeld, Z., Fan, C., Luebbert, L., Blom, A. E. M., Cohen, B. N., Marvin, J. S., Borden, P. M., Kim, C. H., Muthusamy, A. K., et al. (2022). Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands. Elife 11: e74648. Scheepers, G. H., Lycklama A. N. J. A. and Poolman, B. (2016). An updated structural classification of substrate-binding proteins. FEBS Lett 590(23): 4393-4401. Shivange, A. V., Borden, P. M., Muthusamy, A. K., Nichols, A. L., Bera, K., Bao, H., Bishara, I., Jeon, J., Mulcahy, M. J., Cohen, B., et al. (2019). Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors. J Gen Physiol 151(6): 738-757. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234. Unger, E. K., Keller, J. P., Altermatt, M., Liang, R., Matsui, A., Dong, C., Hon, O. J., Yao, Z., Sun, J., Banala, S., et al. (2020). Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning. Cell 183(7): 1986-2002 e1926. Article Information Copyright Beatty et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Neuroscience > Development Molecular Biology > Protein Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,552	https://bio-protocol.org/en/bpdetail?id=4552&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed In vitro Assay to Evaluate Cation Transport of Ionophores HU Huriye D. Uzun MV Melissa Vázquez-Hernández JB Julia E. Bandow TP Thomas Günther Pomorski Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4552 Views: 1047 Reviewed by: David PaulJohn P Phelan Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Proteomics Sep 2022 Abstract Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions. Graphical abstract: Keywords: Calcein Cobalt Copper Fluorescence quenching Large unilamellar vesicles Background Ion homeostasis is a fundamental requirement for all living cells, as it controls diverse biological processes, including cell signaling, maintenance of cell volume and osmotic pressure, and regulation of cellular pH. Ions also act as enzyme cofactors, providing force through concentration gradients, and serve as corepressors for protein regulators orchestrating, for instance, the oxidative stress response. Ion homeostasis in cells depends upon lipid membranes, which function as ion permeability barriers. This allows cells to maintain highly asymmetric concentrations of cations and anions across both plasma membranes and intracellular organelles. Major ionic gradients across the plasma membrane include sodium (Na+), potassium (K+), calcium (Ca2+), and chloride (Cl-). Ionophores, which transport ions across biological membranes, offer a way to manipulate cellular ion composition. Examples of commonly used ionophores include valinomycin, which transports potassium (K+) (Su et al., 2019), or calcimycin (A23187) and ionomycin, which transport zinc (Zn2+), manganese (Mn2+), and Ca2+ (Erdahl et al., 1994, 1995, 1996), or 4-bromocalcimycin (4Br-A23187), which is highly selective for the transport of Zn2+ and Mn2+, compared to Ca2+ (Erdahl et al., 1996). Ion transport and specificity of ionophores are typically characterized in vitro, in model systems comprising model membranes and a single ion species available for transport. A common cell membrane model is based on large unilamellar vesicles (LUVs) formed from one lipid type or lipid mixtures of different compositional complexity. The vesicles are typically prepared from a lipid solution in a volatile organic solvent. A thin lipid film on the inside of a glass flask is formed by slow evaporation of the organic solvent. The subsequent hydration of the lipid film results in the formation of large multilamellar vesicles, which are converted to unilamellar vesicles of defined size by a freeze-thaw and extrusion method (Olson et al., 1979; Hope et al., 1986; Mayer et al., 1986; Kunding et al., 2008). This approach has been used to encapsulate radioactive ions (e.g., 22Na+, 45Ca2+, and the K+ mimic 86Rb+) inside the liposomes to study their efflux upon dilution of the liposomes into an isotope-free solution (e.g., Hamilton and Kaler, 1987 and 1990). A drawback to this procedure is the inconvenience associated with handling radioactive reagents. Alternative assays are based on the encapsulation of water-soluble ion-sensitive dyes into the vesicle. Ion permeability is then monitored by fluorescence spectroscopy. The protocol presented here utilizes calcein as a fluorescent dye, which can be quenched by Cu2+, Ni2+, Co2+, and Fe3+ at neutral pH (Černiauskas et al., 2017; Picard et al., 2000), and was recently used by us to reveal 4-Br-A23187 as a potent copper ionophore (Senges et al., 2022). Given the broad range of ions that can quench calcein fluorescence, the assay should be useful for determining the selectivity and mechanisms of ionophore-mediated cation transport. Furthermore, the impact of ionophores on membrane integrity can be tested using a calcein leakage assay by loading the vesicles with a self-quenching concentration of calcein (> 750 µM), as described previously (Maherani et al., 2013; Dutta et al., 2020; Bae et al., 2021; Senges et al., 2022). This versatile protocol can be fitted to other ion-sensitive dyes, although encapsulation efficiency should be tested for each dye. The assay can also be adapted to study metal transport by reconstituted membrane proteins (Billesbølle et al., 2020). Materials and Reagents Ice bucket (e.g., Magic Touch 2TM ice bucket with lid; Sigma-Aldrich, catalog number: BAM168072002) Glass bead, 3 mm (Merck, catalog number: 104015) Microcentrifuge tubes of 1.5 mL capacity (SARSTEDT AG & Co. KG, catalog number: 72.690.001) Polyethersulfone membrane with a pore size of 0.2 μm (Filtropur, SARSTEDT AG & Co. KG, catalog number: 83.1826.001) Polypropylene tubes of 15 mL and 50 mL capacity (e.g., Falcon tubes, SARSTEDT AG & Co. KG, catalog numbers: 62.554.502 and 62.547.254) Round bottom glass tubes (16 × 150 mm, Carl Roth, catalog number: NY90.1) Magnetic bars ROTILABO® Micro (diameter 2 mm, length 5 mm, Carl Roth, catalog number: 0955.2) Syringe filter, Filtropur S, membrane: Polyethersulfone (PES), filtration surface: 6.2 cm2, pore size: 0.2 µm, for sterile filtration (Sarstedt, catalog number: 83.1826.001) Syringe sterile dimethyl sulfoxide (DMSO; Thermo Scientific, catalog number 036480.AP) 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC; Avanti Polar Lipids, catalog number: 850375C) Aluminum foil Calcein (Merck, catalog number: 1461-15-0) Calcium chloride (Grüssing, catalog nummer: 102331000U) Chloroform, ethanol-stabilized and certified for absence of phosgene and HCl (VWR, catalog number: 22711.290) Deionized water HEPES (Carl Roth, catalog number: 6763.3) Liquid nitrogen Methanol (VWR, catalog number: 20834.291) N2 gas (ALPHAGAZ 1 N2, 99.999%, Air Liquide, Germany) Potassium chloride (Sigma-Aldrich, catalog number: 7447-40-7) Potassium hydroxide (Fisher Scientific, catalog number: P250-1) SephadexTM G-50 fine (GE Healthcare Bio-Sciences AB, catalog number: 17-0042-01) Triton X®-100, extra pure (Carl Roth, catalog number: 3051.3) 4-Br-A23187 (Cfm Oskar Tropitzsch, Marktredwitz, catalog number: 76455-48-6) Buffer A (200 mL) (see Recipes) Calcein stock in buffer A (16.6 mM, 1 mL) (see Recipes) CuCl2 stock (2 mM) (see Recipes) G50-Gel in buffer A (see Recipes) HEPES, pH 7.4 (0.5 M, 200 mL) (see Recipes) Ionophore stock (6 mM) (see Recipes) KCl (0.5 M, 200 mL) (see Recipes) KOH (1 M) (see Recipes) Lipid stock in chloroform (see Recipes) Loading buffer (1 mL) (see Recipes) Triton X-100 solution (20% w/v) (see Recipes) Equipment Forceps (e.g., VWR, catalog number: 232-0032) Analytical balance (e.g., Sartorius Entris-I II, 220 g/0.1 mg; Buch Holm, catalog number: 4669128) Centrifuge with rotor for 15 mL polypropylene tubes (e.g., Eppendorf 5810 R) Flow cabinet to work with organic solvents Fluorometer (PTI QuantaMaster 800 fluorometers) with integrated FelixGX software, equipped with single cuvette Peltier K-155-C temperature control and magnetic stirrer (Horiba) Freezer (-20 °C) Hamilton 700 Series Syringes of 10 µL, 100 µL, 250 µL, 500 µL, and 1,000 µL (Hamilton® syringe, 700 and 1000 Series) Macro polystyrene cuvettes (SARSTEDT AG & Co. KG, Germany, maximum 4.5 mL volume, catalog number: 67.745) Magnets 5 × 2 mm (Merck, catalog number: Z328839) Mini-Extruder Set (Avanti Inc., catalog number: 610023) 1 mL gas-tight syringes (Avanti no: 610017) 10 mm filter supports (Avanti no: 610014) 19 mm Nucleopore tracketch membrane with a pore size of 0.2 μm (Schleicher & Schuell) pH-meter (pH-Meter 761 Calimatic, Knick) Pipettes P20, P200, P1000 (GILSON®, catalog numbers: FD10001, FD10005, and FD10006) Pipette tips 2 µL, 20 µL, 200 µL, and 1,000 µL (SARSTEDT AG & Co. KG, catalog numbers: 70.1130.212, 70.3021, 70.760.002, and 70.3050.020) Refrigerator (4 °C) Scissors Rotavapor® R-100 Evaporator with I-100 Controller and V-100 vacuum pump (Flawil, Switzerland) Vortexer (Vortex Genie 2TM, BENDER & HOBEIN AG, Switzerland) Water bath (e.g., WPE45 Memmert, Germany) Software FelixGX software for the control of the PTI QuantaMaster 8000 fluorometer and accessories Microsoft Excel for Microsoft 365 MSO (Version 2205) Procedure Preparation of the lipid film Clean the Hamilton syringes by flushing them five times with chloroform:methanol (1:1, volume/volume) under a fume hood. Note: Chloroform is a hazardous solvent. Conduct all work in a fume hood, while wearing appropriate personal protective equipment. Using Hamilton syringes, transfer 200 µL of a 25 mg mL-1 DOPC stock solution (see Recipe 9) into a round bottom glass tube placed on ice. Note: Avoid any use of plasticware when handling organic solvents. Evaporate the organic solvent at room temperature (RT) under in a rotary evaporator at the reduced pressure of 250 mbar overnight, followed by evaporation at ~10 mbar for 15 min, see Figure 1. The dried lipid film can be stored at -20 °C. Figure 1. Scheme of lipid film preparation. (A) The desired amount of lipid (I) in chloroform is transferred to a glass tube. Subsequently, the solvent is evaporated in a rotary evaporator under the reduced pressure of 250 mbar overnight (ON), resulting in a thin lipid film on the sides of the glass tube (II). (B) The glass tube containing the desired lipids in chloroform is connected to the rotary evaporator. Preparation of calcein-loaded LUVs Add 1 mL of loading buffer (see Recipe 10) and a 3-mm glass bead to the lipid film. Vortex for 10 min (see Figure 2A). Transfer the lipid suspension to a new glass tube, without the glass bead. Subject the lipid suspension to five freeze-thaw cycles by placing the tube alternatively into liquid nitrogen for 10 min for freezing and in a water bath at 50 °C for 5 min for thawing. Note: Wear a face shield and insulating gloves when handling liquid nitrogen. Use glass tubes with high thermal shock resistance. Assemble the mini-extruder, consisting of two Hamilton glass syringes, a Teflon cylinder with two drain discs, and a 200-nm polycarbonate membrane in between them, see Figure 2B. Note: We routinely use polycarbonate membranes of 200-nm pore size and perform the extrusion at RT. However, for lipids containing saturated, long-chain fatty acids, which have a high lipid phase transition temperature (Tm), the extruder needs to be heated up to a temperature 5–10 °C above the Tm. This temperature can vary greatly between lipid formulations. A useful table of lipid phase transition temperatures is provided by the manufacturer at https://avantilipids.com/tech-support/physical-properties/phase-transition-temps/. Check the tightness of the mini-extruder by flushing with 1 mL of buffer A (see Recipe 1), i.e., pass back and forth between the syringes three to four times. Continue only if the buffer volume stays the same for each passing. Fill one Hamilton glass syringe with ~1 mL of lipid/calcein solution, and place the filled syringe into one end of the mini-extruder. Carefully place an empty syringe into the opposite end of the mini-extruder. The plunger of the empty syringe should be depressed completely into the syringe barrel. Extrude lipid suspension by passing through the filters a minimum of 11 times, starting in syringe 1 and finishing in syringe 2. Note: The number of passages through the extruder needs to be uneven so that the final liposome sample is collected in syringe 2, which is uncontaminated by residual multilamellar vesicles that have never passed the extruder. Inject the final lipid solution into a 1.5-mL microcentrifuge tube, and store it at 4 °C. Note: Size distribution of the resulting liposomal preparations can be evaluated by dynamic light scattering. When using polycarbonate membranes of 200-nm pore size, we typically obtain a preparation with an hydrodynamic diameter in the range of 168.5 ± 2.5 nm. Immediately wash all parts of the extruders with Milli-Q water, then with 70% ethanol, and dry it thoroughly before storing. Solvent-rinse syringes before storing. When extruding additional vesicles, disassemble and clean all parts of the extruder, and replace the membrane and filter supports. Figure 2. Scheme for preparation of calcein-loaded LUVs. (A) The dry lipid film is hydrated in a loading buffer containing the fluorescent dye calcein (step I) by vortexing for 10 min (step II). The lipid suspension (step III) is subjected to five freeze-thaw cycles using liquid nitrogen (-196 °C, step IV) and a water bath at 50 °C (step V). Next, the suspension is passed 11 times through 0.2-μm nucleopore polycarbonate membranes mounted in a mini-extruder to form LUVs (step VI). (B) Assembly of mini-extruder. Use four filter supports, two on each side. Use two polycarbonate membranes, with blank sides towards each other. Further directions and guidance on proper extruder assembly are provided on the manufacturer’s web site (https://avantilipids.com/divisions/equipment-products/mini-extruder-assembly-instructions; https://www.youtube.com/watch?v=WT6WPvGv5eY). Separation of calcein-loaded LUVs from free dye Prepare two G50 columns, using 2-mL plastic syringes without a plunger, and filter supports as a stopper, see Figure 3A. Place the syringes into disposable 15-mL reaction tubes, add 3 mL of Sephadex G50 fine slurry (see Recipe 4) using a Pasteur pipette, centrifuge at 180 × g for 5 min, and transfer the columns to a new 15-mL reaction tube. Load the sample on the top of one of the G50 columns. Centrifuge at 180 × g and RT for 5 min (see Figure 3B). Transfer the eluate to the top of the second G50 column. Centrifuge again at 180 × g and RT for 5 min (see Figure 3C). Note: Repeat the size exclusion chromatography of the eluate with a new G50 column, if you do not achieve a separation of the free dye (intense yellow part of the column) from the vesicles (colorless lower part of the column). Collect the eluate containing the calcein-loaded LUVs in a new 15-mL Falcon tube. Cover the tube with aluminum foil. Note: Calcein-loaded LUVs could be stored at 4 °C in the dark until the next day. Figure 3. Separation of calcein-loaded LUVs by size exclusion chromatography. (A) For size exclusion chromatography, two filter supports are placed as a stopper in a 3-mL plastic syringe without a plunger, and 3 mL of Sephadex G50 fine slurry is added. (B) Calcein-loaded LUVs are separated from free calcein during the size-exclusion chromatography. The LUVs will elute with the void volume in the early fractions, whereas the non-liposome-associated calcein will elute in later fractions. (C) Image showing a G50 filtration column after the filtration with the trapped free calcein. Free calcein is in the first G50 filtration column after the first separation. After the next separation, the second G50 filtration column contains no free calcein. Monitoring ion permeability Cool buffer A (see Recipe 1), the cuvette, and the calcein-loaded LUV at 10 °C (e.g., by storing in a fridge at 10 °C). Note: To suppress the rate of uncatalyzed transport in the absence of the ionophore, the assay is performed at 10 °C. Liposomes of other lipid compositions might exhibit lower passive permeability and thus allow higher assay temperatures. Turn on the fluorometer and set up the parameters as follows: excitation wavelength 480 nm, emission wavelength 520 nm, and measurement duration approximately 10 min, with 1 s resolution. Adjust slits as necessary; a bandpass of 3 nm is usually sufficient. Cool down the sample holder of the fluorometer to 10 °C. Note: We use Peltier-based temperature control with magnetic stirring, providing temperature stability and full control software during the measurements. Add 20 µL of calcein-loaded LUV in the fluorometer cuvette, and top up to 2 mL using precooled buffer. For probing the impact of ionophores on ion permeability, add 1 µL of ionophore (see Recipe 6, final concentration 3 µM) into the cuvette, and incubate in the fridge for a further 5 min. As a control for quenching of calcein in the presence of ions, prepare one sample by adding 10–20 µL of 20% Triton X-100 (see Recipe 11) into a cuvette, and incubate in the fridge for 5 min. Introduce the cuvette into the fluorometer (remember to include the magnetic stir bar), and start monitoring the emission intensity. Wait until fluorescence is stable (90 s). Measure the fluorescence intensity. After 1.5 min, add 3 µM CuCl2 (3 µL of 2 mM CuCl2, see Recipe 3), and record the fluorescence for another 8.5 min. Note: To ensure your setting and the calcein-loaded LUVs are working properly, run an excitation scan from 450 to 500 nm (emission 520 nm) and an emission scan from 500 to 550 nm (excitation 480 nm) at the start of the measurements. For calcein, the excitation and emission maxima should be at 480 nm and 520 nm, respectively (see Figure 4). Figure 4. Excitation and emission scans of the calcein-loaded LUVs were measured using a fluorometer. In the fluorometer cuvette, add 20 µL of calcein-loaded LUVs, and fill up to 2 mL using precooled buffer A. An excitation scan (black trace) was performed from 450–500 nm (emission 520 nm, slit 3 nm) and an emission scan (grey trace) from 500–550 nm (excitation 480 nm, slit 3 nm). Data analysis Save the measured data as a text document (txt file). Export the data from the text document to Microsoft Excel. Find the maximum measured fluorescence signal (Fmax). For normalization, divide the measured fluorescence signal by Fmax and multiply by 100 (see formula 1). Fnorm =F⁄Fmax*100 (1) F: measured fluorescence signal Fmax: maximum of measured fluorescence signal Fnorm: normalized fluorescence signal Plot Fnorm against time (see Figure 5). Note for data interpretation: In this in vitro quenching assay, insertion of ionophores (here 4-Br-A23187) into LUVs allows the transport of Cu2+ ions into the vesicles, which then quench the encapsulated calcein fluorophore (Figure 5). In the absence of an ionophore, fluorescence intensity decreases linearly with time, indicating a slow permeation of Cu2+ ions into the vesicles (Figure 5B, orange trace). 4-Br-A23187 accelerates the influx of Cu2+ ions into vesicles, as indicated by rapid quenching of the calcein fluorescence (Figure 5B, blue curve). The addition of Triton X-100 disrupts the liposomes and thus serves as a control for ~100% quenching (Figure 5B, grey trace; Figure 5B, blue curve). Figure 5. Copper transport by the ionophore 4-Br-A23187. (A) Schematic of the assay based on calcein-loaded LUVs to measure the transport of divalent cations. Transport of divalent cations into the LUVs leads to time-dependent quenching of calcein fluorescence inside the liposomes. (B) Calcein-loaded LUVs were treated with 3 µM CuCl2 alone (orange trace), or in the presence of 3 µM 4-Br-A23187 (blue trace), or 0.1% Triton X-100 (grey trace). Higher ion permeability in the presence of the ionophore leads to faster calcein fluorescence quenching rates than in the absence of the ionophore (orange trace compared to blue trace). Disruption of the liposomes by Triton X-100 makes all ions accessible to calcein, resulting in ~100% quenching (grey trace). Fluorescence intensity was normalized to the fluorescence before copper addition. Notes All figures were prepared using Biorender.com. Recipes Buffer A (200 mL) Mix 4 mL of 0.5 M HEPES pH 7.4 (final 10 mM) with 60 mL of 0.5 M KCl (final 150 mM). Add 100 mL of deionized water and adjust pH with KOH to 7.4. Top up to a final volume of 200 mL with deionized water, and filter-sterilize with a 0.22 µm filter (Millipore). Calcein stock in buffer A (16.6 mM, 1 mL) Dissolve 10.334 mg calcein to a final volume of 1 mL in buffer A by vortexing. Cover the tube with aluminum foil to protect it from daylight. CuCl2 stock (2 mM) Dissolve 110.98 mg CuCl2 to a final volume of 1 mL in deionized water, to obtain1 M CuCl2. Then, add 2 µL of 1 M CuCl2 into 998 µL of deionized water. G50-Gel in buffer A Add 2.5 g Sephadex G-50 to 50 mL of buffer A, and dissolve it by mixing. The gel has to swell at RT ON, and is then stored at 4 °C. HEPES, pH 7.4 (0.5 M, 200 mL) Dissolve 9.532 g HEPES to a final volume of 170 mL in deionized water. Adjust the pH with 1 M KOH to 7.4, and top it up to 200 mL with deionized water. Ionophore stock (6 mM) Prepare an initial stock of 10 mg 4-Br-A23187 in 1 mL of DMSO. To reach 6 mM 1-Br-A23187, 36.15 µL of the stock solution is diluted with 63.85 µL of filter-sterilized DMSO, resulting in a final volume of 100 µL. Before the experiments, the DMSO is filter-sterilized using a 0.22-μm syringe filter system. KCl (0.5 M, 200 mL) Dissolve 7.455 g KCl to a final volume of 200 mL in deionized water. KOH (1 M) Dissolve 5.611 g of KOH to a final volume of 100 mL in deionized water. Lipid stock in chloroform Lipids are ordered in chloroform at a concentration of 25 mg mL-1, and stored at -20 °C until further use. For longer storage, evaporate the chloroform and store the dried lipid at -20 °C. Before using it, dissolve the 25 mg lipid in 1 mL of chloroform:methanol solution (v:v). Note: Some lipids may have limited or very poor solubility in chloroform:methanol, and require a mixture of chloroform:methanol:water. Loading buffer (1 mL) Take 18.07 µL of 16.6-mM calcein stock (final 300 µM) and top up with buffer A to a final volume of 1 mL. Cover the tube with aluminum foil. Triton 100-X solution (20% w/v) Dissolve 200 mg in 1 mL of deionized water. Acknowledgments This protocol was adapted from our previous work (Senges et al. 2022). JEB gratefully acknowledges funding from the German Research Foundation (BA 4193/6-1, RTG 2341). HDU is a scholar of the Friedrich Ebert Foundation. Competing interests The authors declare that no competing interests exist. References Bae, W., Yoon, T. Y. and Jeong, C. (2021). Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field. PLoS One 16(2): e0247326. Billesbølle, C. B., Azumaya, C. M., Kretsch, R. C., Powers, A. S., Gonen, S., Schneider, S., Arvedson, T., Dror, R. O., Cheng, Y. and Manglik, A. (2020). Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms. Nature 586(7831): 807-811. Černiauskas, V., Gruodis, A., Rodaitė-Riševičienė, R., Saulis, G. (2017). Mechanism of Effective Quenching of Calcein Fluorescence by Iron. Ab initio study. In:Innovative Infotechnologies for Science, Business and Education. 2(23): 10-16. Dutta, S., Watson, B. G., Mattoo, S. and Rochet, J. C. (2020). Calcein Release Assay to Measure Membrane Permeabilization by Recombinant Alpha-Synuclein. Bio-protocol 10(14): e3690. Erdahl, W. L., Chapman, C. J., Taylor, R. W. and Pfeiffer, D. R. (1994). Ca2+ transport properties of ionophores A23187, ionomycin, and 4-BrA23187 in a well defined model system. Biophys J 66(5): 1678-1693. Erdahl, W. L., Chapman, C. J., Taylor, R. W. and Pfeiffer, D. R. (1995). Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophys J 69(6): 2350-2363. Erdahl, W. L., Chapman, C. J., Wang, E., Taylor, R. W. and Pfeiffer, D. R. (1996). Ionophore 4-BrA23187 transports Zn2+ and Mn2+ with high selectivity over Ca2+. Biochemistry 35(43): 13817-13825. Hamilton, R. T. and Kaler, E. W. (1987). Ionic permeability of synthetic vesicles.J Colloid Interface Sci 116(1): 248-255. Hamilton, R. T. and Kaler, E. W. (1990). Alkali metal ion transport through thin bilayers. J Phys Chem 94 (6): 2560-2566. Hope, M. J., Bally, M. B., Mayer, L. D. and Cullis, P. R. (1986). Generation of multilamellar and unilamellar phospholipid vesicles. Chem Phys Lipids (40): 89-107. Kunding, A. H., Mortensen, M. W., Christensen, S. M. and Stamou, D. (2008). A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. Biophys J 95(3): 1176-1188. Maherani, B., Arab-Tehrany, E., Kheirolomoom, A., Geny, D. and Linder, M. (2013). Calcein release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids. Biochimie 95(11): 2018-2033. Mayer, L. D., Hope, M. J. and Cullis, P. R. (1986). Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858(1): 161-168. Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J. and Papahadjopoulos, D. (1979). Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta 557(1): 9-23. Picard, V., Govoni, G., Jabado, N. and Gros, P. (2000). Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool. J Biol Chem 275(46): 35738-35745. Senges, C. H. R., Warmuth, H. L., Vazquez-Hernandez, M., Uzun, H. D., Sagurna, L., Dietze, P., Schmidt, C., Mucher, B., Herlitze, S., Kramer, U., et al. (2022). Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore. Proteomics 22(17): e2200061. Su, Z. F., Ran, X. Q., Leitch, J. J., Schwan, A. L., Faragher, R. and Lipkowski, J. (2019). How Valinomycin Ionophores Enter and Transport K+ across Model Lipid Bilayer Membranes. Langmuir 35 (51): 16935-16943. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Drug Discovery > Drug Screening Biochemistry > Other compound > Ion Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols K+ Release Assay and K+ Measurement in Oocyte Assay Hong Li Nov 5, 2020 2641 Views Detection and Quantification of Calcium Ions in the Endoplasmic Reticulum and Cytoplasm of Cultured Cells Using Fluorescent Reporter Proteins and ImageJ Software Shunsuke Saito and Kazutoshi Mori Aug 20, 2023 1338 Views A Protocol for Custom Biomineralization of Enzymes in Metal–Organic Frameworks (MOFs) Zoe Armstrong [...] Zhongyu Yang Feb 5, 2024 708 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,553	https://bio-protocol.org/en/bpdetail?id=4553&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed In vitro Preparation of Homogenous Actin Filaments for Dynamic and Electrophoretic Light Scattering Measurements EA Ernesto Alva * AG Annitta George * LB Lorenzo Brancaleon MM Marcelo Marucho (contributed equally to this work) Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4553 Views: 686 Reviewed by: Giusy Tornillo Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Polymers (Basel) Jun 2020 Abstract Actin filaments are essential for various biological activities in eukaryotic cellular processes. Available in vitro experimental data on these systems often lack details and information on sample preparation protocols and experimental techniques, leading to unreproducible results. Additionally, different experimental techniques and polymerization buffers provide different, sometimes contradictory results on the properties of these systems, making it substantially difficult to gather meaningful data and conclusive information from them. This article presents a robust, accurate, detailed polymerization protocol to prepare high-quality actin filament samples for light scattering experiments. It has been shown to provide unicity and consistency in preparing stable, dispersed, aggregates-free, homogenous actin filament samples that could benefit many other scientific research groups currently working in the field. To develop the protocol, we used conventional actin buffers in physiological conditions. However, it can easily be adapted to prepare samples using other buffers and biological fluids. This protocol yielded reproducible results on essential actin filament parameters such as the translational diffusion coefficient and electrophoretic mobility. Overall, suitable modifications of the proposed experimental method could generate accurate, reproducible light scattering results on other highly charged anionic filaments commonly found in biological cells (e.g., microtubules, DNAs, RNAs, or filamentous viruses). Graphical abstract: Keywords: Actin filaments Dynamic light scattering Electrophoresis Translational diffusion coefficient Auto-correlation Background Actin filaments (F-actin) are highly charged double-stranded semiflexible polyelectrolytes formed by the polymerization of G-actin subunits. Due to their hydrodynamic, mechanical, and electrostatic properties, these cytoskeleton filaments have the extraordinary ability to dynamically change conformations in response to alterations in G-actin concentration and type of crosslinker/binding proteins, as well as electrolyte concentration. These cytoskeleton properties are crucial for eukaryotic cells to achieve specific biological functions in different cellular compartments, which may vary depending on the cell type and location conditions. While a substantial amount of research has been done in the biochemistry and biophysics fields (Lanni and Ware, 1984; Janmey et al., 1986, 1994; Hou et al., 1990; Käs et al., 1996; Xu et al., 1998; Bonet et al., 2000; Niranjan et al., 2001; Y. H. Wang and Narayan, 2008; Crevenna et al., 2013; Del Rocio Cantero et al., 2020), the underlying principles and molecular mechanisms that support the hydrodynamic, electrostatic, and stability properties of these filaments remain elusive (Lanni and Ware, 1984; F. Wang et al., 1989; Hou et al., 1990; Käs et al., 1996; Bonet et al., 2000). Modern dynamic light scattering (DLS) and electrophoretic light scattering (ELS) instruments are the most accurate, non-invasive, experimental techniques to characterize hydrodynamic and electrostatic properties of polydisperse charged biomolecules even at low concentrations and using small sample volumes. These instruments use advanced technology and multi-functional software to measure the hydrodynamic size, polydispersity, dispersion stability and aggregation, biomolecular charge, and zeta potential of particles and polymers immersed in aqueous biological environments. To assure high accuracy and reproducibility of the results, it is essential to use high-quality samples, accurate instruments, robust software, and optimized measurement protocols. This article presents a complete, accurate, detailed polymerization protocol to prepare high-quality actin filament samples for light scattering experiments. Obtaining actin filament samples that are stable, dispersed, clean from aggregates and impurities, and homogenous could benefit many other scientific research groups in the field. We describe the polymerization procedure for three typical actin buffers in physiological conditions (Janmey et al., 1986, 1994; F. Wang et al., 1989). Nevertheless, this protocol can easily be adapted to prepare samples using other buffers and biological fluids. Additionally, we provide a unique methodology based on fundamental biostatistical tools (McDonald, 2009) and a measurement protocol with optimized configuration to perform DLS and ELS experiments on actin filament samples at critical G-actin concentrations. We measured actin filament’s translational diffusion coefficient and electrophoretic mobility using the same sample and experimental conditions for both DLS and ELS experiments to assure unicity and consistency in the results (Alva et al., 2022). A fitting approach was adequate to characterize other filaments’ essential properties, such as asymmetric length distribution, effective electric charge, hydrodynamic size, effective diameter, dispersion stability, aggregation, and semi-flexibility (Kroy and Frey, 1997; Tassieri et al., 2008; Alva et al., 2022). Additionally, the comparison of the light scattering results obtained for different polymerization buffers elucidated the impact of their chemical composition, reducing agents, pH values, and ionic strengths on the hydrodynamic, electrostatic, and stability properties of these actin structures. This kind of comparison and characterization provides a deeper understanding of how actin filaments behave in various cellular environments and conditions. Overall, suitable modifications of the proposed experimental method might allow other scientific groups to obtain accurate and reproducible light scattering results on other highly charged anionic polyelectrolyte filaments, commonly found in biological cells (e.g., microtubules, DNA, RNA, or filamentous viruses), having hydrodynamic, electrostatic, and stability properties resembling those characterizing actin filaments (Janmey et al., 2014). Materials and Reagents 2 mL glass vial (Agilent, catalog number: 20097845) Polypropylene centrifuge tube, 15 and 50 mL (Corning, catalog number: 430791) Polypropylene microfuge tube, 11 × 38 mm, 1.5 mL (Beckman Coulter, catalog number: 9080511) 12 mm square polystyrene cell cuvettes (Malvern Panalytical, number: DTS0012) 2 mL cryogenic vials (Corning, catalog number: 30721070) Business source stainless steel scissors (Fiskars, catalog number: 01-004250J) Optifit pre-sterilized tips, 10–1,000 μL (Sartorius, catalog number: PR151159) Optifit pre-sterilized tips, 5–350 μL (Sartorius, catalog number: PR149531) Quartz spectrophotometer cell rectangular, stopper, 1 mm (Starna Cells, catalog number: 21Q1) Actin protein (>99% pure): rabbit skeletal muscle; store at 4 °C (Cytoskeleton, catalog number: AKL99) Water for molecular biology (Millipore, catalog number: H20MB1001) Calcium chloride, dried, powder, 97% (CaCl2) (Alfa Aesar, catalog number: 10219230) Magnesium chloride, anhydrous, 99% (MgCl2) (Alfa Aesar, catalog number: W07D102) Potassium chloride (KCl) (VWR, BDH Chemicals, catalog number: 18J2256059) Tris base (Fisher Bioreagents, catalog number: BP152-500) Beta-mercaptoethanol, molecular biology grade (BME) (Millipore, catalog number: 3192024) Adenosine triphosphate (ATP), 100 mM stock (Cytoskeleton, catalog number: BSA04) DL-Dithiothreitol (DTT), molecular grade (Promega Corporation, catalog number: 0000383224) Sodium hydroxide, pellets, 97%, A.C.S. reagent (NaOH) (Sigma-Aldrich, catalog number: 1310-73-2) HCl volumetric standard, 0.1 N solution in water (Sigma-Aldrich, catalog number: 7647-01-0) Precision red protein assay reagent (Cytoskeleton, catalog number: ADV02) 5 3/4” pipets (Fisherbrand, Fisher Scientific, catalog number: 13-678-20A) 70% v/v denatured ethanol solution (Fisher Bioreagents, catalog number: 216731) Orion buffers pH 4.01, pH 7.00, and pH 10.00 (Thermo Scientific, catalog numbers: YX1, YW1, YX1) Universal dip cell kit (Malvern Panalytical, catalog number: ZEN1002) 50 mM CaCl2 (see Recipes) 50 mM MgCl2 (see Recipes) 1.0 M KCl (see Recipes) 102.24 mM KCl (see Recipes) G-actin buffer 1 (pH 7.80) (see Recipes) G-actin buffer 2 (pH 7.66) (see Recipes) G-actin buffer 3 (pH 8.23) (see Recipes) Polymerization buffer 1 (pH 7.56) (see Recipes) Polymerization buffer 2 (pH 7.64) (see Recipes) Polymerization buffer 3 (pH 8.07) (see Recipes) Electrolyte buffer 1 (pH 7.72), buffer 2 (pH 7.66), and buffer 3 (pH 8.06) (see Recipes) Equipment Analytical Explorer Pro-Scale (Ohaus Industrial Scales, model: PX84) Pipette+ (Sartorius, Andrew Alliance Stand+) Smart electronic pipettes: 5–350 μL, 10–1,000 μL, 5–10 mL (Sartorius, Andrew Alliance Stand+) Vortex mixer with standard tube head, 120V (Corning LSE, The Lab Depot, Ref:6775) Magnetic stirrer RT Basic-12 (Thermo Scientific, catalog number: 88880007) Orion Star A211 pH meter, accuracy: ± 0.002 (Thermo Scientific, catalog number: X56954) Allegra 64R benchtop centrifuge machine (Beckman Coulter, product number: 367585) with a F1202 Rotor, 30,000 rpm (Beckman Coulter, catalog number: 19U3138) Zetasizer ULTRA, accuracy MW: ± 10% typical, temperature accuracy: 0.1 °C (Malvern Panalytical, model: ZSU5700) Cary 100 UV-Vis spectrophotometer, accuracy: ~2% (Agilent Technologies, product number: 10069000) Software ZS Xplorer software (Malvern Panalytical, https://www.malvernpanalytical.com/en/products/product-range/zetasizer-range/zetasizer-advance-range/zetasizer-ultra) Microsoft Word 2021 (Microsoft, https://www.microsoft.com/) Cary WinUV Kinetics (Agilent Technologies, https://www.bioprocessonline.com/doc/cary-winuv-software-0001) Cary WinUV Scan (Agilent Technologies, https://www.bioprocessonline.com/doc/cary-winuv-software-0001) Procedure ATP reconstitution Briefly centrifuge to collect the white powder at the bottom of the storage tube. Add 1 mL of cold 100 mM Tris base at pH 7.5 to reconstitute the ATP at 100 mM. Aliquot the ATP into experiment-sized amounts as needed. Snap frozen the ATP using liquid nitrogen. Store at or below -20 °C. Notes: The lyophilized ATP (desiccated to <10% humidity) is stable for six months at 4 °C. The ATP is stable for six months if stored at or below -20 °C. Figure 1. Experimental instrumentation and timeline. (A) Daily timeline. (B) Workplace area and instrumentation. (C) Electronic Pipette+ OneLab, which reduces errors and assures consistency across many tests. (D) The protein solution is transferred to the cell cuvette and placed into the Zetasizer ULTRA holder to begin the diffusion coefficient measurements. (E) Subsequently, the dip cell (ZEN1002) is introduced into the Zetasizer ULTRA holder to perform ELS experiments and obtain the electrophoretic mobility, as well as the phase and frequency shift data plots. Protein reconstitution Notes: Muscle actin is a liable protein; it should be handled with care. To avoid repeated thawing cycles, consider specific experimental sized amounts. Some of the steps considered in this section are recommended by the producer (Cytoskeleton, Inc). In addition to those, we considered the addition of mixing steps (B3 and B5), due to the rapid agglomeration/aggregation of the actin as the buffer is continuously added. Thus, we aim to achieve an aggregate-free solution by considering the following: as actin should be handled with care, the vortex speed is set at low to medium speed (4/10) for a short amount of time. Pipetting should also be carefully handled to avoid high mechanical stress in the protein solution. This step is performed on Day 3 (Figure 1). The aliquots containing the protein and G-actin buffers are stable for six months at -80 °C. Transfer the protein powder into a 3 mL glass vial. Add 100 μL of ultra-pure water into the glass vial containing the protein powder to reconstitute at 10 mg/mL G-actin density. Vortex the solution for 30–40 s at low to medium speed (4/10) to dissolve the protein powder as much as possible. Add 2.4 mL of G-actin buffer (buffers 1, 2, or 3—Recipes section B) to the glass vial containing the protein solution. Vortex one more time for 35–45 s at low to medium speed (4/10) to dissolve the aggregates as much as possible. If aggregates are still present, pipette and mix the solution to achieve a homogeneous protein solution. Aliquot the solution into experimental sized amounts according to the number of experiments needed. Transfer the small-size solutions into cryotube vials. We recommend aliquoting the protein solutions into multiples of 250 μL (1×250 μL, 2×250 μL, 3×250 μL, …). Once the wanted protein solutions are aliquoted, snap-freeze the cryotube vials with liquid nitrogen and immediately store at -80 °C. Actin polymerization Notes: ATP and DTT are initially stored at -20 °C. When ready to prepare the solutions, thaw both solutions. Immediately after use, snap-freeze them and store them back in the freezer. Following the manufacturer’s recommendation, after the 2 h centrifuge process, remove the top 90% (198 μL) of the supernatant from each microcentrifuge tube by following step C11. The amount of translucent pellet left in the microcentrifuge will be 22 μL. This step is performed on Day 4 (Figure 1). A timeline for the actin polymerization steps can be seen in Figure 2. Extract one of the cryotube vials containing 1×250 μL of protein solution from the -80 °C freezer. Wait 5 min for de-frosting at room temperature. Incubate the vial on ice for 1 h to depolymerize actin oligomers that form during storage. Extract 200 μL from the cryotube vial and transfer to a 1.5 mL polypropylene microcentrifuge tube. Add 20 μL of polymerization buffer (1/10th volume) (buffers 1, 2, or 3—Recipes section C) to the microcentrifuge tube containing the 200 μL protein solution to start polymerization. Vortex the solution for 40–50 s at low speed (3–4/10). Incubate the protein solution at room temperature for 1 h. Turn on the centrifuge 10–15 min before the previous step is finalized. Set up the centrifuge at 50,000 × g for 2 h at 4 °C. Note: The acceleration and deceleration should both be set at very low (2/10) speed. Centrifuge the protein solutions for 2 h. Carefully remove the tubes containing the actin protein solutions from the centrifuge and place them on ice. Set the 5–350 μL Pipette+ in titration mode at very low speed (1/10) and extract the supernatant from the solution in the following manner, to avoid any possible stress in the solution that could lead to the breakage of the actin filaments: 100 μL, 50 μL, and 48 μL. Using the same Pipette+ specifications as in the previous step, add 978 μL of electrolyte buffer (buffers 1, 2, or 3—Recipes section D) to the actin protein pellet (22 μL). Store the solution at 4 °C and leave it overnight. Figure 2. Activation, nucleation, elongation, and annealing of actin filaments. (A) Hourly polymerization timeline of F-actin, starting with the incubation of actin protein at room temperature and the addition of the polymerization buffer. After one hour, the centrifugation process begins to separate the supernatant from the pellet (F-actins), and finally, the electrolyte buffer is added. (B) G-actin monomers undergo a polymerization process that starts with the activation. In this step, Mg2+, K+, and Ca2+ bind to G-actin monomers reducing its electrostatic repulsion between monomers and inducing a structural change. (C) Subsequently, the nucleation begins, where G-actin monomers form stable nucleus due to the presence of ATP- and ADP-actin, supporting the addition of more monomers. (D) During the elongation stage, monomers are rapidly added to the nucleus through both ends of the filament (pointed and barbed end). (E) Finally, in the annealing step there is an association and dissociation of G-actin monomers at both ends of the filament. UV-Vis spectrophotometer absorbance Notes: Turn on the UV-Vis spectrophotometer 10–15 min before use. Determine the protein concentration in the supernatant with the precision red protein assay reagent. The goal is to obtain the protein concentration in the pellet and after adding the electrolyte buffers. For more information, see the Data analysis > Protein Concentration Analysis. Pipette 1 mL of precision red protein assay reagent into a 1.5 mL disposable microfuge tube. Add 10 μL of the supernatant protein solution obtained in step C11 and mix by inverting. Incubate at room temperature for 1 min. Transfer the solution into the quartz cuvette. Set the Scan Controls at 0.100 s for Ave Time, 0.12 nm for Data Interval, and 100 nm/min for Scan Rate. Blank the spectrophotometer on precision red protein assay reagent at 580–620 nm and read the absorbance of the protein sample at 600 nm. Dynamic light scattering A measurement consists of several averaged normalized intensity autocorrelation decays, called runs, each lasting 10 s. The number of runs can be determined automatically by the software, based on the scattered light intensity, which depends on the concentration and size of the macromolecule. Alternatively, it can be entered manually by the user. For F-actins, the number of runs is typically five (see more details under Data analysis). Instrument start-up Turn on the computer. Turn on the Zetasizer ULTRA by pressing the power switch located on the back of the instrument. The status light on the Zetasizer ULTRA will initially be red. Open the ZS Xplorer software. The status light on the Zetasizer ULTRA will become green shortly after. Wait 10–15 min for the instrument to stabilize. Standard operating procedure (SOP) Select Analyze > select Explorer > select Project Explorer ‘+’ sign > type the name of the new project. Select Measure (see Figure 3) Name: Type a sample name for this measurement. Cell: Select DTS0012 Material: Select Protein Dispersant: Select Water Project: Select the project created in step 2a. Measurement type: Select Size and 5 runs Measurement Temperature: Select 25 °C Return to default temperature: Select Yes Equilibration time (s): Type 180 Data Processing Analysis Model: Select General Purpose Advanced Settings Angle of Detection: Select Back scatter Positioning Method: Automatically selected as Measure at a fixed position Cell Position: Automatically selected as 5.50 Attenuation: Select Automatic Measurement Process: Select Automatic Use pause after sub runs: Select No Optical Filter: Select No filter Pause between repeats (s): Type 0 Sample preparation for measuring translation diffusion coefficient and correlation functions: Remove the 1 mL aliquots of polymerized F-actins (buffers 1, 2, or 3—Recipes section B) from the 4 °C fridge and bring to room temperature for 10–15 min. In the meantime, clean and sterilize the scissors with water and ethanol. Cut Optifit tips up to a diameter of 3–5 mm from the tip. This action will greatly reduce the breakage of filaments when transferring the aliquot into a cell cuvette. The cell cuvette must be clean (air pressure) and free of scratches since this could lead to erroneous measurements. Set the Pipette+ (5–350 μL) in titration mode at low speed (1–2/10) to extract the protein solution into the cell cuvette. Titrate the protein at a 45° angle mode. The cell should be filled slowly at a 45° angle to avoid creating air bubbles. Do not fully cap the cell. Fully cap only one side of the cell. This action will also reduce the presence of air bubbles during measurements. Open the cell area lid by pressing the button in front of the lid. Push the cell cuvette into the cell holder until it stops. The small triangle at the top of the cell indicates the front of the cell, which should be facing towards the user. Close the cell area lid. During the measurements (every two sets of five runs), open the lid, remove the cell cuvette from the cell holder, and mix the protein solution 2–3 times using a cut tip of 3–5 mm in diameter. Immediately after, place the cell cuvette back in the cell holder and proceed to the next set of experiments. This action will minimize sedimentation. Figure 3. Zetasizer ULTRA. (A) The DLS and ELS Zetasizer ULTRA instrument uses a 633 nm He-Ne laser. It houses a single cuvette and measures light scattered at 173°, 90°, and 17°. It is interfaced to a computer running Windows 10 and controlled with the Zetasizer software. (B) SOP window for ELS measurements. The settings shown here are also presented under the ELS SOP (step I1). (C) SOP window for DLS measurements. The settings shown here are also presented under DLS SOP (step H2). DLS measurements When the project and specifications are set and the sample is loaded, begin the measurements. Click the Play bottom icon located by properties selection, and the initial green light of the Zetasizer ULTRA will turn blue. During DLS measurements, select Multi-view to display the following three windows: 1) “g2(t)-1” versus decay time “t,” which is updated as the run continues (see Figure 5); 2) the intensity fluctuations described in Background; and 3) the intensity distribution. As a series of runs accumulates into a measurement, select the Analyze tab and locate the Project Name to get all the data generated by this measurement. After each run is over, the distribution function is calculated, and many data options are available such as “Intensity vs. Size,” “Intensity vs. Volume,” and “Correlogram.” This process is repeated five times, obtaining five similar correlation functions. After the measurement is complete, the blue light will return to green, meaning that the Zetasizer ULTRA is ready for the next set of measurements. Electrophoresis light scattering A measurement consists of several averaged fast field reversal phase shift, called runs, each lasting 1.4 s. The number of runs can be determined automatically by the software, based on the scattered light intensity, which depends on the concentration and size of the macromolecule. Alternatively, it can be entered manually by the user. For F-actins, the number of runs is typically three (see more details under Data analysis). Standard operating procedure (SOP) Select Analyze > select Explorer > select Project Explorer ‘+’ sign > type the name of the new project. Select Measure (see Figure 3) Name: Type a sample name for this measurement. Cell: Select ZEN1002 Material: Select Protein Dispersant: Select Water Project: Select the project created in step 1a. Measurement type: Select Zeta and 3 runs Measurement Temperature: Select 25 °C Return to default temperature: Select Yes Equilibration time (s): Type 180 Data Processing Analysis Model: Select Auto mode Advanced Settings Attenuation: Select Automatic Measurement Process: Select Automatic with minimum runs set at 10 and maximum runs set at 30 Use pause after sub runs: Select Yes and a pause duration (s) set at 10 Voltage selection: Select Automatic Pause between repeats (s): Type 60 Sample preparation for measuring electrophoretic mobility, frequency, and phase shifts: Once the DLS measurements are over, remove the protein solution from the cell holder and proceed to insert the dip cell (ZEN1002). Before inserting the dip cell, its electrodes must be cleaned with ethanol and water. After every measurement, the electrodes must be cleaned to reduce contamination across samples. The dip cell must be placed into the sample cuvette. Place the ZEN1002 cell into the cell cuvette containing the protein solution. Ensure that the small triangle at the top of the cell still faces the front of the instrument, as indicated in section I. Make sure that the sample does not overflow the cuvette when the ZEN1002 is fully inserted. Holding the base of the ZEN1002 cell cap and the top of the cuvette simultaneously, push the cell into the cell holder until it stops. Open the cell area lid by pressing the bottom in front of the lid. Simultaneously holding the base of the ZEN1002 cell cap and the top of the cuvette, push the cell into the cell holder until it stops. Close the cell area lid. During the ELS measurements, check the dip cell electrodes and sample for any potential contamination. Failing to clean the electrodes after each run could lead to cross-contamination between samples. ELS measurements When the project and specifications are set and the sample is loaded, begin the measurements. Click the Play bottom icon located by properties selection, and the initial green light of the Zetasizer ULTRA will turn blue. During ELS measurements, select Multi-view to display the following three windows: 1) “PALS: phase(rad) vs t”, which is updated as the run continues (see Figure 6); 2) the intensity fluctuations described in Background; and 3) the frequency shift. As a series of runs accumulates into a measurement, select the Analyze tab and locate the ‘Project Name’ to get all the data generated by this measurement. After each run is over, the fast field reversal (FFR) of the phase analysis will be available along with other data options such as the frequency shift. After the measurement is complete, the blue light will return to green meaning that the Zetasizer ULTRA is ready for the next set of measurements. Shutdown Exit ZS Xplorer software (File > Exit). Turn off the Zetasizer equipment. Shut down computer. Data analysis Detailed information on data analyses already appears in the “Materials and Methods” section of the original research article (Alva et al., 2022). DLS analysis Note: The Backscatter angle technique significantly reduces the presence of dust in the signal-to-noise ratio correlation plots. However, a wavering behavior can appear in the correlogram’s baseline due to multiple exponential decays and an increase in the number of fluctuations. In that case, it may lead to aggregation and sedimentation of the sample. Thus, we recommend following Note 12. Once the set of measurements is completed, select the Analyze tab and select the appropriate project name where all the measurements have been saved. Select the Size tab. Under the data options, select the multiple parameters available for each run. For instance, select Size > Z-average, PDI, translational diffusion coefficient, derived mean count rate, or any other size parameter according to the user’s needs (see Figure 4). Figure 4. Table properties. (A) Full display of available parameters and properties that can be selected according to the user’s needs for DLS and ELS. (B) Selected and recommended parameters for DLS measurements. (C). Selected and recommended parameters for ELS measurements. Once the chosen parameters are displayed, select the correlogram as one of the data plots to be displayed for each run. The correlogram is an important data function for analyzing and determining the quality of the sample and measurements. The correlograms must follow an experimental criterium to minimize errors and assure reproducibility. The correlogram plot’s intercepts must be under 1 and within a range in the low polydispersity index (0.4 < PDI < 0.8), indicating a good quality in the polydispersity samples. The resulting derived count rate should be higher than 100 kpcs, the minimum value required to obtain suitable measurements. The correlogram plot should also present a smooth and uniform single exponential decay. However, the correlogram data plots may show multiple exponential decays within a single run due to the sample’s aggregates, sedimentation, and dust. In that case, the data is disregarded. This is also considered under the experimental criterium to reduce error and increase reproducibility (see Figure 5). Figure 5. Correlation coefficient. Illustrative example of the correlation data function [g2-1 vs. time (μs)] plots obtained from size measurements. (A) Data measured from five consecutive, independent experiments. (B) The three correlation data functions selected for data analysis after two measurements (2/5) are disregarded according to the experimental criterium to increase accuracy. ELS analysis Once the set of measurements is completed, select the Analyze tab and the appropriate project name where all the measurements have been saved. Select the Zeta tab. Under the data options, select the multiple parameters available for each run. For instance, select Zeta > zeta potential, electrophoretic mobility, quality factor, conductivity, or any other electrophoretic parameter according to the user’s needs (Figure 4). Once the chosen parameters are displayed, select the phase plot and frequency shift data plots to be displayed for each run. The fast field reversal (FFR) phase plot is an important data function to determine the quality of the sample and measurements. Three independent experiments were tested for each actin filament sample to reduce statistical errors in electrophoretic mobility values. The quality factor is a parameter that derives from the phase analysis plot during the FFR stage of the measurement; it must be higher than 1 to be considered as good quality data. The frequency shift plots should have very low traces of noise since this is another quality data check-up. Also, the phase FFR plot should have a sinusoidal behavior, which also implies a good quality data (see Figure 6). Figure 6. Phase and frequency shifts. Illustrative example of phase and frequency shift plots obtained from electrophoretic measurements. Three independent experiments are measured on the phase (A) and frequency shifts (B) to reduce statistical errors in the electrophoretic mobility values. An evidence of good data quality is displayed in the frequency shift plot, since there are no traces of noise and the curves match very well. Protein concentration analysis Note: We performed the calculation on the pellet concentration to confirm that the samples used in our experiments were in the dilute regime, where the light scattering measurements correspond to single actin filament properties. This condition is achieved when the pellet concentration value “c” = 1.37 μM is lower than 1/<L>3 NA, where <L> and NA are the average filament length and Avogadro’s number, respectively. Using a fitting approach (Alva et al., 2022, Table 8), we obtained <L> ~0.5 μm and 1/<L>3 NA ~13.3 μM. Thus, our experiments were performed in the dilute regime. Calculation of initial concentration (Co) When reconstituting the actin protein by adding 100 μL of ultra-pure water, the formulation is the following: C(concentration)= (Mass of Protein Powder (mg))/Volume=10 mg/mL Adding 2.4 mL of G-actin buffer will change the concentration of protein to 0.4 mg/mL: C=(1 mg)/(0.1 ml+2.4 ml)=0.4 mg/mL From each tube, we extract 200 μL and add 20 μL of polymerization buffer. Thus, the resulting initial concentration of protein for experimental measurements is the following: Co=(0.08 mg)/(0.2 mL+0.02 mL)=0.3636 mg/mL Calculation of mass supernatant concentration (MSN) (Figure 7) Note: In this stage, the spectrophotometric absorbance range is 0.009–0.012. This range comes from the multiple independent measurements performed for all buffers. If the absorbance value is outside this range, it is possible that a percentage of G-actins have not polymerized into filaments. In other words, the centrifugation was incomplete and/or some excess protein concentration is found in the supernatant. Figure 7. Absorbance. Illustrative example of an absorbance data plot obtained from spectrophotometric measurements. The absorbance curves for the total initial protein concentration (black line) and three independent experiments for the supernatant were measured at 600 nm. The average absorbance value is used to determine the mass of the supernatant in step C2 of Data analysis. The average of three independent absorbance values obtained for the supernatant at 600 nm is 0.01055. Calculate the supernatant concentration based on 1.00 OD600 nm = 100 μg protein per mL reagent per cm and multiply this protein concentration by 10 to achieve the protein concentration in μg/mL of the original protein solution. The supernatant concentration is 0.1055 mg/mL in 198 μL. Thus, the following equation is applied to solve for MSN: MSN=Supernatant Concentration Supernatant Volume=0.0209 mg Calculation of pellet concentration (F-actin) (CP) First, the difference between the initial mass concentration (0.08 mg) and the mass of the supernatant concentration (0.0209 mg) can be used to obtain the mass of the pellet, MP, as follows: MP=Mass Initial Concentration-Mass Supernatant Concentration=0.0591 mg Furthermore, once the mass of the pellet (0.0591 mg) is known, the pellet concentration is easily predicted in 22 μL (volume pellet): CP=(Mass Pellet)/(Volume Pellet)=2.687 mg/mL Last, to find the molarity (M) of the pellet, we use the following equation where the pellet concentration (mg/mL) is divided by the molecular weight of actin (43 KDa): Pellet Molarity (MP)= (Pellet Concentration(mg/mL))/(Actin Molecular Weight (Da))=1.37 μM Sample quality control Note: The following steps/comments are intended for new experimenters to correlate the quality of their samples to light scattering measurement data plots. The correlation function is an excellent indicative plot to ensure the system is free from aggregation/sedimentation. As indicated in the DLS Analysis, if multiple exponential decays and oscillated baselines are observed, it will most likely represent the sample’s aggregation and/or sedimentation. A good practice to ensure homogeneity in the sample is to mix and vortex the solution prior to any DLS and ELS measurement to avoid the tendency to sedimentation and ensure good quality data. Knowing the protein concentrations in the dilute regime is highly recommended to ensure dispersion. If increased protein concentration is achieved, longer filaments and higher-order structures of actin may be formed, leading to different results in the DLS measurements from those presented in our article (Alva et al., 2022). From the ELS experiments point of view, high zeta potential values are correlated with high repulsive forces between polymers, leading to a disperse system. If increased polyelectrolyte concentration is achieved, zeta potential values may decrease, and the dispersion may be weakened. Notes It is important to have good laboratory practices that include working in very clean conditions. To minimize any source of contamination, tightly close tubes, vials, and samples. Make sure the cuvettes and vials are clean and dust-free to prevent the presence of contaminants (see Figure 1). The usage of a 15 mL conical tube was vital since it facilitated measuring and adjusting the pH of the buffers (1–3). If the tube is smaller, the pH meter probe will not be able to fit; also, there will not be enough solution to cover the tip of the probe leading to inaccurate pH readings. When adjusting the pH (G-actin and polymerization buffers) use glass pipets to carefully add the base and acid solutions drop by drop into the buffers. The addition of too many drops may increase or decrease the pH significantly, so use caution. It is important to mix well by vortexing the solutions after adding these drops; failing to do so will lead to inaccurate pH readings. To begin the protein reconstitution, the 1 mg G-actin protein powder needs to be extracted. The powder is usually frozen and stuck to the bottom of the vial. We suggest vortexing the protein vial at a low to medium speed (5/10) for 30–45 s to smooth the protein powder and allow easy extraction when the protein is still dry. After adding the G-actin buffer to the 10 mg/mL G-actin density, the protein will agglomerate. Mix the solution well by vortexing and pipetting to lose the agglomerates. These structures are visible, so mix the solution well to create and maintain a homogeneous solution. G-actin proteins may concentrate at the solution’s surface. If so, mix the solution well before aliquoting into experimental samples. When cutting the Optifit tips using sterilized scissors, have a reference mark on the tips to make this process easier and more consistent. The tips need to have a diameter of approximately 3–5 mm to reduce the breakage of the filaments. During the centrifugation step, we used a 50,000 × g spin for 2 h to ensure that short to long filaments pellet. If a faster ultracentrifugation (50,000 × g < speed < 100,000 × g) is available, we recommend a duration of 1.5–2 h. If the ultracentrifugation is able to reach 100,000 × g, we highly recommend a duration of 1 h. We recommend imaging actin filaments by fluorescent labeling of phalloidin and/or similar imaging techniques such as transmission electron microscopy, to ensure the quality of actin final preparation and formation (see Figure 8). Figure 8. Micrograph image of actin filaments. Illustrative micrograph image of actin filaments using the JEOL 1400 transmission electron microscopy. During the TEM sample preparations, the samples underwent a series of steps using 2% uranyl acetate on a formvar-coated grid. The samples were washed 2–3 times with distilled water. WARNING: If the actin filaments or other polymers are exposed to high electrolyte salt concentrations (ionic strength), there is a high possibility that new order structures (bundles and networks) and aggregates are formed in solution. We recommend caution on the type and salt concentrations that are used in solution that could induce bundles for actin filaments (Tang and Janmey, 1996) or other polymers. Examples of different actin preparations: At actin concentrations higher than the critical, the ATP-G-actin increases in solution leading to a significant increase in the elongation rates (Crevenna et al., 2013) and the formation of longer and more semiflexible filaments. As the length of filaments grows, the breakage of filaments may be higher. On the other hand, longer filaments generate smaller diffusion coefficients and higher electrophoretic mobility values. The actin filament elongation kinetics depends on the pH solution. At pH higher than 7, shorter filaments may be formed due to decreased elongation rates (Crevenna et al., 2013). Thus, a higher diffusion coefficient and smaller electrophoretic mobility values would be expected at higher pH level solutions. The polymerization rate depends on the type of divalent cation bound to actin. Thus, if Mg2+ is used as part of the polymerization buffer (instead of Ca2+), it could lead to changes in the mechanical and flexibility properties of actin filaments (Steinmetz et al., 1997). These changes may affect the hydrodynamic and electrostatic properties of actin filaments. Thus, changes in the diffusion and electrophoretic mobility values may be expected when using different metal ions in the polymerization buffers. High concentrations of Ca2+ and Mg2+ in the electrolyte solution may attenuate the repulsive inter-filament interaction and the bundling formation of actin filaments (Castaneda et al., 2018). Thus, the actin preparation under different divalent concentrations may generate actin bundles with different bending, diameter, and length, and consequently, hydrodynamic and electrostatic properties. Capping proteins, namely gelsolin, regulate the assembly and length of actin filaments (Warshavsky et al., 2022). For instance, the lower the ratio between G-actin and gelsolin concentration, the shorter the average filament length, the higher the diffusion coefficient, and the smaller the electrophoretic mobility values. To avoid sedimentation and/or aggregation of protein during light scattering experiments, gently pipette 2–3 times the solution in between runs using the 3–5 mm cut tip. If the sample has a higher protein and/or salt concentration than proposed in this protocol, we highly recommend considering the viscosity parameter by adding this value in the ZS Xplorer software under Measure > Dispersant. The attenuation factor in the DLS instrument uses multiple positions (1–11) to control the beam intensity from 100% to 0.0003%. This feature was set to automatic to allow the ZS Xplorer software to determine the best possible attenuator. For F-actins, the attenuation factor usually varies between 10 and 11. The refractive index and absorption of the material have no bearing on the Z-average, polydispersity, and intensity. We selected protein as the material being measured, consisting of a refractive index of 1.450 and an absorption of 0.001. Similarly, the dispersant’s refractive index was set to 1.33, and viscosity to 0.8872 mPa·s. These values may vary according to different materials and/or electrolytes being measured. The correlation functions were measured at the back-scattering angle (173°), where the incident beam does not have to travel through the entire sample. The effect of multiple scattering and dust is greatly reduced. During DLS experiments, the measurement duration was automatically determined from the detected count rate. The lower the count rate, the longer the measurement duration and the higher the noise. Since F-actins form a polydisperse system of unknown size distribution, the general purpose analysis model using the CONTIN algorithm was selected during the DLS measurements. The ZS Xplorer software measures the sample electrical conductivity of the system when set as automatic. It adjusts the cell voltage to keep a low current flowing, close to 5 ms/cm, in the sample. If this is not considered, the sample temperature may increase near the electrodes, inducing bubble formation and sample degradation, leading to inaccurate results. The fast field reversal (FFR) of the phase analysis light scattering was selected since the mobility measured during this period is due to the electrophoresis of the particles only. It is not affected by electro-osmosis associated with the soft field reversal (SFR). This protocol can also be applied for other manufacturer’s instruments such as Dynamics Mobius from Wyatt Technology and NanoBrook Series from Brookhaven Instruments Corporation. Like ULTRA Zetasizer, these instruments can perform DLS and ELS measurements. The software setup for both instruments is somewhat similar, except the data analysis. We recommend the multimodal analysis for size measurements, specifically for polydisperse systems. The Mobius and NanoBrook Series instruments use different volumes and cuvettes for size and zeta potential measurements. We highly recommend following their websites and manuals for usage of their cuvettes. A time delay between measurements helped to reduce sample heating when long run measurements raised the temperature of the cell to approximately 50 °C, allowing the sample to recover 25 °C between consecutive measurements, reducing critical sample degradation and avoiding increasing mobility with sequential measurements. Standard operating protocol, data analysis (Parker and Lollar, 2021), and analysis of biomolecular preparations to detect aggregation of proteins, glycoproteins, protein-protein complexes, and others, can be found elsewhere (Stetefeld et al., 2016). Recipes Stock buffers 50 mM CaCl2 Reagent Final concentration Amount CaCl2 50 mM 55.50 mg H2O ultra-pure n/a 10 mL Total 50 mM 10 mL 50 mM MgCl2 Reagent Final concentration Amount MgCl2 50 mM 47.61 mg H2O ultra-pure n/a 10 mL Total 50 mM 10 mL 1.0 M KCl Reagent Final concentration Amount KCl 1.0 M 745.5 mg H2O ultra-pure n/a 10 mL Total 1.0 M 10 mL 102.24 mM KCl Reagent Final concentration Amount KCl 102.24 mM 76.22 mg H2O ultra-pure n/a 10 mL Total 102.24 mM 10 mL Notes: 1). To maintain the stock buffers biologically active, they need to be remade on a weekly basis. 2). This step is performed on Day 1 (see Figure 1). G-actin buffers G-actin buffer 1 (pH 7.80) Reagent Final concentration Amount Tris base (100 mM, pH 7.6) 2 mM 100 μL H2O ultra-pure n/a 4,845 μL CaCl2 (50 mM) 0.2 mM 20 μL ATP (100 mM) 0.5 mM 25 μL DTT (100 mM) 0.2 mM 10 μL Total n/a 5 mL G-actin buffer 2 (pH 7.66) Reagent Final concentration Amount Tris base (100 mM, pH 7.6) 2 mM 100 μL H2O ultra-pure n/a 4,860 μL CaCl2 (50 mM) 0.2 mM 20 μL ATP (100 mM) 0.2 mM 10 μL BME (50 mM) 0.1 mM 10 μL Total n/a 5 mL G-actin buffer 3 (pH 8.23) Reagent Final concentration Amount Tris base (100 mM, pH 7.6) 2 mM 100 μL H2O ultra-pure n/a 4,830 μL CaCl2 (50 mM) 0.2 mM 20 μL ATP (100 mM) 0.5 mM 25 μL DTT (100 mM) 0.5 mM 25 μL Total n/a 5 mL Notes: 1). Calibrate the pH meter at three-point calibration using Orion buffers pH 4.01, 7.00, and 10.00 before its usage. 2). To increase/decrease the pH, add drops of a base (0.74 M NaOH) or acid (0.1 N HCl) solution, respectively. Add these drops carefully and constantly check the pH, as it can rapidly change. 3). This step is performed on Day 2 (Figure 1). 4). Have ice ready in a cooler, as all G-actin buffers must be done on ice. Polymerization buffers Polymerization buffer 1 (pH 7.56) Reagent Final concentration Amount H2O ultra-pure n/a 4,050 μL MgCl2 (50 mM) 2 mM 200 μL KCl (1.0 M) 150 mM 750 μL Total n/a 5 mL Polymerization buffer 2 (pH 7.64) Reagent Final concentration Amount H2O ultra-pure n/a 4,050 μL MgCl2 (50 mM) 2 mM 200 μL KCl (1.0 M) 150 mM 750 μL Total n/a 5 mL Polymerization buffer 3 (pH 8.07) Reagent Final concentration Amount H2O ultra-pure n/a 4,550 μL MgCl2 (50 mM) 2 mM 200 μL KCl (1.0 M) 50 mM 250 μL Total n/a 5 mL Notes: 1). This step is also performed on Day 2 (Figure 1). 2). Have ice ready in a cooler, as all polymerization buffers must be done on ice. Electrolyte buffers Electrolyte buffer 1 (pH 7.72), buffer 2 (pH 7.66), buffer 3 (pH 8.06) Reagent Final concentration Amount H2O ultra-pure n/a 10 mL KCl 102.24 mM 76.22 mg Total n/a 10 mL Acknowledgments This work was supported by NIH grant 1SC1GM127187-04. We thank Drs. Carrie Schindler, Anna Morfesis, Ronald Soriano, and Matthew Brown from Malvern Panalytical Instruments Inc. for their assistance and advice in the configuration of the light scattering instrument and data analysis. We also thank Drs. Brian Hoover and Lee Toni from Cytoskeleton Inc. for their assistance and advice in the sample’s preparation and protein concentration analysis. This protocol was derived from previous work (F. Wang et al., 1989; Janmey et al., 1994, 1986). Competing interests The authors declare no conflict of interest. Ethics There are no human subjects or animal studies described in this protocol. References Alva, E., George, A., Brancaleon, L. and Marucho, M. (2022). Hydrodynamic and Polyelectrolyte Properties of Actin Filaments: Theory and Experiments. Polymers 14(12): 2438. Bonet, C., Ternent, D., Maciver, S. K. and Mozo-Villarias, A. (2000). Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH. Eur J Biochem 267(11): 3378-3384. Crevenna, A. H., Naredi-Rainer, N., Schonichen, A., Dzubiella, J., Barber, D. L., Lamb, D. C. and Wedlich-Soldner, R. (2013). Electrostatics control actin filament nucleation and elongation kinetics. J Biol Chem 288(17): 12102-12113. Castaneda, N., Zheng, T., Rivera-Jacquez, H. J., Lee, H. J., Hyun, J., Balaeff, A., Huo, Q. and Kang, H. (2018). Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure. J Phys Chem B 122(14): 3826-3835. Del Rocio Cantero, M., Gutierrez, B. C. and Cantiello, H. F. (2020). Actin filaments modulate electrical activity of brain microtubule protein two-dimensional sheets. Cytoskeleton (Hoboken) 77(3-4): 167-177. Hou, L., Lanni, F. and Luby-Phelps, K. (1990). Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm. Biophys J 58(1): 31-43. Janmey, P. A., Hvidt, S., Kas, J., Lerche, D., Maggs, A., Sackmann, E., Schliwa, M. and Stossel, T. P. (1994). The mechanical properties of actin gels. Elastic modulus and filament motions. J Biol Chem 269(51): 32503-32513. Janmey, P. A., Peetermans, J., Zaner, K. S., Stossel, T. P. and Tanaka, T. (1986). Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy. J Biol Chem 261(18): 8357-8362. Janmey, P. A., Slochower, D. R., Wang, Y.-H., Wen, Q. and Cēbers, A. (2014). Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids. Soft Matter 10(10): 1439-1449. Käs, J., Strey, H., Tang, J. X., Finger, D., Ezzell, R., Sackmann, E. and Janmey, P. A. (1996). F-actin, a model polymer for semiflexible chains in dilute, semidilute, and liquid crystalline solutions. Biophysical Journal 70(2): 609-625. Kroy, K. and Frey, E. (1997). Dynamic scattering from solutions of semiflexible polymers. Physical Review E 55(3): 3092-3101. Lanni, F. and Ware, B. R. (1984). Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery. Biophys J 46(1): 97-110. McDonald, J. H. (2009). Handbook of Biological Statistics. Sparky House Publishing. Baltimore, MD. Niranjan, P. S., Forbes, J. G., Greer, S. C., Dudowicz, J., Freed, K. F. and Douglas, J. F. (2001). Thermodynamic regulation of actin polymerization. J Chem Phys 114(24): 10573-10576. Parker, E. T. and Lollar, P. (2021). Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow. J Biol Chem 296: 100420. Pollard, T. D. (1986). Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol 103(6 Pt 2): 2747-2754. Steinmetz, M. O., Goldie, K. N. and Aebi, U. (1997). A correlative analysis of actin filament assembly, structure, and dynamics. J Cell Biol 138(3): 559-574. Stetefeld, J., McKenna, S. A. and Patel, T. R. (2016). Dynamic light scattering: a practical guide and applications in biomedical sciences. Biophys Rev 8(4): 409-427. Tang, J. X. and Janmey, P. A. (1996). The Polyelectrolyte Nature of F-actin and the Mechanism of Actin Bundle Formation (∗). J Biol Chem 271(15): 8556-8563. Tassieri, M., Evans, R. M., Barbu-Tudoran, L., Trinick, J. and Waigh, T. A. (2008). The self-assembly, elasticity, and dynamics of cardiac thin filaments. Biophys J 94(6): 2170-2178. Wang, F., Sampogna, R. V. and Ware, B. R. (1989). pH dependence of actin self-assembly. Biophys J 55(2): 293-298. Wang, Y. H. and Narayan, M. (2008). pH dependence of the isomerase activity of protein disulfide isomerase: insights into its functional relevance. Protein J 27(3): 181-185. Warshavsky, V., and Marucho M. (2022). Theory of Weakly Polydisperse Cytoskeleton Filaments. Polymers 14(10): 2042. Xu, J., Schwarz, W. H., Kas, J. A., Stossel, T. P., Janmey, P. A. and Pollard, T. D. (1998). Mechanical properties of actin filament networks depend on preparation, polymerization conditions, and storage of actin monomers. Biophys J 74(5): 2731-2740. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Neuroanatomy and circuitry Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,554	https://bio-protocol.org/en/bpdetail?id=4554&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans OP Omar Peña-Ramos ZZ Zheng Zhou Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4554 Views: 937 Reviewed by: Manish ChamoliMadhuja SamaddarAnupama Singh Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Jan 2022 Abstract During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted. Keywords: Apoptosis Apoptotic cell clearance Autophagy Autophagosomes Engulfment Phagocytosis Phagosomes Membrane fusions LC3 LGG-1 LGG-2 Fluorescence mCherry mNeonGreen (mNG) GFP Time-lapse imaging Caenorhabditis elegans Background The phagocytic clearance of apoptotic cells plays an important role in eliminating potentially harmful subjects from the surrounding tissues, and in suppressing autoimmune and inflammatory responses that could be stimulated by the content of broken apoptotic cells. Whereas in simple organisms such as C. elegans multiple types of neighboring cells are capable of engulfing apoptotic cells, in more complex organisms such as mammals there are professional phagocytes that engulf dying cells. These include macrophages and dendritic cells in the immune system, and microglia cells and astrocytes in the brain (Yuan and Yankner, 2000; Poon et al., 2014). Phagocytosis and autophagy are two distinct lysosome-mediated degradation processes. Whereas phagocytosis refers to a cell-eat-cell event, autophagy traps protein aggregates, damaged organelles, and other components to be eliminated within a cell in the double-membrane autophagosomes. The formation of autophagosomes requires the organized, step-wise action of a set of proteins known as autophagy-related (ATG) proteins (Nakatogawa, 2020). Yeast ATG8, mammalian LC3 (microtubule-associated protein 1 light chain 3), and their orthologs in other organisms, such as the nematode C. elegans and the fruit fly Drosophila melanogaster, are attached to both the inner and outer membranes of an autophagosome and have been established as specific markers for autophagosomes (Schaaf et al., 2016). In addition, LC3-associated phagocytosis (LAP) vesicles, which are single-membrane, LC3-labeled vesicles, have been reported to facilitate phagocytosis in the mammalian system (Green et al., 2016; Martinez et al., 2016). In C. elegans embryos, by using electron microscopy, genetic analysis, and the real-time fluorescence microscopy protocol described here, we have discovered that the canonical double-membrane autophagosomes, but not the single-membrane LAP vesicles, fuse to phagosomes and facilitate the degradation of apoptotic cells (Peña-Ramos et al., 2022). We tagged LGG-1 and LGG-2, two C. elegans homologs of mammalian LC3, with fluorescent reporters that are resistant or moderately resistant to acidic pHs, such as mCherry (pKa <4.5) or mNeonGreen (mNG, pKa = 5.1), respectively (Shaner et al., 2004, 2013; Shinoda et al., 2018). We specifically expressed these tagged reporters in engulfing cells by placing each fusion construct under the control of the ced-1 promoter (Pced-1), which is expressed in cell types that act as engulfing cells (Zhou et al., 2001; Lu et al., 2009). We monitored the enrichment of puncta labeled with LGG-1 or LGG-2 on phagosomes. C. elegans embryonic development follows a fixed lineage (Sulston et al., 1983); the identities of apoptotic cells, their engulfing cells, and the moments when the apoptosis events occur are all fixed from embryo to embryo (Sulston et al., 1983). Thus, by monitoring phagosomes containing apoptotic cells with specific identities in different genetic backgrounds, we can avoid variances linked to the different properties of particular engulfing cells. In this protocol, we monitor three phagosomes that contain apoptotic cells: C1, C2, and C3 (Figure 1A–1B). These are located on the ventral surface of an embryo, undergo apoptosis at around 330 min post first embryonic division, and each is immediately engulfed by one particular ventral hypodermal cell (Figure 1A). LGG-1+ or -2+ puncta are observed attaching to the surface of a phagosome as early as 2 min post phagosome formation (Figure 1B). The fluorescence signals elicited from the acid-resistant mCherry::LGG-reporters start to appear inside the phagosomal lumen at approximately 14 min post phagosome formation and continue to accumulate there over time until the phagosomal cargo is completely degraded in approximately 60 min. If the LGG+ vesicles were single-membrane, like an LAP vesicle, no membrane-attached LGG reporter would enter the phagosomal lumen because, as a result of membrane fusion, the reporter molecules would be retained on the phagosomal membrane (Figure 1C(a)). The observed entry of the LGG signal into the phagosomal lumen (Figure 1B) indicates that the LGG-labeled puncta represent the double-membrane vesicles, the autophagosomes (Figure 1C(b)). Figure 1. Vesicles labeled with mCherry::LGG-1 or ::LGG-2 are recruited to phagosome surfaces and subsequently fuse to phagosomes. (A) Diagram illustrating the three phagosomes that contain cell corpses, C1, C2, and C3, where we monitor the dynamic recruitment and fusion of autophagosomes, starting at approximately 330 min post first embryonic division. Both the positions of C1, C2, and C3 (purple dots) and the identities of their engulfing cells are shown. (B) Time-lapse images of the indicated reporters in C3 phagosomes in wild-type embryos. White arrowheads indicate the nascent phagosomes. Yellow arrows mark LGG-labeled puncta on the surface of phagosomes. White arrows indicate the moment when LGG signal can be observed in the phagosomal lumen. All reporters were expressed under the control of Pced-1. Scale bar = 2 µm. (C) Two diagrams illustrating the consequence of two fusion events: between single-membrane vesicles and a phagosome (a), and between double-membrane vesicles and a phagosome (b). The vesicles are first observed attaching to the phagosomal surfaces. After the fusion between the outer membrane of double-membrane vesicles and the phagosomal membrane, the mCherry::LGG-tagged inner membrane is released into the phagosomal lumen (b). The continuing incorporation of these vesicles to phagosomes increases the mCherry signal level in the phagosomal lumen over time (b). If the LGG-1 or LGG-2-labeled vesicles are of a single membrane, no fluorescence signal is expected to enter the phagosomal lumen (a). This protocol, together with our genetic analysis, led us to discover that the signaling pathway led by the phagocytic receptor CED-1 promotes the recruitment of autophagosomes to phagosomes (Peña-Ramos et al., 2022); moreover, the subsequent fusion of autophagosomes with phagosomes requires the functions of the small GTPase RAB-7 and the HOPS complex (Peña-Ramos et al., 2022). Further analysis showed that autophagosomes provide apoptotic cell-degradation activities in addition to and independent of lysosomes (Peña-Ramos et al., 2022). The principles of this protocol Previously, methods for quantifying the efficiency of the interaction between highly dynamic intracellular organelles such as autophagosomes and phagosomes in real-time in C. elegans were not available. The protocol we describe here allows us to quantitatively measure the efficiencies of two separate events: (1) the recruitment and (2) the subsequent fusion of autophagosomes to the phagosomes in wild-type and mutant backgrounds. To evaluate the overall efficiency of the incorporation of autophagosomes into phagosomes, which is the end result of both the recruitment and fusion events, we measured the mCherry::LGG-1 and mCherry::LGG-2 signal intensities in the center of a phagosome over time, in a period starting from the formation of a phagosome (0 min) to 70 min afterward (Figure 2A). To evaluate the specific efficiency of recruitment, we measured the mCherry signal on the surface of a phagosome over time, starting from 0 min (Figure 2A). In wild-type embryos, the mCherry signal is detected evenly distributed in the phagosomal lumen starting at +14 min on average. In order to perform a meaningful comparison between the recruitment-defective mutants and wild-type samples, we choose to measure at the +12 min time point (Figure 2A). In a mutant that is specifically defective in the fusion between autophagosomes and phagosomes, one will observe greatly reduced or absent mCherry signals inside the phagosomal lumen, with the mCherry signal accumulating on the surface of a phagosome over time (Figure 2B). On the other hand, in a mutant that is specifically defective in the recruitment of autophagosomes to phagosomes, at the +12 min, +50 min, and +70 min time points, very few mCherry+ puncta are observed on the phagosomal surfaces; in addition, no or very low mCherry signal is observed inside the phagosomal lumen (Figure 2C). If recruitment is partially defective whereas fusion is normal, the accumulation of the mCherry inside the phagosomal lumen will occur, albeit being delayed and/or at a lower intensity (Figure 2D). However, if recruitment is completely inactive, no autophagosome would be seen attached to phagosomes, further resulting in no mCherry signal inside the phagosomal lumen even if fusion is normal (Figure 2C). Such severe phenotype will make it impossible to determine whether the same mutant also suffers from a fusion defect. Figure 2. Recruitment and fusion of autophagosomes to phagosomes in wild-type and the mutants in which these events are impaired. Diagram illustrating the LGG::mCherry signal (in magenta) pattern in wild-type (A) and three kinds of mutants (B–D) on the surface and in the lumen of a C3 phagosome, 12, 50, and 70 min after phagosome formation. In (A), due to the efficient phagosomal degradation process, the size of a phagosome shrinks over time. In (B–D), the fusion and recruitment mutants are defective in phagosomal degradation, resulting in a much slower reduction of the phagosomal size. Our protocol can be applied to characterize the fusion between autophagosomes and phagosomes that carry any kind of cargo, as well as between autophagosomes and other types of intracellular organelles in living C. elegans embryos, as long as the resolution is high enough to distinguish the surface of an organelle from its lumen. Furthermore, the principle of detecting the membrane fusion event between the double-membrane and single-membrane vesicles can be adapted to study the relationship between autophagosomes and phagosomes or between other intracellular organelles in any experimental systems that allows the expression of transgenic reporters and real-time microscopic recording of fluorescent signals. Materials and Reagents In vivo reporters LGG-1 reporter construct: Pced-1mCherry::lgg-1 (for labeling autophagosomes) LGG-2 reporter construct: Pced-1mCherry::lgg-2 (for labeling autophagosomes) Pseudopod and phagosomal surface reporter construct: Pced-1ced-1::gfp (for labeling nascent phagosomes). The Caenorhabditis elegans strains that carry both reporters ZH2919, genotype: enIs82 [pUNC-76(+)(20 ng/μL), Pced-1ced-1::gfp (5 ng/mL), Pced-1mCherry::lgg-1 (5 ng/μL)] II; unc-76(e911) V ZH2898, genotype: enIs83[pUNC-76(+)(20 ng/μL), Pced-1ced-1::gfp (5 ng/mL), Pced-1 mCherry::lgg-2 (5 ng/μL)] II; unc-76(e911) V In these strains, the recruitment and subsequent fusion of autophagosomes to phagosomes can be monitored by recording the dynamic localization patterns of both the mCherry and GFP reporters. Consumable reagents Microscope slides (Premiere, catalog number: 9101) Coverslips (22 × 22 mm) (Fisher Scientific, catalog number: 12-542B) DeltaVision immersion oil, N = 1.516 (Cytiva, catalog number: 29162940) Handmade worm pick, which is a platinum wire (Alfa Aesar, catalog number: 10287) mounted on a Pasteur pipette (Fisher Scientific, catalog number: 13-678-20B) (Figure 3) High vacuum grease (Fisher Scientific, catalog number: 14-635) Agarose (Fisher Scientific, catalog number: BP160-500) KH2PO4 (MilliporeSigma, catalog number: PX1565) Na2HPO4 (MilliporeSigma, catalog number: 567550) NaCl (Fisher Scientific, catalog number: S671) 4% agarose solution (see Recipes) M9 buffer (see Recipes) Figure 3. A handmade worm pick Equipment Stereomicroscope (Nikon, catalog number: SMZ645) DeltaVision Elite Deconvolution imaging system (GE Healthcare, Inc.), including AP1 F1- DeltaVision microscope (Olympus) equipped with 20×, 63×, and 100× Uplan Apo objectives, excitation/emission filter sets, fluorescent light source, differential interface contrast (DIC) microscopy accessories, motorized stage (X, Y, and Z-axis), and a Coolsnap HQ2 digital camera (Photometrics). For fluorescent imaging, two sets of fluorescence filters (Chroma Inc.) are used, including the GFP filter (excitation wavelength 475/28 nm; emission wavelength 525/50 nm) and the mCherry filter (excitation wavelength 575/25 nm; emission wavelength 632/60 nm). The DeltaVision microscope is kept in a room where the temperature is maintained at 20 °C. Computer for image processing Software SoftWoRx 5.5 software (for the deconvolution and processing of images) (GE Healthcare, Inc.) Microsoft Excel (Microsoft, Inc.) Prism GraphPad (Dotmatics, Inc.) Procedure Mounting embryos on an agar pad Melt 4% agarose solution in a microwave oven. Dispense between 100 and 300 μL of the melted agarose solution in the center of the slide. Be cautious not to generate air bubbles. Immediately, flatten the drop by placing a glass slide perpendicular to the one holding the drop and press gently (Figure 4A). Let it stand for 1–2 min to allow the agarose to solidify. Gently separate the two slides by sliding one against the other. Using a clean glass slide as a blade, trim the flattened agar pad into an approximately 12 × 12 mm square (Figure 4B). Place 3 μL of M9 buffer at the center of the agar pad. The buffer is used to wash and maintain the embryos alive during the procedure. Under the stereotype microscope, collect 50–80 embryos with the worm pick and transfer them to the drop of M9 buffer on the agarose pad. When transferring embryos from a Petri dish to the agar pad on the slide, carry as little bacteria as possible. If too many bacteria are transferred, they will consume oxygen and cause the embryos to die. Gently squeeze a thin line of high vacuum grease around the agarose path (Figure 4C). Place a cover slip over the vacuum grease and press the corners gently to seal the slide (Figure 4D). Figure 4. Construction of an agarose pad. (A) Agar flattened by two cover slips. (B) Agar pad cut into a square, marked by the black arrow. (C) High vacuum grease (black arrow) placed around the agar pad. (D) Agar pad covered with a glass cover slip (yellow arrow) by pressing gently pressing the corners with a piper tip (black arrow). Time-lapse recording of C1, C2, and C3 phagosomes Set up microscope parameters: for DIC images, an exposure time of 0.2 s with a 10% neutral density filter is enough. The percentage of a neutral density filter represents the fraction of light that passes through the filter and illuminates the sample. For recording the mCherry::LGG-1/2 reporters, the exposure parameter for the mCherry channel is 0.1 s with 5% neutral density filter. For recording the engulfment marker ced-1::gfp, the parameter is 0.15 s with a 5% neutral density filter. For optimal imaging, align the DIC light path according to the manufacturer's instructions. Under the 20× objective, find embryos. Under the 100× objective, identify embryos at the age between 280 and 300 min post their first cell division and with their ventral side facing up (Figure 5). Mark these embryos utilizing the “point marking” and “position visiting” functions of the SoftWoRx software. For each strain, we image 15 embryos. Define the position where the z-section recording should start. The serial z-section recording is performed from the embryo's ventral surface and proceeds to the center of the embryo. Set up 12–16 z-sections at 0.5 μm thickness for each section, which is sufficient to cover the entire ventral region. Set up the time-lapse recording parameters. Start recording when the embryo is approximately 320 min post the first cell division; a moment later, the hypodermal cells will engulf apoptotic cells C1, C2, and C3 (Figure 1A). A clue hinting that the engulfment of C1, C2, and C3 is about to happen is the event when the middle part of the embryo slightly contracts towards its center, as shown in Figure 5. The recording time interval is set at every 2 mins for 60 to 120 min or until the embryos reach the 1.5-fold developmental stage, which is at 420 min post the first embryonic division. Keep observing the images during the acquisition process. Adjust the starting focal plane if necessary. Abort the recording if the embryos slow down or arrest their development. Examples of time-lapse images of LGG+ vesicles are recruited to the phagosomal surfaces and subsequently fuse to phagosomes are shown in Figure 1B. Figure 5. DIC image of a C. elegans embryo at 300 min post first cell division. Scale bar is 5 µm. Data analysis Measuring the signal intensity inside the phagosomal lumen To measure the fluorescence signal intensity inside the phagosomal lumen over time in embryos expressing Pced-1mCherry::lgg-1 or -2, the boundary of a phagosome is identified at the 0 min time point when a nascent phagosome forms by the CED-1::GFP marker, which labels the surfaces of the extending pseudopods and nascent phagosomes. At each time point, the total mCherry::LGG-1 or -2 signal intensity within a fixed area (4 × 4 pixels) in the center of a phagosome (Intphagosome) is recorded (Figure 6B), as is the intensity of an area of the same size (4 × 4 pixels) outside the embryo as the background image intensity (Intbackground). The relative image intensity (RInt) at a particular time point (Tn) comparing to the start point (T0) is calculated as RIntTn = (Intphagosome - Intbackground)Tn / (Intphagosome - Intbackground)T0. The RIntTn value of 1.0 indicates no entry of LGG-1- or LGG-2-labeled autophagosomes into the phagosomal lumen. The data collected are plotted in a graph with the X-axis indicating the time points and RIntTn in the Y-axis. In addition, for each strain to be characterized, the RIntT50 (at the +50 min time point) of 15 phagosomes is measured. Standard statistical analysis is performed (Figure 6D). Whether the RIntT50 in a particular mutant strain is significantly different from that in the wild-type is determined by Student’s t-test. Measuring the signal intensity on the surface of a phagosome To evaluate the efficiency of recruitment of autophagosomes to phagosomes, the intensity of mCherry::LGG-1 or -2 is measured on the surfaces of phagosomes. First, the boundary of a phagosome at 0 min is identified by the CED-1::GFP reporter, which labels the surfaces of the extending pseudopods and nascent phagosomes. At a particular time point (Tn), the surface of a phagosome is outlined by two closed polygons (Figure 6A). The total signal intensities, as well as the areas of the polygons, are recorded. The unit signal intensity of the donut-shaped area between the two polygons is calculated as follows: Unit Intensity (UIphagosome) = (Intensityexternal polygon - Intensityinternal polygon) / (Areaexternal polygon - Areainternal polygon). The Unit Background Intensity (UIbackground) was measured from a polygon outside the embryo and calculated as follows: UIbackground = Intensitybackground / Areabackground. At the time point Tn, the relative signal intensity on phagosomal surfaces (RsurfaceTn) = (UIphagosome - UIbackground)Tn / (UIphagosome - UIbackground)T0. The RsurfaceTn value 1.0 indicates no recruitment of the LGG-1- or LGG-2-labeled autophagosomes on phagosomal surfaces compared to the 0 min time point. The data collected are plotted in a graph with the X-axis indicating the time points and Y-axis indicating RsurfaceTn. In addition, for each strain to be characterized, the RsurfaceT12 (at the +12 min time point) of 15 phagosomes is measured (Figure 6C). Standard statistical analysis will be performed. Whether the RsurfaceT50 in a particular mutant strain is significantly different from that of the wild-type will be determined by the Student’s t-test. Figure 6. Two diagrams depicting the areas where the LGG signal intensity is measured in the C3 phagosome. (A) Diagram illustrating where the mCherry signal on the surface of a phagosome is measured over time. The area is delimited by the two black-dotted polygons. (B) Diagram illustrating where the relative mCherry signal in the center of a phagosome is measured over time. (C) Box and whisker plots of the relative mNeonGreen (mNG) signal intensities measured on the surfaces of ten C3 phagosomes 12 min post the formation of nascent phagosomes from wild-type and ced-1(e1735) mutant embryos. This graph is adapted from Peña-Ramos et al. (2022). (D) Box and whiskers plots of the relative mNG signal intensities measured in the center of fifteen C3 phagosomes 50 min post the formation of nascent phagosomes from wild-type and ced-1(e1735) mutant embryos. (C and D) Red dashed lines indicate the position of value 1.0, which represents no signal enrichment relative to the background signal. ***, p < 0.001, Student’s t-test of the ced-1(e1735) mutants compared to the wild-type value. Notes The reporter constructs are available upon request. The strains mentioned in this protocol are available upon request. Although this protocol specifically describes the usage of the DeltaVision Elite Deconvolution imaging system for the real-time recording of autophagosome recruitment and fusion events, it can be easily adapted to other real-time imaging systems such as the spinning disk confocal microscope. In addition, in C. elegans, programmed cell death events also occur in larvae and in the gonad of adult hermaphrodites in addition to embryos. Our protocol can also be applied to study the incorporation of autophagosomes into phagosomes in larvae and adults. Furthermore, besides C. elegans embryos, any experimental system in which real-time imaging can be conducted and that allows the expression of transgenic reporter constructs can adopt this protocol as well. Recipes 4% agarose solution Place 2.0 g of agarose in 50 mL of deionized water and heat up in a microwave until the agarose is completely dissolved. After usage, cap the flask with aluminum foil and store it at room temperature. To reuse the agar solution, melt the solidified solution for 35 s in the microwave. M9 Buffer (1 L) Dissolve 5.8 g of Na2HPO4 (40.9 mM), 0.5 g of NaCl (8.6 mM), 1.0 g of NH4Cl (18.7mM), and 3.0 g KH2PO4 (22.0 mM) in 400 mL of deionized water. Bring the volume to 1 L and autoclave for 40 min. Acknowledgments This work is supported by NIH grant GM067648. This protocol is adapted from Peña-Ramos et al. (2022). Competing interests There are no conflicts of interest or competing interests. References Green, D. R., Oguin, T. H. and Martinez, J. (2016). The clearance of dying cells: table for two. Cell Death Differ 23(6): 915-926. Lu, N., Yu, X., He, X. and Zhou, Z. (2009). Detecting apoptotic cells and monitoring their clearance in the nematode Caenorhabditis elegans. Methods Mol Biol 559: 357-370. Martinez, J., Cunha, L. D., Park, S., Yang, M., Lu, Q., Orchard, R., Li, Q. Z., Yan, M., Janke, L., Guy, C., et al. (2016). Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells. Nature 533(7601): 115-119. Nakatogawa, H. (2020). Mechanisms governing autophagosome biogenesis. Nat Rev Mol Cell Biol 21(8): 439-458. Peña-Ramos, O., Chiao, L., Liu, X., Yu, X., Yao, T., He, H. and Zhou, Z. (2022). Autophagosomes fuse to phagosomes and facilitate the degradation of apoptotic cells in Caenorhabditis elegans. Elife 11: e72466. Poon, I. K., Lucas, C. D., Rossi, A. G. and Ravichandran, K. S. (2014). Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol 14(3): 166-180. Schaaf, M. B., Keulers, T. G., Vooijs, M. A. and Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J 30(12): 3961-3978. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E. and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12): 1567-1572. Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, R. N., Israelsson, M., et al. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10(5): 407-409. Shinoda, H., Ma, Y., Nakashima, R., Sakurai, K., Matsuda, T. and Nagai, T. (2018). Acid-Tolerant Monomeric GFP from Olindias formosa. Cell Chem Biol 25(3): 330-338 e337. Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 100(1): 64-119. Yuan, J. and Yankner, B. A. (2000). Apoptosis in the nervous system. Nature 407(6805): 802-809. Zhou, Z., Hartwieg, E. and Horvitz, H. R. (2001). CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell 104(1): 43-56. Article Information Copyright Peña-Ramos and Zhou. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Developmental Biology > Cell signaling > Apoptosis Developmental Biology > Cell signaling Cell Biology > Cell imaging > Fluorescence Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Labelling of Active Transcription Sites with Argonaute NRDE-3—Image Active Transcription Sites in vivo in Caenorhabditis elegans Antoine Barrière and Vincent Bertrand Jun 5, 2022 1348 Views Aerotaxis Assay in Caenorhabditis elegans to Study Behavioral Plasticity Qiaochu Li [...] Karl Emanuel Busch Aug 20, 2022 1245 Views Preparation of Caenorhabditis elegans for Scoring of Muscle-derived Exophers Katarzyna Banasiak [...] Wojciech Pokrzywa Jan 5, 2023 713 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,555	https://bio-protocol.org/en/bpdetail?id=4555&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Using BODIPY FL-Sphingolipid Analogs to Study Sphingolipid Metabolism in Mouse Embryonic Stem Cells WF Wei Fan XL Xiaoling Li Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4555 Views: 988 Reviewed by: Julie WeidnerAgnieszka ZienkiewiczYu Liu Download PDF Ask a question Favorite Cited by Version history Bio-protocol journal peer-reviewed Nov 20, 2022 \| This version Preprint Jul 07, 2021 Original Research Article: The authors used this protocol in eLIFE May 2021 Abstract Sphingolipids are important structural components of cellular membranes. They also function as prominent signaling molecules to control a variety of cellular events, such as cell growth, differentiation, and apoptosis. Impaired sphingolipid metabolism, particularly defects in sphingolipid degradation, has been associated with many human diseases. Fluorescence sphingolipid analogs have been widely used as efficient probes to study sphingolipid metabolism and intracellular trafficking in living mammalian cells. Compared with nitrobenzoxadiazole fluorophores (NBD FL), the boron dipyrromethene difluoride fluorophores (BODIPY FL) have much higher absorptivity and fluorescence quantum. These features allow more intensive labeling of cells for fluorescence microscopy imaging and flow cytometry analysis. Here, we describe a protocol employing BODIPY FL-labeled sphingolipid analogs to elucidate sphingolipid internalization, trafficking, and endocytosis in mouse embryonic stem cells. Graphical abstract: Keywords: Sphingolipid Sphingomyelin BODIPY Internalization Metabolism Plasma membrane Golgi complex Background Sphingolipids are a group of structurally diverse lipids, first discovered from brain extract in the 1880s. Globally, they act as essential components of the plasma membrane in almost all types of vertebrate cells (Schnaar and Kinoshita, 2015). All sphingolipids share a common sphingoid-based backbone (Figure 1A), which acts as the structural foundation for all sphingolipid derivatives. Adding one fatty acid to this sphingosine base forms ceramide, the central molecule in sphingolipid biology (Figure 1B). Further adding a phosphoryl choline or phosphoryl ethanolamine group to ceramide makes sphingomyelin (SM), the most abundant mammalian sphingolipid (Figure 1C and 1D) (Fan et al., 2021). Figure 1. Chemical structure of sphingolipids. General chemical structure of (A) sphingosine backbone, (B) ceramide, which has an additional fatty acid molecule (highlighted in red) attached to the sphingosine backbone, and (C-D) the two most common types of sphingomyelin, which have one (C) phosphocholine or (D) phosphoethanolamine group (highlighted in blue) attached to the ceramide. In mammalian cells, the endoplasmic reticulum (ER), Golgi complex, and plasma membrane are key subcellular locations hosting sphingolipid metabolism. They accommodate the majority of the two most important and abundant types of sphingolipids: ceramide and SM. Ceramide is the central node of the sphingolipid metabolism network. There are two endogenous ceramide synthesis pathways: the first one is the de novo synthesis in ER. The newly synthesized ceramide is then transported into the Golgi complex for further conversion to more complex forms of sphingolipids, such as SM; the second one is the regeneration from complex sphingolipids in the Golgi complex and plasma membrane, by a class of specific hydrolases and phosphodiesterases, including the plasma membrane-bound sphingomyelin phosphodiesterases (Gault et al., 2010; Heinz et al., 2015; Fan et al., 2021). The sphingolipids are not only important in supporting physical microdomain structures of mammalian cell membranes, but also act as prominent signaling molecules controlling a number of cellular events, such as cell growth, differentiation, and apoptosis (Laude and Prior, 2004). The significance of sphingolipids for human health is best demonstrated by human neural degenerative diseases, including Alzheimer’s, Parkinson’s, and Niemann-Pick disease. These neural degenerative diseases are known to be associated with dysregulation or disturbance of key sphingolipids, such as SM and ceramide (Bienias et al., 2016; Alessenko and Albi, 2020). The elucidation of sphingolipid internalization, trafficking, and endocytosis in live mammalian cells, therefore, is key to the understanding of the underlying mechanisms of these human diseases. Lipid analogs generated by replacing naturally occurring fatty acids in complex lipids with artificial short-chain fatty acids containing fluorophores (FL) have been widely used to study membrane lipid trafficking in mammalian cells (Koval and Pagano, 1991; Hoekstra and Kok, 1992; Rosenwald and Pagano, 1993). For example, the fluorophore boron dipyrromethene difluoride (BODIPY)- (Figure 2A) or nitrobenzoxadiazole (NBD)-labeled SMs and ceramides (Figure 2B and 2C), have been widely applied as molecular probes for lipid trafficking in live mammalian cells. These artificial lipid analogs equipped with BODIPY or NBD fluorophores can be readily integrated into cellular membranes through spontaneous lipid transfer from exogenous sources. Further intracellular distribution of those lipid analogs, or metabolites derived from them, can be visualized and quantified with high resolution fluorescence microscopy (confocal laser scanning microscopy [CLSM]) or fluorescence-activated cell sorting flow cytometry (FACS) (Pagano and Chen, 1998). Figure 2. Chemical structure of sphingolipid analogs with BODIPY fluorophore. The chemical structure of (A) boron dipyrromethene difluoride chelate (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore (BODIPY FL), (B) BODIPY FL C5-Sphingomyelin, and (C) BODIPY FL C5-Ceramide. Both BODIPY and NBD are widely used fluorophores. Compared with NBD, the BODIPY fluorophore has several advantages for studying lipid metabolism and trafficking, specifically in live cells. First, BODIPY has stronger fluorescence and is more photostable than NBD (Johnson et al., 1991). Second, BODIPY’s structure is less polar than that of NBD; therefore, it is more efficiently anchored in the bilayer of the mammalian cell membrane. In contrast, NBD easily loops back to the water/bilayer interface, which may interfere with integration of lipid analogs into the plasma membrane (Chattopadhyay and London, 1987; Wolf et al., 1992; Pagano and Chen, 1998). Third, the emission wavelength of BODIPY fluorescence ranges from 450 (green) to 650 (red) with increased concentration in cells (Pagano et al., 1991). This special feature makes the distribution of a particular BODIPY-labeled lipid analog and its metabolites dynamically visible, allowing easy observation and quantification in live cells under fluorescence microscopy (Pagano et al., 1991). We recently investigated the importance of sphingolipid metabolism in neural differentiation of mouse embryonic stem cells (mESCs) using BODIPY FL sphingomyelin (Fan et al., 2021). Therefore, this protocol describes the application of commercially available BODIPY FL sphingomyelin analogs to visualize and quantify internalization, trafficking, and endocytosis of sphingolipids in mESCs, by FACS and CLSM. The protocol includes detailed methods for preparation of BODIPY FL sphingomyelin analog probes and staining of mESCs using these analogs, for FACS and CLSM analyses. We expect that this protocol will have a wide range of applications in sphingolipid research. Things to consider before starting Choice of BODIPY FL sphingolipids There are many commercially available BODIPY FL sphingolipid analogs, containing different lengths of fatty acid chains or different substitutions on the BODIPY moiety to modify its spectral properties. A five-carbon fatty acid, 5-(5,7-dimethyl-BODIPY)-1-pentanoic acid, has been previously reported to be the most effective one for most applications (Pagano et al., 2000). Moreover, the spectral properties of C5-BODIPY lipid analogs are not sensitive to either pH or membrane potential and curvature (Karolin et al., 1994; Chen et al., 1997). The excitation and emission of bright green-fluorescein generated by the commercially available BODIPY FL dye (Invitrogen) are similar to those of fluorescein (FITC) or Invitrogen Alexa Fluor 488 dye. However, compared with fluorophores Alexa Fluor 488 dye and FITC, BODIPY FL dye has higher extinction coefficient and increased fluorescence quantum yield, and is insensitive to solvent polarity and pH change. Also, due to its hydrophobic properties and long excited-state lifetime (typically, 5 nanoseconds or longer), BODIPY FL dye is very effective in labeling cellular lipids, specifically membrane lipids, and useful for fluorescence polarization-based assays (referred in the commercial product manual of Invitrogen D3522). Expected subcellular locations of BODIPY FL sphingolipid analogs The distribution of N-[5-(5,70-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), a BODIPY FL ceramide analog, and its metabolites during endocytosis in human skin fibroblast cells has been previously traced (Pagano et al., 1991; Pagano and Chen, 1998). In those cells, this fluorescent lipid analog starts to be integrated into the plasma membrane and is then exclusively present in the outer leaflet of the plasma membrane bilayer at low temperature (commonly, at 4 °C) (Martin and Pagano, 1987; 1994; Chen et al., 1997). During subsequent temperature rising to 37 °C, this lipid analog starts to be internalized into lipid vesicles throughout the cytoplasm (Koval and Pagano, 1989; 1990). The whole internalization process is highly sensitive to low temperature, due to its strict energy dependence. As soon as the internalization is activated by high temperature incubation, the internalized fluorescent lipid analogs can either return intact (recycle) back to the plasma membrane, or be transported to lysosome storage, and further hydrolyzed by lysosomal sphingolipid hydrolases (Koval and Pagano, 1990; Mayor et al., 1993; Grassi et al., 2019). In this process, in addition to BODIPY FL in the endosomes, BODIPY FL-ceramide resulting from the hydrolysis of BODIPY FL-C5-DMB-Cer at the plasma membrane are also subsequently transported to the Golgi apparatus, because of its high affinity for ceramides (Pagano, 1990; Rosenwald and Pagano, 1993). Therefore, in mESCs, after the stable state of internalization has been reached upon completion of 30 min incubation at 37 °C, multiple cellular membrane structures—including plasma membrane, Golgi complex, ER, and endosomes—are expected to “lighten up” due to BODIPY fluorescence. Cell type-specific distribution of BODIPY FL sphingolipid analogs It is worth noting that different cell types cultured in different media have distinct patterns of sphingolipid metabolism. For instance, ESCs require some specific features to maintain their pluripotency and unique functions (Tanosaki et al., 2021). Therefore, the endocytosis and metabolism of sphingolipids, which can be reflected by the shape, color, and distribution of these lipid analogs and their metabolites in cells, may differ greatly in different cells. Materials and Reagents mESC culture Sterile serological pipettes (Serological pipettes of 5 mL, 10 mL, 25 mL and 50 mL; Sarstedt, catalog numbers: 86.1253.001, 86.1254.001, 86.1685.001) Sterile Corning® centrifuge tubes (50 mL and 15 mL; Millipore, catalog numbers: CLS430290, CLS430055) Falcon® 5 mL Round Bottom Polystyrene Test Tube (with snap Cap for FACS analysis, Sterile; Falcon, catalog number: 352058) FisherbrandTM Class B Amber Glass Threaded Vials (1.8 mL, 3.7 mL, 7.4 mL and16 mL; Fisher Scientific, catalog number: 03-339-23) 6 wells sterile cell culture dishes (NuncTM Cell-Culture Treated Multidishes; Thermo Fisher Scientific, catalog number:140675) NuncTM Glass Bottom Dishes (Perform high quality imaging in the ease of a 35 mm dish; Thermo Fisher Scientific, catalog number:150682) R1 mESC or ES-E14TG2a (E14) mESC lines (ATCC, catalog number: SCRC-1011, RRID: CVCL_2167; CRL-1821, RRID: CVCL_9108) Gelatin powder (Gelatin from porcine skin; Millipore Sigma, catalog number: G1890) ESGRO Complete Plus Grade medium: a commercially developed serum free complete basal medium. It contains a selective GSK3β inhibitor to enhance viability of mESCs and increase maintenance of the pluripotency (Millipore, catalog number: SF001) Cytiva HyClone Dulbecco's Phosphate Buffered Saline liquid (DPBS buffer; Fisher Scientific, catalog number: SH3002802) Water for cell culture (sterile-filtered, BioReagent, suitable for cell culture; Millipore Sigma, catalog number: W3500) GibcoTM Trypsin-EDTA (0.05%), phenol red (Sigma-Aldrich, catalog number: 25300054) Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061) 0.1% Gelatin solution (see Recipes) Preparation of BODIPY FL-sphingomyelin stock and working solutions BODIPYTM FL C5-Sphingomyelin (Invitrogen, catalog number: D3522) Defatted bovine serum albumin (BSA; Roche, catalog number: 03117057001) Hanks’ Balanced Salt solution (HBSS buffer solution; HBSS, no calcium, no magnesium, no phenol red; Gibco, catalog number: 14175095) HEPES solution (1 M N-2-Hydroxyethylpiperazine-N′-2-ethane sulfonic acid in H2O, Gibco, catalog number: 15630106) Chloroform (anhydrous, ≥99%, contains 0.5–1.0% ethanol as stabilizer; Millipore Sigma, catalog number: 288306) Ethyl alcohol (Absolute alcohol, 200 proof anhydrous CAS#64-17-5; The Warner Graham Company) 70% ethanol (see Recipes) Chloroform:ethanol (19:1 v/v) (see Recipes) HBSS/HEPES buffer (pH7.4) (see Recipes) Equipment Cell counter (Countess 3 Automated Cell Counter; Thermo Fisher) Analytical Nitrogen Evaporator (24 Position N-EVAP Nitrogen Evaporator; Organomation Associate, Inc; model: N-EVAPTM 112 #11250) Forma series 3 water jacketed CO2 incubator (Thermo Fisher) Avanti J-15R Centrifuge with GH-3.8/GH-3.8A rotor (Beckman Coulter) Mini Vortex Mixer (Variable Speed) (Fisher Scientific) Refrigerator (Whirlpool) Zeiss LSM 780 UV inverted confocal microscope with AiryScan (Carl Zeiss Sports Optics, model: LSM 780) Fluorescence-Activated Cell Sorting flow cytometer (BD Bioscience, Brand, model: BD LSRFortessa with HTS option) Software Zeiss Zen (Carl Zeiss Sports Optics, web address: https://www.binran.ru/files/ckp/fluorestsentnaya-mikroskopiya/CKP_LSM780_ZEN2010_Manual_ENG.pdf) BDFACSDivaTM software version 8.0.1 (BD Bioscience, web address: https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software) Procedures Plate and culture mESCs Dissolve gelatin powder in cell culture-grade water to the final concentration of 0.1% (w/v), then sterilize this solution by autoclave. Apply an appropriate amount of 0.1% gelatin solution to cover the entire surface area of cell culture dishes, then incubate the dishes at room temperature (RT) for 30 min to coat the cell growth area. After incubation, aspirate the gelatin solution and wash the coated cell culture dishes with PBS solution twice for 3 min each time, to completely remove the residual gelatin solution. Note: All above procedures (2–3) must be performed in a sterile hood. The resulting gelatin-coated dishes will be set aside in PBS solution for fresh cell plating. They can also be dried then kept at RT for up to 1 month for future use (after rehydration with PBS solution). Completely disassociate pre-cultured mESCs, by adding an appropriate amount of 0.05% trypsin (e.g., an approximate volume of 300 µL for each well of the 6-well plate) to cover the entire cell growth area, then incubate at 37 °C for 3 min. After cells are completely disassociated from dishes, wash them twice with 30 mL of PBS solution in 50-mL Corning tubes, to completely remove residual trypsin. Harvest cells by centrifugation at 300 × g for 3 min. Note: mESC colonies must be completely disassociated with 0.05% trypsin (extra amount of trypsin and extended digestion time may be applied) to ensure all cells are evenly dispersed in cell suspension before re-plating. Failure to complete dissociate cells could induce differentiation of mESCs, and/or formation of big cell clumps that interfere with subsequent analyses. Resuspend mESCs with the ESGRO growth medium, and plate cells on gelatin-coated 6-well cell culture plates for FACS analysis, or NuncTM Glass Bottom Dishes for CLSM visualization, at the density of approximately 5 × 104 cells/cm2. Incubate cells in a cell culture incubator at 37 °C with 95% humidity and 5% CO2. Cells will be stained with BODIPY FL-sphingomyelin analogs after their attachment to dishes, or after overnight incubation. Note: mESCs are very sensitive to environmental perturbations, including chemical and physical stresses. Even very minor agitations may result in apoptosis, differentiation, or morphological changes of mESCs. Therefore, cell culture procedures must be performed in an extraordinarily careful and gentle manner. Prepare the stock and working solutions of BODIPY FL C5-sphingomyelin analogs Stock solution: Mix absolute chloroform and absolute ethanol at a ratio of 19:1 (v/v) (Recipe 3) inside a chemical safety cabinet. To prepare 1 mM BODIPY FL C5-sphingomyelin stock solution, directly inject an appropriate amount of the prepared chloroform/ethanol solvent into the original product vial containing the BODIPY FL C5-sphingomyelin powder. Shake vials until all powders are completely dissolved in the solvent, then transfer the 1 mM stock solution to amber glass round vials. This stock solution must be stored at -20 °C and strictly protected from light. Working solution: Dispense 50 µL of 1 mM BODIPY FL C5-sphingomyelin stock solution from step B1 into an amber glass round vial. Evaporate the organic solvent under a stream of nitrogen, by placing the nozzle (like a needle) of the nitrogen evaporator into the mouth of the amber glass round vial, and spraying the nitrogen gas toward the stock solution, until no liquid phase is observed. After the liquid phase has been completely dried off, add 200 µL of absolute ethanol into the amber glass round vial, to reconstitute the dried BODIPY FL C5-sphingomyelin powder. To prepare 5 µM defatted BSA solution, dissolve an appropriate amount of defatted BSA into 10 mL of Hanks’ buffered salt solution containing 10 mM HEPES (HBSS/HEPES buffer, Recipe 4). Add the prepared 200 μL of BODIPY FL C5-sphingomyelin solution in ethanol from step B2a into the 10 mL of 5µM fatty acid-free BSA solution from step B2b, and mix properly on a vortex mixer to generate the working solution (5 μM BODIPY FL-sphingomyelin + 5 μM BSA). Store the working solution at -20 °C, with strict protection from light. Note: The purpose of defatted BSA in the working solution is to remove all residual pools of fluorescent lipid analogs, which will remain on the surface of plasma membranes after internalization of membrane-inserted lipid analogs, by “back-exchange” effects (Kok et al., 1989; Abreu et al., 2003). Stain mESCs with the working solution of BODIPY FL C5-sphingolipid analogs for confocal imaging analysis Remove all ESGRO growth medium from the overnight cell culture dishes and wash adherent cells three times with HBSS/HEPES buffer at RT, to remove residual ESGRO growth medium and floating dead cells. Apply sufficient working solution (prepared from the previous step) into cell culture dishes to completely cover the entire area where cells grow and incubate cells at 4 °C for 30 min with strict protection from light. The negative control sample will be incubated with the HBSS/HEPES buffer instead of BODIPY FL C5-sphingolipid working solution in this step. Note: During the washing step, PBS solution should be added against the side of the well instead of directly on the top of cells, so that cells will not be dislodged. The healthy mESCs are able to attach to gelatin-coated cell culture dishes. The majority of the cells dislodged during washing are dead or apoptotic cells and must be discarded. After 30 min incubation at 4 °C, completely remove all working solution and wash cells three times with ice-cold ESGRO growth medium. After washing, add adequate fresh RT ESGRO growth medium to cover the cells, and incubate cells in an incubator at 37 °C, 95% humidity, and 5% CO2 for 30 min. Strictly protect the incubation from light. Wash cells with fresh ESGRO growth medium at RT. Stained cells on glass bottom dishes will be directly examined and imaged with the Zeiss LSM 780 UV confocal microscope. Note: Cells can also be counter-stained with other dyes, such as nuclei staining with DAPI or DRQA5 (Fan et al., 2021). The dynamic of BODIPY FL-sphingomyelins in stained mESCs will be analyzed using a Zeiss LSM 780 UV inverted confocal microscope equipped with a cell culture chamber that provides appropriate cell growth conditions (e.g., 37 °C, 95% humidity, and 5% CO2). Time-lapse images of stained mESCs are acquired every 5 min for up to 12 h. An example of the dynamic of BODIPY FL-sphingomyelins in mESCs can be found in our original research article (Fan et al., 2021). Analyze mESCs stained with BODIPY FL C5-sphingolipid analogs by FACS After incubation at 37 °C, as described in step C2, wash cells with HBSS/HEPES buffer three times, to completely remove the ESGRO growth medium. Completely disassociate mESCs cells by adding an appropriate amount of 0.05% trypsin (approximately 300–500 µL for each well of the 6-well plate), then incubating at 37 °C for 3 min. Wash cells twice with 30 mL of HBSS/HEPES buffer in 50-mL Corning tubes to completely remove residual trypsin and harvest cells by centrifuging at 300 × g for 3 min. Note: Extra amount of trypsin and extended digestion time can be applied for this step to complete dissociate large mESC colonies and prevent formation of cell clumps. Discard the supernatant and resuspend the cell pellets in 1 mL of HBSS/HEPES buffer, then transfer the cells into 5-mL round bottom polystyrene test tubes for FACS analysis. Note: Samples must be strictly protected from light in the two steps above. The parameters setup for visualization and quantification analysis by BD LSRFortessa FACS are based on the spectral characteristics of the BODIPY FL-sphingolipid described in Table 1. Table 1. Spectral characteristics of BODIPY FL-sphingolipid (recommended in the manual of Molecular Probes) Label Absorption/Emission (nm) Optical Filter BODIPY FL 505/511 Omega/Chroma XF26, XF115/71010,41012 Data analysis Confocal imaging analysis: the concentration of BODIPY FL-sphingolipid in cellular membranes can be determined from the confocal images based on their spectral properties. Generally, when excited with λex = 488 nm, cell organelles or regions containing high levels of BODIPY FL-sphingolipid analogs fluoresce red (λem ≥ 590 nm), while cell organelles or regions containing low levels of BODIPY FL-sphingolipid analogs fluoresce green (λem = 520–590) (Figure 3). Therefore, the concentration of BODIPY FL-sphingolipid analogs in cellular membranes can be estimated by quantifying the ratio of green to red fluorescence from microscopic images (Pagano et al., 1991; Chen et al., 1997). This method has been previously applied to estimate the concentration of BODIPY FL-lipid analogs in membranes of various cell types (Pagano and Chen, 1998; Marks et al., 2008). Please note that, when cultured in defined serum free medium, BODIPY FL-sphingomyelin stained mESCs only emit green fluorescence, as shown in our original research article (Fan et al., 2021). Figure 3. Schematic illustration of the image acquisition process using the Zeiss LSM 780 inverted confocal microscope FACS data analysis: the FACS data from mESCs stained with BOPDIPY FL C5-sphingomyelin analogs are exported to BDFACSDivaTM software (version 8.0.1) for further analysis (Figure 4). All stained cells are initially gated by plotting forward scatter height (FSC-H) versus forward scatter area (FSC-A), to exclude doublets, then by plotting side scatter area (SSC-A) versus FSC-A to exclude the debris in the cell population. This gating strategy results in a population of “monodispersed state” single cells (Figure 4: the two plots on top of A and B). Next, the single parameter histogram of cell counts versus FSC-A is used to identify and gate the cell population carrying positive BODIPY fluorescence (FITC-A) after the comparison between positive sample (Figure 4B, the two middle plots) and negative control (Figure 4A, the two middle plots). Finally, the average FITC intensity of all counted single cells is calculated (Figure 4A and 4B, plots and tables in the bottom). The relative BODIPY fluorescence is calculated by normalizing the average intensity reading of each group against the wild-type (WT) group. Values are expressed as mean fluorescent intensity (MFI) ± standard error of mean from at least three independent experiments or biological replicates. Figure 4. Determination of fluorescence intensity in mESCs stained with BODIPY FL C5-sphingomyelin by FACS. Data is analyzed with BDFACSDivaTM software version 8.0.1. for (A) unstained negative control and (B) stained positive mESCs. Recipes 0.1% Gelatin solution Reagent Final concentration Amount Gelatin (powder) 0.1%(W/V) 1 g H2O n/a 1,000 mL Total n/a 1,000 mL 70% ethanol Reagent Final concentration Amount Ethanol (absolute) 70% 700 mL H2O n/a 300 mL Total n/a 1,000 mL Chloroform:ethanol (19:1 v/v) Reagent Final concentration Amount Chloroform (absolute) 95% 0.95 mL Ethanol 5% 0.05 mL Total n/a 1 mL HBSS/HEPES buffer (pH 7.4) Reagent Final concentration Amount HBSS 1× 495 mL HEPES buffer 10 mM 5 mL Total n/a 500 mL Acknowledgments We thank Dr. Carl Bortner and Mr. Jeff Tucker for critical reading of the manuscript. The authors also thank Yolanda L. Jones, National Institutes of Health Library, for editing assistance. The research related to this work was supported by the Intramural Research Program of National Institute of Environmental Health Sciences of the NIH Z01 ES102205 (to X. L.). Fan, W., Tang, S., Fan, X., Fang, Y., Xu, X., Li, L., Xu, J., Li, J. L., Wang, Z. and Li, X. (2021). SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc-SMPDL3B. Elife 10: e67452. doi: https://doi.org/10.7554/eLife.67452. Competing interests The authors declare that no competing interests exist. References Abreu, M. S., Estronca, L. M., Moreno, M. J. and Vaz, W. L. (2003). Binding of a fluorescent lipid amphiphile to albumin and its transfer to lipid bilayer membranes. Biophys J 84(1): 386-399. Alessenko, A. V. and Albi, E. (2020). Exploring Sphingolipid Implications in Neurodegeneration. Front Neurol 11: 437. Bienias, K., Fiedorowicz, A., Sadowska, A., Prokopiuk, S. and Car, H. (2016). Regulation of sphingomyelin metabolism. Pharmacol Rep 68(3): 570-581. Chattopadhyay, A. and London, E. (1987). Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 26(1): 39-45. Chen, C. S., Martin, O. C. and Pagano, R. E. (1997). Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells. Biophys J 72(1): 37-50. Fan, W., Tang, S., Fan, X., Fang, Y., Xu, X., Li, L., Xu, J., Li, J. L., Wang, Z. and Li, X. (2021). SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc-SMPDL3B. Elife 10: e67452. Gault, C. R., Obeid, L. M. and Hannun, Y. A. (2010). An overview of sphingolipid metabolism: from synthesis to breakdown. Adv Exp Med Biol 688: 1-23. Grassi, S., Chiricozzi, E., Mauri, L., Sonnino, S. and Prinetti, A. (2019). Sphingolipids and neuronal degeneration in lysosomal storage disorders. J Neurochem 148(5): 600-611. Heinz, L. X., Baumann, C. L., Koberlin, M. S., Snijder, B., Gawish, R., Shui, G., Sharif, O., Aspalter, I. M., Muller, A. C., Kandasamy, R. K., et al. (2015). The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity. Cell Rep 11(12): 1919-1928. Hoekstra, D. and Kok, J. W. (1992). Trafficking of glycosphingolipids in eukaryotic cells; sorting and recycling of lipids. Biochim Biophys Acta 1113(3-4): 277-294. Johnson, I. D., Kang, H. C. and Haugland, R. P. (1991). Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores. Anal Biochem 198(2): 228-237. Karolin, J., Johansson, L. B. A., Strandberg, L. and Ny, T. (1994). Fluorescence and Absorption Spectroscopic Properties of Dipyrrometheneboron Difluoride (Bodipy) Derivatives in Liquids, Lipid-Membranes, and Proteins. J Am Chem Soc 116(17): 7801-7806. Kok, J. W., Eskelinen, S., Hoekstra, K. and Hoekstra, D. (1989). Salvage of glucosylceramide by recycling after internalization along the pathway of receptor-mediated endocytosis. Proc Natl Acad Sci U S A 86(24): 9896-9900. Koval, M. and Pagano, R. E. (1989). Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts. J Cell Biol 108(6): 2169-2181. Koval, M. and Pagano, R. E. (1990). Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts. J Cell Biol 111(2): 429-442. Koval, M. and Pagano, R. E. (1991). Intracellular transport and metabolism of sphingomyelin. Biochim Biophys Acta 1082(2): 113-125. Laude, A. J. and Prior, I. A. (2004). Plasma membrane microdomains: organization, function and trafficking. Mol Membr Biol 21(3): 193-205. Marks, D. L., Bittman, R. and Pagano, R. E. (2008). Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells. Histochem Cell Biol 130(5): 819-832. Martin, O. C. and Pagano, R. E. (1987). Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. Evidence for a protein-mediated and ATP-dependent process(es). J Biol Chem 262(12): 5890-5898. Martin, O. C. and Pagano, R. E. (1994). Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms. J Cell Biol 125(4): 769-781. Mayor, S., Presley, J. F. and Maxfield, F. R. (1993). Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J Cell Biol 121(6): 1257-1269. Pagano, R. E. (1990). The Golgi apparatus: insights from lipid biochemistry. Biochem Soc Trans 18(3): 361-366. Pagano, R. E. and Chen, C. S. (1998). Use of BODIPY-labeled sphingolipids to study membrane traffic along the endocytic pathway. Ann Ny Acad Sci 845: 152-160. Pagano, R. E., Martin, O. C., Kang, H. C. and Haugland, R. P. (1991). A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol 113(6): 1267-1279. Pagano, R. E., Watanabe, R., Wheatley, C. and Dominguez, M. (2000). Applications of BODIPY-sphingolipid analogs to study lipid traffic and metabolism in cells. Methods Enzymol 312: 523-534. Rosenwald, A. G. and Pagano, R. E. (1993). Intracellular transport of ceramide and its metabolites at the Golgi complex: insights from short-chain analogs. Adv Lipid Res 26: 101-118. Schnaar, R. L. and Kinoshita, T. (2015). Glycosphingolipids. (3rd edition). In: Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., Darvill, A. G., Kinoshita, T., Packer, N. H., Prestegard, J. H., et al. (Eds.) Essentials of Glycobiology. 125-135. Tanosaki, S., Tohyama, S., Kishino, Y., Fujita, J. and Fukuda, K. (2021). Metabolism of human pluripotent stem cells and differentiated cells for regenerative therapy: a focus on cardiomyocytes. Inflamm Regen 41(1): 5. Wolf, D. E., Winiski, A. P., Ting, A. E., Bocian, K. M. and Pagano, R. E. (1992). Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer. Biochemistry 31(11): 2865-2873. Article Information Copyright Fan and Li. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Stem Cell > Embryonic stem cell > Cell staining Cell Biology > Cell imaging > Confocal microscopy Cell Biology > Cell staining > Lipid Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Acutely Modifying Phosphatidylinositol Phosphates on Endolysosomes Using Chemically Inducible Dimerization Systems Wei Sheng Yap [...] Maxime Boutry Oct 5, 2024 320 Views Measuring Piezo1 and Actin Polarity in Chemokine-Stimulated Jurkat Cells During Live-Cell Imaging Chinky Shiu Chen Liu [...] Dipyaman Ganguly Oct 5, 2024 257 Views Quantitative Analysis of Kinetochore Protein Levels and Inter-Kinetochore Distances in Mammalian Cells During Mitosis Neeraj Wasnik [...] Sivaram V. S. Mylavarapu Dec 5, 2024 254 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,556	https://bio-protocol.org/en/bpdetail?id=4556&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution XZ Xuepei Zhang OL Olga Lytovchenko SL Susanna L. Lundström RZ Roman A. Zubarev MG Massimiliano Gaetani Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4556 Views: 2326 Reviewed by: Chiara AmbrogioAnna A. Zorina Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Feb 2022 Abstract Chemical proteomics focuses on the drug–target–phenotype relationship for target deconvolution and elucidation of the mechanism of action—key and bottleneck in drug development and repurposing. Majorly due to the limits of using chemically modified ligands in affinity-based methods, new, unbiased, proteome-wide, and MS-based chemical proteomics approaches have been developed to perform drug target deconvolution, using full proteome profiling and no chemical modification of the studied ligand. Of note among them, thermal proteome profiling (TPP) aims to identify the target(s) by measuring the difference in melting temperatures between each identified protein in drug-treated versus vehicle-treated samples, with the thermodynamic interpretation of “protein melting” and curve fitting of all quantified proteins, at all temperatures, in each biological replicate. Including TPP, all the other chemical proteomics approaches often fail to provide target deconvolution with sufficient proteome depth, statistical power, throughput, and sustainability, which could hardly fulfill the final purpose of drug development. The proteome integral solubility alteration (PISA) assay provides no thermodynamic interpretation, but a throughput 10–100-fold compared to the other proteomics methods, high sustainability, much lower time of analysis and sample amount requirements, high confidence in results, maximal proteome coverage (~10,000 protein IDs), and up to five drugs / test molecules in one assay, with at least biological triplicates of each treatment. Each drug-treated or vehicle-treated sample is split into many fractions and exposed to a gradient of heat as solubility perturbing agent before being recomposed into one sample; each soluble fraction is isolated, then deep and quantitative proteomics is applied across all samples. The proteins interacting with the tested molecules (targets and off-targets), the activated mechanistic factors, or proteins modified during the treatment show reproducible changes in their soluble amount compared to vehicle-treated controls. As of today, the maximal multiplexing capability is 18 biological samples per PISA assay, which enables statistical robustness and flexible experimental design accommodation for fuller target deconvolution, including integration of orthogonal chemical proteomics methods in one PISA assay. Living cells for studying target engagement in vivo or, alternatively, protein extracts to identify in vitro ligand-interacting proteins can be studied, and the minimal need in sample amount unlocks target deconvolution using primary cells and their derived cultures. Graphical abstract: Keywords: Chemical proteomics Protein solubility PISA Mass spectrometry Proteomics Target deconvolution Mechanism of action Drug development Background Correct target identification and elucidation of the fine mechanism of action (MoA) of ligands, both covalent and noncovalent, representing research-grade as well as already approved drugs, are both key and major bottlenecks in drug development and drug repurposing (Swinney and Anthony, 2011). The case of cancer drugs is a clear example of this. It is estimated that 97% of the tested anticancer molecules never make it to the clinic, often because the proposed mechanism of action is incorrect and the observed drug effects are due to off-target toxicity (Lin et al., 2019). Moreover, the high rate of clinical trial failures clearly indicates the need to seek more precise information on drug targets and the MoA more strictly related to the drug. Therefore, all methodological efforts in this direction are very relevant and worth pursuing. Among the -omics sciences, genomics and transcriptomics have been applied in drug development because of limited operational cost for comparing normal and diseased states, measuring changes in transcription levels, pharmacogenomics, and stratifying patients in clinical trials. However, only mass spectrometry (MS)-based proteomics—and particularly chemical proteomics—can fill in the gap in knowledge of the drug–target–phenotype relationship, by exploring it at a proteome-wide level, with deep proteome coverage, and unbiasedly. Chemical proteomics encompasses different methods, each attacking the problem from a different angle, and each limited by the original intention and experimental design. The MS-based approaches for the identification of protein interactions using affinity-based purification with chemically engineered ligand probes (Rix and Superti-Furga, 2009) have the strong limitation of the uncertainty in the functional properties of the engineered bait compared to the original ligand. Moreover, even with a well-performing click chemistry used to lock the protein to the bait, the always present promiscuity in binding results in locking background proteins. Additionally, the non-specific noncovalent interactions in the affinity enrichment step lead to an increased background (Wright and Sieber, 2016). The consequent limited specificity of the results called for the development of target deconvolution approaches requiring no chemical modification of the ligand. Therefore, the use of unbiased, proteome-wide, and MS-based proteomics approaches for target deconvolution (Kwon and Karuso, 2018) have recently gained a major place in chemical proteomics. A promising proteome-wide approach for protein target identification in living cells uses the specificity of protein abundance regulation in ligand-induced late apoptosis (Gaetani and Zubarev, 2019). The concept, originally developed for chemotherapeutics, was also successfully applied to metallodrugs and nanoparticles (Chernobrovkin et al., 2015; Lee et al., 2017; Tarasova et al., 2017), and then extended to a vast library of cancer drugs and multiple cell lines (Saei et al., 2019). This last work produced an online tool assisting in the deconvolution of targets for molecules exhibiting significant cell toxicity at 48h, showing specific regulation of drug targets and mechanistic factors in late apoptosis. However, limitations regarding elucidation of direct primary drug targets and mechanisms direct to the general call for orthogonal methods based on alternative principles. In thermal proteome profiling (TPP) (Savitski et al., 2014; Franken et al., 2015), drug targets are identified by the increase in protein melting temperatures (Tm) obtained by curve fitting of all temperature points measured in the tandem mass tag (TMT) batch used for each of the drug- or vehicle-treated biological replicates (2 h treatment). Each biological replicate requires a complete TMT batch, and the overall TPP experiment requires as many TMT batches as the biological samples. Therefore, in TPP, scalability and the use of more than two biological replicates per treatment type is complicated by the high sample amounts and costs; straightforward interpretation of the curves obtained within complex intracellular environment as “protein melting” is questionable; data analysis of multibatch TMT and suboptimal curve fitting damage the depth of proteome coverage, with increasing missing values among replicates resulting in false positive and false negative rates (Brenes et al., 2019). The proteome integral solubility alteration (PISA) assay, commonly named “PISA,” in which this protocol focuses, provides deep and quantitative proteome analysis of the changes in amount of the soluble fraction of each identified protein. Each drug-treated or vehicle-treated sample—thus, each soluble proteome—is first exposed to a gradient of heat as solubility perturbing agent, inducing protein aggregation (Gaetani et al., 2019). Each soluble fraction is isolated, and then deep and quantitative proteomics is applied across all multiplexed biological samples. PISA was proven correct in finding primary and secondary targets in its proof-of-principle work using different drugs with known targets in different cell lines and respective lysates. The method successfully performed target identification and provided elements for MoA elucidation by comparing parallel experiments in living cells and lysates. Indeed, PISA can be applied to living cells for studying target engagement in vivo, or alternatively to protein extracts to identify in vitro ligand-interacting proteins. Further studies applying PISA on an antimicrobial molecule showing anticancer properties were recently published, proving PISA correct also in cases where the target is not known, as it was further validated with follow-up experiments of a different nature (Heppler et al., 2022). Using a statistically relevant number of biological replicates for each tested condition within the same TMT batch, PISA is designed to gain the full robustness and quantification accuracy of TMT multiplexing, and to provide high throughput and high sustainability to target deconvolution, with depth of proteome analysis up to approximately 10,000 proteins in human cells (using current protocols). With up to 18 biological samples per TMT-multiplexed assay, and no thermodynamic interpretation, the PISA assay maximizes throughput, sustainability, proteome coverage, statistical power of analysis (three or more replicates), confidence in results, and several drug molecules analyzed per TMT batch (up to five). The PISA assay conveniently takes advantage of the latest TMTpro technology (Thermo Scientific) (Li et al., 2020b), further extended to 18-plex, allowing for up to nine biological replicates of the ligand compared to vehicle in a single LC–MS/MS experiment. Further developments in TMT technology will immediately increase the PISA throughput. The PISA assay also integrates the advances in deep, quantitative proteomics, including extensive off-line high pH fractionation and nanoscale liquid chromatography (nLC)–MS of all produced fractions. PISA does away with the sigmoidal solubility-temperature curves and Tm derivation, as each sample is split in equal portions that are exposed to different temperatures before being recomposed into one and multiplexed with the other samples. In PISA, the final amount of each protein would correspond to the integral of a putative solubility-temperature dependence curve, regardless of its shape and function. The information lost on the shape of the solubility-temperature dependence curves turned out to be largely irrelevant for drug target identification. The normalized difference between drug- and vehicle-treated samples is called ΔSm; the ratio (R) of the soluble abundances is also used, and sometimes more advantageous for final data analysis. The number of samples analyzed in PISA by MS is the same regardless of the number of temperature points used, while in TPP, these numbers are in a linear dependence. Therefore, PISA can easily afford a larger number of temperature points, increasing precision of comparative quantification across replicates, which is already increased by removal of the curve fitting. Furthermore, to increase the ΔSm (or R) sensed in PISA by specific targets at a specific temperature range, the last can be narrowed around the region with the highest solubility changes, for most proteins of interest (Li et al., 2020a). The original PISA assay makes use of the temperature to challenge protein solubility, as temperature is the most optimized and standardized protein aggregation/precipitation agent, easy to control, and removable from samples; however, different PISA assays using organic solvents (Van Vranken et al., 2021) or kosmotropic salts (Beusch et al., 2022) have also been developed. These temperature-free variants of the PISA assay prove its correct design, to robustly interpret and reproducibly measure the proteome amount changes in the soluble fraction as protein solubility rather than protein melting variations. All in all, PISA increases the throughput by 10–100-fold compared to any other chemical proteomics method, routinely allowing experiments with three or four biological replicates for several compounds simultaneously, and making possible larger studies that would otherwise be prohibitively expensive. Of further relevance, PISA also enables analysis of much reduced sample amounts, and is currently optimized for minimal cell amounts typical for cell culture models of primary cells, iPSC-derived cell cultures, and 3D cell cultures, including organoids. PISA can also be applied to a range of ligand concentrations, with preferential selection of the target binding at a lower concentration (2D PISA) (Gaetani et al., 2019). In PISA, the reproducible protein amount changes between the soluble fractions of the drug-treated samples and controls can represent the target and off-target interactions, factors involved in early MoA, modified proteins, and early complex assembly variations occurred during the applied drug treatment. The major PISA capabilities of target deconvolution and MoA elucidation reside in the versatility to accommodate within the same TMT multiplex a project design tailored for specific needs, for example, with robust comparisons among various compounds, or incubation at various time points, drug concentrations, or cells. For fuller target deconvolution, PISA can also be integrated in one multiplex with its orthogonal method expression proteomics—altogether named PISA-Express. In PISA-Express, both the proteome regulation as cellular adaptation / response to the drug in 24 h/48 h, together with the solubility shift for 30–90 min incubation in PISA are studied. A relevant feature of PISA-Express is the possibility to analyze solubility alteration at longer times of drug incubation, when cellular protein expression is altered by the drug treatment, and the solubility shifts can be normalized to the total protein abundance variation, due to the integration of PISA and expression proteomics in the same analysis (Sabatier et al., 2021). A third analysis dimension—RedOx proteomics—can also be integrated, and allows the 3D PISA profiling into a single TMT multiplex set, which could be a future industrial standard analysis for drug target identification and MoA elucidation. Materials and Reagents Cell culture Tubes for centrifugation 50 mL and 15 mL (Sarstedt, catalog numbers: 62.559 and 62.554.502) Sterile 25 cm2 cell culture flask (Sarstedt, catalog number: 83.3910.002) Cell culture medium [e.g., Dulbecco’s modified Eagle’s medium (DMEM)] (Thermo Fisher Scientific, catalog number: 11685260; original manufacture: Lonza, catalog number: BE12-614F) Fetal Bovine Serum (FBS, Thermo Fisher Scientific, catalog number: 11560636) Penicillin-streptomycin solution (Gibco, catalog number: 15140-122) L-Glutamine (Gibco, catalog number: A2916801) Ca2+Mg2+-free Dulbecco’s PBS (Cytiva, catalog number: 10462372) TrypLE express enzyme solution (Thermo Fisher Scientific, catalog number: 12605036) Dimethyl sulfoxide solution (DMSO, Merck, catalog number: D8418) PISA treatment 0.2 mL PCR tube strips with caps (Thermo Fisher Scientific, catalog number: AB0452) Protein low-binding tubes for centrifugation, 1.5 mL and 2 mL (Thermo Fisher Scientific, catalog numbers: 10708704 and 10718894; original manufacture: Eppendorf, catalog number: 0030108116 and 0030108132) 1.5 mL polypropylene tubes for ultracentrifugation (Beckman Coulter, catalog number: 357448) Ca2+Mg2+-free Dulbecco’s PBS (Cytiva, catalog number: 10462372) 100× protease inhibitor cocktail solution (Thermo Fisher Scientific, catalog number: 78439) Liquid N2 in a container Nonidet P-40 (NP40) (Thermo Fisher Scientific, catalog number: 11596671) Milli-Q water Cell lysis buffer (see Recipes) 20% NP40 (see Recipes) Soluble protein fraction processing Micro-BCA assay kit (Thermo Fisher Scientific, catalog number: 23235) Dithiothreitol (DTT, Merck, catalog number: D0632) Iodoacetamide (IAA, Merck, catalog number: I6125) Acetone (Thermo Fisher Scientific, catalog number: 10417440) 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS, Merck, catalog number: E9502) Urea (Merck, catalog number: U5378) Lyophilized LysC enzyme (FUJIFILM Wako Pure Chemical Corporation, catalog number: 125-05061) Lyophilized sequencing grade modified trypsin enzyme (Promega, catalog number: V5111) Trypsin Resuspension Buffer (Promega, catalog number: V5111) Tandem Mass Tag (TMT) 10-plex, TMTproTM 16-plex, or TMTproTM 18-plex isobaric label reagent set (Thermo Fisher Scientific, catalog numbers: 90110, A44520, or A52045) Water-free acetonitrile (Thermo Fisher Scientific, catalog number: 10222052) 50% hydroxylamine solution (Thermo Fisher Scientific, catalog number: 90115) Trifluoroacetic acid (TFA, Merck, catalog number: 302031) Methanol (Skandinaviska Genetec, catalog number: RH1019/2.5) Acetonitrile (ACN, Thermo Fisher Scientific, catalog number: A955-212) Formic acid (FA, Merck, catalog number: 1002641000) pH indicator paper (VWR, catalog number: 85403.600) C18 desalting columns (Waters, catalog number: WAT054960) 0.5 M DTT solution (see Recipes) 0.5 M IAA solution (see Recipes) 20 mM EPPS buffer (pH = 8.2) (see Recipes) 20 mM EPPS buffer (pH = 8.2) including 8M urea (see Recipes) 50% ACN solution (see Recipes) 0.1% TFA (v/v) solution (see Recipes) 2% ACN (v/v) solution including 0.1% TFA (see Recipes) 2% ACN (v/v) solution including 0.1% FA (LC-MS buffer A) (see Recipes) 50% ACN (v/v) solution including 0.1% FA (see Recipes) 80% ACN (v/v) solution including 0.1% FA (see Recipes) Peptide separation, mass spectrometry, and proteomics data analysis 28%–30% NH4OH water solution (Thermo Fisher Scientific, catalog number: 221228-1L-A) Milli-Q water Acetonitrile (Thermo Fisher Scientific, catalog number: A955-212) Reversed-Phase C18 guard column (Waters, catalog number: 186007769) High pH Reversed-Phase C18 Column (Waters, catalog number: 186003621) 0.1% FA in water (Thermo Fisher Scientific, catalog number: 10188164) 0.1% FA in ACN (Thermo Fisher Scientific, catalog number: 10118464) Nano trap-column (Thermo Fisher Scientific, catalog number: 164535) C18 EasySpray nLC peptide separation column (Thermo Fisher Scientific, catalog number: ES803A) 20 mM NH4OH in H2O (high pH Buffer A) (see Recipes) 20 mM NH4OH in ACN (high pH Buffer B) (see Recipes) 98% ACN in H2O including 0.1% FA (LC-MS buffer B) (see Recipes) Equipment Cell culture 37 °C 5% CO2 incubator (Thermo Fisher Scientific, Forma Steri-cycle i160) Laminar flow cabinet (ninoSAFE, class II) Light microscope (ZEISS, Primo Vert) Cell counter (Bio-Rad, model: TC10) Benchtop centrifuge with swinging-bucket rotor for 15 mL tubes (Eppendorf, model: Centrifuge 5804R) PISA treatment Thermal cycler (Applied Biosystems, SimpliAmp) Ultracentrifuge (Beckman Coulter, model: Optima XPN-80) Fixed-Angle Titanium Rotor for centrifugation (Beckman Coulter, model: Type 45 Ti) Thermomixer (Eppendorf, model: ThermoMixer C) Vortex (Scientific Industries, model: Vortex-Genie 2) Milli-Q water purification system (Millipore, model: IQ 7000) Soluble protein fraction processing Absorbance plate reader (BioTek, Epoch) Benchtop centrifuge for 1.5 mL tubes (Eppendorf, model: Centrifuge 5430R) Manifold for vacuum extraction for small columns (Waters, Extraction Manifold) Speed-Vacuum concentrator (Eppendorf, concentrator plus) Peptide separation, mass spectrometry, and proteomics data analysis Mass spectrometer equipped with an EASY ElectroSpray source (Thermo Fisher Scientific, Orbitrap Q Exactive HF or higher) Note: This protocol refers to Orbitrap Q Exactive HF. However, Orbitrap Fusion, Orbitrap Fusion Lumos, Orbitrap Exploris 480, and Orbitrap Eclipse can be also used. Nanoflow UPLC system with fraction collector and UV detector (Thermo Fisher Scientific, Ultimate 3000) Capillary HPLC system for peptide high pH C-18 fractionation with fraction collector and UV detector (Thermo Fisher Scientific, Dionex Ultimate 3000) Software Absorbance plate reader software (BioTek, Gen5 2.09) Q Exactive HF-Orbitrap MS 2.9 build 2926 (Thermo Fisher Scientific) Thermo Scientific SII for Xcalibur (Thermo Fisher Scientific) MaxQuant software (Max Planck Institute of Biochemistry) Proteome Discoverer 2.5 (Thermo Fisher Scientific) Microsoft Office software (Microsoft) Prism (GraphPad) Procedure Cell culture and ligand treatment Thaw the cells to be studied from the N2 stock vials, and wash them in 10–15 mL of their regular cell culture medium (e.g., DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 2 mM L-Glutamine), and sediment cells down by centrifugation according to cell type-specific recommendations (250 × g for 5 min). Discard the supernatant and resuspend the cells in complete growth medium. The day before the PISA experiment, split the cells in the number of samples as by experimental design, up to 18–25-cm2 cell culture flasks containing 5 mL of complete cell growth medium in each flask, and let the cells attach overnight. Incubate each biological replicate with active ligands to be studied (e.g., drugs, active molecules of interest)—here named “D”—at a biologically active concentration previously tested (e.g., IC50 or similar) using a stock with 1,000–2,000× concentration, with at least three biological replicates per D ligand. Control replicates are to be treated with the volume of the vehicle solution (e.g., DMSO). In the chosen example of PISA multiplexing scheme, five D ligands (D1–D5) with three biological replicates each and three replicates of the vehicle-treated controls are used, for a total of 18 biological samples. Note: The number of biological replicates can be higher, as recommended for lower numbers of D ligands to be tested, depending on the number of molecules or conditions to be tested, and up to 18, by using the TMTproTM 18-plex label reagent set, which can host up to five ligand-treated or vehicle-treated samples in triplicate. Incubate treated cells for 30–90 min in a 5% CO2 cell culture incubator at 37 °C. At the end of the treatment with each D or vehicle, collect cells from each flask using TrypLE, and wash the cell pellets twice with 10 mL of PBS, followed by centrifugation (250 × g for 5 min). Protein extraction for PISA assay samples Resuspend the cell pellet in 750 μL of PBS buffer including protease inhibitors. Gently mix, resuspend cells homogeneously, and distribute 45 μL in each 0.2-mL tube, for a total of 16 temperature points to be used for each sample. Perform step B2 for each sample. Heat each sample at a range of 44 to 59 °C, with 1 °C intervals, and perform the temperature treatment for 3 min at each temperature. Notes: By using a gradient thermal cycler, samples can be treated at different temperatures at the same time. The temperature range can be varied and tuned according to specific expectations or needs, with virtually unlimited number of temperature points. Leave the samples at 23 °C for 6 min. At the end of the 23 °C incubation, snap freeze the samples in liquid N2. Thaw all samples at 37 °C, vortex for 10 s, and snap freeze in liquid N2. Repeat step B7 four times, for a total of five freeze-thaw cycles. For each sample and replicate, combine the contents of 16 tubes corresponding to all temperatures of that replicate into one protein low-binding tube, and add 20% NP40 solution, up to a final concentration of 0.4%, which at this point should not resolubilize insoluble proteins. Incubate all samples shaking at 350 rpm/min at 4 °C, in a thermomixer or a refrigerated room, for 30 min. Perform ultracentrifugation at 150,000 × g at 4 °C for 30 min, and recover the supernatant of each replicate without disrupting the pellet. Soluble protein fraction processing Day 1 Measure the total protein concentration of all samples, using a micro-BCA kit and an absorbance plate reader. Take the volume corresponding to 50 μg for each sample. To equalize volumes, dilute all samples with PBS up to the volume of the sample with the least concentration. Perform reduction by adding 0.5 M DTT solution to a final concentration of 8 mM, and incubate samples at 55 °C for 45 min. Add 0.5 M IAA solution to a final concentration of 25 mM, and incubate samples in darkness at 25 °C for 30 min. Precipitate proteins using cold acetone at -20 °C overnight, at a sample:acetone ratio of 1:6 (v/v). Day 2 Collect the precipitated proteins by centrifugation (10,000 × g, 10 min), remove the supernatant, and air-dry the pellet for 5 min. Solubilize each pellet in 15 μL of EPPS buffer at pH 8.2 including 8 M urea for 10 min. Add 14 μL of EPPS buffer to each sample. Dissolve 20 μg LysC powder using 30 μL of EPPS buffer, then add 1 μL of this LysC enzyme solution (equivalent to 0.67 μg of LysC) to each sample, and allow the digestion to proceed gently mixing at 350 rpm/min at 30 °C for 6 h. Add 90 μL of EPPS buffer to each sample. Dissolve 20 μg lyophilized sequencing grade modified trypsin using 20 μL of trypsin resuspension buffer, then add 1 μL of this trypsin solution (equivalent to 1 μg of trypsin) to each sample, and let the digestion occur gently mixing at 350 rpm/min at 37 °C overnight. Stop the reaction the day after by placing the samples on ice. Take out a volume of 60 μL, corresponding to 25 μg, from each of the digested protein samples. Dissolve 800 μg TMTpro 18-plex reagent using 150 μL of water-free acetonitrile, and label each sample with 25 μL of a different TMTpro 18-plex labeling reagent solution. Let the labeling reaction occur at room temperature for 2 h. Keep the remaining 25 μg of each sample at -80 °C as a backup. The remaining TMTpro 18-plex reagent solution can be stored at -80 °C after speed-vacuum drying. Stop the reaction by adding 50% hydroxylamine solution to quench labeling, to a final concentration of 0.5% hydroxylamine. Pool the labeled samples together in a 2-mL tube and concentrate the obtained multiplexed sample at least two-fold in a speed-vacuum concentrator. Acidify the samples using acidifying solution, to a final concentration of ~1% TFA, to reach a final pH < 3. Perform desalting of the multiplexed sample using a C-18 desalting column and a vacuum manifold. The sample desalting procedure, in sequence, is as follows: Wash the column with 0.45 mL of 100% methanol. Wash with 0.45 mL of 50% ACN (v/v) solution including 0.1% TFA (v/v). Equilibrate with 0.9 mL of 0.1% TFA (v/v); load the sample. Wash with 0.45 mL of 2% ACN (v/v) solution including 0.1% TFA (v/v). Wash with 0.15 mL 2% ACN (v/v) solution including 0.1% FA (v/v). Elute with 0.45 mL of 50% ACN (v/v) solution including 0.1% FA (v/v). Collect the elution in a 2-mL protein low-binding tube. Elute with 0.3 mL of 80% ACN (v/v) solution including 0.1% FA (v/v). Collect the elution in the same 2-mL protein low-binding tube of the previous step. Concentrate the desalted and cleaned sample to dryness, using a speed-vacuum concentrator (overnight concentration is possible). Peptide separation, mass spectrometry, and proteomics data analysis Resuspend the final multiplexed sample in 100 µL of High pH Buffer A, and use 50 μL (equivalent to 225 μg of original starting protein amount) for the next steps. Keep the remaining at 80 °C for backup (it is also possible to dry it again in a speed-vacuum concentrator before storing it, which is advised for long term storage). Inject the resuspended sample for off-line, high pH reversed-phase separation and fractionation into a capillary HPLC system equipped with a 25-cm long, 2.1-mm wide (inner diameter) C18 column, at a flow rate of 200 μL/min. Perform peptide separation with a binary solvent system consisting of high pH Buffer A and high pH Buffer B, using a gradient from 1% to 6% B from 2 min to 6 min, to 26% B in 36 min, to 53% B in 4 min, to 63% B in 3 min, and then at 63% B for 5 min. Monitor the elution by UV at 214 nm (Figure 1). Figure 1. Example of a chromatogram showing the separation of TMT-labeled peptides during high pH reverse phase HPLC, monitored by absorbance at 214 nm (blue), and its gradient (black and dotted line). The light blue area depicts the sample collected in fractions, while the fractions are delimited by light blue and dotted lines. Collect 96 fractions of 100 μL in a ninety-six-well plate, starting at the minute 2.5 after injection. Concatenate the ninety-six 100-μL fractions into 48 concatenated fractions by merging each fraction “n” with “n+48” (for example, 1 with 49, 2 with 50 … and 48 with 96). Dry the final fractions using a speed-vacuum concentrator. Inject the equivalent of 1 μg peptides of each fraction on a reversed-phase C18 nano-LC (nLC) column of a nano-LC–ESI–MS/MS instrument, with a C18 nano-trap column and the column for nLC separation at the temperature of 55 °C during peptide analysis. Perform nLC separation using a binary solvent system, consisting of LC-MS buffer A and LC-MS buffer B, with a gradient of 4% to 26% B from 5 min to 91 min, 26% to 95% B in 9 min, and 5 min in 95% B, prior to equilibration in 4% A (Figure 2). Figure 2. Gradient for nano-LC separation of TMT-labeled peptides before MS/MS analysis Perform quantitative bottom-up proteomics analysis for TMTpro-based MS/MS acquisition. Acquire mass spectra in a mass-to-charge (m/z) range of 375–1,500, with a resolution of 120 000 at m/z 200 for MS1. Set the AGC target to 3 × 106, with a maximum injection time of 100 ms. Select the 17 most intense peptide peaks for peptide fragmentation via HCD, with the NCE value set at 33. The ion selection abundance threshold is set at 0.1% with charge exclusion of z = 1 ions. The MS/MS spectra are acquired at a resolution of 45 000, with an AGC target value of 2 × 105 ions, or a maximum injection time of 120 ms. The fixed first m/z is 110, and the isolation window is 1.6 m/z. The instrument is operated in the positive ion mode for data-dependent acquisition of MS/MS spectra, with a dynamic exclusion time of previously selected precursor ions of 45 s. Data analysis Perform identification and quantification of peptides and proteins using MaxQuant or Proteome Discoverer (preferentially with the most recent versions of these software) as proteomic database search engine software. Search against the UniProt complete proteome database accordingly to the species of origin of the used cell culture (e.g., UP000005640 for human samples). When performing the database search for peptide and protein identification, use: cysteine carbamidomethylation as a fixed modification; TMT-related modifications; deamination on methionine oxidation, arginine, and asparagine as variable modifications; trypsin enzyme specificity with maximum of two missed cleavages. Use a 1% false discovery rate as a filter at both protein and peptide levels. Remove all potential contaminants and reversed-hit peptides and include only proteins with at least two unique peptides in the quantitative analysis. For analysis of the obtained protein data, eliminate the proteins with all missing values in all replicates in any treatment and normalize the quantified abundance of each protein in each sample (labeled with a different TMT) over the total intensity of all proteins for that sample. For each protein in each replicate of D1–D5 ligands, divide the normalized protein abundance by the average abundance of this protein in the vehicle-treated replicates. Measure the average ratio across replicates of each D ligand/vehicle control and calculate the Log2 values of the obtained ratios. Calculate the p-value for the assumption that the above ratio is non-zero using two-tailed Student’s t-test (with equal or unequal variance, depending on F test). For each D ligand, visualize the results by a volcano plot containing the Log2 of the ratio as x-axis and -Log10 of the corresponding p-value as y-axis. For each D ligand, the target candidates are found among the proteins with large absolute Log2 of the ratios and simultaneously large -Log10 of the p-values, also in comparison with what was obtained by the other D ligands, with potentially similar or different activities, induced phenotypes, or mechanisms. Recipes Cell lysis buffer Reagent Final concentration Amount protease inhibitors (100×) 1× 150 μL PBS n/a 1,350 mL Total n/a 15 mL 20% NP40 Reagent Final concentration Amount NP40 (absolute) 20% 3 mL Milli-Q water n/a 12 mL Total n/a 15 mL 0.5 M DTT solution Reagent Final concentration Amount DTT (absolute) 0.5 M 77 mg Milli-Q water n/a Add Milli-Q water to dissolve DTT until the volume is 1 mL Total n/a 1 mL 0.5 M IAA solution Reagent Final concentration Amount IAA (absolute) 0.5 M 93 mg Milli-Q water n/a Add Milli-Q water to dissolve IAA until the volume is 1 mL Total n/a 1 mL 20 mM EPPS buffer (pH=8.2) Reagent Final concentration Amount EPPS (absolute) 20 mM 250 mg Milli-Q water n/a Add 40 mL of Milli-Q water to dissolve EPPS 10 M NaOH solution n/a Adjust the pH until the final pH is 8.2 Milli-Q water n/a Add Milli-Q water until the volume is 50 mL Total n/a 50 mL 20 mM EPPS buffer (pH=8.2) including 8 M urea Reagent Final concentration Amount Urea (absolute) 8 M 480 mg 20 mM EPPS buffer (pH=8.2) 20 mM Add Milli-Q water to dissolve urea until the volume is 1 mL Total n/a 1 mL 50% ACN solution Reagent Final concentration Amount ACN (absolute) 50% (v/v) 25 mL Milli-Q water n/a 25 mL Total n/a 50 mL 0.1% TFA (v/v) solution Reagent Final concentration Amount TFA (absolute) 0.1 % (v/v) 50 μL Milli-Q water n/a Add Milli-Q water until the volume is 50 mL Total n/a 50 mL 2% ACN (v/v) solution including 0.1% TFA Reagent Final concentration Amount TFA (absolute) 0.1% (v/v) 50 μL ACN 2% (v/v) 1 mL Milli-Q water n/a Add Milli-Q water until the volume is 50 mL Total n/a 50 mL 2% ACN (v/v) solution including 0.1% FA (LC-MS buffer A) Reagent Final concentration Amount Acetonitrile including 0.1% FA 2% 1 mL H2O including 0.1% FA n/a 49 mL Total n/a 50 mL 50% acetonitrile (v/v) solution including 0.1% FA Reagent Final concentration Amount Acetonitrile including 0.1% FA 50 % 25 mL H2O including 0.1% FA n/a 25 mL Total n/a 50 mL 80% acetonitrile (v/v) solution including 0.1% FA Reagent Final concentration Amount Acetonitrile including 0.1% FA 0.5 M 40 mL H2O including 0.1% FA n/a 10 mL Total n/a 50 mL 20 mM NH4OH in H2O (high pH Buffer A) Reagent Final concentration Amount 28%–30% NH4OH water solution 20 mM 676 μL Milli-Q water n/a Add Milli-Q water until the volume is 500 mL Total n/a 500 mL 20 mM NH4OH in ACN (high pH Buffer B) Reagent Final concentration Amount 28%–30% NH4OH water solution 20 mM 676 μL ACN n/a Add ACN to dissolve DTT to 500 mL Total n/a 500 mL 98% ACN in H2O including 0.1% FA (LC-MS buffer B) Reagent Final concentration Amount Acetonitrile including 0.1% FA 98% 490 mL H2O including 0.1% FA n/a 10 mL Total n/a 500 mL Acknowledgments The PISA assay was invented, developed, and further optimized as method development of the Chemical Proteomics Core Facility of the Karolinska Institutet, Stockholm (https://ki.se/en/mbb/chemical-proteomics-core-facility), unique national unit of Chemical Proteomics at the Swedish infrastructures SciLifeLab (Science for Life Laboratrory, https://www.scilifelab.se/units/chemical-proteomics/), and BioMS (Swedish National Infrastructure for Biological Mass Spectrometry, https://bioms.se/technologies/chemical-proteomics/). The Chemical Proteomics Unit provides chemical proteomics support to research projects. SciLifeLab, BioMS, and Karolinska Insitutet supported this work. This protocol was adapted from the original proof-of-principle article of the PISA method entitled “Proteome integral solubility alteration: a high-throughput proteomics assay for target deconvolution” (Gaetani et al., 2019). Competing interests The authors declare no competing interests. Ethics The protocol here described is generally applicable on any cell culture, commonly on commercial cell lines, and does not have any ethics requirement per se. It is to be noted that the protocol is also suitable to be applied to studies on primary cells extracted from human and/or animal tissues, and in such cases the use of cells derived from human or animal sources needs the approval by the ethics committee. References Beusch, C. M., Sabatier, P. and Zubarev, R. A. (2022). Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein–Molecule Interactions. Anal Chem 94(19): 7066-7074. Brenes, A., Hukelmann, J., Bensaddek, D. and Lamond, A. I. (2019). Multibatch TMT Reveals False Positives, Batch Effects and Missing Values*. Mol Cell Proteomics 18(10): 1967-1980. Chernobrovkin, A., Marin-Vicente, C., Visa, N. and Zubarev, R. A. (2015). Functional Identification of Target by Expression Proteomics (FITExP) reveals protein targets and highlights mechanisms of action of small molecule drugs. Sci Rep 5(1): 11176. Franken, H., Mathieson, T., Childs, D., Sweetman, G. M. A., Werner, T., Tögel, I., Doce, C., Gade, S., Bantscheff, M., Drewes, G., et al. (2015). Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Nat Protoc 10(10): 1567-1593. Gaetani, M., Sabatier, P., Saei, A. A., Beusch, C. M., Yang, Z., Lundström, S. L. and Zubarev, R. A. (2019). Proteome Integral Solubility Alteration: A High-Throughput Proteomics Assay for Target Deconvolution. J Proteome Res 18(11): 4027-4037. Gaetani, M. and Zubarev, R. A. (2019). Functional Identification of Target by Expression Proteomics (FITExP). Mass Spectrometry-Based Chemical Proteomics. 257-266. Heppler, L. N., Attarha, S., Persaud, R., Brown, J. I., Wang, P., Petrova, B., Tošić, I., Burton, F. B., Flamand, Y., Walker, S. R., et al. (2022). The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase. J Biol Chem 298(2): 101531. Kwon, H. J. and Karuso, P. (2018). Chemical proteomics, an integrated research engine for exploring drug-target-phenotype interactions. Proteome Science 16(1): 1. Lee, R. F. S., Chernobrovkin, A., Rutishauser, D., Allardyce, C. S., Hacker, D., Johnsson, K., Zubarev, R. A. and Dyson, P. J. (2017). Expression proteomics study to determine metallodrug targets and optimal drug combinations. Sci Rep 7(1): 1590. Li, J., Van Vranken, J. G., Paulo, J. A., Huttlin, E. L. and Gygi, S. P. (2020a). Selection of Heating Temperatures Improves the Sensitivity of the Proteome Integral Solubility Alteration Assay. J Proteome Res 19(5): 2159-2166. Li, J., Van Vranken, J. G., Pontano Vaites, L., Schweppe, D. K., Huttlin, E. L., Etienne, C., Nandhikonda, P., Viner, R., Robitaille, A. M., Thompson, A. H., et al. (2020b). TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples. Nat Methods 17(4): 399-404. Lin, A., Giuliano, C. J., Palladino, A., John, K. M., Abramowicz, C., Yuan, M. L., Sausville, E. L., Lukow, D. A., Liu, L., Chait, A. R., et al. (2019). Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. 11(509): eaaw8412. Rix, U. and Superti-Furga, G. (2009). Target profiling of small molecules by chemical proteomics. Nat Chem Biol 5(9): 616-624. Sabatier, P., Beusch, C. M., Saei, A. A., Aoun, M., Moruzzi, N., Coelho, A., Leijten, N., Nordenskjöld, M., Micke, P., Maltseva, D., et al. (2021). An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation. Nature Communications 12(1): 6558. Saei, A. A., Beusch, C. M., Chernobrovkin, A., Sabatier, P., Zhang, B., Tokat, Ü. G., Stergiou, E., Gaetani, M., Végvári, Á. and Zubarev, R. A. (2019). ProTargetMiner as a proteome signature library of anticancer molecules for functional discovery. Nature Communications 10(1): 5715. Savitski, M. M., Reinhard, F. B. M., Franken, H., Werner, T., Savitski, M. F., Eberhard, D., Molina, D. M., Jafari, R., Dovega, R. B., Klaeger, S., et al. (2014). Tracking cancer drugs in living cells by thermal profiling of the proteome. 346(6205): 1255784. Swinney, D. C. and Anthony, J. (2011). How were new medicines discovered? Nature Reviews Drug Discovery 10(7): 507-519. Tarasova, N. K., Gallud, A., Ytterberg, A. J., Chernobrovkin, A., Aranzaes, J. R., Astruc, D., Antipov, A., Fedutik, Y., Fadeel, B. and Zubarev, R. A. (2017). Cytotoxic and Proinflammatory Effects of Metal-Based Nanoparticles on THP-1 Monocytes Characterized by Combined Proteomics Approaches. J Proteome Res 16(2): 689-697. Van Vranken, J. G., Li, J., Mitchell, D. C., Navarrete-Perea, J. and Gygi, S. P. (2021). Assessing target engagement using proteome-wide solvent shift assays. eLife 10: e70784. Wright, M. H. and Sieber, S. A. (2016). Chemical proteomics approaches for identifying the cellular targets of natural products. Nat Prod Rep 33(5): 681-708. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Systems Biology > Proteomics Molecular Biology > Protein > Detection Biochemistry > Protein > Quantification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Genetic Tagging and Imaging of Proteins with iFAST in Candida albicans Jonas Devos [...] Wouter Van Genechten Oct 5, 2024 211 Views Compartment-Resolved Proteomics with Deep Extracellular Matrix Coverage Maxwell C. McCabe [...] Kirk C. Hansen Dec 5, 2024 336 Views An Assay System for Plate-based Detection of Endogenous Peptide:N-glycanase/NGLY1 Activity Using A Fluorescence-based Probe Hiroto Hirayama and Tadashi Suzuki Jan 5, 2025 201 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,557	https://bio-protocol.org/en/bpdetail?id=4557&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Hypochlorite Stress Assay for Phenotypic Analysis of the Halophilic Archaeon Haloferax volcanii Using an Improved Incubation Method and Growth Monitoring PM Paula Mondragon SH Sungmin Hwang AS Amy Schmid Julie A. Maupin-Furlow Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4557 Views: 623 Reviewed by: Gal HaimovichYufang LuWolf Dieter Röther Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in mBio Aug 2022 Abstract The study of haloarchaea provides an opportunity to expand understanding of the mechanisms used by extremophiles to thrive in and respond to harsh environments, including hypersaline and oxidative stress conditions. A common strategy used to investigate molecular mechanisms of stress response involves the deletion and/or site-directed mutagenesis of genes identified through omics studies followed by a comparison of the mutant and wild-type strains for phenotypic differences. The experimental methods used to monitor these differences must be controlled and reproducible. Current methods to examine recovery of halophilic archaea from extreme stress are complicated by extended incubation times, nutrients not typically encountered in the environment, and other related limitations. Here we describe a method for assessing the function of genes during hypochlorite stress in the halophilic archaeon Haloferax volcanii that overcomes these types of limitations. The method was found reproducible and informative in identifying genes needed for H. volcanii to recover from hypochlorite stress. Keywords: Archaea Oxidative stress Redox biology Reactive oxygen species Mutant Phenotype Background Accumulation of reactive species that are redox-active compounds usually leads to cytotoxic activity. Hypochlorite (HOCl) is a reactive species that is particularly cytotoxic as it reacts in vivo with low molecular weight inorganic molecules and organic molecules including the functional groups of lipids, proteins, carbohydrates, and nucleic acids (Panasenko et al., 2013). HOCl levels usually become more abundant during oxidative stress when there is an increase in molecular oxygen (O2) leading to incomplete reactions stopping at reactive oxygen species such as superoxide anions (O2•−), hydrogen peroxide (H2O2), and the highly reactive hydroxyl radical (OH•) (Loi et al., 2015). The rise in H2O2 levels leads to chlorination where H2O2 reacts with Cl– anions leading to the formation of HOCl (Winterbourn and Kettle, 2013). H2O2 + Cl– + H+ → HOCl + H2O HOCl ↔ OCl– + H+ While haloarchaea are of interest to understand how organisms thrive in harsh environments, mechanisms of HOCl stress response are best understood in pathogenesis, as neutrophils of mammalian innate immunity kill exogenous pathogens through an oxidative burst that includes HOCl production (Imlay, 2003, 2008, 2013; Ulfig and Leichert, 2021). Haloarchaea are microorganisms that often dominate hypersaline ecosystems where the concentration of NaCl is greater than in seawater (3.5% w/v NaCl) (Jones and Baxter, 2017). Of note in HOCl stress is the exceedingly high concentrations of chloride ions that are constantly present as exemplified by the hypersaline Lake Tyrrell that fluctuates from 4 to 5 M Cl– (Podell et al., 2014). These conditions lead to an increase in HOCl and consistent exposure of cells to cytotoxic agents. The study of haloarchaea is of interest to the scientific community, as these microorganisms can thrive in such harsh conditions. This inquiry has led to examining the mechanisms used by the haloarchaea to survive stress, including co-expression networks of coordinately regulated genes used to combat or control oxidative stress (Martinez-Pastor et al., 2017). A common method used in biology to determine if a protein plays an important role in the cell is to delete the gene that encodes the protein of interest and observe the mutant strain phenotype. For example, here we are interested in comparing the growth rate and recovery of mutant and wild-type strains of the halophilic archaeon Haloferax volcanii before and after exposure to HOCl stress. For HOCl, supplementation of cultures with sodium hypochlorite (NaOCl) in aqueous solution leads to the spontaneous conversion of NaOCl into sodium hydroxide (NaOH) and HOCl, which can further dissociate into hydroxide (OH–) and hypochlorite (OCl–). NaOCl + H2O ↔ NaOH + HOCl ↔ Na+ + OH– + H+ + OCl– However, cultivating haloarchaea in a controlled environment can be difficult due to their demand for high concentrations of salts (e.g., > 2.5 M NaCl) and thermophilic temperatures (42–55 °C) (Robinson et al., 2005). Both environmental factors lead to evaporation, which exacerbates stress and can extend the time of cultivating cells for HOCl stress assays. Here we describe a newly designed and improved method to examine the response of the haloarchaeon Haloferax volcanii to HOCl stress. Our initial analysis was conducted in a circular rotary shaker (Figure 1A) that yielded variable results. The inconstancy in results was likely due to the extensive incubation times required to detect cell recovery after HOCl exposure in minimal medium, as the conditions were microaerobic and dehydrating. These findings led to a demand for better growth conditions where the recovery of H. volcanii from HOCl stress could be monitored in a reproducible manner. Thus, a new protocol was developed that reduces the lag time of cellular recovery from HOCl stress and allows for experimental reproducibility by cultivating the cells using a mini rotator to improve aeration and redesigning the incubator to promote humidity (Figure 1B). The method is performed in minimal medium to avoid the complexity of antioxidants otherwise present in yeast extract and other common additions to undefined medium. Figure 1. Diagram comparing two different setups for cell growth in culture tubes. A) Old setup with culture tubes oriented upright and agitated by rotary shaking, which causes an uneven distribution of oxygen and irregular growth patterns. B) New setup with culture tubes rotated at an angle to allow for more even aeration. A beaker is filled with water and included at the bottom of the incubator to enhance moisture. Materials and Reagents "Zipper" seal sample bags; thickness, 2 mil, and size, 6 × 9 in. 10 mL Sterile polystyrene disposable serological pipets (Genesee Scientific, catalog number: 12-104) 15 mL Conical centrifuge tubes, racked (polypropylene, Olympus Plastics, catalog number: 28-101) 2.5” Toothpicks, needle point, autoclavable (sterile) (LevGo, catalog number: 18250-NP) 200 µL XTIP4 barrier tips, low binding, racked, pre-sterilized (RNase and DNase free) (Genesee Scientific, catalog number: 24-712) 250 mL Erlenmeyer flasks (Pyrex, manufacturer number: 4980250/EMD) (Fisher Scientific, catalog number: S63271) 500 mL Erlenmeyer flasks (Pyrex, manufacturer number: 4980500/EMD) (Fisher Scientific, catalog number: S63273) Disposable culture tubes, borosilicate glass 13 × 100 mm (Fisher Scientific, catalog number: 14-961-27) Disposable plastic cuvettes semi-micro, 1.5 mL (Fisher Scientific, catalog number: 14955127) Disposable petri dishes, polystyrene, sterile, semi-stackable (100 × 15 mm) (VWR, catalog number: 25384-088) Plain PTFE stir bars, length: 70 mm; diameter: 10 mm (Fisher Scientific, catalog number: 16255801) Plastic caps (to cap culture tubes) (DWK Life Sciences, manufacturer number: 7366013) (Fisher Scientific, catalog number: 14-957-91) Poxygrid 96-place test tube rack; for 13–16 mm tubes (Bel-Art, catalog number: F18765-0001) PYREX griffin low form 1 L beaker, double scale, graduated (Pyrex, manufacturer number: 10001L/EMD) (Fisher Scientific, catalog number: S14276) Agar ash 2.0%–4.5% (Sigma-Aldrich, catalog number: A7002-250G) Aluminum foil roll (used to cover opening for the Erlenmeyer flasks for sterilization and growing strains) (Fisher Scientific, catalog number: 01-213-105) Ammonium chloride (NH4Cl) (Fisher Scientific, catalog number: A661-500) Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500) Copper sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C3036) D-Biotin (Fisher Bioreagents, catalog number: 58-85-5) Glycerol for molecular biology (Fisher Bioreagents, catalog number: BP229-4) Haloferax volcanii strains used including the H26 parent and SH125 mutant (ΔoxsR) that are previously described (Mondragon et al., 2022) Iron sulfate heptahydrate (FeSO4·7H2O) (Alfa Aesar, catalog number: 7782-63-0) L–Shaped cell spreader (Fisher Scientific, catalog number: 14-665-230) Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M0250-KG) Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Science Education, catalog number: S25414A) Manganese chloride tetrahydrate (MnCl2·4H2O) (Fisher Chemical, catalog number: M87-100) Nanopure water (purified from Barnstead/Sybron Nanopure II 4-Module Water Purification System) Novobiocin sodium salt (≥93% HPLC) (Sigma-Aldrich, catalog number: 74675-1G) Potassium chloride (KCl) (Fisher Chemical, catalog number: P217-3) Potassium phosphate dibasic anhydrous (K2HPO4) (Fisher Chemical, catalog number: P288-500) Potassium phosphate monobasic (KH2PO4) (Fisher Chemical, catalog number: P285-500) Potassium sulfate (K2SO4) (Fisher Chemical, catalog number: P304-500) Sodium chloride (NaCl) certified ACS crystalline (Fisher Scientific, catalog number: S271-10) Sodium hypochlorite (NaOCl) reagent grade, available chlorine 10%–15% (Sigma-Aldrich, catalog number: 425044-250mL) Thiamine (Sigma cell culture, catalog number: T3902) Tris-base (Fisher Bioreagents, catalog number: BP152-500) Tryptone (Fisher Bioreagents, catalog number: BP1421-500) Uracil, 99+% (Acros organics, catalog number: 66-22-8) Yeast extract molecular genetics powder (Fisher Bioreagents, catalog number: BP1422-500) Zinc sulfate heptahydrate (ZnSO4·7H2O) (Fisher Scientific, catalog number: 7446-20-0) Concentrated salt water (SW) stock solution at 30% (w/v) (see Recipes) GMM base (see Recipes) Supplements (see Recipes) 0.5 M Potassium phosphate buffer (KPB), pH 7.5 for 100 mL total 1.5 mg/mL Uracil Thiamine and biotin solution for 10.8 mL total Hv-minimal salts for 12 mL total Trace elements for 100 mL total Volume of supplements to add to GMM base for total of 1 L of GMM + uracil (see Recipes) For GMM + uracil agar plates a total of 500 mL (see Recipes) For ATCC 974 medium plates a total of 500 mL (see Recipes) Equipment Cimarec basic stirring hotplate (ThermoFisher Scientific, catalog number: SP194715) Genesys 40 visible spectrophotometer (ThermoFisher Scientific, catalog number: SP194715) Genie 2 vortex mixer (Fisher Scientific, catalog number: 12-812) Heratherm incubator (Thermo Scientific) I24 Incubator shaker series (New Brunswick Scientific) Mini rotator (20° angle, 2–80 rpm with disk and clamps, 120 V, Glas-Col Terre Haute, USA) Pipette (10–100 µL, Rainin, pipet-lite) Spectronic 20+ spectrophotometer (ThermoSpectronic, Filter: 600–950 nm) Software Microsoft Excel (version 16.16.27) Procedure Preparation of the strains Using a sterile toothpick, pick a small aliquot of the frozen H. volcanii strain from a -80 °C glycerol stock and streak onto a glycerol minimal medium (GMM) plate. Repeat this for each strain including the parent, mutant, and complement strains. The stocks consist of stationary phase cells frozen in GMM with 20% (v/v) glycerol. Incubate the plates for 5 days in a closed plastic zippered bag at 42 °C, until colonies appear. Using a sterilized toothpick, collect five isolated colonies and put them into 20 mL of GMM in a 250 mL Erlenmeyer flask. This approach avoids dilution stress. Grow the cells at 42 °C (200 rpm, rotary shaking) using the I24 Incubator shaker. After 48 h of incubation, prepare a 1:5 dilution of each strain by sterile transfer of 0.2 mL culture and 0.8 mL fresh GMM in disposable plastic cuvettes. Measure the optical density at 600 nm (OD600) using a Genesys 40 visible spectrophotometer using uninoculated GMM to blank the instrument. Multiply the value obtained on the instrument by 5 to determine the original OD600. The cells should be at an OD600 of 0.8 to 1.0, which is equivalent to late log phase. Dilute the cells with fresh GMM to an OD600 of 0.1 in 65 mL of final volume in a sterile 125 mL Erlenmeyer flask. For example, for cells at an OD600 of 0.89, mix 7.3 mL of cell culture with 57.7 mL of fresh GMM. Gently swirl the diluted cells to mix. Using 10 mL sterile pipets, transfer aliquots (5 mL) of this cell suspension into 12 loosely capped sterile 13 × 100 mm glass culture tubes per strain type. Include tubes with 5 mL of fresh uninoculated GMM as negative controls. Incubate the cell cultures for 12 h at 42 °C with aeration using a mini rotator (Glas-Col from Terre Haute in the USA) fitted with a culture rack (see details below). Use the maximum percent speed setting of 50 for the rotation. Perform the incubation in a Heratherm incubator. Measure the OD600 of the culture by directly inserting the culture tube with the plastic cap into the Spectronic 20+ spectrophotometer (refer to Figure 2 to use the Spectronic 20+ spectrophotometer before measuring samples). Use the uninoculated controls to determine the background signal. The cells should reach log phase, which is estimated at an OD600 of 0.4–0.6. Make sure to close the lid of the instrument prior to taking the OD600 measurements. Figure 2. Steps to set up the Spectronic 20+ spectrophotometer before measuring samples. 1) Turn on the spectrophotometer by turning the Power switch/Zero control to the right. 2) Make sure the filter lever is set to 600–900 nm and the red light is turned on. 3) The wavelength should be adjusted to 600 nm. 4) Allow for the spectrophotometer to warm up for 15 minutes. 5) Adjust the Power switch/Zero control to percent transmittance to 0. 6) Place into the sample compartment the 13 × 100 mm glass culture tube with only the medium (GMM) that will serve as a blank to 7) adjust the Transmittance/ Absorbance control (100%T/0A) to 100/0. Note: Do not remove plastic cap; the cap and the tube should both fit in the compartment. Setup for incubation Incubate the culture tubes in a Heratherm incubator with the following setup (Figure 3): Place the culture tubes on a 96-place test tube rack attached to a mini rotator. Place the mini rotator with the culture tubes on the top shelf of the incubator. On the bottom shelf of the incubator, fill a 1 L beaker with distilled water up to the 1,000 mL mark to maintain moisture in the incubator. Turn on rotator and slowly increase the maximum percent speed to 50. Note: The same incubation setup is used after supplementation of NaOCl. Figure 3. Schematic of the workflow for setting up for incubation. Circled numbers refer to the steps in the text. Preparation of NaOCl Upon arrival of shipment, dispense the NaOCl reagent in 10 mL aliquots in 15 mL conical tubes and store at -80 °C (time of NaOCl storage was not monitored). For each experiment, thaw 10 mL of frozen NaOCl stock to room temperature. Briefly vortex the solution once it is thawed. Then, transfer 5 mL of the thawed NaOCl solution to a fresh 15 mL conical tube, which serves as the 16.2 M NaOCl stock. Perform a serial dilution of the 16.2 M NaOCl stock to a final concentration of 2.025 M as follows: dispense 2.5 mL of nanopure water to three 15 mL conical tubes, perform a 1:2 dilution by transferring 2.5 mL of the 16.2 M stock to the first of the three tubes and mixing the solution to generate an 8.1 M stock, repeat the 1:2 dilutions two more times to a final concentration of 2.025 M (Figure 4A). Figure 4. Schematic of the workflow for preparation and supplementation of NaOCl. A) Preparation of NaOCl; B) Supplementation of NaOCl. Circled numbers refer to the steps in the text. Supplementation of NaOCl (Figure 4B) Randomly select half of the 12 culture tubes for each strain for supplementation with or without 5 mM NaOCl. Supplement the first set of 6 culture tubes with 12.3 µL of nanopure water. Attention: Vortex each individual cell culture tube immediately after addition of the water. Supplement the second set of 6 cell cultures with 12.3 µL of 2.025 M NaOCl for a final concentration of 5 mM. Attention: Vortex each individual cell culture tube immediately after addition of the NaOCl. Monitoring of cell growth after supplementation with NaOCl Remove all cell culture tubes from the mini rotator every 24 h of incubation at 42 °C after supplementing with NaOCl. Examine the culture tubes to see if there are marks visible on the outside of the tube. If marks are present on the outside of the tubes, use a Kimwipe with ethanol to remove the marks. This action will allow the OD600 to be more accurately measured using the Spectronic 20+ spectrophotometer. Place each culture tube directly into the spectrophotometer individually to determine the OD600 value. If using the Spectronic 20+ spectrophotometer, make sure to close the lid before recording the OD600. Repeat these OD600 measurements for 10 days. Further analysis of strains that may exhibit two distinct responses in hypochlorite stress Exposing cells to second round of hypochlorite stress (Figure 5). For further analysis of the biological significance of a mutation, pool tubes with a similar growth pattern and treat as independent groups. For example, the mutant strain of this example is treated as three independent groups after the first stress assay. The first group consists of culture tubes pooled for the mock control. The second group is the pool of cell cultures that did not grow under 5 mM NaOCl. The third group is the pool of cell cultures that grew in the presence of 5 mM NaOCl. Measure the OD600 for the two groups, by using the same measuring method from step A5. Repeat steps A6 and A7. Incubate the cell cultures for 10 h as in Section B. Repeat all steps from Sections C–E. Figure 5. Schematic of the workflow for preparing strains for second incubation with hypochlorite. Circled numbers refer to the steps in the text. Plate count assay After 10 days of monitoring the growth of each cell culture tube, randomly choose 2 or 3 technical replicates of each sample type to perform a serial dilution in 18% concentrated salt water (SW) (Figure 6). First transfer 1 mL of cell culture into a sterilized 1.5 mL microcentrifuge tube. Then, transfer 50 µL of this cell culture to a 1.5 mL microcentrifuge tube with 950 µL of 18% SW, and mix the sample by pipetting up and down. Repeat step b three more times to generate a serial dilution series of 1:20, 1:400, 1:8,000, and 1:16,000. Plate 100 µL of the diluted samples on ATCC 974 agar medium plates using an L-shaped cell spreader. Allow the plates to air dry for ~15 min. Place the air-dried plates in a plastic bag and incubate at 42 °C for 5 days. When the colonies are visible, choose a plate that appears to have between 30 and 300 colonies. Count the total colonies. Calculate the CFUs per mL (CFUs/mL) of the sample by multiplying the number of colonies times the dilution factor of the counted plate. Note that because only 100 µL of cells were plated, a multiple of 10 must be included in the calculations of CFU per mL in addition to the original dilution factors (DF) that range from 400 to 16,000. Figure 6. Schematic workflow for plate count assay. Circled numbers refer to the steps in the text. Data analysis Record and plot the OD600 units over time (hours) in Microsoft Excel. Use the scatter plot to compare growth patterns of the different strains and culture conditions including the mock (H2O) and experimental sets (exposed to 5 mM NaOCl). An example is shown in Figure 7. Figure 7. Scatter plots comparing the growth curve from “New setup” of Figure 1 Calculate the growth rate and doubling time of each strain. Plot time (h) against LN(OD of 600) to better identify the exponential phase (straight line). Choose the best two (or three) points that give the best straight line (Figure 8). Figure 8. Plot time (h) against LN(OD of 600) to better identify the exponential phase (straight line) Calculate the growth rate by adding a linear trendline by going to Chart Design < Press Add Chart Element < Go to Trendline < Choose Linear < Go to More Trendline Options < Select Display Equation on chart (Figure 9). This will give you an equation for the trendline that includes the slope of the line that is the growth rate in generation per hour (gen/h). Note: The growth rate for each 12 technical replicates is calculated separately. Then, the average growth rate (µ) is calculated based on all growth rates from each replicate. Figure 9. Finding the growth rate. After selecting More Trendline Options make sure the option is set to Linear (A) and select Display Equation on chart (B). The slope of the trendline is the growth rate in generation per hour (gen/h). To calculate the doubling time (k), use the average growth rate in the following formula: k = 1/µ Calculate the standard deviation (±) for each average growth rate and doubling time. For the strains supplemented with 5 mM NaOCl, the exponential phase will be seen much later than the mock control (H2O). As shown in Figure 10, the two points that made a straight line were selected after adding the 5 mM of NaOCl. Figure 10. Finding the points to make trendline for strains after adding 5 mM of NaOCl. Make sure to select points that make a straight line. Calculating Area Under the Curve (A.U.C.) by using Trapezoidal Rule For each technical replicate find the area under the curve by finding the area of each trapezoid. For example, first trapezoid is between x = 1 and x = 2 by using the following formula (Figure 11A): area = (y1 + y2)/2(x2 - x1) Calculate the area for the other trapezoids (Figure 11B). Then, add the areas of all trapezoids to find the sum that will be the A.U.C. for the growth curve (Figure 11C). Repeat the above steps for each technical replicate for each strain, then find the average and standard error. For helpful reference to guide A.U.C. calculations see Github link https://rdrr.io/github/leonpheng/lhtool/man/AUC.html. Plot the average of A.U.C. of each strain in the bar graph and add the technical replicates individually and convert them to a scatter plot (Figure 11D). Use the average for each experiment to calculate if it has equal variance by F-test. If so, use the two-tailed Student’s t-test analysis to find if there are significant differences between strains. Note: If you find that the variances are unequal, you can do the two-tailed, unpaired Welch's t-test instead of the Student's t-test. Figure 11. Calculating the Area Under the Curve (A.U.C.) for a Haloferax volcanii strain (H26). A) Find the area of the trapezoid by using area = (y1 + y2)/2*(x2 - x1). B) Calculate the area for the other trapezoids. C) Find the sum of all areas to find the area under the curve. D) Example of bar graph showing the A.U.C. and the significant differences between strains by using the Student’s t-test analysis. Recipes The first set of recipes are for glycerol minimal media plus uracil (GMM + uracil) and were modified from the Halohandbook (Dyall-Smith, 2009). Concentrated salt water (SW) stock solution at 30% (w/v) (1 L) Use nanopure water as approximately 70% of the total volume of the solution (~700 mL), and warm it on a hot plate. Note: A microwave could also be used to warm up the water. When the water gets warm, add the following salts one by one allowing each to completely dissolve: NaCl 240 g MgCl2·6H2O 30 g MgSO4·7H2O 35 g KCl 7 g 1 M Tris base (with pH of 7.5, the pH is adjusted with HCl) 20 mL GMM base (1 L) Nanopure water 20.4 mL 30% SW 647.2 mL 1 M Tris base (with pH of 7.5, the pH is adjusted with HCl) 32.4 mL Autoclave solution before use. Supplements 0.5 M KPB, pH 7.5 for 100 mL total 1 M K2HPO4 83.4 mL 1 M KH2PO4 16.6 mL pH should be approximately 7.5. Note: When mixing compounds, add in small volumes of the 1 M KH2PO4 to the 1 M K2HPO4 while monitoring the pH. When all 1 M KH2PO4 has been added, the pH should be approximately 7.5. Do not add HCl or NaOH to adjust pH to 7.5. Filter the solution before use and store at room temperature. 1.5 mg/mL Uracil The Halohandbook suggests making a 50 mg/mL solution of uracil by dissolving in DMSO. However, DMSO is a reactive compound that may expose the cells to oxidative stress. To better control the hypochlorite stress conditions, uracil is dissolved at 1.5 mg/mL in nanopure water overnight at 42 °C with rotary shaking at 200 rpm using an I24 Incubator shaker. After ~16 h the dissolved uracil is filtered and stored at room temperature. 1 mg/mL of Novobiocin Novobiocin is used to select for cells transformed with plasmids that harbor the novobiocin resistance marker. Plasmids are often used to carry the wild-type gene of interest and demonstrate that the mutation can be complemented. Dissolve the novobiocin at 10 mg/mL in nanopure water and then dilute 10-fold to a working stock of 1 mg/mL. Store excess reagent in aliquots at -20 °C for future use. Thiamine and biotin solution for 10.8 mL total 1 mg/mL thiamine 9.6 mL 1 mg/mL D-biotin 1.2 mL Filter solution before use and store at 4 °C. Hv-minimal salts for 12 mL total 1 M NH4Cl 5.0 mL 0.5 M CaCl2 6.0 mL Trace elements 1.0 mL Fill up to 12 mL with nanopure water. Filter the solution. Make Hv-minimal salts fresh before use. Trace elements for 100 mL total Dissolve the following elements in ~70 mL of nanopure water: MnCl2·4H2O 36 mg ZnSO4·4H2O 44 mg FeSO4·7H2O 230 mg CuSO4·5H2O 5 mg Fill up to 100 mL with nanopure water and filter before storing at 4 °C. Volume of supplements to add to GMM base for total of 1 L of GMM + uracil Add the calculated amount of GMM base to a sterile flask (total GMM minus total volume of all supplements). Then add the following volumes of each supplement: 1 M glycerol 20 mL 0.5 M KPB, pH 7.5 1.9 mL 1.5 mg/mL uracil 33.4 mL Thiamine and biotin solution 0.882 mL Hv-minimal salts 11.8 mL Note: For the complement and empty vector strains, GMM + uracil is supplemented with 0.3 µg/mL of novobiocin by using a stock solution of 1 mg/mL of novobiocin. For GMM + uracil agar plates a total of 500 mL Autoclave the volume of GMM base (total GMM minus total volume of all supplements) with 7.5 g of agar with a stirring rod. Cool the solution to 60°C for 30 min. After cooling the medium, add the same supplements while the GMM base with agar are stirring on the plate: 1 M glycerol 10 mL 0.5 M KPB, pH 7.5 0.950 mL 1.5 mg/mL uracil 16.7 mL Thiamine and biotin solution 0.441 mL Hv-minimal salts 5.9 mL Pour the medium in petri dishes (~20 mL for each plate). Allow the medium to solidify and cool before use. Note: For the complement and empty vector strains, GMM + uracil plates are supplemented with 0.3 µg/mL of novobiocin by spreading the volume of 1 mg/mL of novobiocin onto the dry GMM + uracil plate. The novobiocin solution is allowed to dry on the plates before inoculating the strain from the -80 °C glycerol stock. The following recipe is for 1 L of ATCC 974 medium plates that was modified: Nanopore water ~700 mL NaCl 125 g MgCl2·6H2O 50 g K2SO4 5 g CaCl2·2H2O 0.134 g Tryptone 5 g Yeast extract 5 g Adjust to pH 6.8 with 1 M KOH Agar 15 g Fill up to 1,000 mL with nanopore water and autoclave. Pour the medium in petri dishes (~20 mL for each plate). Allow the medium to solidify and cool before use. Note: For strains that contain plasmids, novobiocin with the same concentration as for GMM + uracil was used. Acknowledgments Funds awarded to JMF and AS to develop systems biology tools were through the Bilateral NSF/BIO-BBSRC program (NSF 1642283). Funds awarded to JMF to determine archaeal redox regulation were through the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE-FG02-05ER15650) and to provide evolutionary insight in biological systems were through the National Institutes of Health (NIH R01 GM57498). Competing interests The Authors declare that there are no conflicts of interest. References Dyall-Smith, M. (2009). The Halohandbook: Protocols for Halobacterial Genetics v.7.2. Imlay, J. A. (2003). Pathways of oxidative damage. Annu Rev Microbiol 57: 395-418. Imlay, J. A. (2008). Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 77: 755-776. Imlay, J. A. (2013). The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium.Nat Rev Microbiol 11(7): 443-454. Jones, D. L. and Baxter, B. K. (2017). DNA repair and photoprotection: Mechanisms of overcoming environmental ultraviolet radiation exposure in halophilic archaea. Front Microbiol 8: 1882. Loi, V. V., Rossius, M. and Antelmann, H. (2015). Redox regulation by reversible protein S-thiolation in bacteria. Front Microbiol 6: 187. Martinez-Pastor, M., Tonner, P. D., Darnell, C. L. and Schmid, A. K. (2017). Transcriptional regulation in archaea: From individual genes to global regulatory networks. Annu Rev Genet 51: 143-170. Mondragon, P., Hwang, S., Kasirajan, L., Oyetoro, R., Nasthas, A., Winters, E., Couto-Rodriguez, R. L., Schmid, A. and Maupin-Furlow, J. A. (2022). TrmB family transcription factor as a thiol-based regulator of oxidative stress response. mBio: e0063322. Panasenko, O. M., Gorudko, I. V. and Sokolov, A. V. (2013). Hypochlorous acid as a precursor of free radicals in living systems.Biochemistry (Mosc) 78(13): 1466-1489. Podell, S., Emerson, J. B., Jones, C. M., Ugalde, J. A., Welch, S., Heidelberg, K. B., Banfield, J. F. and Allen, E. E. (2014). Seasonal fluctuations in ionic concentrations drive microbial succession in a hypersaline lake community. ISME J 8(5): 979-990. Robinson, J. L., Pyzyna, B., Atrasz, R. G., Henderson, C. A., Morrill, K. L., Burd, A. M., Desoucy, E., Fogleman, R. E., Naylor, J. B., Steele, S. M., et al. (2005). Growth kinetics of extremely halophilic Archaea (family Halobacteriaceae) as revealed by Arrhenius plots. J Bacteriol 187(3): 923-929. Ulfig, A. and Leichert, L. I. (2021). The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens. Cell Mol Life Sci 78(2): 385-414. Winterbourn, C. C. and Kettle, A. J. (2013). Redox reactions and microbial killing in the neutrophil phagosome.Antioxid Redox Signal 18(6): 642-660. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > Microbial physiology > Stress response Environmental science Biological Sciences > Microbiology Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols An Assay to Determine NAD(P)H: Quinone Oxidoreductase Activity in Cell Extracts from Candida glabrata Anamika Battu [...] Rupinder Kaur Nov 5, 2021 1761 Views Quantitative and Anatomical Imaging of Human Skin by Noninvasive Photoacoustic Dermoscopy Zhiyang Wang [...] Sihua Yang Apr 5, 2022 1146 Views Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique Federico Berdun [...] Eduardo Zabaleta Mar 5, 2024 658 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,558	https://bio-protocol.org/en/bpdetail?id=4558&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource This protocol has been updated. See the update notice. Peer-reviewed Efficient Generation of Genome-wide Libraries for Protein–ligand Screens Using Gibson Assembly TS Tamara Sternlieb ML Mira Loock MG Mengjin Gao IC Igor Cestari Published: Vol 12, Iss 22, Nov 20, 2022 DOI: 10.21769/BioProtoc.4558 Views: 1643 Reviewed by: Kristin L. ShinglerEmmanuel Orta-Zavalza Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in ACS Infectious Diseases Apr 2023 Abstract Genome-wide screens using yeast or phage displays are powerful tools for identifying protein–ligand interactions, including drug or vaccine targets, ligand receptors, or protein–protein interactions. However, assembling libraries for genome-wide screens can be challenging and often requires unbiased cloning of 105–107 DNA fragments for a complete representation of a eukaryote genome. A sub-optimal genomic library can miss key genomic sequences and thus result in biased screens. Here, we describe an efficient method to generate genome-wide libraries for yeast surface display using Gibson assembly. The protocol entails genome fragmentation, ligation of adapters, library cloning using Gibson assembly, library transformation, library DNA recovery, and a streamlined Oxford nanopore library sequencing procedure that covers the length of the cloned DNA fragments. We also describe a computational pipeline to analyze the library coverage of the genome and predict the proportion of expressed proteins. The method allows seamless library transfer among multiple vectors and can be easily adapted to any expression system. Keywords: Genome-wide library Gibson assembly Yeast surface display High-throughput screen Nanopore sequencing Genomics Protozoan Trypanosoma Background Protein–ligand interactions are essential for nearly all cellular processes, including gene expression regulation, pathogen–host interactions, and the identification of drug or vaccine targets. Defining interacting partners can be challenging and usually relies on in vitro biochemical approaches or genetic manipulation of the organism of interest. For many non-model organisms, genetic manipulation tools are still scarce or of laborious application, thus limiting the approaches available for discovering protein–ligand partners or protein interaction networks. Genome-wide libraries are powerful tools for exploratory studies in protein–ligand interactions (Bharucha and Kumar, 2007; Bidlingmaier and Liu, 2011). Once incorporated into an expression system, libraries can encode all possible polypeptides of an organism without bias associated with its life cycle stage or culture conditions. Generating a eukaryote genome-wide library requires cloning approximately 105–107 different DNA fragments. Hence, the method must be robust to ensure that all possible coding sequences are included and in frame with the proteins of the expression system. The most common methods for generating genomic libraries involve DNA fragmentation by enzymatic digestion or sonication, followed by cloning the DNA fragments into restriction enzyme–digested or T/A-based plasmid vectors. Adapters or barcode sequences are often added to the DNA fragments to help with library amplification and sequencing. There are several expression systems for interaction screenings. Baker's yeast—Saccharomyces cerevisiae—has been widely used as an expression system due to its versatility for genetic manipulation, high levels of protein expression, inducible gene expression, and the presence of eukaryotic post-translational modifications (Boder and Wittrup, 1997; Bidlingmaier and Liu, 2011). Yeast surface display (YSD) is one of the most successful screening systems for discovering protein–ligand interactions (Gibson et al., 2009; Bidlingmaier and Liu, 2011; Gu et al., 2014; Danecek et al., 2021). In this system, library proteins are expressed in frame with proteins of an endogenous surface anchoring system (e.g., Aga1p-Aga2p) and then displayed in approximately 105 copies of a single protein per cell (Inoue et al., 1990). The yeast population can express a whole genomic library and is usually screened using fluorescence or magnetic-activated cell sorting to enrich yeast cells that react with the ligands. The library DNAs from the sorted yeasts are then sequenced to identify the coding sequences for the ligand–interacting partners. Hence, the YSD methodology coupled with a strategy to enrich or isolate the positive interactions can lead to the discovery of protein–ligand partners while overcoming the limitations of genetic manipulation of non-model organisms. We present here an efficient method to construct genome-wide libraries. In our approach, the genomic DNA is fragmented to the average gene size, end-repaired, and A-tailed for the ligation of adapter sequences for PCR amplification (Figure 1). The fragments are PCR-amplified with a pair of primers that hybridizes with the adapters and contains a ~20 bp overlapping sequence to the receptor vector for Gibson assembly. The Gibson assembly entails an isothermal reaction to join DNA molecules using a ~20 bp overlapping sequence between DNA fragments (e.g., library fragments and plasmid vector). This reaction includes a 5' exonuclease that generates 3’ overhangs in the joining molecules. After DNA molecules annealing, a DNA polymerase fills existent gaps in the annealed strands, and a DNA ligase seals strand nicks completing the process (Kieke et al., 1997). Using the Gibson method, the amplicons originating from adapter-ligated genomic fragments are joined to a linearized vector in a highly efficient one-step library assembly. Our protocol was designed to facilitate library transfer between expression systems and streamline library sequencing by Oxford nanopore technology to speed up the screening process. The nanopore sequencing technology has the advantage of generating sequences covering the entire cloned fragment; however, the protocol can be adapted to other sequencing methods. Our protocol details the library assembly, optimized library sequencing, and a computational pipeline to analyze the library coverage of the genome and evaluate library diversity and completeness. We also include a computational tool that predicts the library polypeptides expressed in frame with proteins of the expression system. Figure 1. Diagram of genome-wide library preparation protocol and quality control steps Materials and Reagents Serological pipet, 10 mL (Drummond, catalog number: 6 000 010) Labcon Eclipse refill universal-fit pipette tips with TubeGuard 0.1–10 µL (Labcon Eclipse, catalog number: 1036-260) VWR refill pipette tips 100/200 µL (VWR, catalog number: 89079-476) VWR refill pipette tips 1,000 µL (VWR, catalog number: 89079-470) 15 mL Centrifuge Tube, Bulk, Sterile, 25/pk, 500/cs (Montreal Biotech, catalog number: MBI601052C) 50 mL Centrifuge Tube, Bulk, Sterile, 25/pk, 500/cs (Montreal Biotech, catalog number: MBI602052C) 150 mm × 15 mm tissue culture-treated dishes (Fisher Scientific, catalog number: FB0875714) Microtube-50 AFA fiber screw-cap vials (Covaris, catalog number: 520166) Syringe Filter PES 0.22 µm 30 mm diameter, sterilized by Gamma irradiation (Ultident, catalog number: 229747) NEB® 5-alpha F'Iq Competent Escherichia coli (High Efficiency) (New England Biolabs, catalog number: C2992H) Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, catalog number: M1378-01) NEBNext® end-repair module (New England Biolabs, catalog number: E6050S) Monarch® DNA elution buffer (New England Biolabs, catalog number: T1016L) Ligation sequencing kit (Oxford Nanopore Technologies, catalog number: SQK-LSK110) Native barcoding expansion 1-12 (Oxford Nanopore Technologies, catalog number: EXP-PBC001) Blunt/TA ligase master mix (New England Biolabs, catalog number: M0367S) NEBNext FFPE DNA repair mix (New England Biolabs, catalog number: M6630S) NEBNext Quick Ligation Module – 20 rxs (New England Biolabs, catalog number: E6056S) Taq DNA polymerase with ThermoPol buffer (New England Biolabs, catalog number: M0267S) dNTPs mixture 10 mM (BioBasic, catalog number: DD0056) Agarose (Bishop Canada Inc., catalog number: AGA002.250) NEBuilder HiFi DNA assembly master mix (New England Biolabs, catalog number: E2621S) Tryptone (BioBasic, catalog number: TG217(G211)) Yeast extract (BioBasic, catalog number: G0961) Agar A (Biobasic, catalog number: FB0010) Sodium chloride (Fisher Scientific, catalog number: S271-1) Sodium Hydroxide (white pellets) (Fisher Scientific, catalog number: BP359-212) Hydrochloric acid (Fisher Scientific, catalog number: A144-212) Boric Acid (Omnipure, catalog number: 2710) Ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich, catalog number: E9884-100G) Manganesium chloride Hexahydrate (Fisher Scientific, BP214-500) Calcium chloride dihydrate (Sigma Aldrich, catalog number: 223506-500G) PIPES, Sesquisodium Sodium Salt (Fisher Scientific, catalog number: BP304-100) Potasium hydroxide pellets (Fisher Scientific, catalog number: P250-500) Trizma Base (AMRESCO, catalog number: 0497-5KG) Ampicillin, trihydrate (BioBasic, catalog number: AB0064) pUC-19 plasmid (NEB, catalog number: N3041S) Covaris AFA-grade water (800 mL) (D-mark Biosciences, catalog number: Cov-520101) Lambda DNA/Hind III plus marker (BioBasic, catalog number: M105R-2) Ecostain (BioBasic, catalog number: DT81413) Cell scraper (Cole-Parmer, catalog number: UZ01959-14) EZ-10 spin column plasmid DNA miniprep kit (BioBasic, catalog number: BS414) NucleoBond® Xtra maxi plus EF (Takara, catalog number: 740426.10) pYD1 plasmid (Addgene, catalog number: 73447) Restriction enzymes: Bam HI (New England Biolabs, catalog number: R0136S) Hind III (New England Biolabs, catalog number: R0104S) Xho I (New England Biolabs, catalog number: R0146S) NEBufferTM r3.1 (New England Biolabs, catalog number: B6003S) Flow cell (R9.4.1) (Oxford Nanopore Technologies, catalog number: FLO-MIN106D) Luria-Bertani (LB) medium with agar and ampicillin (see Recipes) Ampicillin stock solution (100 mg/mL) (50 mL) (see Recipes) Tris-Borate-EDTA buffer (10 L) (see Recipes) 10× TBE stock solution (1 L) (see Recipes) SOB medium (1 L) (see Recipes) Inoue solution (1 L) (see Recipes) 0.5 M PIPES buffer (100 mL) (see Recipes) 70% ethanol (see Recipes) Equipment Pipettes 0.2–2 µL (Gilson, catalog number: FA10001M), 2–20 µL (Gilson, catalog number: FA10003M), 20–200 µL (Gilson, catalog number: FA10005M), and 100–1,000 µL (Gilson, catalog number: FA10006M) Portable Pipet-Aid® XP pipette controller (Drummond, catalog number: 4-000-101). Focused-ultrasonicator (Covaris, M220, catalog number: 500295) 12-position magnetic rack for 1.5 mL tubes (Promega, catalog number: Z5342) T100 thermal cycler (Bio-Rad, catalog number: 1861096) Isotemp 215 water bath (Fisher, catalog number: 15-462-15) Avanti® J-E centrifuge, 50 Hz, 200 V, 24 A (Beckman Coulter, catalog number: 369005) Microcentrifuge 5418/5418R (Eppendorf, catalog number: EP5401000137) Variable speed rocking platform shaker (VWR, catalog number: 75832-308) NAPCO analog display automatic CO2 water-jacketed incubator (Napco, catalog number: 102219021) Innova® 44 incubator shaker (New Brunswick, catalog number: M1282-0000) Water system ultrapure (Millipore Synergy, catalog number: C9202) Electrophoresis unit: Thermo Scientific Power Supply 400 mA 300 V (Fisher, catalog number: S65533Q) NanoDrop ND-1000 (Thermo Fisher, catalog number: 2353-30-0010) ChemiDocMP imaging system (BioRad, catalog number: 12003154) Oxford nanopore MinION Mk1C sequencer (Oxford Nanopore Technologies, catalog number: MIN-101C) Software Minimap2 (Lemos Duarte et al., 2021) (https://github.com/lh3/minimap2) Samtools (Li, 2018) (https://www.htslib.org/) DeepTools (Liao et al., 2014) (https://deeptools.readthedocs.io/en/develop/index.html) Libframe (This work) (https://github.com/cestari-lab/Libframe-tool) Subread (Pointer et al., 2014) (http://subread.sourceforge.net/featureCounts.html) Circlize (Ramirez et al., 2016) (https://jokergoo.github.io/circlize/) Procedure DNA fragmentation and size selection Prepare three microtube-50 AFA fiber screw-cap vials, each containing 5 µg of genomic DNA in 55 µL of AFA-grade water. Sonicate using a focused-ultrasonicator for 3 s at 200 cycles per burst, 25 peak incidence power (W), and 10% duty factor at 7 °C, to obtain fragments of 0.5–3 kb (average gene range). Optimization of this step may be necessary depending on organism genome size, base composition, or DNA extraction method (see Note 1). Combine the fragmented DNA from the three vials for a total of 165 µL in a 1.5 mL Eppendorf tube and analyze 30 µL in 1% agarose/TBE gel with 5 µL of ecostain. Load a well with 1 µg of non-fragmented DNA for comparison (optional). Run at 90 V for 30 min or two-thirds of the gel length and visualize the DNA in a ChemiDocMP imaging system. The fragmented DNA should migrate as a smear between 0.5–3 kb with peak intensity at 1.5 kb (Figure 4A). The peak intensity of fragments may vary depending on fragmentation conditions and genome composition. Size select the DNA to obtain fragments above 500 bp by adding 95 µL of Mag-Bind® TotalPure NGS beads to 135 µL of fragmented DNA (ratio 0.7×, beads volume: fragmented DNA volume). Incubate for 10 min at room temperature with rotation at 50 rpm (see Note 2). Quickly spin (approximately 10 s, 850 × g) the microfuge tube containing beads and DNA mixture, transfer the tube to a magnetic rack, and incubate it at room temperature until the beads separate from the solution (approximately 1 min). Pipette and discard the supernatant. Slowly add 200 µL of 70% ethanol over the beads and incubate for 30 s or until beads separate from the solution. Collect and discard the ethanol. Repeat this wash step twice. Air-dry the beads and mixture for 2 min at room temperature. Do not dry to the point of cracking the beads. Add 55 µL of elution buffer to the beads, mix gently by flicking the tube, and incubate for 1 min at room temperature. Transfer the Eppendorf tube to a magnetic rack to separate the beads from eluted DNA. Collect the supernatant, now containing the DNA, into a new 1.5 mL microtube tube. Discard the tube containing the beads. Quantify the DNA concentration and purity using NanoDrop. Size selection should recover between 50% and 70% of the DNA. The ideal 260/280 and 260/230 absorbance ratios are 1.8 and 2.0, respectively. Analyze 5 µL of recovered DNA in 1% agarose/TBE gel with 5 µL of ecostain, as described in step A2 to verify size selection and DNA recovery. End-repair reaction and adapter ligation Add 1 µg of DNA (from step A8) to a 0.2 mL tube and mix with 10 µL of NEBNext® end-repair buffer (10×) and 5 µL of end-repair enzyme mix. Add water for a final volume of 100 µL. Incubate for 60 min at 20 °C in a thermocycler. Clean up the end-repaired DNAs by adding 70 µL of Mag-Bind® TotalPure NGS beads (ratio of 0.7×, beads volume:end repair reaction volume). Follow the steps described in steps A3 to A8. Elute the DNA in 31 µL of elution buffer. Quantify the DNA (1 µL of the sample) using NanoDrop. Expected DNA recovery is approximately 90%. Add the remaining 30 µL of end-repaired DNA (approximately 1 µg) into a 0.2 mL tube. Add 20 µL of the Oxford nanopore barcode adapters (BCA reagent from the ligation sequencing kit) to the tube and 50 µL of 2× Blunt/TA ligase master mix. Pipette gently to mix the reaction. Incubate at 25 °C for 2 h in a thermocycler. Clean up the barcode adapter-ligated DNA by adding 70 µL of Mag-Bind® TotalPure NGS beads (ratio of 0.7×, beads volume:barcode adapter ligation reaction volume), as described in steps A3 to A8. Elute the DNA in 31 µL of elution buffer. Quantify the DNA using NanoDrop. Expected DNA recovery is approximately 90% of the input material (step B5). Primer design and library amplification This step describes the design of primers and fragment amplification by PCR for library cloning into the expression vector using Gibson assembly. Figure 2 shows the primers' sequences and a diagram of the pYD1 plasmid used in this step. The primers should anneal to the barcode adapter ligated to the library fragments in step B5 (Figure 2A, sequences denoted by As) and have overhangs complementary to the expression vector (sequences indicated by Xs). Each primer overhang sequence should be ~20 bp, and the primer melting temperature should be approximately 50 °C. If desired, add restriction enzyme sites in the primers to reconstitute any restriction sites in the plasmid. The restriction site used to linearize the vector will be lost if not reconstituted in the primer (Figure 2A). Figure 2. Design of primers for Gibson assembly. (A) Scheme of forward and reverse primers used for Gibson assembly. (B) Diagram of Bam HI-digested pYD1 vector, barcode adapter-ligated fragment (DNA fragments), and primers. (C) Forward (For) and reverse (Rev) primers used to amplify barcode adapter-ligated fragments for Gibson assembly (see pairing regions in B). The sequences in orange correspond to a ~20 bp vector sequence complementary to the pYD1 vector, the black letters are the Bam HI restriction sites, and the ~20 bp nanopore barcode adapter annealing sequences are in green. F and R: forward and reverse primers, respectively, that pair to the vector flanking the cloning site and are used to amplify cloned fragments by PCR for cloning validation or sequencing. Prepare six PCR reactions, using the barcode adapter-ligated fragmented DNA as template, as follows: 10 µL of ThermoPol buffer 5×, 200 µM dNTPs, 500 nM primer mix (mix of forward and reverse primers), and 1.25 units of Taq DNA polymerase. Add the barcode adapter-ligated DNA (20 ng) and adjust the final volume to 50 µL with water. Amplify the reaction in a thermocycler with an initial denaturation at 95 °C for 30 s, then 14 cycles at 95 °C for 30 s, 49 °C for 30 s, and 68 °C for 3 min. Add a final extension of 5 min at 68 °C and store the reaction at 4 °C (see Note 3). Combine the PCR reactions and purify the amplicons by adding 210 µL of Mag-Bind® TotalPure NGS beads to the 300 µL of reaction (ratio of 0.7×, beads volume: end repair reaction volume). Follow the procedure described in steps A3–A8. Elute the DNA in 31 µL of elution buffer. Quantify the DNA using NanoDrop. Expected DNA recovery is approximately 80–140 ng/µL. Linearize the vector Digest 4 µg of pYD1 vector DNA by adding 3 µL of 10× reaction buffer r3.1, 2 µL (20 units) of Bam HI restriction enzyme, and water to 30 µL final volume. Incubate at 37 °C for 16 h. Note: Do not dephosphorylate the vector, as it will interfere with the Gibson assembly reaction (see Note 4). Purify the linearized DNA by adding 15 µL of Mag-Bind® TotalPure NGS beads (ratio of 0.5×, beads volume:digestion reaction volume). Follow the procedure described in steps A3 to A8. Elute the DNA in 31 µL of elution buffer. Quantify the DNA using NanoDrop. Expected DNA recovery is approximately 90%. Gibson assembly reaction and efficiency assessment Add 10 µL of NEBuilder HiFi DNA assembly master mix to a 0.2 mL tube. Then add 50 ng (16 fmol) of digested vector (from step D3), 30 ng (32 fmol, see Note 5) of amplified fragments (from step C3), and water to a total of 20 µL reaction. The concentrations indicated result in a 1:2 ratio of vector:PCR fragments. Incubate the Gibson assembly reaction for 1 h at 50 °C in a thermocycler. Mix 1 µL of the Gibson assembly reaction with 50 µL of competent NEB 5-alpha F'Iq E. coli cells, defrosted in ice, in an ice-cold 15 mL Falcon tube. Incubate on ice for 30 min. We recommend performing additional transformations with 100 pg of pUC19 and 50 µL of competent cells to estimate the efficiency of the competent cells, and with 50 ng of the pYD1 vector as a control for no fragment insertion (to be used in step E9). See Notes 6 and 7. Incubate the mix at 42 °C for 30 s, immediately transfer to ice, and incubate for 2 min. Add 950 µL of SOB medium, incubate at 37 °C, and shake for 1 h. Plate 500 µL of transformed cells into 150 × 15 mm Petri dishes containing LB medium with agar and ampicillin at 50 µg/mL for plasmid selection. For the pUC19 transformed cells, make serial dilutions (1:10, 1:100, and 1:1,000) and plate 500 µL into Petri dishes. Incubate overnight at 37 °C. Quantify the number of colonies obtained with the transformations to estimate the number of Gibson assembly reactions and transformations necessary for library assembly. Use the pUC19 transformation to estimate the efficiency of the competent cells. A total of 4,000–6,000 colonies per plate should be expected for the Gibson reaction transformation using competent cells with an efficiency of 109 CFU/µg of pUC19 DNA. See Note 8. To check the efficiency of the Gibson assembly, select 10–20 colonies from the transformation for colony PCR to amplify the cloned fragments using primers pairing to the vector and flanking the library fragments (Figure 2B and 2C, primers F and R). In 0.2 mL tubes, prepare 22 PCR reactions using 4 µL of ThermoPol buffer 5×, 200 µM dNTPs, 500 nM primer mix (forward and reverse), and 1.25 units of Taq DNA polymerase in 20 µL. We recommend preparing a master PCR reaction mix, then splitting reactions into 21 tubes. Pick a colony with a 10 µL pipet tip and mix the colony with the reaction in the PCR tube. Prepare one reaction with a colony from pYD1-transformed (no library) bacteria as a control. Amplify the reaction with an initial denaturation at 95 °C for 2 min, then 35 cycles at 95 °C for 30 s, 54 °C for 30 s, and 68 °C for 3 min. Add a final extension of 5 min at 68 °C, and store the reaction at 4 °C. Load 20 µL of the reaction to a 1% agarose/TBE gel with 5 µL ecostain, as described in step A2, to analyze the amplified DNA. The amplicons should appear as a single band with a size in the range of the selected fragments (0.5–3 kb). A higher than 90% efficiency in the assembly should be expected, i.e., 18 positive colonies out of 20. Otherwise, optimize the Gibson assembly reaction (step E1, see Note 9). To check if fragments cloned are from the desired organism genome, pick ten colonies from the transformation to 15 mL cell culture tubes with 2 mL of LB medium and 50 µg/mL of ampicillin antibiotic for plasmid selection. Incubate at 37 °C, shaking at 225 rpm for 18–24 h. Isolate the plasmid from colonies using a mini-prep kit and perform Sanger sequencing using primers pairing with the vector and flanking the library sequences (Figure 2C, primers F and R). Perform sequence alignment with the basic local alignment search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the organism genome or the non-redundant database from the National Center for Biotechnology Information. The sequences should align with sequences from the organism's genome used for the initial DNA fragmentation. Calculation of library size and large-scale transformation Calculate the required number of cloned fragments (N) for each genome sequence to be represented in the library with a probability (P) of 99% using the Carbon and Clarke equation: N=ln⁡(1-P)/ln(1-F) , where F=i/g F is the quotient between insert fragment size (i) and genome size (g) in bp. We recommend multiplying the final value by six to increase the chance that a fragment will be cloned in frame with proteins from the expression system, e.g., Aga2p in the pYD1 vector. Divide the calculated number of required fragments (N) by the number of colonies obtained in step E8 to estimate the number of transformations and Gibson assembly required. One Gibson assembly reaction yields 20 transformations and results in 40 transformation plates. For a 44 Mb haploid genome (e.g., the protozoan Trypanosoma cruzi Sylvio X10/1 strain haploid genome) and 6-fold of the minimum calculated number of fragments at 1.5 kb mean size, expect to harvest approximately 811,000 colonies. Hence, 203 plates at 4,000 colonies each and approximately five Gibson assembly reactions. Transform the Gibson assembly mix as described in steps E3–E7 to achieve the number of calculated fragments. Consider each colony a cloned fragment if assembly efficiency equals 100%. Extraction of library plasmids DNA Scrape all colonies from the plates and combine them in a 50 mL Falcon tube. Adjust the volume to 50 mL using LB medium with 50 µg/mL of ampicillin. If not extracting DNA immediately, keep the suspension at 4 °C for a maximum of one week. Dilute the culture in LB with 50 µg/mL of ampicillin to obtain an OD between 1 to 2. Incubate at 37 °C for 3 h for cell recovery. Isolate the plasmid DNAs using the NucleoBond® Xtra maxi plus EF according to the manufacturer's instructions. Quantify the plasmid DNA on the NanoDrop. The expected DNA yield should be approximately 0.5–2 mg. Digest 2 µg of plasmid DNA by mixing the DNA with 3 µL of 10× NEBufferTM r3.1 and 0.5 µL of Bam HI (5 units) at a final reaction volume of 30 μL. Incubate at 37 °C for 2–18 h. Other restriction enzymes that cut in the multiple cloning site, e.g., Hind III and Xho I, can also be used. Load the 30 µL reaction to a 1% agarose/TBE gel with 5 µL of ecostain, as described in step A2, to analyze the digested DNAs. The gel should show a 5 kb band (digested pYD1) and a smear between 500 and 3,000 bp (digested library fragments) with a peak around 1.5 kb (Figure 4A). Preparation of the library for Oxford nanopore DNA sequencing Prepare six PCR reactions, each with 20 ng of plasmid, 10 µL of ThermoPol buffer 5×, 200 µM dNTPs, 400 nM barcode primers (included in the Native Barcoding Expansion 1-12 from Oxford Nanopore), 1.25 units of Taq DNA Polymerase, and water for a 50 µL final reaction volume. Run the PCR reactions in a thermocycler with an initial denaturation at 95 °C for 30 s, then 16 cycles at 95 °C for 30 s, 60°C for 30 s, and 68 °C for 3 min. Add a final extension of 5 min at 68 °C and store the reaction at 4 °C. Mix the PCR reactions to obtain a total of 300 µL reactions. Add 210 µL of Mag-Bind® TotalPure NGS beads (0.7× ratio, beads volume:PCR reaction volume) and purify the DNA according to the steps described in steps A3 to A8. Elute the DNA in 61 µL of elution buffer. Quantify the DNA using NanoDrop. The expected yield is approximately 30–60 ng/µL. Add 1 µg of DNA to a 0.2 mL tube and mix with 3.5 µL of NEBNext® end-repair buffer and 3 µL of NEBNext end-repair enzyme mix, 3.5 µL NEBNext® FFPE DNA Repair Buffer, 3 µL NEBNext® FFPE DNA Repair mix, and 1 µL (approximately 10 ng) of CS DNA (control DNA, ~3 kb phage lambda DNA fragment). Add water for a final volume of 100 µL. Incubate for 60 min at 20 °C in a thermocycler. Clean up the end-repaired DNAs using Mag-Bind® TotalPure NGS beads with a ratio of 0.7× (beads volume:end repair reaction volume) by adding 70 µL of beads and follow the steps A3 to A8. Elute the DNA in 61 µL of elution buffer. Quantify the DNA using NanoDrop. Expected DNA recovery is approximately 90%. Add the end-repaired DNA (0.5–1 µg) in 60 µL volume to a 0.2 mL tube, then mix with 25 µL of Oxford nanopore ligation buffer (LNB), 10 µL of NEBNext Quick T4 DNA ligase, and 5 µL of Adapter mix (AMX) (LNB and AMX are reagents included in the Ligation sequencing kit from Oxford Nanopore). Mix by carefully pipetting the reaction up and down. Incubate for 2 h at 20 °C in a thermocycler. Add 40 µL of Mag-Bind® TotalPure NGS beads to the ligation reaction (0.4× ratio, beads volume:ligation reaction volume). Mix for 10 min with rotation at room temperature. Quickly spin the sample for 10 s and separate the beads from the solution by incubation on a magnetic stand for 2 min at room temperature. Remove and discard the supernatant. Add 250 µL of short fragmentation buffer and mix gently by flicking the tube. Repeat steps H11 and H12. Then, separate the beads from the solution as described in step H11. Air-dry the beads for 30 s at room temperature. Do not over-dry. Add 12 µL of Oxford nanopore elution buffer. Expect a yield of approximately 10–40 ng/µL, i.e., 200–1,500 fmol of DNA. Between 5 and 55 fmol of DNA are used for sequencing. See Note 5. Load 5–55 fmol of the prepared library into the flow cell (R9.4.1) according to manufacturer’s instruction. Sequence the DNA following the manufacturer's instructions. Data analysis Computational analysis of sequenced library The data used in these analyses were deposited in the Sequence Read Archive (SRA) with BioProject number PRJNA851089. The Minknow software from the MinION Mk1C device creates sequencing report files that summarize the nanopore sequencing results. The .txt summary file can be used to extract information on sequence quality, reads' length, and barcodes. Sequences are produced in fast5 format and then basecalled using the Guppy tool through the MinION device during or after sequencing. For our experiments, we chose high accuracy basecalling through MinION, which generates the fastq files. The following steps describe the tools and scripts used for data analysis using fastq as input files in the pipeline. We also describe the scripts used for calculating genome mapping, visualizing mapped reads, and predicting the peptides produced by the libraries using the Libframe tool we developed. For a schematic summary of these steps and an illustration of the results, see Figures 3 and 4. Note: Before running scripts, check the available computational resources, i.e., memory and processors. We recommend connecting to a server and submitting jobs via Bash scripts. The resources necessary for the job will vary according to the number and size of files and the tools used. The scripts below were submitted to a server (Compute Canada) using Linux Ubuntu in Windows Subsystem for Linux. A general file name is given for simplicity, e.g., "GenomeOfReference.fasta" for the reference genome or "reads-map_v1.sam" for the mapped output file. Figure 3. Flow chart of the computation analysis steps Decompress the fastq files and map the reads to the genome using minimap2. Mapping conditions can be changed to improve the data mapping to the genome. See minimap2 manual (https://github.com/lh3/minimap2). #Before mapping, files in the .fasta.tar.gz format must be decompressed using the bash command tar xvzf yourdata.fastq.tar.gz -C pathdirectory #This script aligns the reads obtained from the ONT sequencing to the reference genome, creating a .sam file. module load minimap2/2.24 minimap2 -ax map-ont -k11 -m30 -w7 -I4G -t16 -2 GenomeOfReference.fasta \ /~/data/fastq/.fastq.gz \ >/~/data/analysis/reads-map_v1.sam Obtain the mapping statistics and convert .sam files to .bam files. Then, sort and index .bam files. The proportion of mapped reads will vary according to the data quality and assembly of the reference genome. #Samtools commands extract statistics from the read mapping and will create the bam binary files for further processing. module load samtools samtools flagstat /~/data/analysis/reads-map_v1.sam > /~/data/analysis/reads-map_v1-flagstat.txt samtools stat /~/data/analysis/reads-map_v1.sam > /~/data/analysis/reads-map_v1-stat_lib.txt samtools view -S -b /~/data/analysis/reads-map_v1.sam > /~/data/analysis/reads-map_v1.bam samtools sort /~/data/analysis/reads-map_v1.bam > /~/data/analysis/reads-map_v1_sorted.bam samtools index /~/data/analysis/reads-map_v1_sorted.bam > /~/data/analysis/reads-map_v1_sorted.bam.bai Perform coverage analysis to determine the read coverage distribution to the genome. Then, perform read counts to determine the number of reads mapped per gene. #Using the DeepTools module to perform further analysis and generate visualizations. module load python/3.8.2 #For these steps, a virtual environment was created source ~/ENV/bin/activate #plotCoverage generates a curve plot depicting the reads per base and the mean coverage of the genome. plotCoverage -b /~/data/analysis/reads-map_v1_sorted.bam --labels reads-map_v1 --plotFileFormat pdf --outRawCounts Reads-V1_Lib.txt --numberOfProcessors 8 #Load required packages for readcount analysis (package subread). Read count was used to quantify the number of reads per gene. module load gcc/7.3.0 module load StdEnv/2020 module load subread/2.0.3 #featureCounts counts mapped reads for genomic features such as genes, exons, promoters, etc. The script below will generate the number of read counts for each exon. featureCounts -LMO -a GenomeOfReference.gtf -o reads-map_v1_counts.txt -F "exon" -g "gene_id" -s 0 -T4 reads-map_v1_sorted.bam Process the data for visualization of library coverage and analysis of potential biases to genome regions. #bamCoverage generates a coverage track by normalizing and binning the reads, producing a .bw file. The file can be analyzed in a genome visualization tool (e.g., an integrated genome viewer such as https://igv.org/app/) for visual validation and to check for potential bias in library coverage. bamCoverage -b reads-map_v1_sorted.bam -o reads-map_v1.bw --binSize 50 --normalizeUsing RPKM --extendReads 1000 --outFileFormat bigwig --numberOfProcessors 10 #computeMatrix creates a matrix of scores per region that is necessary as an intermediate for the visualization of the data as a heatmap. computeMatrix scale-regions -b 500 -a 500 -m 3000 -R GenomeOfReference.gtf -S reads-map_v1.bw --skipZeros --sortRegions no --transcriptID 'exon’ –o matrix_reads-map_v1-sr1.gz #plotHeatmap generates the heatmap to visualize gene coverage using the matrix file from computeMatrix. plotHeatmap -m matrix_reads-map_v1-sr1.gz --outFileName reads-map_v1_heatmap.png --colorMap RdBu --whatToShow 'heatmap and colorbar' --zMin -4 --zMax 4 Generate graphs to visualize the read coverage over the genome using a circular plot. This step requires loading R installed in Linux. The same script can be used for the analysis in RStudio for Windows. #Circlize package was used to generate the circular visualization of genome coverage. First, load R. module load r #Initiate R R #Install required packages install.packages("circlize") #Loading necessary libraries library(dplyr) library(circlize) #Reading data from the plotCoverage output file (reads per base) Lib_Cov.df = read.table('Reads-V1_Lib.txt', sep = "\t") #Order the table according to read counts Lib_Cov.ordered<-Lib_Cov.df[order(Lib_Cov.df$V4), ] #Filter the data frame to have only the reads from complete chromosomes. This is a helpful step for partially sequenced or partially assembled reference genomes. Lib_Cov.chrom <-dplyr::filter(Lib_Cov.ordered, grepl('CHR', V1)) %>% #Converting the scale to log2 to improve visualization (optional) dplyr::mutate(V4 = log2(V4))%>% # Converting all cells that became –inf during log conversion to NA. dplyr::mutate_if(is.numeric, list(~na_if(., -Inf))) #Generate the circlize graph #This first row inicializes the circular graph circos.initializeWithIdeogram(Lib_Cov.chrom, plotType = NULL, circos.par(gap.degree = 8)) #This chunk generates the most outer ring with the chromosomes tracks circos.track(ylim = c((0, 1), panel.fun = function(x, y) { chr = CELL_META$sector.index #Uses the chromosome names as labels xlim = CELL_META$xlim ylim = CELL_META$ylim circos.rect(xlim[1], 0, xlim[2], 1, col = "lightblue") #Changes the color of the chromosome rectangles circos.text(mean(xlim), mean(ylim), chr, cex = 1.5, col = "white", facing = "bending.inside", niceFacing = TRUE) #Changes the color of the text and the way it is oriented inside the rectangles. }, track.height = 0.15, bg.border = NA) #This chunk adds the counts track circos.genomicTrack(Lib_Cov.chrom, panel.fun = function(region, value, ...) { circos.genomicPoints(region, value, type = "segment", lwd = 2, col = "magenta", cex = 0.5, ...) circos.yaxis("right", labels.cex = 0.4, col = "grey", labels.col = "darkgrey") }) #This adds a text in the middle of the circle text(0, 0, "Genome\ncoverage", cex = 1.5) Determine the proportion of sequences in the library that are in frame with proteins in the expression system (e.g., Aga2p-Xpress in pYD1) and calculate their predicted peptide lengths and amino acid sequences. Note: The Libframe tool used in this step was developed in Python and is used for pYD1. If using a different expression system, the code can be modified to replace the Xpress tag sequence with any sequence that should be in frame with the cloned fragments. The code is available at https://github.com/cestari-lab/Libframe-tool. #The libframe script finds the Xpress tag sequence in the reads and translates library nucleotide sequences in-frame with the Xpress tag until a STOP codon is found. #Load python and activate the environment module load python/3.8.2 source ~/ENV/bin/activate #Install biopython pip install biopython #Decompress the .fasta.gz files into .fasta files and concatenate all the multiple FASTA files. gunzip .fastq.gz cat *.fastq > newfilename.fastq #Execute libframe. It will output a text file with peptides' length and sequence. It takes three commands: 1) input fastq file; 2) name for the output file (.txt); and 3) the minimum length of a peptide. The Xpress tag has 8 aa (24 bases), plus linker (6 bases, 2 aa), and restriction site (Bam HI, 6 bases, 2 aa), resulting in the 12 aa sequence DLYDDDDKVPGS. Hence, we recommend the minimum value of 13 for a peptide. python3 ./libframe.py path/to/fastq/file/.fastq path/to/output/file/.txt 13 #Example of results: #Protein length is 127 aa and sequence is: DLYDDDDKVPGSTSHAPGRHGGRRIRLELHLDNFKLLPQLCSSVSADGSPAVPLQPVLPGIRQMLHHSVSIKCADARVARFLWPCPYALHCALLSITSEFAAACGSTIWISVVEFCEISSTVAAARV #Protein length is 16 aa and sequence is: DLYDDDDKVPGSLCWC #Protein length is 75 aa and sequence is: DLYDDDDKVPGSFLLVLILRRLLGCLTLIRAERHNRPQQGFVRRTAGVFHPSVATKSTCVFHFCISMYGRICGLL #Protein length is 25 aa and sequence is: DLYDDDDKVPGSFVGADIAGDRAGK #Protein length is 73 aa and sequence is: DLYDDDDKVPGSFLLVLILRRLHGCLTLCLRVHTLDTKQPEYPLLGGISRAPAHKGAALGGSFSSGCLHAEEV Anticipated results The protocol initiates with the sonication of genomic DNA using a Covaris M220 to obtain DNA fragments between approximately 0.5 and 3 kb. Optimization of sonication conditions may be necessary depending on the genome or sonicator used. We analyzed the sonicated genomic DNA on a 1% agarose/TBE gel to confirm that the DNA was fragmented in the expected length. Our fragmentation conditions resulted in 92% of the T. cruzi genome fragments ranging between 401 bp and 2,000 bp (Figure 4A and 4C). We then used magnetic beads to size select fragments above 500 bp (Figure 4A and B). We advise checking the manufacturer's instructions for size selection if another brand of magnetic beads is used. The size selected DNAs should migrate as a smear similar to the sonication range (Figure 4A) on a 1% agarose/TBE gel. For library preparation steps, we found that end-repair and A-tailing of DNA fragment greatly improve subsequent ligations, even if PCRs are performed with a DNA polymerase that A-tails DNA. After each library preparation step (e.g., DNA end-repair, A-tailing, adapter ligation, PCR reactions, or vector linearization) DNAs were cleaned up with magnetic beads and the recovered DNA quantified using NanoDrop. The 260/280 and 260/230 ratios are ideally 1.8 and 2.0, respectively. Minor variations (approximately 10%) of these values are well tolerated in the assay. More considerable variations indicate potential contaminants that might affect subsequent reactions, especially the Gibson assembly. We found that DNAs purified using magnetic beads result in higher efficiency of Gibson assembly reaction than DNAs purified using silica-based columns and chaotropic buffers. To improve DNA purity, we recommend performing the DNA–beads washes with an ethanol volume equal to the reaction volume plus the beads volume, making sure to extract all the ethanol from the beads and air-drying the DNA before elution. Before scaling up Gibson reactions, we recommend checking the efficiency of assembly. We performed PCR reactions with approximately 20 transformed colonies and analyzed the amplicons on a 1% agarose/TBE gel. Only proceed with the scale-up of library transformation if a minimum of 90% of the colonies show amplicons migrating in the expected size range (size of DNA fragments). We have often obtained 100% efficiency in the Gibson assembly reaction. To troubleshoot the Gibson assembly efficiency, we suggest performing ligations with various ratios of DNA fragments to vector, e.g., 1:2, 1:4, 1:6, and 1:8 (vector:DNA fragments). Most of our libraries worked well in the 1:2 to 1:4 ratio. It is also vital to Sanger sequence a few library clones before scaling up the library transformation to ensure that the library contains sequences from the desired organism. After scaling up the transformations of the library, all colonies should be scraped and combined, and plasmids extracted. To ensure that the fragments were cloned, we performed plasmid DNA digestion to release the cloned fragments. Figure 4 shows results from our T. cruzi genome-wide library in the pYD1 vector. The library was analyzed on a 1% agarose/TBE gel and showed a 5 kb DNA band indicating digested pYD1 and a smear around 1.5 kb indicating digested library fragments (Figure 4A). Our approach was designed for Oxford nanopore sequencing, as it produces long reads covering the entire sequence of the cloned fragment; however, the protocol can be adapted to other sequencing technologies. The extent of the sequencing data for analysis depends on the genome and library sizes. The T. cruzi Sylvio X10/1 strain has a haploid genome of 44 Mb. Our library included four million genomic fragments with an estimated mean length of 1.5 kb resulting in a 30-fold of the genome. In our example in Figure 4, we obtained approximately 8.7 Gb of DNA sequencing, resulting in approximately 8.2 million reads and 147× coverage of the genome (Figure 4D and 4E), which is sufficient for data analysis. After library sequencing and alignment of reads to the genome, we usually obtained 85%–99% genome mapping with minimap2. However, the percentage of genome mapped will vary according to the quality of the reference genome assembly. We recommend checking mapping per chromosome or contigs if the reference genome is not well assembled. We verified any potential bias in cloned fragments or the possible lack of sequences using a circular plot to visualize the reads mapped to the genome (Figure 4D). For a detailed analysis, we used a genome browser (e.g., integrated genome browser). We noted a high number of reads mapping to repetitive regions of large gene families, e.g., mucin or mucin-associated genes in T. cruzi (Figure 4D), which most likely reflects the quality of the genome assembly in these regions rather than bias in library cloning. We found that all the reference genome sequences were present in the library. We performed a plot heatmap analysis using DeepTools and confirmed that all gene sequences were covered by library reads (Figure 4F). Counting the number of reads per exon (featureCounts, package subread) may also help to determine how enriched a gene is in the library. To evaluate the potential peptides generated by our library, we developed a tool that uses the nanopore reads (in fastq format) and searches for sequences in frame with the expression system, e.g., Aga2p and Xpress tag in the pYD1 vector. The Libframe tool predicts the library amino acid sequences and calculates their lengths using the available nanopore data. Using our data set, we found that approximately 90% of predicted peptides had between 21 and 160 amino acids (aa) in length (Figure 4G). The aa length reflects the size of library fragments and the presence of premature stop codons. In genomic libraries, not all DNA sequences are from coding genes, and fragments can be cloned in multiple frames and orientations. Hence, it is important to generate libraries that are at least 6-fold of the genome to ensure that all coding sequences are included (e.g., the T. cruzi library has 4 million fragment sequences and 30-fold of the genome). Overall, our protocol details a step-by-step methodology to efficiently assemble genome-wide libraries into expression vectors for protein–ligand interaction studies. Figure 4. Analysis of T. cruzi genome-wide library for yeast surface display. (A) Left, genomic DNA sonicated using Covaris M220. DNA was resolved in 1% agarose/TBE gel. S1: incidence power 25, 2 duty factor, 200 cycles per burst, for 20 s; S2: incidence power of 10, duty factor 2, 700 cycles per burst, for 30 s. Middle, 1% agarose/TBE of sonicated genomic DNA size selected using magnetic beads to obtain fragments above 500 bp (S). Right, T. cruzi library cloned in pYD1 vector undigested (U) or digested (D1 and D2) with Hind III and Xho I restriction enzymes. D1: 1.0 µg of DNA; D2: 1.5 µg DNA. (B) Image of microcentrifuge tubes containing 100 µL of sonicated DNA sample and magnetic beads (0.7×) mixed before beads separation (mixed) and after separation in a magnetic rack (magnetization). Front and side views are indicated. (C) Analysis of fragment sequence length. Data were obtained from the Oxford nanopore sequencing summary file. (D) Circlize plot created using the Circlize R package shows the read coverage over the genome. Outer green tracks represent the chromosomes; inner orange dots represent mapped reads. Below is a snapshot of chromosome 5 (Chr 5) visualized using the integrated genome browser. The green plot shows read coverage, and the black bars below indicate genes. A 100 kb segment of the Chr 5 (inset above) is also shown. (E) Library coverage analysis using plotCoverage function (DeepTools) depicting read coverage per fraction of genome after mapping. (F) Heatmap of reads per gene generated by the computeMatrix and plotHeatmap functions. All genes were resized to the same length. A 0.5 kb from the start (S) and after the end of the gene (E) are shown. (G) Distribution of predicted library proteins in frame with proteins from the expression system, i.e., Aga2p and Xpress tag. Data were analyzed using the Libframe tool. Notes If using a standard probe sonicator, we recommend optimizing the sonication conditions starting with 50% frequency, 1,000 J, and three pulses of 5 s with 15 s rest between pulses. Perform the procedure in ice. The user should refer to Omega Bio-Tek “Protocols and resources” section to select the ratio of beads during the size selection process according to the University of Oregon, Genomics & Cell Characterization Core Facility protocol (Weitzman, 2018). We recommend first performing a PCR reaction with 35 cycles and analyzing the amplicons in an agarose gel to check the efficiency of amplification. It should result in a smear ranging from 0.5 to 3 kb (Figure 4A). Perform a gradient PCR to optimize annealing temperature if necessary. If no amplicons are observed, check for failed end-repair or adapter ligation reactions, and repeat section B. Once the amplification is confirmed, proceed with the multiple 14-cycle PCR amplification reactions, which should provide enough DNA to perform the next steps of the protocol. The number of PCR cycles should be kept between 14 and 16 to prevent bias in the library due to PCR amplification of selected sequences, or the introduction of PCR artifacts. We recommend checking plasmid complete digestion before library assembly by performing agarose gel electrophoresis. Restriction enzyme–digested plasmids can be purified from agarose gel using silica-based columns; however, they should always be cleaned up using magnetic beads before Gibson assembly. We observed that the presence of impurities common to silica-based DNA purification methods with chaotropic buffers decreases the efficiency of the Gibson assembly. Calculation of fmol based on average fragment size of 1.5 kb. Chemical-competent Escherichia coli cultures were obtained using the low-temperature method (Ueda and Tanaka, 2000). We used the NEB® 5-alpha F'Iq E. coli strain for preparing the competent cells. Theefficiency should be 1 × 109–5 × 109 CFU/µg of pUC19 plasmid DNA. Bacterial electroporation protocols usually produce highly efficient transformations using purified plasmids (1010 CFU/μg of pUC19 DNA). However, the electroporation of Gibson assembly reactions will result in lower efficiencies than chemical transformation. We noticed that the Gibson reaction components interfere with the electroporations. Hence, we recommend using chemical transformations. We also do not recommend using more than 1 µL of the Gibson assembly reaction for the chemical transformations. Transformation efficiency (TE) is calculated as: Whereas CFU is colony-forming units, NT is the number of transformants, VT (µL) is the volume of transformation in µL, DNA (µg) is the amount of DNA in µg added to the cells, VCP (µL) is the volume of cells plated in µL, and DF is the cell dilution factor in plating. Poor quality DNAs can affect the Gibson assembly reaction. Ensure 260/280 and 260/230 ratios are in the recommended ranges. To troubleshoot the Gibson assembly efficiency, we recommend performing ligations with various ratios of fragments to vector, e.g., 1:2, 1:4, 1:6, and 1:8 (vector:DNA fragments). Recipes Luria-Bertani (LB) medium with agar and ampicillin Prepare LB broth by mixing 10 g of Bacto-tryptone, 5 g of Bacto-yeast extract, and 5 g of NaCl; dilute to 900 mL of distilled water. Adjust to pH 7.25 with 1 N NaOH or 1 N HCl as necessary, and then add distilled water to the final volume of 1 L. Add 15 g Bacto-agar to the LB broth and autoclave immediately. Wait to cool to approximately 40–50 °C and add 1 mL of ampicillin stock solution (100 mg/mL). Then, pour 40 mL into 150 mm × 15 mm Petri dishes. Keep Petri dishes at 4 °C for a maximum of one month. Ampicillin stock solution (100 mg/mL) (50 mL) Dissolve 5 g of ampicillin in 45 mL of distilled water. Adjust the volume with distilled water to 50 mL. Filter using a 0.2 µm syringe filter. Prepare 1 mL aliquots and keep them at -20 °C. Tris-Borate-EDTA buffer (10 L) Prepare TBE buffer by mixing 1 L 10× TBE stock solution (see below) with 9 L distilled water to a final volume of 10 L. 10× TBE stock solution (1 L) Prepare TBE solution by mixing 108 g of Trizma base, 55 g of boric acid, and 8.2 g of EDTA to 800 mL of distilled water. Adjust to pH 8.3 with 1 N NaOH or 1 N HCl as necessary. Add distilled water to a final volume of 1 L. Filter using a 0.2 µm syringe filter. Keep at room temperature. SOB medium (1 L) Prepare SOB medium by mixing 5 g of NaCl, 20 g of tryptone, 5 g of yeast extract, and 2.5 mL of 1 M KCl, and dilute to 800 mL of distilled water. Add distilled water to a final volume of 1 L. Autoclave immediately. Keep at 4 °C for up to six months. Inoue solution (1 L) 86.62 mM MnCl2, 19.82 mM CaCl2, 250.84 mM KCl, 10 mM PIPES buffer Prepare Inoue solution by mixing 10.9 g of MnCl2, 2.2 g of CaCl2, 18.7 g of KCl, and 20 mL of 0.5 M piperazine-1,2-bis [2-ethanesulfonic acid] (PIPES) buffer (see below). Add MilliQ water to final volume of 1 L and filter-sterilize using 0.2 µm filter. 0.5 M PIPES buffer (100 mL) Dissolve 15.1 g of PIPES in 80 mL of MilliQ water by throwing in pellets of KOH until the solution clears up. Adjust to pH 6.7 with 1 N KOH or 1 N HCl. Add MilliQ water to the final volume of 100 mL and aliquot in 10 mL tubes. Store at -20 °C. 70% ethanol Mix 100 mL of MilliQ water with 368 mL of ethanol (95%). Adjust the volume by adding MilliQ water to a final volume of 500 mL. Keep it at 4 °C. Acknowledgments This work was funded by the Canadian Institutes of Health Research (CIHR PJT-175222, to IC), the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2019-05271, to IC), the Canada Foundation for Innovation (JELF 258389, to IC), and by McGill University (130251, to IC). We thank Andressa Lira and Sahil Sanghi for their technical support at the early stages of this work. This research was enabled in part by computational resources provided by Calcul Quebec (https://www.calculquebec.ca/en/) and Compute Canada (www.computecanada.ca). This protocol is derived from the original research paper by Rhiannon Heslop et al. (2023). Competing interests The authors declare that they have no conflicts of interest with the contents of this article. References Bharucha, N. and Kumar, A. (2007). Yeast genomics and drug target identification. Comb Chem High Throughput Screen 10(8): 618-634. Bidlingmaier, S. and Liu, B. (2011). Construction of yeast surface-displayed cDNA libraries. Methods Mol Biol 729: 199-210. Boder, E. T. and Wittrup, K. D. (1997). Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6): 553-557. Danecek, P., Bonfield, J. K., Liddle, J., Marshall, J., Ohan, V., Pollard, M. O., Whitwham, A., Keane, T., McCarthy, S. A., Davies, R. M., et al. (2021). Twelve years of SAMtools and BCFtools. Gigascience 10(2). Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd and Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5): 343-345. Gu, Z., Gu, L., Eils, R., Schlesner, M. and Brors, B. (2014). circlize Implements and enhances circular visualization in R. Bioinformatics 30(19): 2811-2812. Inoue, H., Nojima, H. and Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene 96(1): 23-28. Kieke, M. C., Cho, B. K., Boder, E. T., Kranz, D. M. and Wittrup, K. D. (1997). Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng 10(11): 1303-1310. Lemos Duarte, M., Trimbake, N. A., Gupta, A., Tumanut, C., Fan, X., Woods, C., Ram, A., Gomes, I., Bobeck, E. N., Schechtman, D., et al. (2021). High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics. Commun Biol 4(1): 238. Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34(18): 3094-3100. Liao, Y., Smyth, G. K. and Shi, W. (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30(7): 923-930. Pointer, K. B., Clark, P. A., Zorniak, M., Alrfaei, B. M. and Kuo, J. S. (2014). Glioblastoma cancer stem cells: Biomarker and therapeutic advances. Neurochem Int 71: 1-7. Ramirez, F., Ryan, D. P., Gruning, B., Bhardwaj, V., Kilpert, F., Richter, A. S., Heyne, S., Dundar, F. and Manke, T. (2016). deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res 44(W1): W160-165. Smith, A. M., Ammar, R., Nislow, C. and Giaever, G. (2010). A survey of yeast genomic assays for drug and target discovery. Pharmacol Ther 127(2): 156-164. Ueda, M. and Tanaka, A. (2000). Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18(2): 121-140. Weitzman, M. (2018). Evaluation of Omega Mag-Bind® TotalPure NGS Beads for DNA Size Selection. Genomics & Cell Characterization Core Facility, University of Oregon Technical Note. Heslop, R., Gao, M., Brito Lira, A., Sternlieb, T., Loock, M., Sanghi, S. R. and Cestari, I. (2023). Genome-Wide Libraries for Protozoan Pathogen Drug Target Screening Using Yeast Surface Display. ACS Infect. Dis: e2c00568. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Systems Biology > Interactome > Protein-ligand interaction Microbiology > Heterologous expression system > Saccharomyces cerevisiae Drug Discovery > Drug Design Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Determination of Boron Content Using a Simple and Rapid Miniaturized Curcumin Assay Thotegowdanapalya C. Mohan and Alexandra M. E. Jones Jan 20, 2018 7548 Views Analyzing the Functionality of Non-native Hsp70 Proteins in Saccharomyces cerevisiae Laura E. Knighton [...] Andrew W. Truman Oct 5, 2019 3466 Views Purification of Human Cytoplasmic Actins From Saccharomyces cerevisiae Brian K. Haarer [...] Jessica L. Henty-Ridilla Dec 5, 2023 521 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,559	https://bio-protocol.org/en/bpdetail?id=4559&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa William J. Nicolas GJ Grant J. Jensen EM Elliot M. Meyerowitz Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4559 Views: 1052 Reviewed by: Shyam Solanki Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Current Biology Jun 2022 Abstract Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall. Keywords: FIB-milling Plant cell wall Onion Cryo-ET Cellulose Cell morphology Background Cryo-electron tomography (cryo-ET) relies on the ability to observe a thin vitrified or cryo-immobilized sample. Vitrified water is a metastable state of water achieved when the freezing rate is high enough that the water molecules do not have time to reorganize in a hexagonal lattice crystal (Dubochet, 2007). This state of water, also called amorphous ice, can also withstand the vacuum of the cryo-electron microscope (cryo-EM). In turn, the sample is observed in a near-native state, as all cellular processes were immobilized at freezing time. At ambient pressure, vitrification is achieved by plunge freezing the sample in liquid propane, ethane, or a mix of both. The freezing rates achievable with these cryogens at ambient pressure limit vitrification thickness to approximately 10 μm. After proper vitrification, samples that exceed 500 nm in thickness need to be thinned down. The go-to method nowadays is cryo-focused ion beam (cryo-FIB) milling (Villa et al., 2013). Originally used in the material science field, this technique uses a focused gallium beam to erode the sample away and create thin lamellae, approximately 200 nm thick, amenable to cryo-ET. Onion epidermal cell wall peels have been used in atomic force microscopy (AFM) studies to study the arrangement of the cellulose fibers in the superficial inner layers of the cell wall (Kafle et al., 2013; T. Zhang et al., 2014, 2017). They are a model for the structural and mechanical study of the primary cell wall (Y. Zhang et al., 2021). Despite being a high-resolution imaging method allowing observations in the native state, AFM can only image the few superficial layers of the cell wall. These cell wall peels happen to be between 5 and 10 μm in thickness, which is compatible with plunge freezing but still too thick for direct imaging by cryo-EM. Performing cryo-FIB milling on the cell wall peels allow i) screening and tilt series acquisition by cryo-ET, and ii) access to the deeper layers of the cell wall, previously unattainable by AFM. This protocol describes the sample preparation, data acquisition, and processing involved in our study called “Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks” (Nicolas et al., 2022). We are confident that this system, in conjunction with cryo-ET and cryo-FIB milling, can become a powerful method to study the effects of mechanical stress and various treatments on the cell wall, while allowing a better understanding of the interaction of the different components present in the cell wall and their mechanical impact there. Materials and Reagents Plant material Plastic Petri dishes (RPI, catalog number: 160265) Sharp knife Double edge razor blades (EMS, catalog number: 72002-01) Glass slides (EMS, catalog number: 71863-01) Cover slips (VWR, catalog number: 48366-067) Fresh white onions from the local supermarket (see Note 1) HEPES powder (RPI, catalog number: H75030-50.0) CAPS powder (Sigma, catalog number: C2632-100G) KOH powder (Sigma, catalog number 06103-1KG) Tween-20 (RPI, catalog number: P20370-0.5) Deionized (DI) water BAPTA (Sigma-Aldrich, catalog number: C2632-100) Pectate lyase from Aspergillus (Megazyme, catalog number: E-PCLYAN2) NaOH powder (Sigma, catalog number RDD007-1KG) HEPES 20 mM buffer solution (see Recipes) CAPS 50 mM buffer solution (see Recipes) Pectate lyase at 4.7 U/mL (5 mL) (see Recipes) BAPTA 2 mM (see Recipes) Plunge freezing Glass slides (EMS, catalog number: 71863-01) Grid boxes (Thermo Fisher) (see Note 2) London Finder Quantifoil grids NH2 R2/2 copper 200 mesh (EMS #LFH2100CR2) (see Note 3) Cut up blotting pads (Whatman, catalog number: 1540-055) Liquid nitrogen Liquid ethane/propane (60/40 ratio) Ethanol 70% Grid clipping FIB autogrids (Thermo Fisher, catalog number: 1205101) C-rings (Thermo Fisher) Magnifying lens (Intertek LTS-F21-61) Clipping pens (Thermo Fisher) Sharpie pen (red or black) Autogrid grid boxes (Thermo Fisher) Lid pen (Thermo Fisher) Cryo-FIB milling Liquid nitrogen Argon gas Shuttle (Quorum Tech, catalog number: 12406) Cryo-ET screening and tilt-series acquisition Grid cassette (Thermo Fisher) NanoCab (Thermo Fisher) Equipment Plant material Any light microscope equipped with phase contrast Plunge freezing EMS tweezers (EMS style 2, 5, 3X, and 7) Vitrobot tweezers (Inox-Medical LS 11253-27) Flexible LED lamp Large forceps and small rubber band (EMS style 7330) Vitrobot Mark II (Thermo Fisher) Glow discharger (EMS 100X, not sold anymore; alternatively, Pelco easyGlow) Liquid nitrogen storage system (Worthington HC35) LN2 containers (spearlab.com) Grid clipping Autogrid tweezers (PELCO 5046-SV) Clipping station (Thermo Fisher) Cryo-FIB milling Versa 3D DualBeam FIB-SEM (FEI) Quorum PP3000T transfer system (Quorum Tech) Cryo-ET screening and tilt series acquisition Titan Krios 300 kV microscope (Thermo Fisher) Gatan K3 direct director (Gatan) Post-column GIF energy filter (Thermo Fisher) Software SerialEM (https://bio3d.colorado.edu/) (Mastronarde, 2003, 2005), to control the TEM Gatan DM3 (https://www.gatan.com/products/tem-analysis/gatan-microscopy-suite-software), to control the K3 detector Etomo (https://bio3d.colorado.edu/imod/download.html) for tomogram reconstruction and 3dmod for tomogram visualization (Kremer et al., 1996; Mastronarde, 1997; Mastronarde and Held, 2017) EMAN2 (https://blake.bcm.edu/emanwiki/EMAN2) for Convolutional Neural Network (CNN) training and application (Chen et al., 2017; Tang et al., 2007, p. 2), Amira and the Xtracing plugin (Thermo Fisher) (https://www.thermofisher.com/us/en/home/electron-microscopy/products/software-em-3d-vis/amira-software.html) for segmentation (Rigort et al., 2012) ARETOMO for automated alignment and reconstruction (https://msg.ucsf.edu/software) Custom scripts derived from Dimchev et al. (2021) R and R studio to analyze the data (https://cran.r-project.org/index.html, https://www.rstudio.com/) Procedure Preparation of the onion cell wall peels Note: This protocol involves the use of liquid nitrogen and other cryogenic liquids that could lead to severe burning if not handled properly. Please refer to your institution’s safety guidelines for manipulation of these liquids. For all the following steps described in this manuscript, samples after vitrification should always be handled with pre-cooled tools, so always precool the tools before touching the grids or grid boxes to avoid devitrification of the sample. Gather fresh, hard, hydrated white onions (see Note 1). Cut off the tips of the onion and take off the dried, brownish superficial layers until the first fresh layer is reached (Video 1, step 1). This is layer one (Figure 1A). Figure 1. Anatomy of an onion scale and generation of the epidermal cell wall peels. (A) Global view of a longitudinally cut onion. (B) Side and top views (left and middle, respectively) of an onion scale. Black lines indicate the first incisions to be done to cut away the tips. Red lines indicate the incisions to be done to create the rectangular cell wall peels. Right, top view of three separate rectangular-like scale explants with the black lines indicating the incisions to be made to create handles that will be used to generate the peels in the next step. (C) Top row: left shows top views of three rectangular-like scale explants. Here, the abaxial epidermis is facing upwards (glowing, waxy side) and is the side to be used to generate the cell wall peel. Middle shows a piece of onion scale in the process of being peeled. The clear membrane is the cell wall peel. Right shows two cell wall peels with the two handles at their extremity incubating in HEPES buffer. Bottom row: side view diagrams of the peeling process. Left shows a scale just incised, so that the abaxial epidermal layer has not been cut (black dashed lines). The two side squares are the handles to pull on in order to create the cell wall peel. Middle shows the beginning of the process where the handle is pulled away from the main body of the scale to tear away the abaxial epidermal layer. Right shows the epidermal peel where the abaxial epidermal cells were ripped in half. The two handles on the side are used to manipulate the peel. (D) Contrast phase image of a successfully peeled abaxial epidermal cell wall. The anticlinal cell walls show jagged edges. See also Video 1. Video 1. Peeling of the onion. The peeling procedure to create the cell wall peels from intact onions is described; it is divided into five steps: i) preparation of the onion, ii) sampling the scales, iii) preparing the scales, iv) peeling the cell wall, and v) screening of the cell wall peel. Create two deep longitudinal incisions going down to the core of the onion. These incisions should cover approximately a quarter of the onion (90°) (Video 1, step 2). Take apart the scales from this excised part. The scales are concave (Figure 1B) and the abaxial side, where the peels will be created, faces outside (convex part). Keep note of the order of the scales. Cut off the tips of the scale (a few centimeters from the tips) and keep the central large part of the scale (Figure 1B, black dashed lines, and Video 1, step 3). Cut this central part longitudinally in several bands, each approximately 3 cm long and 1 cm wide (Figure 1B, red dashed lines, and Video 1, step 3). There should now be several rectangular excisions from the scale (3 × 1 cm). The waxy, hydrophobic side is the epidermis and the more porous side will be discarded later. For each band, create two transversal incisions 0.5 cm away from the extremities on the adaxial porous side. These incisions should go through the entire scale, except the waxy abaxial thin membrane, i.e., the epidermal cell wall (Figure 1B and C and Video 1, step 4). The excised scales should now have two floppy extremities, here called handles (Video 1, step 4). Grab one of the handles, fold over towards the epidermal cell wall and pull by brief powerful strokes to rip the abaxial epidermal cells in half. A Velcro sound should be heard (Video 1, step 4). A continuous membrane between the two thick handles indicates successful cell wall peeling (Figure 1C). Cut off one of the handles and mount the peel between glass slide and cover slip. Screen the peel by light microscopy to control its quality. Only peels with close to 100% ripped cells are kept for freezing (Figure 1D, Video 1, step 5). Incubate the peels in HEPES 20 mM buffer (see Recipe 1) for 20 min to clean out cellular remnants off the cell wall peel. Plunge freezing of the cell wall peels Note: The onion cell wall peels need to be presented parallel to the FIB beam for optimal milling. It is therefore important to keep track of the direction of the cells within the peel during freezing, clipping, and mounting of the grids in the FIB-SEM, despite not being able to see the cells (see Note 4). Turn on the Vitrobot and prepare the liquid nitrogen and ethane/propane cup (Video 2, step 1). Video 2. Plunge freezing of the cell wall peels. The cell wall peel vitrification procedure is described: i) preparing the freezing well, ii) glow discharging the grids, iii) cutting up the cell wall peel to fit on an EM grid, iv) setting the cell wall peel explant on the EM grid, and v) grid blotting and plunge freezing. Figure 2. Plunge freezing of the cell wall peels. (A) Global view of the freezing setup: 1- Vitrobot, 2- Freezing well, 3- Various tweezers and forceps, 4- Petri dish with HEPES buffer and cell wall peels, 5- Glass slide where the cell wall peel will be dissected, 6- Light shining tangential to the table, 7- Liquid nitrogen dewar, 8- Liquid nitrogen container for grid boxes, 9- Propane/ethane filling station, and 10- Desk lamp shining at the well for extra visibility while transferring vitrified grids to the grid boxes. (B) Top view of a cell wall peel, where one handle (left one) has been removed. Black dashed lines indicate the first incisions to be made. Red dashed lines indicate the latter incisions to be made to generate small rectangles to be positioned on the EM grid. (C) Top view of a London Finder Quantifoil grid with a cell wall peel positioned along the grid notch. The right inset shows that the long axis of the onion cells is more or less parallel to the direction of the main notch. See also Video 2. Glow-discharge the grids at 20 mA for 1 min (Video 2, step 2). Gather all necessary tools and equipment to prepare the rectangles of the cell wall next to the plunge freezer (Figure 2A). Prepare labeled grid boxes (see Note 2) and long forceps with blotting pad at the end. Place the cell wall peel on a glass slide with a drop of HEPES buffer. Never let the peel dry out. Shine a bright light tangential to the glass slide towards the experimenter in order to see the edges of the thin cell wall peel (Video 2, step 3). Optionally, if certain extremities of the peel look sub-optimal, cut and discard them, so that the region to be used is accessible now. Use the Vitrobot tweezers to securely grab the Quantifoil EM grid at the triangular notch (Figure 2C shows the Quantifoil notch, Video 2, step 3). Using a sharp razor blade, apply constant pressure on the peel extremity to make clean incisions, parallel to the long side of the peel, approximately 1 cm long if possible. Reiterate this process on the whole width of the peel in order to have clean parallel incisions equal in length (Figure 2B black dashed lines, Video 2, step 3). Make clean incisions perpendicular to the ones generated in step B9 starting on one side of the peel. This will free a small rectangle of approximately 3 × 1 mm that can fit on an EM Quantifoil grid (Figure 2B red dashed lines, Video 2, step 3). With the other hand and another pair of tweezers, grab the very tip of the short end of one of the rectangular peels and drag it on the grid so that the long side of the peel is parallel to the grid notch (Figure 2C, Video 2, step 4). Load the Vitrobot tweezers in the Vitrobot chamber by clipping it on the lowered rod and press on the pedal to raise the tweezers in the chamber (Video 2, step 4). Blotting settings: humidity 50%, 20 °C. Grids are first manually back blotted for approximately 6 s and then the pedal is pressed to initiate two iterations of automatic blotting front and back (5 s, maximal blot force of 25 and drain time of 3 s) (Video 2, step 5). Store the grids in the grid boxes (see Note 2). Iterate steps B8 to B15 for making more grids. When done, store the grid boxes in the liquid nitrogen storage dewar (Video 2, step 5). Grid clipping Note: Detailed clipping will not be described as this has been done elsewhere (Wagner et al., 2020) and can be found online (https://em-learning.com/totara/dashboard/index.php). It is however emphasized in Note 4 how to align the milling notch of the autogrid with the long side of the onion peel. Load the C-rings in the clipping pens. Prepare as much as necessary. Optionally, if using the Thermo Fisher FIB autogrids, mark the sides of the grid on the opposite side from the notch (for easily locating the notch in future steps) using a Sharpie. Place the FIB autogrids in the clipping station. Cool down the clipping station and place the lid to avoid ice contamination to accumulate in the station. Perform all subsequent operations with the lid on. Clip each grid, carefully aligning the milling notch of the autogrid with the long side of the peel, making sure that the peel is facing down in order to have it exposed to the FIB/SEM beams in the latter steps (Figure 3). Figure 3. Clipping vitrified grids with FIB autogrids. Top and side view (left and right, respectively) of a clipped grid. Top view emphasizes how the cell wall peel (and the notch of the grid) needs to be aligned with the notch of the autogrid to allow milling parallel to the cell wall peel. The black dots are landmarks visible by the naked eye to allow orienting the FIB autogrid. The side view emphasizes that the peel needs to be exposed on the rounded part of the autogrid. When clipping in the clipping station, this means that the grid needs to be facing down. This allows the sample to be exposed to the FIB beam. Cryo-FIB milling Note: The process of cryo-FIB milling is also thoroughly described elsewhere (Schaffer et al., 2015; Wagner et al., 2020). Emphasis will be put on a few key points relevant if using the cryo-transfer chamber Quorum PP3000T and a Versa 3D DualBeam FIB-SEM. Optionally, if GIS coating is intended, tune the temperature of the GIS needle to 26 °C. Cool down the loading well with the shuttle inserted vertically (Figure 4A and 4B). Figure 4. Loading of the clipped grids in the FIB/SEM with the Quorum PP3000T system. (A) Global view of the cryo-FIB/SEM setup. CHE3010T is the liquid nitrogen tank that cools down the flowing nitrogen, keeping the sample at cryogenic temperature. (B) Top view of the well where the grids are loaded in the shuttle. The shuttle is mounted on a hinge system and is seen vertical in this example. (C) Top: Picture of the shuttle that can fit two grids. Bottom: Top view of the grid loaded in the shuttle (left) and simplified diagram of the clamping system (right). Notice the milling notch and peel are facing upwards. This is crucial for successful milling. (D) FIB views of the milling process of a lamella in the periclinal cell wall. The lamella is boxed in orange. What is seen above and below the lamella is in the background. (E) SEM view of the final lamella shown in (D). Notice that the lamella is surrounded by dark holes above and below it. This ensures that the electron beam in the TEM will have a clear path through the lamella to the detector. Transfer grid boxes with clipped grids to the well. Load the grids in the two slots (it is also possible to load the grids one by one to avoid too much in-chamber ice contamination during milling) and make sure milling notch is positioned vertically (see Note 4, Figure 4C). This will ensure that the peel is presented parallel to the FIB beam. Pump down the well, load the shuttle on the transfer rod, and connect the rod to the cryo-preparation chamber. Note: Cryogenic temperature during the transfer of the rod from the well to the preparation chamber is solely maintained by the vacuum. It is imperative to go as quickly as possible during this step to avoid devitrification. Platinum sputter coat at 15 mA for 60 s. Transfer the shuttle from the preparation chamber to the SEM chamber. Screen the grid and identify areas of interest. For each spot, save the x and y coordinates, proceed to setting the stage to eucentric height, and update their z coordinate (see Note 5). For each spot, set up beam coincidence and update their z coordinate (see Note 5). Optionally, if using the GIS needle for adding an extra coating layer of platinum, proceed as follows: Go to a spot away from the loaded grids. For convenience, save the coordinates as “flush out spot” in order to call this position easily in the future. Lock stage position. Take a demagnified picture to make sure the stage is out of the field of view. Insert GIS needle and open valves for approximately 15 s as a flush out step. Take a picture to make sure needle is inserted. Retract needle and unlock stage position. Take a picture to make sure needle is retracted. Recall milling position. Set Z height to eucentric +2 mm. Lock stage position. Insert GIS needle and open valves for approximately 3–5 s. Retract needle and take picture to make sure it retracted. Unlock stage position. Note: In between each milling step, astigmatism and focus must be checked away from the milling region. Set stage angle as low as possible without hindering the field of view of the FIB beam (see Note 6). Set the SEM imaging conditions to 5 kV and 27 pA in order to accurately assess lamella thickness. Set beam intensity to 1 nA and milling pattern to “Clean Cross Section” (CCS) and select the “Si-ccs” application with a dwell time of 1 μs and a Z size between 1 and 3 μm (Figure 4D and Table 1), until the initial lamella is approximately 3 μm thick. Set beam intensity to 0.5 nA (Figure 4D and Table 1). Note: According to Schaffer et al. (2017), in order to have a homogenously thick lamella, the lamella has to be rocked back and forth when milling the underside and the topside, respectively. Increase angle +1° and shave off approximately 1 μm of the top of the lamella (Table 1). Increase angle -1° and shave off approximately 1 μm of the bottom of the lamella (Table 1). The lamella should be approximately 1 μm thick (Figure 4D). Set beam intensity to 100 pA (Table 1). Increase angle +0.5° and shave off approximately 200 nm of the top of the lamella (Table 1). Increase angle -0.5° and shave off approximately 200 nm of the bottom of the lamella (Table 1). The lamella should be approximately 600 nm (Figure 4D). Set beam intensity to 30 pA (Table 1). Set the angle to the original milling angle and shave off the top of the lamella until it reaches its final thickness of approximately 200 nm (Figure 4D). Optionally, to reach final thickness, beam intensity can be set to 10 pA. Note: It is important to have set beam coincidence in order to be able to monitor lamella thickness, especially towards the final stages of milling where the lamella is getting thin and very fragile (see Note 7 for more details). Iterate steps D12 to D23 for other milling targets on the grid. When switching to other grid, set the stage angle to 0 and pan over to the other grid. Note: For ease of navigation save left and right grid slot positions in order to easily recall these positions. Iterate steps D8 to D23 for screening and milling the other grid. When done screening both grids, set the stage angle to 0° and come back to loading position. Note: For ease of navigation, loading position should also be saved for easy recall. Close valves of the electron and FIB beam. Fetch the shuttle with the transfer rod and transfer it to the cryo-preparation chamber. Transfer back the shuttle into the precooled preparation well. Secure the grids back into the grid box. For loading and screening/milling new grids, iterate steps D4 to D31. Cryo-ET screening and tilt series acquisition Note: Only the key points of loading the grids for observing lamellae will be emphasized. In-depth information can be found online (https://em-learning.com/login/index.php). This run-through is relevant for the use of the Titan Krios cryo-TEM operated through SerialEM, a highly modular, scriptable software (Mastronarde, 2003). The steps detailed here to acquire the tilt series are meant for data acquisition on lamella as performed in Nicolas et al. (2022). For a more global understanding of what the different used options do and how this integrates in the spectrum of possible tomography workflows, please refer tohttps://bio3d.colorado.edu/SerialEM/hlp/html/main_index.htmand the SerialEM video serieshttps://www.youtube.com/playlist?list=PLGggUwWmzvs-DV4jCapSl5XQ-hpAXtzMb. Precool the loading station and the NanoCab (Figure 5A, Video 3, steps 1 and 2). Figure 5. Loading the FIB milled grids in the Krios. (A) Global view of the loading station. (B) Top: Top view of the well of the loading station showing the autogrid forceps (red), open grid box containing three grids (blue), grids from the previous user (yellow), and the Krios cassette (orange outline). Bottom: Diagrams of the cassette from the top and side (left and right, respectively), with a magnified view of one grid slot to show the orientation of the grid in the cassette. Green arrow shows how the sample side of the autogrid should face towards the tip of the Krios cassette (as opposed to the side grabbed by the handle, blue arrow); the two dots are the two landmarks on the FIB autogrid indicating the axis of the notch. They should be positioned horizontally in the cassette. (C) Low dose control panel in SerialEM. Red square ticked indicates low dose mode is active. Green rectangle are the radio buttons to visualize the focus and trial spots. Blue rectangle allows to switch between the different imaging modes defined as “View,” “Focus,” “Trial,” “Record,” and “Search.” (D) Atlas montage of the whole grid. (E) View image (×2,250 magnification) of a lamella. See also Video 3. Video 3. Loading of the milled grids in the Krios. Loading procedure is divided into five steps: i) cooling down of the loading station, ii) cooling down of the NanoCab, iii) transfer of the grid boxes in the loading station, iv) loading of the grids in the Krios cassette, and v) loading of the Krios cassette in the Titan Krios microscope. Note that the experimenter in this video is not wearing the recommended protective eyewear and insulating gloves. Transfer grid box with milled grids in transfer station (Video 3, step 3). Put the grids in the slots of the cassette, so that the long axis of the lamellae is perpendicular to the tilt axis of the grid holder, leaving slot #1 free (commonly used slot for cross-grading grid) (see Note 8, Figure 5B, and Video 3, step 4). Using the handle in the loading station, clamp the cassette and push it in the NanoCab (Video 3, step 4). Transfer the NanoCab to the docking station of the microscope and load the cassette (Video 3, step 5). When the cassette is loaded, let the cartridge gripper cool down to < -165° and start an inventory of the cassette. Load a grid. In the Low Dose panel, tick Low Dose Mode (Figure 5C, red square). Generate a low magnification (search mode, x82) atlas of the grid to spot the lamellae (Figure 5D): Navigator > Open (will open a new Navigator panel); Navigator > Montage & Grids > Setup Full Montage (see Note 9); select the place holder of the file and the name of the atlas file. Montage Controls > Start. Spot good looking lamella: no hexagonal ice due to failed vitrification, no cubic ice due to devitrification, and electron-lucent (clear looking). Acquire a view image (×2,250) of the lamella (Figure 5E). Make sure the lamella appears vertical (perpendicular to the tilt axis). Take a view image of the lamella and make sure it is centered. Set the stage to eucentric height: Tasks > Eucentric – Rough. In the Navigator panel, select the object corresponding to the lamella and Update Z. Note: Regularly save the navigator panel in case SerialEM crashes: Navigator > Save as… Before going any further, go into an empty area near the lamella, Navigator panel > Add Stage Position to record this empty area for tuning the beam. Go to the working magnification (record or preview), center/tune the energy filter, and perform a gain/dark reference in counting mode using Gatan DM3 software. Tasks > Setup Autocenter… to set up the Autocenter beam function. Get out of low dose mode and follow instructions. Get back into low dose mode once this is set up. Now Tasks > Autocenter Beam will center the beam by focusing the beam and doing edge detection. Define areas of interest where tilt series will be acquired by using the Anchor Maps function in the Navigator panel (see Note 10). If correctly done, the anchor maps should appear as blue outlines on the view image of the lamella. Camera & script panel > view to have a global view of the lamella with acquisition areas (blue rectangles). Navigator panel > select the first Sec 0 – AnchorHM.mrc item, tick Tilt series > Set tilt series acquisition parameters in the pop-up panel, and define tilt series parameters (see Note 11, Figure 6A, green rectangles for mandatory parameters and blue rectangles for optional control of dose spread along the tilt series). Figure 6. Setting up tilt series acquisition parameters. (A) Tilt series acquisition window. The green rectangles are the bare minimum parameters to be set up. In this example, a tilt series ±50° with a base increment of 3° using a dose symmetric scheme (see dose symmetric pop-up window in red and description below) with a target defocus of -8 μm. Optionally, the exposure can be set to vary according to tilt angle in order to distribute the dose optimally (blue rectangles). In the dose symmetric panel, at least two of the parameters need to be set (green rectangles). In this example, initial group size was set to 5 and alternation is set to end at 30°. This means that from 0 to ±30°, the stage will rock back and forth every five tilt images. After 30°, the stage will go linearly from ±30° to ±50°. (B) Acquire at items window. The typical checkboxes to activate are highlighted in red. This means that the stage will recall the position of the area to acquire, align it using the anchor maps as reference (realign to item), auto center the beam, do rough and fine eucentricity on the position, do autofocus, and start the tilt series. Valves will be closed when all tilt series are acquired. (C) Slice of a cryo-tomogram acquired on a cell wall lamella. The long rod-like densities are the cellulose fibers. (D) Slice of a probability map generated by the EMAN2 CNN trained to recognize the cellulose fibers on tomograms such as (C). (E) Template matching–based vector field generated by AMIRA from the CNN maps such as (D). These vector fields were used to extract per-fiber parameters used for analysis. Tick Edit focus and check whether the focus/trial spot is not overlapping with another tilt series to be acquired and that it is on the tilt axis. Repeat steps E19 to E20 to set up remaining tilt series. To edit the parameters of select tilt series, Navigator panel > TS Params. When all tilt series are set up, Navigator > Acquire at items and tick Rough eucentricity, Fine eucentricity, Autocenter beam, Realign to item, and Autofocus; Acquire tilt series and tick Close column valves at end if needed (Figure 6B, red squares) Reiterate steps E12 to E23 on the subsequent lamellae. Data analysis Note: Detailed description of tomogram reconstruction, CNN training, and fiber tracing exceeds the scope of this protocol paper, which focuses on the sample preparation and data acquisition. Instead, we invite the reader to visit the following pages for the different data analysis steps: Tomogram reconstruction For ETOMO reconstruction tutorial, please visit (https://bio3d.colorado.edu/imod/doc/patchTrackExample.html). An example of a reconstructed tomogram of the onion cell wall is shown in Figure 6C. For ARETOMO automated alignment and reconstruction tutorial and manual, please visit https://drive.google.com/drive/folders/1Z7pKVEdgMoNaUmd_cOFhlt-QCcfcwF3 and refer to Zheng et al. (2022) (see Note 12). See also the following script for batch contrast transfer function correction and reconstruction using ETOMO and ARETOMO: http://dx.doi.org/10.13140/RG.2.2.31326.31043. For EMAN2 automated alignment and reconstruction tutorial, please visit https://blake.bcm.edu/emanwiki/EMAN2/e2tomo (see Note 12). CNN training and application For training and application of CNNs with EMAN2, please visit https://blake.bcm.edu/emanwiki/EMAN2/Programs/tomoseg for a complete tutorial. An example of CNN segmented map of the onion cell wall is shown in Figure 6D. Tracing of fibers with Amira – Xtracing plugin For understanding the significance of the parameters used to trace the fibers, please refer to Rigort et al. (2012). For a complete run-through to detect fibers in a tomogram, please refer to the Amira help documentation for the Xtracing extension. An example of a fiber-traced map of the onion cell wall is shown in Figure 6E. Notes White, fresh, and hard onions are preferrable. If possible, choosing big onions will allow for more material. In this study, the onions were usually bought the day prior to freezing and stored in the fridge. As effective peeling is not necessarily consistent between onions, it is therefore best to acquire a couple onions each time in case one is not peeling well. EM grid boxes come in multiple shapes and forms. Classically they accommodate four grids. To avoid contamination, the best are the ones with a notched lid that allows opening one slot at a time (Mitegen #M-CEM-CGBSW1 and/or M-CEM-71166-10). Autogrid grid boxes number 1 through 4 are good for storing clipped grids (Mitegen M-CEM-7AGB). Different types of grids were used. The goal in mind was to avoid accumulation of unblotted water underneath the peel, causing an increase of the total thickness of the sample to be milled and, in turn, hindering lamellae generation. Non-filmed grids (only copper grids) were tested. Despite giving extremely good blotting results, the cell wall is not supported by carbon, which caused instability during the final stages of milling, translated by a vibration of the final lamellae, impossible to thin down to final thickness. Quantifoil R17/5 (17 μm diameter holes spaced approximately 5 um) gave really good blotting results and lamella polishing stability. We therefore advise the use of Quantifoil R 17/5 grids (EMS # Q210CR175) or R 2/2, which is also a good compromise (EMS # Q250CR1). Onion epidermal cells elongate in an anisotropic fashion, resulting in rectangular-like shapes. Having the peel presented to the FIB gun so that the long side of the rectangle-like cells is parallel to the FIB beam path allows more space for milling on the periclinal cell wall compared to when it is presented perpendicular to the FIB beam. The small rectangular-shaped cuts of cell wall (section B9 and 10) are dragged on the London Finder Quantifoil grid so that the long arrow (notch) on the grid is parallel to the long side of the rectangular peel (Figure 2C). Then, during the clipping stage, it is important to align the arrow on the Quantifoil grid with the notch of the autogrid (Figure 3). Eucentric height is important in order to be able to tilt the stage without having significant x and y shift of the milling target. It can be first set by aiming at a contamination at the working magnification. Start at 0° and set the scan time to very fast. Increase the tilt angle by 5° increments until 20° and recenter the target for each increment using the Z slider. It is important to not shift in x and y during the procedure but just move the stage in z. For further tuning, beam coincidence is established in order to have the milling target at the height where the SEM and FIB beam coincide. This is very useful for continuous monitoring with the SEM beam of the milling progress. To do so, center on a feature with SEM beam, image with the FIB beam, and center on the same feature by changing the Z-height of the sample (the offset should only be vertical). Then, recenter with SEM beam on feature using x and y shifts and repeat operation until the two images coincide on the same spot. Note that this needs to be done on each target. After beam coincidence has been set, decrease the angle of the stage by 1° increments and check the FIB image. As long as the image is clear with no distortions or dark objects in the foreground, continue decreasing the angle. As soon as the bottom of the image suffers from distortions or a dark object is occluding the bottom of the image, the angle is too low. The last angle with a clear image is the shallowest angle usable for milling. While some exclusively use the FIB view to monitor lamella thickness, SEM view informs on thickness homogeneity and overall shape and length on the lamella. It is important to monitor the thickness of the lamella using a combination of voltage and current that creates the smallest interaction volume possible. Around 5 kV – 27 pA is appropriate. At this setting, an approximately 200 nm thin lamella will appear homogenously black (Figure 4E). When this tint is reached, the lamella is considered ready for cryo-ET. For on-lamella tilt series acquisition on a complete ±60° range, it is necessary to position the short side of the lamella parallel to the tilt axis of the goniometer in the microscope. In Thermo Fisher microscopes that are loaded with the standard grid cassette, this is done by positioning the FIB milling notch or the sputter-coating marks (only possible if the sample is sputter coated in PP3000T type shuttle), or the two black contiguous dots of the FIB autogrid sideways in the loading cassette (Figure 5B). The magnifications used for this study are the following: ×82 (search mode), ×2,250 (view mode), and ×26,000 (record, preview, trial, and record modes). To manage multiple magnifications, the Navigator > Imaging states can be used to store imaging states (magnification, illuminated area, 1st condenser lens parameters, etc.). To modify on the fly an imaging mode: Go into this imaging mode in the Low Dose Control panel > “Go to (view mode of choice).” Camera&Script panel > click on the corresponding imaging mode to set the microscope accordingly and record an image > Low Dose Control panel > tick “continuous update” > modulate C2 with Intensity knob and check dose rate > untick "continuous update.” To set up anchoring images, File > Open New and create two files, AnchorHM (for high magnification maps) and AnchorLM (for low magnification maps). Go to the first area of interest (Navigator panel > Go to XY). Buffer Control panel > Go through open files with “To file” to set the active file as AnchorHM (visible in the top window bar of SerialEM). Camera&Script panel > Preview. Buffer Control panel > “Save Active”. Navigator panel > New map. Buffer Control panel > “To file” to set the active file as AnchorLM. Camera&Script panel > View. Buffer Control panel > “Save Active”. Navigator panel > New map. Navigator panel > Tick “for anchor state”. Go to second area of interest (Navigator panel > Go to XY). Buffer Control panel > “To file” to set the active file as AnchorHM. Acquisition panel > Preview. Buffer Control panel > “Save Active”. Navigator panel > New map. Navigator panel > Anchor map. This will automatically set AnchorLM as active file. Take a View image, save it, and make a new anchor map. Go to third area of interest (Navigator panel > Go to XY). Make sure the active file is AnchorHM. Camera&Script panel > Preview. Navigator panel > Anchor map. Repeat the last four bullet points for all the other areas of interest. When setting up the tilt series acquisition parameters, the main goal is to expose the area of interest to the lower total dose possible, which damages the sample over time, but also to be in the optimal dose range for the detector in order to count electrons accurately (approximately 15 e-/px/s dose rate after the sample on a K3 detector in Correlated Double Sampling mode, CDS). For a tilt series acquired on an approximately 200 nm thick lamella, 80 e-/A2 total dose works well. To compute the total dose the sample will be exposed to, we use the following equation: The before sample dose rate is measured by going to an empty area near the lamella and acquiring a Preview or Record image to read out the dose rate in e-/px/s. Exposure time is set by going to Acquisition panel > Setup parameters > Exposure time. This parameter can be modified accordingly in order to lower the total dose. Number (#) of exposures is defined by the tilting range and the tilt increment: Tilting range is usually set to ±60° or ±50°. Increment set to 3°. Pixel size depends on the magnification. The smaller the pixel size (increased magnification), the higher the total dose will be. The following Excel tool allows to conveniently calculate the total dose: DOI: 10.13140/RG.2.2.31306.85442. For the tilt scheme, the associated study to this protocol used a bidirectional scheme starting from 0°. Nowadays, it is standard to acquire using a dose symmetric scheme (Figure 6A, red inset) in order to prioritize lower tilts before high resolution details are destroyed by the electron beam. The following default parameters for the dose symmetric scheme work well: Initial group size: 5. Stop alternating directions beyond 30° from initial angle. Reorder file in background when finished. Recipes HEPES buffer (HEPES 20 mM + Tween-20 0.1%, pH 6.8, 50 mL) 238 mg of HEPES powder 50 μL of Tween-20 100% Quantity sufficient for 50 mL with DI water pH 6.8 with KOH 1 M This buffer can be prepared ahead of time and stored at room temperature. Make sure it is clear of precipitants before use. CAPS buffer (50 mM, pH 10, 50 mL) 553 mg of CAPS powder Quantity sufficient for 50 mL with DI water pH 10 with NaOH 1 M This buffer can be prepared ahead of time and stored at room temperature. Make sure it is clear of precipitants before use. Pectate lyase at 4.7 U/mL (5 mL) 8 μL of the commercial solution (kept at 4 °C) in 5 mL of CAPS buffer 50 mM (see Recipe 2) Incubate peels for three hours. Enzymatic solution was always prepared fresh and kept on ice before use. BAPTA 2 mM (50 mL) 48 mg of BAPTA powder Quantity sufficient for 50 mL with DI water This solution was always prepared fresh and could be kept at room temperature for the duration of the experiment. Acknowledgments The work related to this protocol was supported by the Howard Hughes Medical Institute (HHMI) and grant R35 GM122588 to Grant J Jensen, and Austrian Science Fund (FWF): P33367 to Florian KM Schur. Cryo-EM work was done in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication. Competing interests There are no conflicts of interest or competing interests. References Chen, M., Dai, W., Sun, S. Y., Jonasch, D., He, C. Y., Schmid, M. F., Chiu, W. and Ludtke, S. J. (2017). Convolutional neural networks for automated annotation of cellular cryo-electron tomograms. Nat Methods 14(10): 983-985. Dimchev, G., Amiri, B., Fassler, F., Falcke, M. and Schur, F. K. (2021). Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data. J Struct Biol 213(4): 107808. Dubochet, J. (2007). The physics of rapid cooling and its implications for cryoimmobilization of cells. Methods Cell Biol 79: 7-21. Kafle, K., Xi, X., Lee, C. M., Tittmann, B. R., Cosgrove, D. J., Park, Y. B. and Kim, S. H. (2014). Cellulose microfibril orientation in onion (Allium cepa L.) epidermis studied by atomic force microscopy (AFM) and vibrational sum frequency generation (SFG) spectroscopy. Cellulose 21(2): 1075-1086. Kremer, J. R., Mastronarde, D. N. and McIntosh, J. R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1): 71-76. Mastronarde, D. N. (1997). Dual-axis tomography: an approach with alignment methods that preserve resolution. J Struct Biol 120(3): 343-352. Mastronarde, D. N. (2003). SerialEM: A Program for Automated Tilt Series Acquisition on Tecnai Microscopes Using Prediction of Specimen Position. Microsc Microanal 9(S02): 1182-1183. Mastronarde, D. N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152(1): 36-51. Mastronarde, D. N. and Held, S. R. (2017). Automated tilt series alignment and tomographic reconstruction in IMOD. J Struct Biol 197(2): 102-113. Nicolas, W. J., Fassler, F., Dutka, P., Schur, F. K. M., Jensen, G. and Meyerowitz, E. (2022). Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks. Curr Biol 32(11): 2375-2389 e2376. Rigort, A., Gunther, D., Hegerl, R., Baum, D., Weber, B., Prohaska, S., Medalia, O., Baumeister, W. and Hege, H. C. (2012). Automated segmentation of electron tomograms for a quantitative description of actin filament networks. J Struct Biol 177(1): 135-144. Schaffer, M., Engel, B. D., Laugks, T., Mahamid, J., Plitzko, J. M. and Baumeister, W. (2015). Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography. Bio-protocol 5(17): e1575. Schaffer, M., Mahamid, J., Engel, B. D., Laugks, T., Baumeister, W. and Plitzko, J. M. (2017). Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. J Struct Biol 197(2): 73-82. Tang, G., Peng, L., Baldwin, P. R., Mann, D. S., Jiang, W., Rees, I. and Ludtke, S. J. (2007). EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157(1): 38-46. Villa, E., Schaffer, M., Plitzko, J. M. and Baumeister, W. (2013). Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography. Curr Opin Struct Biol 23(5): 771-777. Wagner, F. R., Watanabe, R., Schampers, R., Singh, D., Persoon, H., Schaffer, M., Fruhstorfer, P., Plitzko, J. and Villa, E. (2020). Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography. Nat Protoc 15(6): 2041-2070. Zhang, T., Mahgsoudy-Louyeh, S., Tittmann, B. and Cosgrove, D. J. (2014). Visualization of the nanoscale pattern of recently-deposited cellulose microfibrils and matrix materials in never-dried primary walls of the onion epidermis. Cellulose 21(2): 853-862. Zhang, T., Vavylonis, D., Durachko, D. M. and Cosgrove, D. J. (2017). Nanoscale movements of cellulose microfibrils in primary cell walls. Nature Plants 3(5): 17056. Zhang, Y., Yu, J., Wang, X., Durachko, D. M., Zhang, S. and Cosgrove, D. J. (2021). Molecular insights into the complex mechanics of plant epidermal cell walls. Science 372(6543): 706-711. Zheng, S., Wolff, G., Greenan, G., Chen, Z., Faas, F. G. A., Bárcena, M., Koster, A. J., Cheng, Y. and Agard, D. (2022). AreTomo: An integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction. bioRxiv: 2022.2002.2015.480593. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant cell biology > Cell wall Cell Biology > Cell imaging > Electron microscopy Biophysics > Electron cryotomography Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach Clément Chambaud [...] Lysiane Brocard Jan 20, 2023 1118 Views Fast and High-Resolution Imaging of Pollinated Stigmatic Cells by Tabletop Scanning Electron Microscopy Lucie Riglet and Isabelle Fobis-Loisy Nov 20, 2024 409 Views Cryo-SEM Investigation of Chlorella Using Filter Paper as Substrate Peng Wan [...] Jinghan Wang Dec 20, 2024 229 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,560	https://bio-protocol.org/en/bpdetail?id=4560&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Assessing the in vitro Binding Affinity of Protein–RNA Interactions Using an RNA Pull-down Technique AC Anand Chopra FB Feras Balbous KB Kyle K. Biggar Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4560 Views: 2313 Reviewed by: Gal HaimovichMarion HoggJian Chen Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nucleic Acids Research Apr 2020 Abstract RNA is a vital component of the cell and is involved in a diverse range of cellular processes through a variety of functions. However, many of these functions cannot be performed without interactions with proteins. There are currently several techniques used to study protein–RNA interactions, such as electrophoretic mobility shift assay, fluorescence anisotropy, and filter binding. RNA-pulldown is a technique that uses biotinylated RNA probes to capture protein–RNA complexes of interest. First, the RNA probe and a recombinant protein are incubated to allow the in vitro interaction to occur. The fraction of bound protein is then captured by a biotin pull-down using streptavidin-agarose beads, followed by elution and immunoblotting for the recombinant protein with a His-tag–reactive probe. Overall, this method does not require specialized equipment outside what is typically found in a modern molecular laboratory and easily facilitates the maintenance of an RNase-free environment. Graphical abstract Keywords: RNA–protein interactions Affinity Streptavidin–biotin pull-down Nab3 protein snR47 Background DNA is the fundamental molecule containing the genetic information of the cell. However, RNA and its interactions with proteins play an essential functional role in using this genetic information and translating it to biological implication(s). RNA is less stable than DNA, is often single-stranded, and forms secondary and tertiary structures such as hairpins, helices, stem-loops, and others. The structure and sequence of an RNA molecule are essential for the formation of specific protein–RNA complexes (Singh et al., 2019). Further, there are thousands of proteins in the cell responsible for recognizing specific RNAs, and these interactions may be critical for carrying out their role: they may promote or repress RNA degradation, as well as regulate the splicing, transport, and translation of mRNA strands (Corley et al., 2020). Many proteins are subjected to dynamic post-translational modifications (PTMs), which impact the structure and function of the proteins being modified (Ramazi and Zahiri, 2021). These PTMs include, but are not limited to, phosphorylation, acetylation, ubiquitination, and methylation. Further, any mutation in protein sequence may block PTMs and prevent protein–RNA interactions (Zhang et al., 2020). Similarly, RNA may be subjected to chemical modifications that impact its structure. These RNA modifications include methylation of adenosine (N6-methyladenosine), guanine carbonyl addition (8-oxo-7,8-dihydroguanosine), and acetylation of cytidine (N4-acetylcytidine), among others (Boo and Kim, 2020). For these reasons, the study of protein–RNA interactions is necessary for improving our understanding of how RNA plays a role in vital cellular processes. Traditionally, electrophoretic mobility shift assays (EMSA), fluorescence anisotropy, and filter binding have been used to study protein–RNA interactions (Majumder and Palanisamy, 2021). In contrast to traditional techniques, the proposed RNA pull-down method obtains in vitro binding affinity measurements (i.e., KD) for protein–RNA interactions (Figure 1A and 1B). Furthermore, RNA pull-down offers an approach in which an RNase-free environment is easy to maintain and uses standard immunoblotting for detection of bound protein. This contrasts with other protocols such as fluorescence anisotropy, which depend on specialized equipment, or other techniques, which are difficult to maintain an RNase-free environment. RNA pull-down can provide valuable information regarding protein–RNA binding affinities and analyze the effect of chemical modifications on these interactions. We have successfully applied this protocol to study the binding of nuclear polyadenylated RNA-binding (Nab) 3 protein RNA-recognition motif (RRM) with RNA and how point mutations to the lysine-363 residue affect this protein–RNA interaction (Lee et al., 2020). Materials and Reagents PCR tubes (Bio Basic Canada Inc., catalog number: BP541-S100) P200 tip (Bio Basic Canada Inc., catalog number: BT224-YS) Eppendorf tube (UltiDent Scientific, catalog number: 87-B150-C) 96-well microplate (Corning, catalog number: 351172) PVDF membrane (GE Healthcare, catalog number: 10600023) 15 mL Falcon tube Streptavidin agarose (Millipore Sigma, catalog number: 69203) Bovine serum albumin (BSA) (Bioshop, catalog number: ALB001.100) RNA probe (Sigma, sequence: 5’-[Btn]UUUCUUUUUUCUUAUUCUUAUU-3’; see Note 1) DEPC (Bio Basic, catalog number: 1609-47-8) Nonfat dry milk (Carnation, catalog number: n/a) Clarity Western ECL substrate (Bio-Rad, catalog number: 170-5060) Tris (Bioshop, catalog number: 77-86-1) Tris-HCl (Bioshop, catalog number: 1185-53-1) Hydrochloric acid (HCl) (Anachemia, catalog number: CA11020-884) Sodium hydroxide pellets (Bioshop, catalog number: SHY700.2) NaCl (Bioshop, catalog number: SOD002.1) DTT (Bioshop, catalog number: DTT001.25) EDTA, disodium dihydrate (Bioshop, catalog number: EDT001.500) Glycerol (Bioshop, catalog number: 56-81-5) SDS (BioShop, catalog number: SDS001) Tween-20 (Bioshop, catalog number: TWN510.500) Bromophenol blue (Sigma, catalog number: B-6896) 2-Mercaptoethanol (Sigma, catalog number: M3148) HisProbe-HRP conjugate (Thermo Fisher Scientific, catalog number: 15165) Protein assay dye reagent concentrate (Bio-Rad, catalog number: 5000006) Stock solutions (see Recipes) Pull-down buffers & solutions (see Recipes) Western blotting & other buffers (see Recipes) Equipment 100 mL beaker 500 mL beaker Magnetic stirrer Sorvall Legend Micro 21 centrifuge (Thermo Scientific, catalog number: 75772436) Mini centrifuge (Fisher Scientific, catalog number: 05-090-100) Gel Doc XR+ imaging system (Bio-Rad, model: Universal Hood II) Thermocycler (Bio-Rad, model: ICycler Thermal Cycler) Rotator (Kylin-Bell Lab Instruments, model: BE-1100) -80 °C freezer (Thermo Scientific, model: 990) Software ImageJ 1.50c software GraphPad Prism v7.00 Procedure The procedure below outlines three main steps: (A) bead preparation, (B) protein–RNA binding reactions, and (C) streptavidin–biotin pull-down and detection. This procedure was originally applied to study the binding affinity of the interaction between Nab3 RRM and a biotinylated small nucleolar RNA (e.g., snR47) probe (Lee et al., 2020). If this procedure is to be used with other protein–RNA interactions, then we recommend taking into consideration how variables (e.g., RNA probe length, RNA structure, buffer conditions, temperature, etc.) may affect the RNA–protein interaction and adjust accordingly (see Notes 2 and 4). Further, the protein source here is the recombinant His-tagged Nab3 RRM, expressed and purified from E. coli. A pure protein source is preferred if obtaining binding affinity measurements is desired (see Note 2). Bead preparation Cut off the end (2–3 mm on average) of a P200 tip and transfer 150 μL (i.e., 75 μL dry bead volume) of streptavidin-agarose to an Eppendorf tube (see Note 3). Centrifuge beads at 500 × g for 2 min and discard the supernatant (see Note 5). Wash with 1 mL of wash buffer for 5 min with end-to-end rotation. Spin down beads at 500 × g for 2 min and discard the supernatant. Repeat steps A3–A4 twice. Add 300 μL of wash buffer containing 5% BSA to the beads and incubate for 4 h with end-to-end rotation. Spin down beads at 500 × g for 2 min and discard supernatant. Wash with 500 μL of wash buffer for 5 min with end-to-end rotation. Spin down beads at 500 × g for 2 min and discard supernatant. Repeat steps A8–A9 five times but retain the supernatant of the fifth wash for a Bradford assay. In triplicates, add 10 μL of wash buffer (as a blank) or the supernatant retained in the previous step to 190 μL of diluted Bradford reagent in a 96-well microplate and measure absorbance at 595 nm. Proceed to step A13 if the supernatant is indistinguishable from the blank. If not, repeat steps A8–A9 until this result is obtained. Resuspend beads in 260 μL of wash buffer. Distribute 20 μL of beads (approximately 5 μL dry bead volume) into individual PCR tubes using a P200 tip with the end cut off. Keep the prepared bead suspension at 4 °C or on ice until needed. Protein–RNA binding reactions Remove RNA probe, protein stock, and protein storage buffer from -80 °C and leave to thaw on ice. Prepare binding reactions in PCR tubes as 10 μL reactions according to Table 1. Adjust volumes to achieve desired protein or RNA concentrations. Table 1. Protein–RNA binding reactions. The example below shows an initial experimental setup for preparing binding reactions with a fixed RNA probe concentration of 25 μM, varying protein concentration from 1 to 50 μM, and reaction controls. 5× binding buffer (μL) Final protein concentration (μM) 100 μM protein stock (μL) Protein storage buffer (μL) Final RNA probe concentration (μM) 250 μM RNA probe (μL) DEPC water Control 1 2 0 0 7 25 1 0 Control 2 2 50 5 2 0 0 1 Reaction 2 1–50 0.1–5 2–6.9 25 1 0 Incubate in a thermocycler for 30 min at 15 °C. Place the reactions on ice and immediately proceed to Section C. Streptavidin–biotin pull-down and detection Note: Unless otherwise stated, all centrifugations should occur on the mini centrifuge (2,200 × g) at room temperature. Transfer each binding reaction or control to a separate PCR tube containing prepared beads. Incubate at 4 °C for 2 h with end-to-end rotation. Spin down beads for 2 min and discard the supernatant. Wash beads with 100 μL of wash buffer for 5 min with end-to-end rotation. Repeat step C3. Repeat steps C4–C5 four times. Add 20 μL of Laemmli buffer with 2-mercaptoethanol to elute the bound protein. Mix and heat tubes in a thermocycler at 95 °C for 5 min. Spin down beads for 2 min. Load the entire sample (supernatant) on a standard SDS-PAGE gel (here, a 17% gel was used, which is specific for the recombinant protein of interest due to its small size; see Notes 6 and 7). Run the gel at 120 V for approximately 2 h (until the dye front reaches the bottom). Transfer proteins onto a PVDF membrane at 180 mA for 2 h. Block membrane in blocking buffer for 1 h. Wash membrane five times for 5 min with TBST (see Recipes). Incubate membrane with appropriate antibody or probe (HRP-conjugated HisProbe) for 4 h or overnight at 4 °C. Wash membrane five times for 5 min with TBST. Image membrane by standard chemiluminescent detection with ECL substrate. Representative data Figure 1. Assessing the binding of His-Nab3 RRM (329–419) to biotinylated snR47 RNA probe. (A) Chemiluminescent detection of bound His-tagged recombinant protein (1–50 μM) to 25 μM RNA probe using HRP-conjugated HisProbe. (B) Dose-responsive binding curve of variable His-Nab3 RRM (329–419) to the biotinylated snR47 RNA probe. KD is shown in the figure panel. (C) Chemiluminescent detection of bound His-Nab3 RRM wildtype and mutant proteins (10, 25, 50 μM) to 25 μM RNA probe using HRP-conjugated HisProbe. Data analysis Densitometry Start ImageJ and open the file containing the raw blot image. Convert the image to 8 bit grayscale by selecting “Image,” “Type,” then “8-bit.” Set the measurement criteria by selecting “Analyze” and then “Set Measurements.” In the pop-up window, ensure that only “Mean gray value” is checked and click “OK.” Draw a rectangle around the largest band on the blot. Click “Analyze,” then “Measure” (or Ctrl+M), then record the value. Without adjusting the size of the rectangle, move the rectangle to the next band and repeat step A5. Continue measuring the density of the protein band for each sample lane, including the lane for the zero protein condition (this will serve as a background measurement). Subtract the background value from all other values. Normalize each condition to the maximum binding signal (i.e., Bmax). Curve fitting and KD estimation Start GraphPad Prism. In the pop-up window under “New table & graph” select “XY.” Under “Enter/import data,” for “X” select “Numbers” and for “Y” select “Enter and plot a single Y value for each point.” Then click “Create.” In “Data Tables” enter the protein concentrations and the corresponding normalized binding values. In “Graphs” visualize the binding curve and ensure that the binding profile reaches saturation. Under “Analysis” select “Fit a curve with nonlinear regression.” In the pop-up window under “Dose-response–Stimulation” choose the “[Agonist] vs. normalized response–Variable slope” equation. Navigate to the “Results” sheet and locate the EC50 value (in this case, where background has been subtracted off, the EC50 represents the KD). Notes Several variables must be taken into consideration when designing the RNA probe. The snR47 RNA probe sequence used in the procedure outlined herein was previously used to characterize the protein–RNA interaction between the Nab3 RRM and snRNAs (Hobor et al., 2011). The probe sequence was originally used for fluorescence anisotropy and was 5’-labelled with fluorescent markers. Here, we replaced the fluorophore with biotin to enable the pull-down with streptavidin-coated beads. If aiming to assess the binding affinity between protein–RNA interactions that were previously characterized with fluorescence anisotropy by using this RNA pull-down, then we recommend using the same probe sequence and replacing the fluorophore with biotin on the same end of the RNA. For less characterized RNA–protein interactions, we recommend experimenting with using 5’-biotin or 3’-biotin labels, as well as experimenting with sequence length and the number of binding sites within probe sequence. We have only performed this procedure using pure recombinant protein prepared from E. coli. Pure sources of native protein can be obtained from other sources [such as recombinants from Sf9 cells or immunoprecipitation from cells (with native elution)]. If aiming to obtain binding affinity measurements (KD) by performing experiments shown in Figure 1A and 1B, then a relatively pure quantifiable source of protein is needed. If aiming to simply assess relative RNA binding of a protein, as shown in Figure 1C, then it is theoretically possible to use other more impure protein sources such as a cell lysate or in vitro translated protein. As an initial experiment, we recommend performing a titration of the sample, given that expression level of the protein of interest may vary. The bead preparation section outlines a standard procedure for equilibration and blocking of beads. For the volume of beads used, we recommend adjusting this parameter based on the volume of beads needed and the amount of biotinylated RNA probe used. Generally, we aim to use approximately 5 μL of dry bead volume per binding reaction. At a binding capacity of >85 nmol biotin/mL, 5 μL of dry beads is sufficient to capture >425 pmol biotin. At a biotinylated RNA probe concentration of 25 μM in a 10 μL reaction, 250 pmol of probe is present and thus the number of beads used is sufficient to capture all probe molecules. The binding reaction conditions provided in this protocol are amenable to assessing the binding of Nab3 RRM (329–419) to the snR47 RNA probe. If another interaction is of interest, we recommend using buffer conditions that have been used previously in literature. The probe and protein concentration ranges used here are a good starting point for assessing biological interactions; however, these variables may need to be optimized. If increasing the concentration of biotinylated RNA probe, ensure that enough streptavidin-agarose beads are being used, so that the amount of RNA captured is not limited by the binding capacity of the beads. Other variables such as binding reaction temperature and time should be taken into consideration, as these may influence degradation of the RNA probe used. We and others have used the snR47 probe sequence in Nab3-RNA binding reactions at 15 °C and 25 °C (Hobor et al., 2011), respectively. Finally, temperature and certain buffer components, such as DTT, may influence secondary and tertiary structures formed. The Nab3 RRM binding to snR47 does not involve secondary or tertiary RNA structures; however, if such structures are required for the protein of interest, then we suggest adjusting temperature and binding reactions accordingly. To ensure that no beads are lost during the removal of the supernatant, a needle can be used to remove the supernatant. The parameters of SDS-PAGE (e.g., gel percentage) and transfer (e.g., time) may need to be changed depending on the size of the protein of interest. To ensure that the method of detection performs as expected, an additional lane can be loaded containing the recombinant protein of interest. Generally, loading 10% of the inputted protein should be sufficient for detection by immunoblotting. Dry SDS in powdered form is very dangerous to work with; it is recommended to wear appropriate personal protective equipment such as face mask and safety goggles when weighing the powder. As an alternative, it is preferred to purchase ready-made 10% and 20% SDS solutions. Recipes Stock solutions Tris, 1 M pH 8.0 Add 121.14 g of Tris to a 1 L beaker Add 800 mL of distilled water Stir with a magnetic stirrer until dissolved Adjust pH to 8.0 with HCl Top up volume to 1 L in a graduated cylinder Autoclave at 121 °C for 20 min Store at room temperature Tris, 1 M pH 6.8 Add 121.14 g of Tris to a 1 L beaker Add 800 mL of distilled water Stir with a magnetic stirrer until dissolved Adjust pH to 6.8 with HCl Top up volume to 1 L in a graduated cylinder Autoclave at 121 °C for 20 min Store at room temperature SDS, 10% w/v Add 10 g of SDS to a 100 mL beaker (see Note 8) Add 100 mL of distilled water Stir with a magnetic stirrer until dissolved Store at room temperature Tris-HCl, 1 M pH 7.8 Add 157.6 g of Tris-HCl to a 1 L beaker Add 800 mL of distilled water Stir with a magnetic stirrer until dissolved Adjust pH to 7.8 with HCl Top up volume to 1 L in a graduated cylinder Autoclave at 121 °C for 20 min Store at room temperature NaCl, 4 M Add 116.88 g of NaCl to a 500 mL beaker Add 350 mL of distilled water Stir with a magnetic stirrer until dissolved Top up volume to 500 mL in a graduated cylinder Autoclave at 121 °C for 20 min Store at room temperature DTT, 1 M Add 1.54 g of DTT to a 15 mL Falcon tube Add 7.5 mL of distilled water Invert or vortex until dissolved Top up volume to 10 mL Make 0.5–1 mL aliquots in Eppendorf tubes and store at -20 °C EDTA, 0.5 M pH 8 Add 93.06 g of EDTA to a 500 mL beaker Add 300 mL of distilled water Stir with a magnetic stirrer and slowly add approximately 20 g of sodium hydroxide pellets to dissolve Adjust pH to 8 Top up volume to 500 mL in a graduated cylinder Autoclave at 121 °C for 20 min Store at room temperature DEPC H2O Add 1 mL of DEPC to 999 mL of distilled water Stir overnight at room temperature on a magnetic stir plate Autoclave at 121 °C for 20 min Store at room temperature Pull-down buffers & solutions Protein storage buffer Reagent Final concentration Amount Tris (1 M, pH 8.0) 20 mM 20 mL NaCl (4 M) 250 mM 62.5 mL Glycerol 5% (v/v) 50 mL DTT (1 M) 1 mM 1 mL H2O n/a 866.5 mL Total n/a 1 L 5× binding buffer Reagent Final concentration Amount Tris-HCl (1 M, pH 7.8) 50 mM 0.5 mL NaCl (4 M) 250 mM 0.625 mL EDTA (0.5 M) 5 mM 0.1 mL Glycerol 25% (v/v) 2.5 mL DTT (1 M) 12.5 mM 0.125 mL DEPC H2O n/a 6.15 mL Total n/a 10 mL Wash buffer Reagent Final concentration Amount Binding buffer (5×) 1× 2 mL Tween-20 0.1% 10 μL DEPC H2O n/a 8 mL Total n/a 10 mL RNA probe Depending on the amount of RNA probe received, dissolve in enough volume of DEPC water to a final concentration of 250 μM. Quickly aliquot and store at -80 °C until use. Protein stock Depending on the concentration of pure recombinant protein, dilute the protein to a final concentration of 100 μM using protein storage buffer (see Note 2). Western blotting & other buffers Diluted Bradford reagent Note: Prepare just before use. Add 1 mL of protein assay dye reagent concentrate to a 15 mL Falcon tube. Add 4 mL of distilled water and invert to mix. TBST, pH 7.6 Dissolve 3.152 g of Tris-HCl and 8.006 g of NaCl in 800 mL of distilled water Adjust the pH to 7.6 with sodium hydroxide Add 1 mL of Tween-20 and mix Top up the volume to 1 L with distilled water Store at room temperature Blocking buffer Note: Prepare just before use. Dissolve 1 g of nonfat dry milk in 10 mL of TBST Invert or vortex to dissolve Diluted HisProbe-HRP conjugate in TBST Note: Prepare just before use. In a 15 mL Falcon tube, dilute 1 μL of HisProbe-HRP conjugate in 5 mL of TBST Store at 4 °C or on ice until just before use Store the used solution at 4 °C Laemmli buffer, 2× Reagent Final concentration Amount Tris (1 M, pH 6.8) 120 mM 1.2 mL Glycerol 20% v/v 2 mL SDS (10% w/v) 4% w/v 4 mL Water n/a 2.8 mL Bromophenol blue 0.02% w/v 2 mg Total n/a 10 mL Laemmli buffer with 2-mercaptoethanol In a 1.5 mL Eppendorf tube, mix 50 μL of 2-mercaptoethanol and 950 μL of Laemmli buffer, 2× Store at room temperature Acknowledgments This research was supported by Discovery Grants from the Natural Science and Engineering Council (NSERC) of Canada to K.K.B (grant no. RGPIN-2016-06151). Anand Chopra held a Canada Graduate Scholarship-Doctoral (CGS-D), from the NSERC of Canada. Feras Balbous held an Undergraduate Student Research Award from the NSERC of Canada. This protocol was used in Lee et al. (2020). Competing interests The authors declare no competing interests. References Boo, S. H. and Kim, Y. K. (2020). The emerging role of RNA modifications in the regulation of mRNA stability. Exp Mol Med 52(3): 400-408. Corley, M., Burns, M. C. and Yeo, G. W. (2020). How RNA-Binding Proteins Interact with RNA: Molecules and Mechanisms. Mol Cell 78(1): 9-29. Hobor, F., Pergoli, R., Kubicek, K., Hrossova, D., Bacikova, V., Zimmermann, M., Pasulka, J., Hofr, C., Vanacova, S., Stefl, R. (2011). Recognition of transcription termination signal by the nuclear polyadenylated RNA-binding (NAB) 3 protein. J Biol Chem 286(5): 3645-3657. Lee, K.Y., Chopra, A., Burke, G.L., Chen, Z., Greenblatt, J.F., Biggar, K.K., Meneghini, M.D. (2020) A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation. Nucleic Acids Res. 48(6): 2897-2911. Majumder, M. and Palanisamy, V. (2021). Compendium of Methods to Uncover RNA-Protein Interactions In Vivo. Methods Protoc 4(1): 22. Ramazi, S. and Zahiri, J. (2021). Posttranslational modifications in proteins: resources, tools and prediction methods. Database (Oxford) 2021. Singh, J., Hanson, J., Paliwal, K. and Zhou, Y. (2019). RNA secondary structure prediction using an ensemble of two-dimensional deep neural networks and transfer learning. Nat Commun 10(1): 5407. Zhang, N., Lu, H., Chen, Y., Zhu, Z., Yang, Q., Wang, S. and Li, M. (2020). PremPRI: Predicting the Effects of Missense Mutations on Protein-RNA Interactions. Int J Mol Sci 21(15): 5560. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biochemistry > RNA > RNA-protein interaction Molecular Biology > RNA > RNA-protein interaction Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae Congwei Wang [...] Anne Spang Oct 5, 2018 5762 Views Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC Harunori Yoshikawa [...] Angus I. Lamond Aug 5, 2021 4426 Views An Aptamer-based mRNA Affinity Purification Procedure (RaPID) for the Identification of Associated RNAs (RaPID-seq) and Proteins (RaPID-MS) in Yeast Rohini R. Nair [...] Jeffrey E. Gerst Jan 5, 2022 2999 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,561	https://bio-protocol.org/en/bpdetail?id=4561&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Improved Macrophage Enrichment from Mouse Skeletal Muscle LK Linda K. Krasniewski DT Dimitrios Tsitsipatis EI Elizabeth K. Izydore CS Changyou Shi YP Yulan Piao MM Marc Michel PS Payel Sen MG Myriam Gorospe CC Chang-Yi Cui Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4561 Views: 1060 Reviewed by: Meenal SinhaSabine Le Saux Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Version history Bio-protocol journal peer-reviewed Dec 05, 2022 \| This version Preprint Jan 07, 2023 Original Research Article: The authors used this protocol in eLIFE Oct 2022 Abstract Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45+/CD11b+ macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing. Keywords: Macrophage CD11b Skeletal muscle Aging Senescence Background Macrophages were discovered by Metchnikoff and colleagues more than a century ago as professional phagocytes (Underhill et al., 2016). Later studies revealed that macrophages constitute a heterogenous class of cells that exert diverse functions in tissues throughout the body (Wynn et al., 2013). Macrophages can be divided into two major types: tissue-resident and non-tissue-resident macrophages (Ginhoux and Guilliams, 2016). The former can be further divided into two distinct populations: embryo-derived self-renewing and bone marrow–derived non-self-renewing macrophages. Typical self-renewing macrophages derived from the embryonic yolk sac or fetal liver include microglia, Kupffer cells, alveolar macrophages, and Langerhans cells. Bone marrow–derived non-self-renewing resident macrophages, which must be replenished by circulating monocytes at tissue-specific levels, include tissue-resident macrophages in the intestine, pancreas, and dermis (Chakarov et al., 2019). Non-tissue-resident macrophages are derived from bone marrow progenitor cells and infiltrate tissues following injury or infection (Kratofil et al., 2017). Macrophages are highly versatile cells, capable of ingesting and digesting pathogens as well as necrotic and infected cells, activating T and B lymphocytes, and inducing or suppressing inflammation (Shapouri-Moghaddam et al., 2018). The functional diversity of macrophages is well represented by their dynamic polarization abilities. Depending on signals from the local environment, macrophages can be polarized toward functionally opposite roles: pro-inflammatory M1 or anti-inflammatory M2 subtypes (Mills et al., 2000; Martinez et al., 2008). Cytokines produced by Th1 (T helper type 1) lymphocytes, including interferon γ (IFNγ or IFNG) and tumor necrosis factor (TNF), polarize macrophages to the M1 subtype, while cytokines produced by Th2 lymphocytes, such as interleukin (IL) 4 and IL13, promote macrophage M2 polarization (Mills et al., 2000; Martinez et al., 2008). Polarized M1 macrophages induce inflammation, destroy pathogens, and clean up cell debris, partly through upregulation of the nitric oxide synthase (NOS) pathway (Rath et al., 2014). By contrast, M2 macrophages suppress inflammation and promote tissue repair, partially through upregulation of the arginase pathway (Rath et al., 2014). While M1 and M2 are well-known macrophage subtypes, more recent single-cell studies have identified additional subtypes in several mouse tissues (Chakarov et al., 2019; Jaitin et al., 2019). These subtypes share similarities and differences with M1 and M2, which further reveal the heterogeneity and versatility of macrophages. Macrophages adapt to individual tissues and largely act in a tissue-dependent manner. Macrophages from different tissues possess distinct gene expression profiles and transcriptional regulatory pathways (Gautier et al., 2012). Recent studies suggest that local environmental factors in each tissue contribute to the tissue specificity of resident macrophages (Gosselin et al., 2014; Lavin et al., 2014). For instance, tumor growth factor β (TGFβ or TGFB) promotes the development of microglia by affecting the enhancer/promoter landscape of brain macrophages, while retinoic acid determines peritoneal macrophage specificity (Hoeksema and Glass, 2019). These studies have further uncovered the capacity of macrophages to adapt to local environments and acquire tissue-specific identities. Skeletal muscle contains numerous diverse resident macrophages, localized in the perimysium and endomysium (Cui et al., 2019), where they resolve infections and repair injury (Arnold et al., 2007; Tidball, 2011 and 2017). For example, when skeletal muscle is damaged, monocytes from the bloodstream differentiate and polarize into pro-inflammatory M1 macrophages, which eliminate pathogens and clean up tissue debris. Subsequently, M1 macrophages convert to M2 macrophages to suppress inflammation and repair tissues along with resident M2 macrophages (Yang and Hu, 2018; Cui and Ferrucci, 2020). Recently, skeletal muscle macrophages have been associated with physiological adaptations to exercise that differ between young and elderly individuals (Walton et al., 2019; Jensen et al., 2020), although the full spectrum of macrophage subtypes in skeletal muscle and their functions are only partially known. We have recently found that macrophages residing in human and mouse skeletal muscle are mostly of the M2 subtype (Cui et al., 2019); however, recent single-cell analyses from skeletal muscle and other tissues suggest that the identities of skeletal muscle macrophages are likely more complex (Chakarov et al., 2019; Jaitin et al., 2019; Wang et al., 2020). Furthermore, skeletal muscle macrophages have been shown to have mixed origins, including the embryonic yolk sac, fetal liver, and adult bone marrow (Wang et al., 2020). It remains unclear whether macrophages from various origins behave differently, and the function of each macrophage subtype in skeletal muscle physiology and aging is yet to be elucidated. To answer these questions, it is critical to isolate macrophages from skeletal muscle. There are a few methods for macrophage enrichment from skeletal muscle. One protocol for stem cell isolation from mouse skeletal muscle (Liu et al., 2015) was also effective for macrophage isolation from skeletal muscle (Kosmac et al., 2018). In this study, we sought to formulate a simpler protocol that was effective for the isolation of a macrophage-enriched cell fraction from young and old mouse skeletal muscle. We tested a commercial skeletal muscle dissociation kit combined with a programmable tissue dissociator and added a debris removal step. This kit-based approach allowed us to reduce cell isolation steps and obtain cleaner and more consistent cell preparations. The new protocol was effective for the enrichment of macrophages from skeletal muscle and enabled the characterization of resident macrophages by flow cytometry and single-cell transcriptomic analyses. Materials and Reagents 100 mm FalconTM bacteriological Petri dishes with lid (Fisher Scientific, catalog number: 08-757-100D) GentleMACS C-tubes (Miltenyi Biotec, catalog number: 130-093-237 or 130-096-334) 5 mL FalconTM polypropylene round-bottomed tubes (Corning, catalog number: 352063) 15 mL FalconTM tubes 50 mL FalconTM tubes 1.5 mL EppendorfTM tubes Polystyrene containers; “sticky” for cells but could be used after BSA coating PluriStrainer, 50 µm (pluriSelect, catalog number: 43-50050-01) 10 mL syringe, Luer-Lok tip (BD, catalog number: 309604) 20G × 1.5" blunt tip dispensing fill needles (CML Supply, catalog number: 901-20-150) CountessTM cell counting chamber slides (Invitrogen, catalog number: C10228) Skeletal muscles from the hind limbs of 3-month-old and 18-month-old C57BL/6J mice (see below) 70% ethanol DMEM (ThermoFisher, catalog number: 11965-092). Store at 4 °C. Shelf life is 12 months from date of manufacture Fetal bovine serum, heat inactivated (ThermoFisher, catalog number: 10438-026). Store at -20 °C. Shelf life is two years from date of manufacture Penicillin and streptomycin solution (10,000 U/mL) (100×) (Thermo Fisher Scientific, catalog number: 15140122). Store at -20 °C. Shelf life is 12 months from date of manufacture PBS, pH 7.2 (ThermoFisher, catalog number: 20012-027), free of Ca2+ and Mg2+. Store at 4 °C RNAseZAPTM zaps (Ambion, catalog number: 9786-9788) Skeletal Muscle Dissociation kit (Miltenyi Biotec, catalog number: 130-098-305). Make aliquots for enzymes P, D, and A, and store them at -20 °C immediately after arrival. Shelf life is six months after aliquot Debris removal solution (Miltenyi Biotec, catalog number: 130-109-398). Protect from light and store at 4 °C. Expiration date is labeled on the vial Red blood cell lysis solution (Miltenyi Biotec, catalog number: 130-094-183). Protect from light and store at 4 °C. Expiration date is labeled on the vial Auto MACSTM rinsing solution (Miltenyi Biotec, catalog number: 130-091-222). Protect from light and store at room temperature. Expiration date is labeled on the bottle MACSTM BSA stock solution (Miltenyi Biotec, catalog number: 130-091-376). Protect from light and store at 4 °C. Expiration date is labeled on the bottle. After opening, the solution should be used within three days 0.5 M EDTA (ThermoFisher, catalog number: 15575020). Store at room temperature Trypan Blue solution (Invitrogen, catalog number: T10282). Store at room temperature Antibodies: All antibodies and isotype controls from Biolegend should be stored undiluted at 4 °C and protected from light. Do not freeze. Shelf life is approximately two years PE anti-mouse/human CD11b antibody (Biolegend, catalog number: 101208, Clone M1/70) PE Rat IgG2b, κ Isotype Ctrl antibody (Biolegend, catalog number: 400607, Clone RTK4530) APC anti-mouse CD45 antibody (Biolegend, catalog number: 103111, Clone 30-F11) APC Rat IgG2b, κ Isotype Ctrl antibody (Biolegend, catalog number: 400611, RTK4530) FITC anti-mouse CD206 (MMR) antibody (Biolegend, catalog number: 141703, Clone C068C2) FITC Rat IgG2a, κ Isotype Ctrl antibody (Biolegend, catalog number: 400505, Clone RTK2758) TruStain FcXTM (anti-mouse CD16/32) antibody (Fc blocker) (Biolegend, catalog number: 101319). Store undiluted at 4 °C and protected from light Fixable Viability Dye eFluorTM 780 (Invitrogen, catalog number: 65-0865-14). Store at -80 °C and protect from light and moisture. Expiration date is labeled on the vial DMEM-I and DMEM-II medium (see Recipes) Digestive enzymes (see Recipes) PEB buffer for cell isolation and flow cytometry (see Recipes) Reagent combination to label macrophages for flow cytometry (see Recipes) Equipment Dissection tools: forceps, scalpels, and scissors Pipettes -80 °C freezer, -20 °C freezer, 4 °C refrigerator GentleMACSTM Octo Dissociator with heaters (Miltenyi Biotec, catalog number: 130-096-427) CountessTM II FL automated cell counter (Invitrogen, catalog number: AMQAF1000) BD FACSCantoTM II Cell Analyzer (BD, catalog number: REF338960) Centrifuge 5702R (Eppendorf, catalog number: 022626205) Centrifuge 5415R (Eppendorf, catalog number: 22-62-140-8) Procedure Note: The following protocol was designed for isolating mononuclear cells from skeletal muscle from two adult C57BL/6J mice. Muscle preparation Decontaminate the tools for muscle dissection, including forceps, scalpels, and scissors, with RNaseZAPTM wipes and rinse thoroughly with double-distilled water (ddH2O). Decontaminate the procedure area by spraying with 70% ethanol. Prepare three 100 mm Petri dishes, each containing 10 mL DMEM-I (see Recipes). Place on ice for washing and trimming of isolated muscles. To collect dissected muscles from two mice, prepare two 50 mL Falcon tubes, each containing 5 mL of DMEM-I, and place on ice. Sacrifice a mouse by cervical dislocation or CO2 asphyxiation and spray 70% ethanol over the entire mouse. Incise the skin at the ankle and peel off the skin from the hind limb. Dissect out all muscles surrounding the femur, tibia, and fibula (Figure 1A), and place into the 100 mm dishes containing DMEM-I. Cut out the fat, blood vessels, and tendons under a dissection microscope. Collect the trimmed muscles in the 50 mL Falcon tube containing DMEM-I; weigh the tube before and after sampling and record the muscle weight. Two hind limbs from an adult mouse provide approximately 1 g of muscle. Collect the muscles from the second mouse following the same procedure. Cut and slice the harvested muscles with scissors on ice, leaving 1–2 mL of DMEM-I on the Petri dish and removing the rest. Holding one end of a piece of muscle with forceps, use the scissors to cut the muscle into small pieces (approximately 1 mm3 fragments). After cutting, gather all the muscle pieces in the center of the dish and mince with scissors for an additional 2–3 min [Figure 1B (a)]. Mononuclear cell isolation Transfer the minced muscle tissues from one mouse into two C-tubes containing the enzyme mix (5 mL each) and close the tubes tightly. Mount the C-tubes onto the GentleMAC Octo Dissociator with heaters [Figure 1B (b)]. Choose the 37C-mr-SMDK-2 program. Start the digestion, which takes approximately 1.5 h. After digestion, briefly centrifuge the C-tubes [Figure 1B (c)]. At this step, combine the two C-tubes containing muscle suspensions that were dissected from the same mouse (5 + 5 = 10 mL) into one tube. Use a 10 or 15 mL syringe and a 20G blunt needle (pre-washed with 70% ethanol and distilled water or with DMEM-I) to slowly aspirate and eject the muscle suspension 10 times. Eject the suspension toward the wall of the tube to avoid foaming. This step further breaks down the tissue debris and increases cell yield. Add 10 mL of DMEM-II to each suspension to dilute and deactivate the enzymes. Pipette the suspension onto a cell strainer (50 µm mesh size) and collect the flowthrough into a 50 mL Falcon tube. Wash each strainer with 10 mL of DMEM-II twice. Use a 200 µL pipette to collect any remaining liquid from the underside of the strainer. Each tube will contain 40 mL cell suspension. Discard the strainer and centrifuge the cell suspension at 600 × g for 15 min at room temperature. A clear pellet will be visible at the bottom of the tube [Figure 1B (d)]. Discard the supernatant completely. Dissolve the pellet in 1 mL DMEM-II and transfer to a 1.5 mL Eppendorf tube. Centrifuge at 500 × g and 4 °C for 10 min in a bench-top centrifuge. Discard the supernatant and resuspend the pellet in 0.5 mL cold PBS. Place on ice. Debris removal Note: This step efficiently removes fiber debris from the digested skeletal muscles and significantly improves the purity of mononuclear cells (Figure 1C). Carefully transfer the cell suspension to a 15 mL Falcon tube containing 5.7 mL of cold (4 °C) PBS (0.5 mL of cell suspension + 5.7 mL of PBS = 6.2 mL). Add 1.8 mL of cold debris removal solution; mix well by slowly pipetting up and down 10 times using a 10 mL pipette. Slightly tilt the tube and very slowly and gently overlay the cell suspension with 4 mL of cold PBS. Ensure that the PBS and the cell suspension phases do not mix. Centrifuge at 3,000 × g and 4 °C for 10 min. Discard the supernatant completely; this contains tissue debris [Figure 1C (a)]. The cell pellet at the bottom contains the mononuclear cells [Figure 1C (b)]. Add cold PBS to a final volume of 15 mL. Gently invert the tube three times. Do not vortex. Centrifuge at 1,000 × g and 4 °C for 10 min. Discard the supernatant completely. Resuspend cells in 0.5 mL of PEB (see Recipes) by pipetting gently and slowly. Place on ice. Red blood cell lysis Make 10 mL of 1× red blood cell lysis solution by diluting 10× lysis solution with double-distilled water (ddH2O). Do not use deionized water (including deionized DEPC water). Store at room temperature. Mix one volume of cell suspension with 10 volumes of 1× red blood cell lysis solution (e.g., 0.5 mL of cell suspension + 5 mL of 1× lysis solution) in a 15 mL tube. Vortex for 5 s and incubate for 2 min at room temperature. Longer incubation may damage the cells. Centrifuge at 500 × g for 10 min at room temperature [Figure 1C (c)]. Discard the supernatant completely and resuspend the pellet in 0.5 mL of PEB. Place cells on ice. Mix 5 µL cells with 5 µL of Trypan Blue and count the number of cells using the CountessTM machine. Approximately two million live mononuclear cells can be obtained from a mouse using the above procedure. Figure 1. Outline of macrophage isolation from mouse skeletal muscle. (A) Hind limb after muscle harvest. (B) Minced muscle from mouse hind limbs (a), cell dissociator (b), digested muscle in a C-tube (c), and cell pellet after centrifugation (d). (C) Debris removed by the debris removal solution (a), cell pellet after debris removal (b), cell pellet after red cell removal (c), and cells after sorting (d, 20× brightfield image). Flow cytometry analysis of isolated mononuclear cells For analytical studies, evenly divide approximately two million cells into nine 1.5 mL Eppendorf tubes (50 μL in each), including a tube with unstained live cells, a tube with heat-induced dead cells, and tubes with isotype controls, single antibody controls, or combined antibodies. Add viability dye eFluor 780, Fc blocker, isotype control, or antibodies to each tube as explained below. Procedures for staining Immunofluorescence staining for surface marker proteins enables us to identify specific immune cell populations. To confirm the enrichment of macrophages, we stained using three antibodies that recognized leukocytes (CD45), myeloid lineage cells including macrophages (CD11b), and M2 macrophages (CD206). See Table 1 for reagent combination. Add 50 µL of PEB to tube 1 (unstained) and place on ice. Incubate tube 2 at 65 °C for 10 min to generate the dead cell control. Subsequently, add 950 µL of PEB to bring the volume to 1 mL. Place on ice. Add 950 µL of PEB to the remaining tubes to bring the volume to 1 mL. Add 1 µL of viability dye (eFluor 780) to tubes 2, 3, 5, 7, and 9 (tubes 3, 5, and 7 are IgG isotype controls). Mix immediately by inverting the tubes 5–10 times. Incubate for 10 min at 4 °C. Centrifuge tubes 3, 5, 7, and 9 at 500 × g for 5 min, discard the supernatants, and resuspend in 1 mL of PEB. Centrifuge tube 2 at 12,000 × g and 4 °C for 5 min, discard the supernatant, and resuspend the pellet in 100 µL of PEB. Place tube 2 on ice. Add 1 µL of Fc blocker to tubes 3–9 and gently vortex for 1 s; repeat three times. Incubate for 5 min at room temperature (~25 °C). Centrifuge tubes 3–9 at 500 × g and 4 °C for 5 min. Discard supernatants and resuspend the pellet in 100 µL of PEB. Add 1 µL of IgG-PE, 1 µL of IgG-APC, or 1 µL of IgG-FITC to tubes 3, 5, and 7, respectively. Add 1 µL of CD11b-PE antibody, 1 µL of CD45-APC antibody, or 1 µL of CD206-FITC antibody to tubes 4, 6, and 8, respectively (tubes 4, 6, and 8 are for compensation). Add 1 µL of CD11b-PE, 1 µL of CD45-APC, and 1 µL of CD206-FITC to tube 9. Gently vortex for 1 s and repeat three times. Incubate for 40 min at 4 °C. Centrifuge tubes 3–9 at 500 × g and 4 °C for 5 min, discard the supernatant and resuspend the pellet in 500 µL of PEB. Gently vortex for 1 s and repeat three times. Spin down tubes 3–9 at 500 × g and 4 °C for 5 min, aspirate the supernatant and resuspend the cells in 100 µL of PEB. Cells are ready for flow cytometry analysis. Figure 2 shows the results of flow cytometry analysis of isolated mononuclear cells. Figure 2. Flow cytometry analysis of isolated mononuclear cells. (A) Isolated mononuclear cells from young and old skeletal muscle were gated in a Forward/Side scatter plot. (B) Gated cells (from A) were further analyzed for CD45 and CD11b expression. CD45+/CD11b+ cells were separated from the main cell population (left, gate Q6). The double-positive cells accounted for 7.58% of the total cells in the young preparations and 6.28% in the old preparations (left). CD45+ cells accounted for 9.87% of the total cells in the young preparations and 8.24% in the old preparations (center). CD11b+ cells accounted for 9.21% of the total cells in the young preparations and 7.15% in the old preparations (right). CD11b+ cells were clearly separated from the main population and almost all CD11b+ cells were CD45+. (C) Putative M2 macrophage marker CD206 was highly expressed (gate Q2) in 27.7% of CD11b+ cells (gates Q2+Q3) in the young preparations (left) and in 32.1% of CD11b+ cells in the old preparations (right). BD FACSCantoTM II cell analyzer and FlowJo 10 software were used for analysis. Notes Here, we tested a commercial skeletal muscle dissociation kit combined with a programmable tissue dissociator and added a debris removal step. This protocol allowed the isolation of high-purity skeletal muscle macrophages. We generally obtain approximately two million live mononuclear cells from two hind limbs of a mouse, among which ~5–9% of cells are CD11b+/CD45+. This number is sufficient for FACS and single-cell transcriptomics analyses. Several steps in our protocol were included to improve the purity and yield of macrophages. Firstly, the combination of tissue weight and enzyme mix affects cell yield. In our experience, 0.5 g muscle per 5 mL enzyme mix provided superior muscle digestion and cell yield as compared with 1 g muscle per 5 mL enzyme mix. Hence, we use two C-tubes, each containing approximately 0.5 g muscle and 5 mL enzyme mix, for one mouse. Secondly, we compared two digestion programs—37C-mr-SMDK-1 (1 h) and 37C-mr-SMDK-2 (1.5 h)—in a GentleMAC octo dissociator with heaters. The 1.5-h program provided a more thorough digestion and better cell yield. Thirdly, the debris removal solution effectively removed most of fiber debris, which allowed for much cleaner cell preparations. We highly recommend the debris removal steps. Perfusing mice with saline solution through the inferior vena cava before sacrifice can be performed to reduce the presence of circulating immune cells in the skeletal muscle preparation. Splenocytes, which can be easily harvested from the same mouse, are another option for generating dead cells. Staining for additional markers, e.g., F4/80 and MHCII, will help to confirm macrophage specificity. Recipes Medium DMEM-I DMEM containing 1× penicillin and streptomycin solution. Store at 4 °C. Use within one month of preparation. DMEM-II DMEM-I supplemented with 5% heat-inactivated fetal bovine serum. Store at 4 °C. Use within one month of preparation. Digestive enzymes A skeletal muscle dissociation kit (see Reagents) was used for mononuclear cell isolation. Upon arrival of the kit, reconstitute and aliquot enzymes D, P, and A, and store at -20 °C. Prepare an enzyme mix for cell dissociation. Remove the enzymes from the -20 °C freezer and place at room temperature (~25 °C) for 5–10 min. An enzyme master mix will be prepared first. This master mix must be freshly prepared each time. Each GentleMACS C-tube will include 4.7 mL of DMEM supplemented with: 1× antibiotics 200 μL of enzyme D 50 μL of enzyme P 36 μL of enzyme A Total 5 mL Make 20 mL of enzyme master mix for four C-tubes to digest muscles from two mice, as follows: 20.21 mL of DMEM 860 μL of enzyme D 215 μL of enzyme P 154.8 μL of enzyme A (one tube × 4.3) Total 21.65 mL Resuspend the enzyme master mix. Add 5 mL of enzyme mix to each of the four GentleMACS C-tubes and place on ice. Use two C-tubes to digest skeletal muscles from one mouse. Given that approximately 1 g of skeletal muscle can be obtained from two hind legs of a mouse, approximately 0.5 g muscle tissues will be digested in 5 mL of enzyme mix in each C-tube. Buffers for flow cytometry analysis PEB buffer: 1,450 mL of Auto MACS rinsing solution 150 mL of MACS BSA stock solution 24 mL of 0.5 M EDTA The PEB buffer contains 1% BSA and 10 mM of EDTA Store at 4 °C. Use within three months of preparation. Reagent combination to label macrophages for flow cytometry Evenly divide approximately two million cells into nine 1.5 mL Eppendorf tubes (50 μL in each). Tube 1 contains unstained live cells and tube 2 contains heat-induced dead cells. Tubes 3, 5, and 7 contain isotype controls, and tubes 4, 6, and 8 contain single antibody controls. Tube 9 contains the combined antibodies. Add eFluor 780, Fc blocker, isotype control, or antibodies to each tube as described in Table 1. See Procedures for staining (Section F) for more details. Table 1. Reagent combination for staining Tubes Viability Dye (eFluor 780) Fc blocker Isotype control Antibody 1 2 1 µL 3 1 µL 1 µL 1 µL (IgG-PE) 4 1 µL 1 µL (CD11b-PE) 5 1 µL 1 µL 1 µL (IgG-APC) 6 1 µL 1 µL (CD45-APC) 7 1 µL 1 µL 1 µL (IgG-FITC) 8 1 µL 1 µL (CD206-FITC) 9 1 µL 1 µL 1 µL (CD11b-PE) 1 µL (CD45-APC) 1 µL (CD206-FITC) Acknowledgments This work was supported by the Intramural Research Program of the National Institute on Aging. The authors thank Dr. Ramaiah Nagaraja for helpful technical assistance. This protocol derives from the main paper (Krasniewski et al, 2022). Competing interests The authors declare no competing interests. Ethics Animal use was approved by the Animal Care and Use Committee of the National Institute on Aging, NIH. Approved ID is 476-LGG-2024, which is effective until 2024. References Arnold, L., Henry, A., Poron, F., Baba-Amer, Y., van Rooijen, N., Plonquet, A., Gherardi, R. K. and Chazaud, B. (2007). Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med 204(5):1057-1069. Chakarov, S., Lim, H. Y., Tan, L., Lim, S. Y., See, P., Lum, J., Zhang, X. M., Foo, S., Nakamizo, S., Duan, K., et al. (2019). Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches.Science 363(6432): eaau0964. Cui, C. Y., Driscoll, R. K., Piao, Y., Chia, C. W., Gorospe, M. and Ferrucci, L. (2019). Skewed macrophage polarization in aging skeletal muscle. Aging Cell 18(6): e13032. Cui, C. Y. and Ferrucci, L. (2020). Macrophages in skeletal muscle aging. Aging (Albany NY). 12(1):3-4 Gautier, E. L., Shay, T., Miller, J., Greter, M., Jakubzick, C., Ivanov, S., Helft, J., Chow, A., Elpek, K. G., Gordonov, S., et al. (2012). Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol 13(11): 1118-1128. Gosselin, D., Link, V. M., Romanoski, C. E., Fonseca, G. J., Eichenfield, D. Z., Spann, N. J., Stender, J. D., Chun, H. B., Garner, H., Geissmann, F., et al. (2014). Environment drives selection and function of enhancers controlling tissue-specific macrophage identities. Cell 159(6): 1327-1340. Ginhoux, F. and Guilliams, M. (2016). Tissue-Resident Macrophage Ontogeny and Homeostasis. Immunity 44(3): 439-449. Hoeksema, M. A. and Glass, C. K. (2019). Nature and nurture of tissue-specific macrophage phenotypes. Atherosclerosis 281: 159-167. Jaitin, D. A., Adlung, L., Thaiss, C. A., Weiner, A., Li, B., Descamps, H., Lundgren, P., Bleriot, C., Liu, Z., Deczkowska, A., et al. (2019). Lipid-Associated Macrophages Control Metabolic Homeostasis in a Trem2-Dependent Manner. Cell 178(3): 686-698 e614. Jensen, S. M., Bechshoft, C. J. L., Heisterberg, M. F., Schjerling, P., Andersen, J. L., Kjaer, M. and Mackey, A. L. (2020). Macrophage Subpopulations and the Acute Inflammatory Response of Elderly Human Skeletal Muscle to Physiological Resistance Exercise. Front Physiol 11: 811. Kosmac, K., Peck, B. D., Walton, R. G., Mula, J., Kern, P. A., Bamman, M. M., Dennis, R. A., Jacobs, C. A., Lattermann, C., Johnson, D. L., et al. (2018). Immunohistochemical Identification of Human Skeletal Muscle Macrophages. Bio-protocol 8(12): e2883. Kratofil, R. M., Kubes, P. and Deniset, J. F. (2017). Monocyte Conversion During Inflammation and Injury. Arterioscler Thromb Vasc Biol 37(1): 35-42. Krasniewski, L. K., Chakraborty, P., Cui, C. Y., Mazan-Mamczarz, K., Dunn, C., Piao, Y., Fan, J., Shi, C., Wallace, T., Nguyen, C., et al. (2022). Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations.eLife 11: e77974. Lavin, Y., Winter, D., Blecher-Gonen, R., David, E., Keren-Shaul, H., Merad, M., Jung, S. and Amit, I. (2014). Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159(6): 1312-1326. Liu, L., Cheung, T. H., Charville, G. W. and Rando, T. A. (2015). Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc 10(10): 1612-1624. Mills, C. D., Kincaid, K., Alt, J. M., Heilman, M. J. and Hill, A. M. (2000). M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 164(12): 6166-6173. Martinez, F. O., Sica, A., Mantovani, A. and Locati, M. (2008). Macrophage activation and polarization. Front Biosci 13: 453-461. Rath, M., Muller, I., Kropf, P., Closs, E. I. and Munder, M. (2014). Metabolism via Arginase or Nitric Oxide Synthase: Two Competing Arginine Pathways in Macrophages. Front Immunol 5: 532. Shapouri-Moghaddam, A., Mohammadian, S., Vazini, H., Taghadosi, M., Esmaeili, S. A., Mardani, F., Seifi, B., Mohammadi, A., Afshari, J. T. and Sahebkar, A. (2018). Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 233(9): 6425-6440. Tidball, J. G. (2011). Mechanisms of muscle injury, repair, and regeneration. Compr Physiol 1(4): 2029-2062. Tidball, J. G. (2017). Regulation of muscle growth and regeneration by the immune system. Nat Rev Immunol 17(3): 165-178. Underhill, D. M., Gordon, S., Imhof, B. A., Nunez, G. and Bousso, P. (2016). Elie Metchnikoff (1845-1916): celebrating 100 years of cellular immunology and beyond. Nat Rev Immunol 16(10): 651-656. Walton, R. G., Kosmac, K., Mula, J., Fry, C. S., Peck, B. D., Groshong, J. S., Finlin, B. S., Zhu, B., Kern, P. A. and Peterson, C. A. (2019). Human skeletal muscle macrophages increase following cycle training and are associated with adaptations that may facilitate growth. Sci Rep 9(1): 969. Wang, X., Sathe, A. A., Smith, G. R., Ruf-Zamojski, F., Nair, V., Lavine, K. J., Xing, C., Sealfon, S. C. and Zhou, L. (2020). Heterogeneous origins and functions of mouse skeletal muscle-resident macrophages. Proc Natl Acad Sci U S A 117(34): 20729-20740. Wynn, T. A., Chawla, A. and Pollard, J. W. (2013). Macrophage biology in development, homeostasis and disease. Nature 496(7446): 445-455. Yang, W. and Hu, P. (2018). Skeletal muscle regeneration is modulated by inflammation. J Orthop Translat 13: 25-32. Article Information Copyright Krasniewski et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Immunology > Immune cell isolation > Macrophage Cell Biology > Cell isolation and culture Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages Gretchen L. Seim [...] Jing Fan Jul 20, 2020 6883 Views Production, quantification, and infection of Amazonian Phlebovirus (Bunyaviridae) Carolina Torturella Rath [...] Ulisses Gazos Lopes Jul 5, 2021 2664 Views Differentiation of Bone Marrow Monocytes into Alveolar Macrophages-like Cells through Co-culture with Lung Epithelial Cells and Group 2 Innate Lymphoid Cells Pauline Loos [...] Laurent Gillet Sep 20, 2023 833 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,562	https://bio-protocol.org/en/bpdetail?id=4562&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Human Auto-IgG Purification from High Volume Serum Sample by Protein G Affinity Purification SS Serena Sensi * AG Andreas Goebel * (*contributed equally to this work) Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4562 Views: 1098 Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Pain Dec 2019 Abstract Immunoglobulins are proteins produced by the immune system, which bind specifically to the antigen that induced their formation and target it for destruction. Highly purified human immunoglobulins are commonly used in research laboratories for several applications, such as in vitro to obtain hybridomas and in vivo animal immunisation. Several affinity purification methods are used to purify immunoglobulins from human serum, such as protein A/G Sepharose beads, polyethylene glycol, and caprylic acid ammonium sulphate precipitation. Here, we provide a detailed protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. The protocol is divided into four main steps (column preparation, serum running, wash, and elution) for IgG purification, and two extra steps (protein dialysis and sucrose concentration) that should be performed when buffer exchange and protein concentration are required. Several IgG affinity purification methods using protein A or G are available in the literature, but protein A has a higher affinity for rabbit, pig, dog, and cat IgG, while protein G has a higher affinity for mouse and human IgG. This affinity-based purification protocol uses protein G for a highly specific purification of human IgG for animal immunization, and it is particularly useful to purify large amounts of human IgG. Graphical abstract IgG purification protocol. The IgG purification protocol consists of four main steps (column preparation, serum running, wash, and elution) and two extra steps (protein dialysis and concentration). a. Diluted serum is added to the protein G beads and IgG binds to the Fc receptors on protein G beads. b. Beads are washed in Hartman’s solution to fully remove the complex protein mixture (multicolour shapes, as depicted in the graphical abstract). c. IgG (orange triangles, as depicted in the graphical abstract) are removed from protein G with glycine and collected in Tris buffer. d. The IgG is transferred into a semi-permeable membrane (‘snake skin’) and allowed to dialyse overnight for buffer exchange with a physiological solution (Hartmann’s). Keywords: Antibody Human IgG Affinity purification Liquid chromatography Protein G Immunization Background Immunoglobulins (Ig) or antibodies (Ab) are glycoproteins found in serum and tissue fluids, which are produced in large amounts after contact with immunogenic foreign molecules. They are Y-shaped and consist of five classes or isotypes: IgG, IgM, IgA, IgE, and IgD. Immunoglobulin G (IgG) is a type of antibody normally found in blood and extracellular fluid that is predominant in adaptive immune responses. The IgG isotype is 75% of normal serum immunoglobulins (14 mg/mL) and is divided into four sub-classes, called IgG1, IgG2, IgG3, and IgG4, in the following proportion: 70%, 20%, 8%, and 2%, respectively. All antibodies have the same basic structure divided into variable and constant regions; the variable region is antigen specific and contains two fragment antigen binding (Fab) sites, while the constant region, also known as fragment crystallizable (Fc), is isotype specific and stimulates antigen destruction. Human-derived IgGs are a subgroup of immunoglobulins that are commonly used for a variety of research methods or as effective therapeutics in inflammation, cancer, as well as autoimmune and infectious diseases. For in vitro methods, antibodies can be used directly in their crude form (serum) or in a pure form (IgG); however, purified antibodies are more advantageous than the whole serum because they reduce cross-reactivity, can be easily stored, and are stable for longer periods. Several autoimmune syndromes have been successfully "transferred" in animals immunized with highly purified human autoantibodies (Cuhadar et al., 2019; Goebel et al., 2021; Helyes et al., 2019; Pohoczky et al., 2022). Several protocols for antibody purification are available in the literature, and these are divided in two main groups: non chromatographic techniques, such as polyethylene glycol, and caprylic acid ammonium sulphate precipitation, and chromatographic techniques, such as affinity chromatography with protein A/G beads. Non-chromatographic techniques depend on the combination of several factors, and are therefore complex, laborious, and expensive, especially due to the usage of polyethylene glycol. Affinity chromatography with commercially available protein-A or protein-G beads is the standard methodology for purification of antibodies from serum, for the relative simplicity and reliability of results. While protein A has a higher affinity for rabbit, pig, dog, and cat IgG1, IgG2, and IgG4, protein G has a higher affinity for mouse and human IgG1, IgG2, IgG3, and IgG4. Furthermore, protein G beads have a wider binding range than protein A beads because they can bind to intact IgG as well as antibody fragments F(ab) 2 and Fc regions, while protein A binds strongly to the Fc part and weakly to the Fab region. Here, we provide a detailed and easy to follow protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. Differently from previously published methods, this protocol is specifically designed for purification of large amounts of human serum (20 mL), whereby serum is diluted before purification, and Hartmann’s solution is used as a binding buffer instead of PBS, to keep neutral pH and physiological ionic strength(Cuhadar et al., 2019; Goebel et al., 2021; Helyes et al., 2019; Pohoczky et al., 2022). These steps significantly improved the human IgG purification. Furthermore, this protocol contains useful comments regarding factors affecting IgG binding and stability. This protocol could also be used for purification of antibodies from hybridoma culture supernatant or ascites. Materials and Reagents Nunc 96-Well U Bottom (Thermo Fisher, catalog number: 268200) Syringes PlastipakTM 20 mL Luer-LokTM (BD, catalog number: 302237) 0.2 µm filters (Sigma-Aldrich, Millipore, catalog number: SLGP033RS) 50 mL centrifuge tubes (Sigma-Aldrich, Greiner, catalog number: T2318-500EA) 1.5 mL Eppendorf Safe-Lock Tubes (Eppendorfs, catalog number: 0030120086) SnakeSkinTM Dialysis Tubing, 10K MWCO, 22 mm (Thermo Fisher, catalog number: 68100) SnakeSkinTM Dialysis Clips (Thermo Fisher, catalog number: 68011) Protein G Sepharose 4 Fast Flow, 5 mL (Sigma-Aldrich, catalog number: GE17-0618-01) storage at 4 °C Econo-pac chromatography columns, package of 50 (Bio-Rad, catalog number: 7321010) Two-way Stopcocks (Bio-Rad, catalog number: 7328102) Compound Sodium Lactate Solution for Infusion BP (Hartmann's Solution for infusion) in Viaflo, 1,000 mL (Baxter, catalog number: FKE2324) Bradford Reagent (Sigma-Aldrich, catalog number: B6916) Ethanol, Absolute, Molecular Biology Grade, 500 mL (ThermoFisher, catalog number: 16606002) Sucrose, Molecular Biology Grade (AlfaAesar by Thermo Fisher Scientific, catalog number: J65148.A1) Trizima Base (ThermoFisher, catalog number: BP152-500) Glycine (Sigma-Aldrich, catalog number: 50046) HCl (Sigma-Aldrich, catalog number: H1758) Neutralisation Buffer: Trizma base 1 M (pH 8.0) (see Recipes) Elution Buffer: Glycine 100 mM (pH 2.3) (see Recipes) Storage buffer: 20% Ethanol (see Recipes) Washing buffer: Hartmann's Solution for Infusion (Compound Sodium Lactate) in Viaflo, 1,000 mL (see Recipes) Equipment pH Meter Mettler ToledoTM FiveEasyTM F20 pH/mV Meter (Mettler Toledo, catalog number: 15543360) Centrifuge 5810R (Eppendorf, catalog number: 5811000465) PIPETMAN Classic Starter Kit, P20, P200, P1000 (Gilson, catalog number: F167300) Laboratory stand Model SBS-LS-100 (Expondo, catalog number: EX10030473) Borosilicate Glass Tall Form Beakers 2 L (ThermoFisher, catalog number: 15499083) Procedure Preparation of the protein G column Resuspend two vials of protein G Sepharose (5 mL each) in 50 mL of Hartmann’s solution in a 50-mL tube. Centrifuge at 41 × g for 5 min, gently pour off the Hartmann’s solution by decantation, and add fresh Hartmann’s solution to top up the 50-mL tube; repeat twice. Wash the column with 100% ethanol, and then with Hartmann’s solution. Add protein G solution to the column with a plastic Pasteur pipette until the column is full. The two-way stopcock should be closed (horizontal) to avoid the loss of protein G. The length of the packed protein G column should be 6–8 cm (the empty colums is 12cm)". Open the two-way stopcock (vertical) and let all the solution slowly run down (to allow the protein G to deposit at the bottom of the column). Do not permit protein G to dry, always leave at least 1 mL of Hartmann’s solution on top of the beads (Figure 1A). Running the sample through the protein G column Centrifuge the serum at 2,000 × g for 15 min to get rid of particulates before dilution. A second centrifugation may be required to achieve this. Dilute the serum 1:3 in Hartmann’s solution: 20 mL of serum and 40 mL of Hartmann’s solution, for a total of 60 mL of diluted serum. Add the diluted serum (60 mL) to the column with a plastic Pasteur pipette. Run the serum slowly through the column, adjusting the stopcock to half closed for 1 drop/second, and setting 1 min on a timer (1 mL of IgG should pass through the column in 1 min). Firstly, top up the column with serum, then open the stopcock and start the purification. Continue topping up the column until all serum is transferred into the column. Run the diluted serum slowly (1 mL/min) through, and collect the flow through in a 50-mL tube. Run the flow through through the column again, then discard or store the flow through for further analysis (Figure 1B). Washing any non-specific binding with HS Run 60–100 mL of Hartmann’s solution through the column, to wash away any non-specific binding (Figure 1C). Check Hartmann’s flow through for IgG, using the Bradford reagent: Resuspend 20 μL of eluted flow through in 100 μL of Bradford reagent, using a well of a 96-well plate. Continue adding Hartmann’s solution until no blue colour is detected, then start eluting with glycine (Procedure D). The blue colour means that there are still other unbound protein in the column (not IgG) that should be removed before elution. Elution with glycine pH 2.3 and Tris pH 8.0 Place 30 Eppendorf tubes (1.5 mL) on ice. Add 100 μL of 1 M Tris, pH 8.0 into each tube (Figure 1D). Add 30 mL of 100 mM glycine pH 2.3 to the column, and elute 900 μL of flow through into each prepared tube. Run glycine slowly through the column, adjusting the stopcock to half closed for 1 drop/second and setting 1 min on a timer (1 mL of IgG should pass through the column in 1 min). Firstly, top up the column with glycine, then open the stopcock and start elution from the first tube. When the column needs to be topped up, close the stopcock and add glycine, as above. Shake each fraction, to neutralize glycine with Tris and to protect the sample. Usually, there is no IgG in the first four tubes. Check the presence of IgG in all tubes using the Bradford assay: Resuspend 20 μL of eluted solution from each tube with 100 μL of Bradford reagent using 30 wells of a 96-well plate. The last tube should have no blue colour. Pool the highly concentrated fractions, as indicated by the intense blue colour, into one 50-mL tube. For 20 mL of serum (diluted in 40 mL of Harmann’s = 60 mL of overall solution), the yield should be approximately 20 tubes. Run 60 mL of Hartmann’s Solution (1 mL/min) to remove any residual glycine from the column. Check if there is no glycine in the flow through with the Bradford assay, as in step D3. Store the column at 4 °C in 20% ethanol to prevent contaminations. The same column can be used up to five times. Figure 1. IgG purification steps. A. Column preparation. Wash the column with 20 mL of 100% ethanol and then 20 mL of binding buffer (HS), and then add the protein G Sepharose slurry from the vials to the column. B. Serum running. Slowly apply diluted serum to the column twice (1 mL/min). C. column washing. Wash protein G beads in 100 mL of binding buffer. D. Elute the bound IgG fraction by adding 20 mL of 100 mM glycine pH 2.3 to the column, and collect in 20 tubes (1.5-mL Eppendorf tubes) pre-filled with Tris buffer. E. IgG dialysis. Dialyse purified IgG at 4 °C overnight in Hartmann’s solution using a 10 kDa dialysis membrane. F. IgG concentration. Move the IgG from the beaker into a tray, covered with sucrose and left until has reached the right concentration; the sucrose starts to slowly liquefy after approximately 20 min and the sample is concentrated 2x after 1 h. G. IgG aggregates. IgG aggregates (red circle) may form during overnight dialysis, but these can be removed with a spin at 657 × g for 10 min. Dialysis Fill a beaker with 1 L of Hartmann’s solution. Cut approximately 10 cm of snakeskin dialysis tubing, fold over one end, and secure with a clip. Then, pipette the IgG solution into it, leaving approximately an extra third volume of air. Fold over the top and secure with a clip. Place the dialysis bag into the beaker and leave it on a stirrer at 4°C, allowing the sample to dialyse overnight (Figure 1E). Sucrose concentration Move the dialysis preparation from the beaker into a tray, cover with sucrose, and leave until the sample has reached the right concentration—the exact time will depend upon the starting concentration of the dialysed sample, as it may take a couple of hours and it may need sucrose changing after 1 h (Figure 1E). Transfer the sample into a 50-mL tube. Centrifuge the sample at 657 × g for 10 min and transfer the IgG solution into a fresh tube to remove aggregates. Aggregates may still form later but they can be removed with another spin at 657 × g for 10 min (Figure 1G). Filter the sample through a 0.2 μm filter. Store the IgG sample at 4 °C. Data analysis IgG concentration is measured using a Nanodrop 2000 after purification, after dialysis, and after sucrose concentration. Open the Nanodrop 2000 software and select Protein 280. Then select "IgG" as sample type. Pipette 1.5 μL of Hartmann’s solution onto the lower measurement pedestal, then click on "Blank" to measure the blank. Clean residual Hartmann’s solution with a tissue. Pipette 1.5 μL of IgG solution onto the lower measurement pedestal, then click on "measure" to measure the sample. An OD280 graph is then provided, together with IgG concentration (Figure 2). Human IgG purified from serum has a concentration from 7 mg/mL to 10 mg/mL, while human IgG purified from plasma has a concentration between 5 mg/mL and 7 mg/mL. The OD280 graph is used to assess the specificity and quantitative yield of the purification procedure. In particular, OD260/280 ratio is used to check sample purity and possible contamination by nucleic acids (absorbance at 260). An OD260/280 less than 0.60 indicate pure protein yield. Hartmann’s solution (salt buffer) is used as IgG buffer to help minimize the presence of nucleic acids. Figure 2. Purified IgG OD280 graph. Red arrows indicate salts (absorbance at 230), DNA and RNA (absorbance at 260), and IgG (absorbance at 280). The high salt peak is caused by salts in the Hartmann’s solution (IgG buffer). OD260/280 ratio is 0.51, IgG concentration is 10.10 mg/mL. Notes Factors affecting IgG binding and stability Due to issues related to temperature fluctuations in the laboratory during summer (defect in the air-conditioning), serendipitously initially IgG were purified either at high room temperature (30 °C) or at standard room temperature (20 °C). Subsequently, the IgG solution purified at 30 °C was found not to be active by collaborating groups when used in in vivo experiments, suggesting that the very high room temperature may have negatively affected either IgG binding to protein G or IgG stability, as the manufacturer recommended room temperature (20 °C) as optimal. Another factor that affects IgG binding and stability is pH. Low pH (2.5–3.0) is commonly used during protein G elution to break the ionic and hydrogen bonds between the antigen and antibody. IgGs used for this project were eluted at a lower pH 2.3, to achieve a more complete elution yield. Other factors that may negatively affect IgG binding to protein G include protein G beads freezing (freezing may cause detachment of the protein G from the agarose beads) and column flow rate (high flow rate leading to IgG loss, as IgG would not have sufficient time to bind to Fc receptors on protein G). Finally, according to the manufacturer’s instructions, any element of drying of the protein G resin will result in a detachment of the protein G from agarose beads, with significant loss in IgG yield. Recipes Neutralisation Buffer: Trizma base 1 M (pH 8.0) Dissolve 60.57 g of Trizma base in 400 mL of ddH2O placed on a beaker with a magnetic stirrer. Adjust pH to 8.0 by adding HCl. Adjust the volume to 500 mL using ddH2O. Store at room temperature for up to 1 year. Elution Buffer: Glycine 100 mM (pH 2.3) Dissolve 3.785 g of glycine in 400 mL of ddH2O placed on a beaker with on a magnetic stirrer. Adjust pH to 2.3 by adding HCl. Adjust the volume to 500 mL using ddH2O. Store at 4 °C for up to 3 months. Storage buffer: 20% Ethanol Mix 20 mL of 100% ethanol with 80 mL of ddH2O. Store at 4 °C. Washing buffer: Hartmann's Solution for Infusion (Compound Sodium Lactate) in Viaflo, 1,000 mL Acknowledgments This work was supported by grants from the Pain Relief Foundation, Liverpool, UK. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics Plasma samples used for IgG purification were obtained after plasma exchange therapy (PTE) and no ethics approval was required for the use of waste plasma (15/NW/0467, North West – Haydock Research Ethics Committee). Sera samples used for IgG purification were obatined under ethical permission and individual consent for autoantibody research (12/EE/0164, East of England). References Cuhadar, U., Gentry, C., Vastani, N., Sensi, S., Bevan, S., Goebel, A. and Andersson, D. A. (2019). Autoantibodies produce pain in complex regional pain syndrome by sensitizing nociceptors. Pain 160(12): 2855-2865. Goebel, A., Krock, E., Gentry, C., Israel, M. R., Jurczak, A., Urbina, C. M., Sandor, K., Vastani, N., Maurer, M., Cuhadar, U., et al. (2021). Passive transfer of fibromyalgia symptoms from patients to mice. J Clin Invest 131(13). Helyes, Z., Tekus, V., Szentes, N., Pohoczky, K., Botz, B., Kiss, T., Kemeny, A., Kornyei, Z., Toth, K., Lenart, N., et al. (2019). Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms. Proc Natl Acad Sci U S A 116(26): 13067-13076. Pohoczky, K., Kun, J., Szentes, N., Aczel, T., Urban, P., Gyenesei, A., Bolcskei, K., Szoke, E., Sensi, S., Denes, A., et al. (2022). Discovery of novel targets in a complex regional pain syndrome mouse model by transcriptomics: TNF and JAK-STAT pathways. Pharmacol Res 182: 106347. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Immunology > Antibody analysis > Antibody detection Biochemistry > Protein > Isolation and purification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy Wenxin Zhang [...] Sharon A. Tooze Jan 5, 2024 650 Views Chromogranin B Purification for Condensate Formation and Client Partitioning Assays In Vitro Anup Parchure and Julia Von Blume Oct 20, 2024 286 Views Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization Jasmeet Kaur Shergill and Domenico Azarnia Tehran Jan 20, 2025 237 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,563	https://bio-protocol.org/en/bpdetail?id=4563&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Infection of the Developing Central Nervous System of Drosophila by Mammalian Eukaryotic and Prokaryotic Pathogens BB Billel Benmimoun BW Bente Winkler PS Pauline Spéder Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4563 Views: 627 Reviewed by: Nafisa M. JadavjiKai Yuan Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Communications Nov 2020 Abstract Pathogen invasion of the central nervous system (CNS) is an important cause of infection-related mortality worldwide and can lead to severe neurological sequelae. To gain access to the CNS cells, pathogens have to overcome the blood–brain barrier (BBB), a protective fence from blood-borne factors. To study host–pathogen interactions, a number of cell culture and animal models were developed. However, in vitro models do not recapitulate the 3D architecture of the BBB and CNS tissue, and in vivo mammalian models present cellular and technical complexities as well as ethical issues, rendering systematic and genetic approaches difficult. Here, we present a two-pronged methodology allowing and validating the use of Drosophila larvae as a model system to decipher the mechanisms of infection in a developing CNS. First, an ex vivo protocol based on whole CNS explants serves as a fast and versatile screening platform, permitting the investigation of molecular and cellular mechanisms contributing to the crossing of the BBB and consequences of infection on the CNS. Then, an in vivo CNS infection protocol through direct pathogen microinjection into the fly circulatory system evaluates the impact of systemic parameters, including the contribution of circulating immune cells to CNS infection, and assesses infection pathogenicity at the whole host level. These combined complementary approaches identify mechanisms of BBB crossing and responses of a diversity of CNS cells contributing to infection, as well as novel virulence factors of the pathogen. Graphical abstract Procedures flowchart. Mammalian neurotropic pathogens could be tested in two Drosophila central nervous system (CNS) infection setups (ex vivo and in vivo) for their ability to: (1) invade the CNS (pathogen quantifications), (2) disturb blood–brain barrier permeability, (3) affect CNS host cell behaviour (gene expression), and (4) alter host viability. Keywords: Central nervous system Infection Drosophila Blood–brain barrier Brain culture Pathogens Host–pathogen interactions Background Infections of the central nervous system (CNS) are devastating yet remain relatively rare due to the presence of specific protective layers. In particular, the blood–brain barrier (BBB) is a selective physical and chemical filter, and performs its neuroprotective role by controlling molecular import into the CNS and blocking the entry of circulating cells (Daneman and Prat, 2015). In higher vertebrates, brain microvascular endothelial cells, harbouring intercellular tight junctions, form the core structure of the BBB. In addition, pericytes, astrocytes, and an extracellular matrix basal membrane regulate BBB integrity and functions (Obermeier et al., 2013; Daneman and Prat, 2015; Saunders et al., 2016). However, several pathogens have developed strategies to overcome the BBB and invade the CNS (Coureuil et al., 2017). To study interactions between pathogens and the BBB, various cell culture systems have been developed, from simple endothelial monolayers to more complex multicellular cultures, combining endothelial cells and pericytes (Sivandzade and Cucullo, 2018). While these in vitro models have proposed a number of BBB crossing mechanisms, they remain limited in recapitulating the 3D architecture of the BBB and its interactions with the complexity of CNS cells (Sivandzade and Cucullo, 2018; Jackson et al., 2019). Moreover, organoids also present architectural limitations (Bergmann et al., 2018). To bypass this restriction, a number of animal models, in particular rodents and zebrafish, have been used (Dando et al., 2014; Jackson et al., 2019), allowing the investigation of infection mechanisms in vivo. However, in addition to cost and ethical issues, their genetic and cellular complexity prevents systematic, screen-based approaches. Separating the systemic from the local impact of these immune challenges is also difficult in such context, and infections can have lethal consequences for the host. In addition, culturing CNS tissue of these animal models is still difficult. Drosophila melanogaster represents an original and powerful model to study CNS infection, from identifying novel mechanisms of BBB-pathogen interactions to understanding the extent of consequences on the diversity of CNS cell populations. The fly BBB is composed of two glial layers exhibiting conserved neuroprotective mechanisms with the mammalian BBB. The subperineurial glia (SPG), forming an epithelium-like structure with septate junctions, represent the core structure and physical filter of the BBB (Stork et al., 2008; Hindle and Bainton, 2014; Weiler et al., 2017; Babatz et al., 2018). The perineurial glia, a hemolymph sensor, and the extracellular matrix both cover the SPG (DeSalvo et al., 2014; Parkhurst et al., 2018). Many transporter proteins expressed by the BBB to allow the selective shuttling of diverse solutes and xenobiotics are actually conserved, including solute carriers, ATP-binding cassette transporters, and lipoprotein receptors (DeSalvo et al., 2014; Hindle and Bainton, 2014; Weiler et al., 2017). Moreover, many aspects of mammalian neurogenesis are conserved in flies (Mira and Morante, 2020), enabling us to probe the impact of infection on such process. Drosophila neural stem cells self-renew via asymmetric cell division, switch from quiescence to proliferation, and are present within a specific microenvironment sharing neurogenic features and common constituents with the mammalian niche, including glial populations and the BBB (Homem and Knoblich, 2012; Otsuki and Brand, 2017). Finally, due to Drosophila unrivalled genetics, each of the BBB cell layers and the different CNS populations can be manipulated independently, in parallel with each other, and in a temporally controlled and spatially restricted manner. These features provide a simpler, genetically tractable model to study CNS infection, from pathogen entry to downstream consequences for the host. We have devised a double, complementary approach to generate CNS infection in Drosophila: An ex vivo protocol that mimics CNS infection. Here, an intact larval CNS explant is cultured in a specific medium inoculated with pathogens (Benmimoun et al., 2020). This explant setup is first designed to allow fast screening of pathogens, mutant strains, and culture conditions for the generation and outcome of CNS infection. In this frame, it has first allowed the identification of a number of prokaryotic and eukaryotic neurotropic pathogens, known to trigger meningitis and/or encephalitis in mammals, that are able to cross the fly BBB. On the prokaryotic side, this included Streptococcus agalactiae (Group B Streptococcus, GBS), Streptococcus pneumoniae, Listeria monocytogenes, and the strictly human Neisseria meningitidis. The eukaryotic Candida albicans and Candida glabrata were also found to reach the Drosophila CNS, but not Cryptococcus neoformans. This ex vivo platform was further used to screen a collection of GBS mutant strains, by which surface lipoproteins were identified—more precisely the lipoprotein Blr—as virulence factors crucial for BBB crossing (Benmimoun et al., 2020). Finally, its combination with Drosophila genetics revealed the Drosophila lipoprotein receptor (LpR2), which is expressed on the surface of the BBB layer on the SPGs, as a host receptor for Blr, and important for CNS invasion by GBS (Benmimoun et al., 2020). Another asset of the explant protocol is to study the effect of pathogenic infection on the different cell populations outside of the systemic context (for example, outside of systemic inflammation, a common hallmark of infection), potentially revealing other layers of response that could be otherwise hidden. For example, the lactic acidosis generated in the milieu by GBS could be quenched by buffer solutions, revealing the contribution of this phenomenon to BBB weakening (Benmimoun et al., 2020). An in vivo setup through direct microinjection of the pathogen into the fly circulatory system (Benmimoun et al., 2020; Winkler et al., 2021). This approach aims to evaluate the impact of infection i) on the CNS, this time including the contribution of systemic components, such as circulating immune cells, and ii) at the level of the whole organism, by assessing a diversity of host–pathogen interactions, in particular virulence. Comparison with the explant system allows to confirm or infirm the importance of molecular and cellular mechanisms in a whole-body context, offering the possibility to discriminate between local and systemic effects, and to identify the hierarchy between and dominance of specific mechanisms. This protocol was first used to determine the conservation of GBS Blr and Drosophila LpR2 interactions in BBB crossing mechanism and CNS invasion, initially identified through the explant setup (Benmimoun et al., 2020). Moreover, assessing survival overtime of the injected larvae in this setup demonstrated that Blr is a virulence factor in Drosophila, a property further validated in a mouse model of GBS hematogenous brain infection (Benmimoun et al., 2020). This whole in vivo CNS infection protocol also participated in revealing a new inflammatory mechanism, in which glial cells direct CNS infiltration by macrophages in response to GBS infection (Winkler et al., 2021). Here, we detail these two complementary platforms to elicit infection of the Drosophila developing CNS by mammalian neurotropic pathogens. By taking advantage of the Swiss knife of Drosophila genetics, the architectural preservation of the CNS tissue, and the similarity of many fundamental cellular and molecular mechanisms with mammals, these approaches allow more systematic screening of wild-type and mutant pathogen strains, as well as targeted genetic manipulations, to infer precise causal relationships between cellular layers or between molecular pathways during infection. Ultimately, these setups can be used to study all steps of the CNS infection, from crossing of the BBB to downstream consequences on the host, including neuroinflammation and impact on neurogenesis. Although these methods are described for prokaryotic and eukaryotic pathogens, they could be adapted for other pathogens (virus, parasites) as well as for studying systemic extrinsic stresses, and for investigating the entry of therapeutic molecules into the CNS. Materials and Reagents Fly strain and Drosophila larval culture mdr65-mtd: Tomato (Benmimoun et al., 2020 and Figure 1) Figure 1. Confocal image of top view (A) and cross-section with orthogonal views (B) of the Drosophila BBB in a portion of the ventral nerve cord Egg laying plates (50 mm, Deep) (Thermo Scientific, catalog number: 124-17), see Figure 2A Fly egg laying cages (homemade, see Figure 2B) Larval collection plates (60 mm Petri dishes) (Corning-Falcon, catalog number: 353004), see Figure 2C Figure 2. Fly larval collection and culture materials Active dry yeast (Genesee Scientific, catalog number: 62-103) Corn flour (Limagrain, catalog number: WFMZ01HS) Agar (Sigma-Aldrich, catalog number: A7002-1KG) Sugar (Dominique Dutscher, catalog number: 067508B) Organic grape juice Propionic acid (Sigma-Aldrich, catalog number: P5561-1L) Methyl-4-hydroxybenzoate (Sigma-Aldrich, catalog number: H3647) Ethanol (Sigma-Aldrich, catalog number: 24105) Egg laying medium (see Recipes) Fly food (see Recipes) Pathogen preparation Pathogen growth media [Brain Heart Infusion broth (BHI), Yeast extract Peptone Dextrose (YPD), or de Man Rogosa Sharpe (MRS)] (BD Difco, Millipore, catalog numbers: 237500, 242820 or 288130) Round-bottom tubes with cap (Fisher Scientific, Falcon, catalog number: 352059) Micro cuvette for spectrophotometer (Dutscher, Greiner Bio-One, catalog number: 613101) Microcentrifuge tubes (Fisher Scientific, Eppendorf, catalog number: 10708704) Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408) Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024) Drosophila dissection Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20) Tissue culture dishes (Fisher Scientific, Corning, Falcon, catalog number: 3530001) Glass cavity dish (Atom Scientific, catalog number: SDCE4040-1) Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408) Ethanol (Sigma-Aldrich, catalog number: 493511) Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024) CNS explant culture 24-well cell culture plate (Fisher Scientific, Corning Falcon, catalog number: 353047) Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024) l-Glutamine (Fisher Scientific, Gibco, catalog number: 25030-032) Sodium l-ascorbate (Sigma-Aldrich, catalog number: A4034) Fetal bovine serum (Sigma-Aldrich, catalog number: F4135) CNS culture medium I (see Recipes) CNS culture medium II (see Recipes) In vivo CNS infection Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20) Pipette Drummond Scientific 3.5’’ (Dutsher, Drummond Scientific, catalog number: 3-000-203-G/X) Nano-injector Nanoject III (Dutsher, Drummond Scientific, catalog number: 075250) Mineral oil (Sigma-Aldrich, catalog number: M5904) Microscope slides (Fisher Scientific, Fisherbrand, catalog number: 10219280) Permeability assay Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024) Dextran, Texas Red, lysine fixable 10 KDa (ThermoFisher, catalog number: 11530236, Invitrogen D1863) Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408) 4% methanol-free formaldehyde (ThermoFisher Scientific, catalog number: 28908) Immunohistochemistry 4% methanol-free formaldehyde (ThermoFisher Scientific, catalog number: 28908) Bouin’s solution (Sigma-Aldrich, catalog number: HT10132) Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408) Triton X-100 (Sigma-Aldrich, catalog number: T9284) Bovine serum albumin (Sigma-Aldrich, catalog number: A3608) Normal goat serum (Invitrogen, catalog number: 10000C) Mowiol mounting medium (homemade), or equivalent Microscope slides (Fisher Scientific, Fisherbrand, catalog number: 10219280) Coverslips (Fisher Scientific, Fisherbrand, catalog number: 15707592) Anti-GBS antibody (Benmimoun et al., 2020) Gene expression Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20) Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408) Glass cavity dish RNeasy Mini kit (QIAGEN, catalog number: 74104) QuantiTect Rev. Transcription Kit (QIAGEN, catalog number: 205311) TaqMan gene expression assay (ThermoFisher, catalog number: 4331182) TaqMan Universal PCR Master Mix II (ThermoFisher, catalog number: 4440042) 96-well PCR plates (e.g., Bio-Rad, catalog number: MLL9601) Seals for PCR plate (e.g., Bio-Rad, catalog number: MSB1001) Equipment Incubator with regulated temperature and humidity (Memmert, HPP110, or equivalent) Agitator Titramax 100 (Heidolph Instruments, catalog number: 544-11200-00) Spectrophotometer (Pharmacia, Novaspec II) Centrifuge (Sigma Bioblock scientific, 1-15K) Microbiological Safety Post (HeraSafe KS9) Flaming/Brown micropipette puller (Sutter Instrument Model P-97) Nano-injector Nanoject III (Dutsher, Drummond Scientific, catalog number: 075250) Laser scanning confocal microscope [Zeiss LSM 880 with Zen software (2012 S4)] Real-Time PCR machine (e.g., ThermoFisher, StepOne Real-Time PCR System, catalog number: 4376357) Software Zen software (2012 S4) Fiji (ImageJ version 2020 2.1.0/1,53c and version 1.52p; available at: https://imagej.net/software/fiji/) GraphPad Prism software [version 7 and version 2020 8.4.2 (464)] R, SPSS, or comparable software Procedure Drosophila larval culture Set fly crosses for 2–3 h in cages with grape juice egg-laying plates (supplemented with a bit of yeast paste, see Figure 2A and 2B) at appropriate temperature in order to collect fly embryos (Figure 3A). After 21 h at 25 °C (or 48 h at 18 °C), remove the already hatched larvae from the plate. Then, at each hour, collect and transfer equivalent numbers (approximately 100) of hatching first instar larvae to standard food plates at appropriate temperature (29 °C for RNAi knockdown, for example) (Figures 2C and 3B-C). Let the larvae grow and then collect them from the appropriate developmental stage to perform experiments. Of note, pathogenic infection has so far been done with third instar larvae (+72 h at 25 °C) (Figure 3D). Figure 3. Schematic representation of Drosophila larval culture procedure Pathogen preparation Set an overnight bacteria or a 36 h yeast preculture from -80 °C glycerol stocks on appropriate growth medium (BHI, YPD, MRS, …) and at adequate temperature (30 °C or 37 °C) (Figure 4A). a) For bacteria, dilute the overnight preculture at 1/20 in corresponding growth medium, and allow them to grow till the mid-exponential phase (~2 h 30 min at 37 °C for GBS) (to adapt depending on the bacterial strains) (Figure 4B). b) For fungi, dilute the overnight preculture at OD600 = 0.2, and allow them to grow at 30 °C for 5–6 h until OD600 of 1 (to adapt depending on the strains) (Figure 4B). Pellet the pathogens, after OD600 measurement (Figure 4C), by centrifugation at 3,500 × g and 4 °C for 5 min. Wash pathogenic culture in PBS, then pellet the pathogens by centrifugation at 3,500 × g and 4 °C for 5 min. Do this step twice (Figure 4D). Wash pathogenic culture in Drosophila Schneider’s medium, then pellet the pathogens by centrifugation at 3,500 × g and 4 °C for 5 min (Figure 4E). a) For the ex vivo CNS infection protocol: Resuspend the pathogens in 750 µL of Drosophila Schneider’s medium (Figure 4F), to prepare a 10× infectious dose and keep them at 4 °C until use (for example: 10 × 108 CFU/mL for Streptococcus agalactiae, Streptococcus pneumoniae, Listeria innocua, and Listeria monocytogenes; 10 × 107 CFU/mL for Neisseria meningitidis, Candida albicans, and Candida glabrata, and 10 × 105 CFU/mL for Cryptococcus neoformans). b) For the in vivo CNS infection protocol: Resuspend the pathogens in 100 µL of Drosophila Schneider’s medium (Figure 4F), to reach the infectious dose in the hemolymph (estimated at 2 μL) by injecting 20 nL of concentrated pathogen (for example: 4.4 × 1010 GBS/mL Streptococcus agalactiae). Note: Pathogen concentration was calculated by OD600 correlation (Streptococcus agalactiae, Streptococcus pneumoniae, Listeria innocua, and Listeria monocytogenes: 1 OD600 = 8.8 × 108 CFU/mL; Neisseria meningitidis: 1 OD600 = 109 CFU/mL; Candida albicans and Candida glabrata: 1 OD600 = 3 × 107 CFU/mL; Cryptococcus neoformans: 1 OD600 = 6 × 107 CFU/mL). Figure 4. Schematic representation of pathogen preparation procedure CNS explant culture and ex vivo CNS infection Wash staged larvae successively in PBS and ethanol 70% v/v in water and transfer them into cold Drosophila Schneider’s medium inside dissection wells. Dissect larvae by (a) cutting at approximately a quarter from the posterior spiracle, to minimise damage to motor nerves, (b) turning the anterior part inside-out to expose the CNS [Figure 5A and see video (Hafer and Schedl, 2006)]. Keep all larval tissues except for the gut (to avoid contamination with intestinal symbiotic pathogens). Transfer eight larvae to one well (24-well cell culture plate) and culture them under gentle rotary agitation (275 rpm) in 750 μL of CNS medium I at 30 °C and 60% humidity (Figure 5B). Note: A temperature of 30 °C was chosen as a compromise temperature, to allow Drosophila development while culturing mammalian pathogens closer to their usual environment. Dilute 1/10 of the prepared 10× infectious dose of each pathogen in the CNS culture medium I (Figure 5C), to reach the appropriate infectious dose (see step B6a). Replace the infected culture medium with Drosophila Schneider’s medium supplemented with 2 mM l-Glutamine, 0.5 mM sodium l-ascorbate, and 1% fetal bovine serum. Place the culture at 30 °C and 60% humidity under gentle rotary agitation (275 rpm) for a defined time (3 h for GBS; the same time must be kept for the control condition without pathogenic inoculum). Replace the medium after 3 h and every 10 h, with a fresh culture medium (Drosophila Schneider’s medium supplemented with 2 mM l-Glutamine, 0.5 mM sodium l-ascorbate, and 1% fetal bovine serum) Note: In these conditions, CNS explants can be kept for up to 48 h, at 30 °C. Proceed with the treatment required for each different analysis (D, E, and F). Figure 5. chematic representation of the ex vivo CNS infection procedure Pathogenic microinjection and in vivo CNS infection Pull micropipettes for injection (Heat: 350–360, Pull: 60–80, Velocity: 100, Time: 150). This step can be done in advance. Fill the completely prepared micropipette with mineral oil, using a needle and syringe (Figure 6A). Attach the micropipette filled with mineral oil to the injector head (Figure 6A). Eject part of the mineral oil and replace it by filling the micropipette with prepared concentrated pathogens (Figure 6A) (this will avoid contamination of the injector head with pathogens, by forming two distinct phases in the micropipette). Wash staged larvae successively in PBS and ethanol 70% v/v in water and transfer them into cold Drosophila Schneider’s medium inside dissection wells. One by one, dry the larvae, place them on one slide, and gently inject 20 nL of Drosophila Schneider’s medium (mock control) or prepared pathogen in its posterior part, using the nanoinjector (Figure 6B). This injection should be done very gently to only puncture the cuticle without affecting other tissues. We have chosen to inject the posterior part as it is far from the CNS. Keep injected larvae on standard fly food plates in an incubator at 30 °C and 60% humidity until analysis. Notes: All injected larvae will present a melanization spot, which seals and heals the cuticle punctured by the injection. Mock injection results in lethality per se, due to a combination of unsuccessful healing of the punctured cuticle, potential damage to tissues neighbouring the injection point, and potential temperature-induced stress (30 °C). In our hands, approximately 10% of GBS-injected larvae survive 4 h post-injection (all animals died between 4 and 6 h post-injection), while approximately 40% of mock-injected larvae survive 4 h post-injection (Benmimoun et al., 2020). Figure 6. Schematic representation of the in vivo CNS infection procedure Blood–brain barrier permeability assay Prepare a solution of 10 KDa dextran Texas Red (50 mM) in CNS culture medium II. Change CNS culture medium and incubate CNS explants with this solution under agitation (275 rpm) for 30 min. CNS explants are immediately fixed 4 × 5 min (to wash out excess dextran) in 4% methanol-free formaldehyde. Wash 3 × 10 min with PBS. Dissect the CNS and mount samples in mounting medium. Visualise with a laser scanning confocal microscope, with an optimal distance between each slice of 0.38 μm. Immunohistochemistry This protocol is a classic, general protocol to perform immunochemistry on the Drosophila larval CNS. For infections, we have used it to assess pathogen localization inside the CNS, by using specific antibodies against the strains of interest (Benmimoun et al., 2020), as well as cell fate (anti-Deadpan, anti-Repo, and anti-ElaV antibodies), morphology, and proliferation (anti-phosphohistone 3). It can also detect fusion proteins (anti-GFP antibody). A variation (using Bouin’s solution, see below) exists for antigens and antibodies, which do not work well with the classical formaldehyde fixation (for example, anti-Neurexin-IV antibody, Babatz et al., 2018). Fix (inside-out larvae ex vivo, or dissect and turn inside-out in vivo injected larvae). In 4% methanol-free formaldehyde at room temperature for 30 min, or overnight at 4 °C. In Bouin’s solution at room temperature for 3 min. Wash 3 × 10 min in PBS. Permeabilize 3 × 10 min in PBS-Triton 0.3%. Incubate with primary antibodies at 4 °C in blocking solution (PBS-Triton 0.3%, bovine serum albumin 5%, normal goat serum 2%) for 18–36 h. Of note: medium acidification could occur upon infection for some pathogenic strains (e.g., lactic pathogens, such as GBS or S. pneumoniae). If so, all GFP fusions should be detected with an anti-GFP antibody. Wash 3 × 10 min with PBS-Triton 0.3%. Incubate with secondary antibodies in blocking solution at room temperature for 3 h, or at 4 °C for 18–24 h. Wash 3 × 10 min with PBS-Triton 0.3%. Dissect the CNS and mount samples in Mowiol mounting medium. Visualise with a laser scanning confocal microscope, with an optimal distance between each slice of 0.38 μm (Figure 7). Figure 7. Confocal images showing GBS (attached to the BBB or inside the brain) using a specific GBS antibody Pathogenic load (CFU quantifications) Without tissue fixation, wash five injected flies (by condition) on paper with ethanol 70%. Open and bleed the larvae in 10 μL of PBS, and pool their hemolymph (keep on ice). Dissect, transfer, and homogenise the CNS in 10 μL of PBS (keep on ice). Transfer and homogenise the rest of the larval carcass (other tissues except for the gut) in 10 μL of PBS (keep on ice). Prepare seven serial tenfold dilutions with 2 μL in 20 μL of PBS for each tissue tested (hemolymph, CNS, carcasses). Plate 2 x 4 μL for each dilution on pathogen culture medium agarose plate(s). Incubate the plates at 37 °C for 16 h prior to the quantifications. Note: While these steps are described for the in vivo infection, only CNS bacterial load is determined for ex vivo infection. For this, follow steps 3 and 5–6 only. qPCR for gene expression Dissect 9–12 CNS with fine forceps at the desired time (3 h or 6 h post infection for GBS) and transfer each dissected CNS directly into the lysis buffer of the RNeasy Mini kit on ice. Isolate RNA according to the manufacturer’s instructions. Directly proceed with the cDNA synthesis. Use 450–600 ng RNA per reaction. Store the remaining isolated RNA at -80 °C. Set up qPCR reactions with a final volume of 10 µL. Seal plates carefully and spin them down in a centrifuge at 3,000 rpm for approximately 2 min. Run qPCR. Analyse qPCR data (see Data analysis). Notes: 50 ng of cDNA for each reaction is generally sufficient to get reasonable cycle threshold (Ct) values. Rpl32 can be used as a housekeeping gene. Data analysis Pathogen quantifications in infected Drosophila CNS After imaging (immunohistochemistry) Manually determine the exact number of pathogens for each analysed CNS by counting each individual bacterium contained within the boundary of the BBB (mdr65-mtd:: Tomato) through confocal acquisition. Compare between pathogen entry into the CNS using Student’s t-test (two conditions) or one-way ANOVA test followed by Tukey’s post-hoc analysis (more than two conditions), when values follow a normal distribution (assessed by Shapiro–Wilk normality test). Otherwise, using non-parametric Mann–Whitney tests (two conditions) or Kruskal–Wallis tests (more than two conditions). After pathogenic load (CFU quantifications) Count the bacterial colonies (CFUs) for each dilution (when possible, i.e., when isolated single colonies are detectable). Calculate an average of CFU/µL (bacterial load) by condition. Represent the bacterial loads per larva in a log10 scale. For in vivo infection, calculate the ratios from raw counting and represent them on a log10 scale: Ratio CNS/hemolymph = log10 (cfu per CNS/cfu per hemolymph) Ratio CNS/other tissues = log10 (cfu per CNS/cfu per other tissues) Ratio CNS/(hemolymph + other tissues) = log10 [cfu per CNS/(cfu per hemolymph + cfu per other tissues)]. Blood–brain barrier permeability assay Calculate Texas Red pixel intensity of three selected equal-sized areas (300 × 300 µm) from each CNS. Calculate the average of the mean pixel intensity. Calculate background intensity (empty equal-sized area 300 × 300 µm). Calculate permeability index by subtracting background intensity from the average of the mean pixel intensity. Compare between permeability index using Student’s t-test (two conditions) or one-way ANOVA test followed by Tukey’s post-hoc analysis (more than two conditions) when values follow a normal distribution (assessed by Shapiro–Wilk normality test). Otherwise, using non-parametric Mann–Whitney tests (two conditions) or Kruskal–Wallis tests (more than two conditions). Quantification of gene expression levels Perform qPCR with at least three biological and three technical replicates. Calculate the average for the technical replicates for each sample. Calculate the ∆Ct, by subtracting the average Ct of the housekeeping gene from the gene of interest. Calculate the ∆∆Ct, by calculating the average of the ∆Ct of the control group (uninfected) as a calibrator. Then, subtract the calibrator from each calculated ∆Ct of each biological replicate. For easier representation of the expression level, the data can be normalised to the uninfected control, by calculating the fold gene expression levels. For this, calculate two to the power of negative ∆∆Ct (2-∆∆Ct) for each biological replicate. For statistical analysis, first test the calculated fold gene expression levels for normal distribution (e.g., by using the Shapiro–Wilk test). When normally distributed, perform a Student’s t-test; if not normally distributed, perform a Mann–Whitney U Test. Represent data in a box plot. Survival of in vivo infected Drosophila Score survival overtime (each 30 min) and compare survival curves using the log-rank test. When more than two conditions are considered, p-values should be adjusted by determining their statistical significance (alpha = 0.05) through stacked p-values analysis, through the Holm–Sidak method. Represent data as Kaplan–Meier curves with error bars corresponding to standard errors (SE). Recipes Egg laying medium Reagent Final concentration Amount Agar 22.2 g/L 22.2 g Sugar 25 g/L 25 g Organic grape juice 25% v/v 250 mL Methyl-4-hydroxybenzoate (20% in ethanol) 0.25% v/v 12.5 mL Purified water (reverse osmosis) n/a 900 mL Total n/a ~1 L Fly food Reagent Final concentration Amount Active dry yeast n/a 300 g Corn flour n/a 211 g Agar n/a 46 g Sugar n/a 300 g Propionic acid 0.40% v/v 25 mL Methyl-4-hydroxybenzoate (20% in ethanol) 0.25% v/v 75 mL Purified water (reverse osmosis) n/a 6.3 L Total n/a 6 L (after cooking) CNS culture medium I Reagent Final concentration Amount l-Glutamine (200 mM) 2 mM 100 μL Sodium l-ascorbate (500 mM) 0.5 mM 10 μL Drosophila Schneider’s medium n/a 9.89 ml Total n/a 10 mL CNS culture medium II Reagent Final concentration Amount l-Glutamine (200 mM) 2 mM 100 μL Sodium l-ascorbate (500 mM) 0.5 mM 10 μL Fetal bovine serum 1% 100 μL Drosophila Schneider’s medium n/a 9.79 mL Total n/a 10 mL Acknowledgments We thank S. Dramsi, C. d’Enfert, G. Janbon, F. Leulier, M.-K. Taha, O. Disson, M. Lecuit, and F. Schweisguth for reagents and strains. We thank S. Liégeois for his help on hemolymph injection of Drosophila larvae and CFU counting. We thank D. Briand for his technical help on qPCR. This work has been funded by a starting package from Institut Pasteur/ LabEx Revive and a JCJC grant from Agence Nationale de la Recherche (NeuraSteNic, ANR- 17-CE13-0010-01) to P.S. B.B. has been supported by a Roux-Cantarini (Institut Pasteur) and a LabEx Revive post-doctoral fellowships. This protocol was adapted from previous work (Benmimoun et al., 2020; Winkler et al., 2021). Competing interests The authors declare no conflicts of interest. References Babatz, F., Naffin, E. and Klambt, C. (2018). The Drosophila Blood-Brain Barrier Adapts to Cell Growth by Unfolding of Pre-existing Septate Junctions. Dev Cell 47(6): 697-710 e693. Benmimoun, B., Papastefanaki, F., Perichon, B., Segklia, K., Roby, N., Miriagou, V., Schmitt, C., Dramsi, S., Matsas, R. and Speder, P. (2020). An original infection model identifies host lipoprotein import as a route for blood-brain barrier crossing. Nat Commun 11(1): 6106. Bergmann, S., Lawler, S. E., Qu, Y., Fadzen, C. M., Wolfe, J. M., Regan, M. S., Pentelute, B. L., Agar, N. Y. R. and Cho, C. F. (2018). Blood-brain-barrier organoids for investigating the permeability of CNS therapeutics. Nat Protoc 13(12): 2827-2843. Coureuil, M., Lecuyer, H., Bourdoulous, S. and Nassif, X. (2017). A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers. Nat Rev Microbiol 15(3): 149-159. Dando, S. J., Mackay-Sim, A., Norton, R., Currie, B. J., St John, J. A., Ekberg, J. A., Batzloff, M., Ulett, G. C. and Beacham, I. R. (2014). Pathogens penetrating the central nervous system: infection pathways and the cellular and molecular mechanisms of invasion. Clin Microbiol Rev 27(4): 691-726. Daneman, R. and Prat, A. (2015). The blood-brain barrier. Cold Spring Harb Perspect Biol 7(1): a020412. DeSalvo, M. K., Hindle, S. J., Rusan, Z. M., Orng, S., Eddison, M., Halliwill, K. and Bainton, R. J. (2014). The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes. Front Neurosci 8: 346. Hafer, N., and Schedl, P. (2006). Dissection of larval CNS in Drosophila melanogaster. J Vis Exp 9: (1):85. Hindle, S. J. and Bainton, R. J. (2014). Barrier mechanisms in the Drosophila blood-brain barrier. Front Neurosci 8: 414. Homem, C. C. and Knoblich, J. A. (2012). Drosophila neuroblasts: a model for stem cell biology. Development 139(23): 4297-4310. Jackson, S., Meeks, C., Vezina, A., Robey, R. W., Tanner, K. and Gottesman, M. M. (2019). Model systems for studying the blood-brain barrier: Applications and challenges. Biomaterials 214: 119217. Mira, H. and Morante, J. (2020). Neurogenesis From Embryo to Adult - Lessons From Flies and Mice. Front Cell Dev Biol 8: 533. Obermeier, B., Daneman, R. and Ransohoff, R. M. (2013). Development, maintenance and disruption of the blood-brain barrier. Nat Med 19(12): 1584-1596. Otsuki, L. and Brand, A. H. (2017). The vasculature as a neural stem cell niche. Neurobiol Dis 107: 4-14. Parkhurst, S. J., Adhikari, P., Navarrete, J. S., Legendre, A., Manansala, M. and Wolf, F. W. (2018). Perineurial Barrier Glia Physically Respond to Alcohol in an Akap200-Dependent Manner to Promote Tolerance. Cell Rep 22(7): 1647-1656. Saunders, N. R., Habgood, M. D., Mollgard, K. and Dziegielewska, K. M. (2016). The biological significance of brain barrier mechanisms: help or hindrance in drug delivery to the central nervous system? F1000Res 5. Sivandzade, F. and Cucullo, L. (2018). In-vitro blood-brain barrier modeling: A review of modern and fast-advancing technologies. J Cereb Blood Flow Metab 38(10): 1667-1681. Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J. and Klambt, C. (2008). Organization and function of the blood-brain barrier in Drosophila. J Neurosci 28(3): 587-597. Weiler, A., Volkenhoff, A., Hertenstein, H. and Schirmeier, S. (2017). Metabolite transport across the mammalian and insect brain diffusion barriers. Neurobiol Dis 107: 15-31. Winkler, B., Funke, D., Benmimoun, B., Speder, P., Rey, S., Logan, M. A. and Klambt, C. (2021). Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages. Sci Adv 7(44): eabh0050. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Development Microbiology > Microbe-host interactions > Ex vivo model Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Evaluating Baseline and Sensitised Heat Nociception in Adult Drosophila Josephine N. Massingham [...] G. Gregory Neely Jul 5, 2021 2275 Views Preference Test of Plutella xylostella Larvae upon DMNT Treatment Chen Chen [...] Peijin Li Nov 5, 2021 1399 Views Aerotaxis Assay in Caenorhabditis elegans to Study Behavioral Plasticity Qiaochu Li [...] Karl Emanuel Busch Aug 20, 2022 1245 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,564	https://bio-protocol.org/en/bpdetail?id=4564&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Fluorescence Time-lapse Imaging of Entosis Using Tetramethylrhodamine Methyl Ester Staining EB Emir Bozkurt HD Heiko Düssmann JP Jochen H. M. Prehn Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4564 Views: 656 Reviewed by: Ralph Thomas BoettcherRakesh BamOlga Kopach Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Cell Biology Nov 2021 Abstract Entosis is a process where a living cell launches an invasion into another living cell’s cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining. Keywords: Entosis Entotic cell death TMRM Venus Time-lapse imaging Confocal fluorescence microscopy Background Entosis is a form of cell–cell interaction in which a living cell (inner cell) orchestrates its invasion into another cell (outer cell) by a Rho/ROCK signaling–dependent mechanism (Overholtzer et al., 2007). Entosis results in the formation of a unique morphological microscopic structure, also known as cell-in-cell structure or bird’s eye structure, which is characterized by evidence of an inner cell, a visible entotic vacuole between inner and outer cells, and an outer cell with a crescent-shaped nucleus as it is pushed towards the periphery (Fais and Overholtzer, 2018). Entosis spontaneously—but rarely—occurs in cultured cells in vitro; nevertheless, it has important consequences for both inner and outer cells. From one perspective, entosis seems to be an assisted-suicide mechanism for the inner cells, as the vast majority of these undergo lysosomal degradation by a mechanism known as entotic cell death (Florey et al., 2011). However, inner cells can also survive and be released, and even complete full cell division inside outer cells. Intriguingly, outer cells also benefit from this invasion as they gain survival advantage under stress conditions (Hamann et al., 2017) or become resistant to apoptosis (Bozkurt et al., 2021). Entosis-like structures are more frequently observed in cancers compared to normal tissues, and the presence of these structures is associated with poor outcome and disease recurrence in cancer (Mackay et al., 2018; Bozkurt et al., 2021). Quantification of entosis relies on morphological detection of the events using microscopy images. Currently, it is not possible to automatically quantify entosis events when both inner and outer cells are alive. Thus, events are first detected with the aid of nuclear, cytoplasmic, and/or membrane staining, and then manually quantified (Overholtzer et al., 2007). When inner cells undergo entotic cell death, however, quantifying entosis events is relatively easy due to lysosomal acidification of inner cells. Lysosomal marker Lamp1 or autophagic marker LC3 can be fluorescently expressed in the cells to quantify the dynamics of entotic cell death (Florey et al., 2011; Overholtzer et al., 2007). However, overexpression of artificially produced proteins might also affect the rate of entosis events. Probably the most practical method to label entotic cell death is using lysosomal dyes such as LysoTracker, which freely passes the cell membrane and is sequestered inside acidic organelles. Alternatively, fluorogenic cathepsin substrates or acridine orange have been used to detect entosis and entotic cell death (Overholtzer et al., 2007; Garanina et al., 2017). Recently, we reported a novel alternative approach for detecting entosis events and measuring the dynamics of entotic cell death by using tetramethylrhodamine methyl ester (TMRM) (Bozkurt et al., 2021). This cationic cell-permeable dye is sequestered by active mitochondria and has been widely used to analyze real-time changes in mitochondrial events such as loss of mitochondrial membrane potential during apoptosis as well as fusion/fission (Dußmann et al., 2003; Cho et al., 2019). Here, we provide a detailed protocol for performing time-lapse microscopy in any cell line to detect entosis events and entotic cell death by using TMRM staining. Our approach allows simultaneous detection of mitochondrial and entotic events as well as quantification of the real-time kinetics in living cells at the single-cell level. Materials and Reagents WillCo-dish® 12 mm glass bottom dishes (WillCo Wells B.V., catalog number: HBST-3512) Cell culture flask, T-75, surface: standard, filter cap (Sarstedt, catalog number: 83.3911.002) Living cells (in this protocol, HCT116-Venus cells (Bozkurt et al., 2021) were used; however, this method is applicable to any living cell) Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7525), store at -20 °C. L-glutamine (Sigma-Aldrich, catalog number: G7513), store at -20 °C Mineral oil, suitable for mouse embryo cell culture (Sigma-Aldrich, catalog number: M5310), store at room temperature Penicillin–streptomycin (Sigma-Aldrich, catalog number: P0781), store at -20 °C Roswell Park Memorial Institute 1640 medium (RPMI medium) (Sigma-Aldrich, catalog number: R0883), store at 4 °C Tetramethylrhodamine, methyl ester, perchlorate (TMRM) (Thermo Fisher Scientific, catalog number: T668), store at 4 °C Equipment Inverted confocal laser scanning microscope (Carl Zeiss Ltd, LSM 710) equipped with a 40×/1.3 NA plan apochromat oil immersion objective, a microscope incubator chamber (37 °C with 5% CO2), and a motorized stage Cell culture hood (Heraeus, HERAsafe, Type HS12) Centrifuge (Eppendorf, 5810, catalog number: 5810000060) Water bath (GFL Type 1004) Humidified CO2 incubator (New Brunswick/Eppendorf, Galaxy 170S) Software ZEN 2009 version 6,0,0,303 configuration 5, with MTS 2009-2010 version 32.007 Fiji/ImageJ (National Institutes of Health, https://imagej.nih.gov/ij/) PlotTwist, a web app for plotting continuous data (https://huygens.science.uva.nl/PlotTwist/) (Goedhart, 2020) Procedure Cell seeding and staining Prepare complete cell culture RPMI (use 4 mL per WillCo-dish for calculations) by adding 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Preheat the complete cell culture media in a 37 °C water bath. Prepare staining solution by adding 30 nM TMRM to 2 mL of complete cell culture RPMI. Keep the staining solution warm in a 37 °C water bath. Seed 7,500 cells in 1.5 mL of staining solution on sterile 12 mm glass bottom WillCo dishes and let them adhere overnight at 37 °C with 5% CO2. Note: Staining should be added at the seeding stage and left in the media throughout the experiment. Cover the WillCo-dish with embryo-tested sterile mineral oil. Note: Do not put the lid of the WillCo-dish after adding mineral oil. Image acquisition Carefully mount the WillCo-dish on an LSM 710 confocal laser scanning microscope. Note: Make sure that there is no dirt or air bubbles in the immersion medium between objective lens and coverslip as it would interfere with the image acquisition and the function of the definite focus. Start ZEN software. Select 40× 1.3 NA oil objective using touchscreen. Randomly select multiple positions with Definite Focus active. Choose 488 nm laser, adjust to <5%, and select detection range of 490–544 nm or 505–560 nm for imaging Venus; use 561 nm laser, adjust to <0.5%, and select detection range of 590–644 nm for imaging TMRM. Set digital gain to 1 and the offset to 0 for both. Activate the transmitted light detector and make sure to have the required prism and analyzer in the beam path. The % transmission of the acousto-optic tunable filter is based on the 488 nm line of an argon laser, which is <10 mW; the 0.5% is based on a 561 nm diode-pumped solid-state laser, which is 40 mW when the lasers are new. These settings should be tested on the cells chosen for the experiment. Tolerance to laser irradiation can vary and the calibration of the confocal microscope and the age of lasers are major factors that contribute to the power in the object plane, hence, photo-damage to the cells. Here, proliferation of cells and no change in mitochondrial membrane potential (as detected with TMRM) was observed for 24 h under control conditions. Set the zoom to 1 and use 1024 × 1024 pixel resolution for a field of view of 212 μm2. Set the optical slice thickness to 1.5–2 μm, use bidirectional scan, and make sure bidirectional scan and pinhole are calibrated. Then, save this configuration for the use with the Multiple Time Series Macro. Lateral and axial resolution is set at a compromise between optimal resolution and imaging conditions for cells. If the signal-to-noise ratio (S/N) is too low, it might be required to open the pinhole further. Start Multiple Time Series Macro. Make sure the locations are transferred and select the proper configuration for each location. Run the autofocus test for each location and adjust the offset accordingly. Define the storage name and location on the hard disk drive (HDD) and make sure to have at least 30% free defragmented HDD. Also, make sure to have enough available storage to end the experiment with at least 25% free HDD capacity. Select the time interval between repetition of imaging. This is usually around 5 to 10 min depending on the number of locations (keep number of locations less than 12). Also, select the number of experiment repetitions. Acquire images for at least 12 h. Keep in mind that a shorter time interval will also increase the photo-damage to the cells; the time interval should be short enough to see the order of events you are interested in and to track the objects. After finishing the experiment, remove the dish, clean the objective, and follow the switching off procedure of the LSM 710. Copy the data to a server or mobile HDD and generate a second confirmed copy before deleting the data from the image acquisition PC. Image processing Download and install Fiji/ImageJ software. Start Fiji/ImageJ. Watch “Step-by-step video guide for image processing” (Video 1). Video 1. Step-by-step video guide for image processing Open the file with LSM extension by clicking File – Open. Note: You can simply drag and drop the file into Fiji/ImageJ software. Open Brightness/Contrast (B&C) panel by clicking Image – Adjust – Brightness/Contrast (or simply use shortcut Ctrl + Shift + C). Open Channels Tool by clicking Image – Color – Channels Tool (or simply use shortcut Ctrl + Shift + Z). Change Lookup Tables by clicking Image – Lookup Tables and set TMRM as red and Venus as green. Note: Setting Lookup tables is just for visualization of the events; any color combination can be chosen as it will not affect the results. Adjust B&C as desired. Note: Adjusting B&C to auto usually gives a very good result. Unmark DIC channel on Channels Tool and slowly scroll throughout the fields to locate entotic events. In case of an event of entotic cell death, a big circular structure displaying TMRM accumulation over time, in parallel with reduction in Venus fluorescence, will appear in the field (see Figure 1 and Video 2). Figure 1. Representative field of view and kinetics of an entosis event in untreated HCT116 cells. (A) Representative time-lapse microscopy images of an entosis event in HCT116-Venus cells. DIC, Venus (green), and TMRM (red) are shown. White-dashed and yellow-dashed lines indicate an inner cell (IC) and outer cell (OC), respectively. (B) Quantification of single-cell kinetics of TMRM and Venus fluorescence intensity in an inner cell during entosis and entotic cell death. Video 2. Representative time-lapse video showing an entosis event in untreated HCT116 cells. DIC, Venus (green), and TMRM (red) are shown. Frame rate: 20 frames per second; the interval between two frames is 4 min. White arrow depicts an entosis event. Once an event is found, carefully follow the event by scrolling back to confirm that a cell is invading into another cell. Open ROI (Region of Interest) Manager by clicking Analyze – Tools – ROI Manager. Carefully draw a region around the inner cell by using polygon selections. Use the magnifying glass tool to correct the edges. Use right click to connect the first and the last point of the selections. Note: Instead of using the magnifying glass tool, simple press Shift and use the mouse scroll to zoom in and out and press Space, and use left click on the field to move freely. Optional: At this stage, working on a smaller field of view is easier and uses less computer power. To create a smaller field of view, click rectangle tool, draw a region that covers the event, and click Image - Duplicate, then click OK. Notes: Holding Shift while drawing a region will create a square field. Simply use the shortcut Shift + D to duplicate the region. Save this region by clicking File - Save (or use shortcut Ctrl + S) for further analysis. Click Add on ROI manager (or use shortcut t) to add selection, click to the next slice, modify the selection (or draw a new one), and then click Add on ROI manager. Repeat this step until all steps of the entosis event are recorded. (Pressing Alt + mouse scroll will move to the next slice.) Select all regions by pressing Ctrl + A on ROI Manager, then save the regions by clicking More – Save on ROI Manager. Note: Regions can be updated anytime by clicking on a region first, then clicking update after modifying the region. Regions can also be opened later by clicking More – Open on ROI Manager. Click Analyze – Set Measurements and mark mean gray value. To measure mean fluorescence intensity (MFI), select TMRM channel on the image, select all regions by pressing Ctrl + A on ROI Manager, and click Measure. Rename and save the results file as TMRM. Repeat this step to measure Venus and rename the file as Venus. Optional: It is possible to create a time-lapse video using Fiji/ImageJ. Before generating the video format, we suggest putting a time stamp by clicking Image – Stacks – Time Stamper. Also make sure to add any annotation or label before generating the video. To create a time-lapse video from the experiment, click File – Save As – AVI, select Compression format and frame rate, and click OK. Optional: It is possible to create a montage from time-lapse images. Click Image – Stacks – Make Montage, complete the new setting window as desired, and click OK. Data visualization and analysis Open PlotTwist, click Data Upload, and click Download (tidy) data (.csv). Carefully copy and paste the results of TMRM and Venus into the template. Click Plot under Data and select Data as Dots. Mark the Plot thickness; set Visibility of the data as 0.4 and set Visibility of the statistics as 0.0; mark Change scale, set Range x-axis (min,max) scale as 0,850 and Range y-axis (min,max) as 0,2000; mark Use color for the data and click color palette Tol; bright; set Height (catalog number: pixels) as 480 and Width (catalog number: pixels) as 480; under Labels mark Add treatment/condition and Bar&Box; set Range of grey box (from,to) to the time at which TMRM intensity starts increasing and Venus intensity starts decreasing; add text as Start of inner cell degradation and mark add Title; mark Change axis labels and Change x-axis as Time (min) and y-axis as Mean fluorescence intensity (a.u.); mark add labels to objects. To reuse the same settings in the future, click on Clone current setting tab, copy the link, and paste it to a file as PlotTwist _Settings. Notes Raw data for Figure 1A can be found in Supplementary_File 1. PlotTwist settings for the graph shown in Figure 1B can be found in Supplementary_File 2. This link can be directly copied and pasted to any web browser to generate the same graph. Acknowledgments This work was supported by Science Foundation Ireland (16/RI/3740, 16/US/3301, 18/RI/5792) and the Health Research Board (16/US/330, TRA/2007/26). This research was funded in whole or in part by Science Foundation Ireland. This protocol is related to our previous published study in Journal of Cell Biology (Bozkurt et al., 2021, doi:10.1083/jcb.202010030). Competing interests The authors declare no competing interests. References Bozkurt, E., Düssmann, H., Salvucci, M., Cavanagh, B. L., Van Schaeybroeck, S., Longley, D. B., Martin, S. J. and Prehn, J. H. (2021). TRAIL signaling promotes entosis in colorectal cancer. J Cell Biol 220(11): e202010030. Cho, H. M., Ryu, J. R., Jo, Y., Seo, T. W., Choi, Y. N., Kim, J. H., Chung, J. M., Cho, B., Kang, H. C. and Yu, S.-W. (2019). Drp1-Zip1 interaction regulates mitochondrial quality surveillance system. Mol Cell 73(2): 364-376. e368. Düßmann, H., Rehm, M., Kögel, D. and Prehn, J. H. (2003). Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis. J Cell Sci 116(3): 525-536. Fais, S. and Overholtzer, M. (2018). Cell-in-cell phenomena in cancer. Nat Rev Cancer 18(12): 758-766. Florey, O., Kim, S. E., Sandoval, C. P., Haynes, C. M. and Overholtzer, M. (2011). Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes. Nat Cell Biol 13(11): 1335-1343. Garanina, A. S., Kisurina-Evgenieva, O. P., Erokhina, M. V., Smirnova, E. A., Factor, V. M. and Onishchenko, G. E. (2017). Consecutive entosis stages in human substrate-dependent cultured cells. Sci Rep 7(1): 1-12. Goedhart, J. (2020). PlotTwist: A web app for plotting and annotating continuous data. PLoS Biol 13;18(1): e3000581. Hamann, J. C., Surcel, A., Chen, R., Teragawa, C., Albeck, J. G., Robinson, D. N. and Overholtzer, M. (2017). Entosis is induced by glucose starvation. Cell Rep 20(1): 201-210. Mackay, H. L., Moore, D., Hall, C., Birkbak, N. J., Jamal-Hanjani, M., Karim, S. A., Phatak, V. M., Piñon, L., Morton, J. P. and Swanton, C. (2018). Genomic instability in mutant p53 cancer cells upon entotic engulfment. Nat Commun 9(1): 1-15. Overholtzer, M., Mailleux, A. A., Mouneimne, G., Normand, G., Schnitt, S. J., King, R. W., Cibas, E. S. and Brugge, J. S. (2007). A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell 131(5): 966-979. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > Cell death > Cell biology assays Cell Biology > Cell isolation and culture > Monolayer culture Cell Biology > Cell imaging > Live-cell imaging Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Primary Neuronal Culture and Transient Transfection Shun-Cheng Tseng [...] Eric Hwang Jan 20, 2025 341 Views Versatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells Angélique Pipier and David Monchaud Feb 5, 2025 199 Views Real-time IncuCyte® Assay for the Dynamic Assessment of Live and Dead Cells in 2D Cultures Arlene K. Gidda [...] Sharon M. Gorski Feb 5, 2025 71 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,565	https://bio-protocol.org/en/bpdetail?id=4565&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Analysis of N6-methyladenosine RNA Modification Levels by Dot Blotting YD Yu Du MX Mingyue Xia ZH Zhigang Hu Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4565 Views: 2382 Reviewed by: Chiara AmbrogioWilly R Carrasquel-UrsulaezTanxi CaiYoshihiro Adachi Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE May 2022 Abstract N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNAs (mRNAs), affecting their fold, stability, degradation, and cellular interaction(s) and implicating them in processes such as splicing, translation, export, and decay. The m6A modification is also extensively present in non-coding RNAs, including microRNAs (miRNAs), ribosomal RNAs (rRNAs), and transfer RNAs (tRNAs). Common m6A methylation detection techniques play an important role in understanding the biological function and potential mechanism of m6A, mainly including the quantification and specific localization of m6A modification sites. Here, we describe in detail the dot blotting method for detecting m6A levels in RNA (mRNA as an example), including total RNA extraction, mRNA purification, dot blotting, and data analysis. This protocol can also be used to enrich specific RNAs (such as tRNA, rRNA, or miRNA) by isolation technology to detect the m6A level of single RNA species, so as to facilitate further studies of the role of m6A in biological processes. Keywords: Dot blot N6-methyladenosine RNA m6A RNA modification mRNA Non-coding RNAs METTL3 Background N6-methyladenosine (m6A) is the most prevalent internal RNA modification in eukaryotic mRNAs and long non-coding RNAs (Jia et al., 2013). The m6A modification refers to the methylation of the nitrogen atom at position 6 of the RNA adenosine, mainly located on the common motif of RRm6ACH (R denotes A or G, H denotes A, C, or U) (Csepany et al., 1990). The reversible activity of m6A modification is regulated by the combined action of methylases and demethylases (Niu et al., 2013). The m6A writers with methyltransferase activity consist of three individual proteins: METTL3, METTL14, and WTAP (Wiedmer et al., 2019); FTO and ALKBH5 are m6A demethylases (Nachtergaele and He, 2018). Another protein family is the m6A readers, which can recognize the m6A modification to modulate the fate of mRNA (Harcourt et al., 2017). Recent studies suggest that m6A plays a pivotal role during various biological processes including virus infection (Winkler et al., 2019), stress (Engel et al., 2018), heat shock (Zhou et al., 2015), and DNA damage (Xiang et al., 2017; E. Li et al., 2022). In mammals, m6A methylation plays a variety of key roles including embryonic development, neurogenesis, circadian rhythm, stress responses, sex determination, and tumorigenesis (Sun et al., 2019). The m6A modification is vital during stem cell proliferation, with METTL3 depletion reducing the differentiation of embryonic stem cells (Batista et al., 2014). The correlation between the level of m6A modification and clinicopathological features has been shown in diverse tumors, which may provide prognostic value in these diseases. Detection of m6A modification in vitro can help identify the precise regulatory forms and synergistic roles of m6A modifications in cancer and other diseases. Also, detection of m6A is important for studying its biological functions and mechanisms. Currently, a variety of methods have been developed to identify m6A modifications in cells, which can be divided into three categories: semi-quantitative, quantitative, and precise location detection. Semi-quantitative methods include dot blot technology (Z. Li et al., 2017), methyl-sensitivity of MazF RNA endonucleases (Imanishi et al., 2017), and immuno-northern blotting (Mishima et al., 2015). Quantification methods include RNA photo-crosslinkers and quantitative proteomics (Arguello et al., 2017), electrochemical immunosensors (Yin et al., 2017), and support vector machine–based methods (Chen et al., 2016). Precise location detection includes methylated RNA immunoprecipitation sequencing (MeRIP-seq) (Liu et al., 2018), m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) (Linder et al., 2015), and high-performance liquid chromatography (HPLC) (Rana and Tuck, 1990). Although many methods have been developed to detect m6A methylation, in many cases dot blot hybridization remains the method of choice for analyzing the global changes of m6A levels in total RNA or single RNA species. The dot blotting technique significantly saves time because it does not require chromatography, gel electrophoresis, or complex gel closure procedures, and is relatively low in cost (Wang et al., 2018). In this protocol, we describe in detail how to detect m6A content in mRNA by dot blot (Figure 1). Figure 1. Schematic diagram of the major steps of dot blot analysis for the separation and purification of m6A in mRNA Materials and Reagents 100 mm culture dish (Corning, Falcon, catalog number: 353003) Pipette tips (Axygen, catalog numbers: T-300, T-200Y, T-1000B) 1.5 mL RNase/DNase-free microcentrifuge tube (Axygen, catalog numbers: MCT-50-C) Cell scraper (any brand) TRIzol reagent (Invitrogen, catalog number:15596026) Chloroform (General-Reagent, catalog number: G75915B) Isopropanol (Sangon Biotech, catalog number: A507048) Ethanol absolute (Sangon Biotech, catalog number: A500737) RNase-free water GenElute mRNA miniprep kit (Sigma, catalog number: DMN10) AmershamTM HybondTM-N+ membrane (GE Healthcare, catalog number: RPN203B ) N6-methyladenosine/m6A rabbit mAb (ABclonal, catalog number: A19841) HRP goat anti-rabbit IgG (H+L) (ABclonal, catalog number: AS014) ECL western blotting substrate (Thermo Fisher Scientific, catalog number: 32106) Saran wrap (any brand) Tween-20 (Sigma-Aldrich, catalog number: P1379) Non-fat milk (any brand) Na2HPO4 (Sangon Biotech, catalog number: A501727) KH2PO4 (Sangon Biotech, catalog number: A100781) KCl (Sangon Biotech, catalog number: A100395) NaCl (Sangon Biotech, catalog number: A501218) 10× phosphate-buffered saline (PBS) (see Recipes) 1× phosphate-buffered saline (PBS) (see Recipes) 1× phosphate-buffered saline/0.1% Tween-20 (PBST) (see Recipes) Blocking buffer (see Recipes) Antibody dilution buffer (see Recipes) Methylene blue dye buffer (Solarbio, catalog number: G1301) Equipment Refrigerated centrifuge (Eppendorf, model: 5424 R) NanoDrop (Thermo Fisher Scientific, model: ND-1000 spectrophotometer) UV cross-linker or UV torch with 254 nm wavelength UV (UVP, model: CL1000) Chemiluminescent imaging system (Tanon, model: Tanon 5200) Camera (any brand) Software GraphPad Prism (GraphPad Software) ImageJ (https://imagej.nih.gov/ij) Procedure Isolation of total RNA For mammalian cells we recommend using approximately 1 × 107 cells for mRNA purification. Remove the medium and rinse the cells twice with 1–2 mL of ice-cold PBS. Remove PBS and lyse the cells directly in the culture dish by adding 1 mL of TRIzol reagent per 100 mm culture dish and scraping with a cell scraper. Pass the cell lysate through a pipette several times. Then, transfer to 1.5 mL RNase-free microcentrifuge tubes. Vortex thoroughly. Incubate the homogenized samples for 5 min at room temperature. Add 0.2 mL of chloroform, vortex for 30 s, and incubate for 2–3 min at room temperature. Centrifuge at 12,000 × g and 4 °C for 15 min. Carefully transfer the upper, aqueous phase to a fresh tube without disturbing the interface. Add 0.5 mL of isopropanol to the aqueous phase and mix well. Incubate the samples for 10 min at room temperature. Centrifuge at 12,000 × g and 4 °C for 10 min. Decant the supernatant and completely remove any traces of liquid by aspiration. Wash the pellet with 1 mL of ice-cold 75% ethanol. Centrifuge at 12,000 × g and 4 °C for 15 min. Remove all traces of ethanol. Air-dry or vacuum-dry the RNA pellet for 5–10 min. Resuspend the pellet in 10–30 μL of RNase-free water. mRNA purification Isolate mRNA from total RNA using the GenElute mRNA miniprep kit following the manufacturer’s instructions (other brands can also be used for the purification of mRNA). Mix the oligo (dT) beads thoroughly by vortexing and inverting until resuspended and homogenous. Add the resuspended oligo (dT) beads to the total RNA, cap the tube, and mix thoroughly by vortexing. Incubate the mixture at room temperature for 10 min. No mixing or rocking is necessary. Resuspend the pellet in 350 μL of wash solution by vortexing. Transfer the suspension into a GenElute spin filter–collection tube assembly by pipetting. Spin for 1–2 min at maximum speed. Remove the spin filter, discard the flow through liquid, then place the spin filter back into the same collection tube. Pipette 350 μL of low salt wash solution into the column. Spin for 1–2 min at maximum speed. Remove the spin filter, discard the flow through liquid, then place the spin filter back into the same collection tube. Pipette another 350 μL of low salt wash solution into the column. Spin for 1–2 min at maximum speed. Transfer the spin filter into a fresh collection tube. Pipette 10–20 μL of preheated (65 °C) elution solution onto the spin filter, ensuring that it comes into contact with the bead–mRNA complex. Incubate for 2–5 minutes at 65 °C. Spin for 1–2 min at maximum speed. Save the flow through liquid; it contains most of the purified mRNA. Determine the concentration of purified mRNA with NanoDrop. Dilute different concentrations of mRNA to 50 ng/μL. Dot blotting The isolated mRNA is first denatured by heating at 95 °C for 5 min to disrupt the secondary structure. Chill on ice for 5 min immediately after denaturation to prevent re-formation of the mRNA secondary structure. Have the nitrocellulose membrane ready. Using a narrow-mouth pipette tip, drop 2 μL of mRNA sample directly onto the Hybond-N+ membrane at the center of the grid (Figure 2). Figure 2. Dot sample example Air dry at room temperature for 5 min. Crosslink spotted RNA to the membrane using the UV cross linker (irradiate under ultraviolet lamp with 254 nm wavelength for 5 min). Incubate the membrane with blocking buffer for 1 h at room temperature. After blocking, incubate the membrane with anti-m6A antibody (1:1,000 dilution) in 5 mL of antibody dilution buffer overnight at 4 °C with gentle shaking. Wash three times with PBST (3 × 5 min). Incubate the membrane with HRP goat anti-rabbit IgG (1:10,000 dilution) in 5 mL of antibody dilution buffer for 1 h at room temperature with gentle shaking. Wash three times with PBST (3 × 5 min). Incubate with ECL substrate for 1 min (according to the manufacturer’s instructions, 0.125 mL ECL solution per cm2 of the membrane is recommended), cover with Saran wrap aiming to remove the excess solution from the surface, and expose the film in the darkroom (Figure 3B). Try different exposure times to get clear results. After exposure, transfer the membrane to a solution containing 10 mL of 0.02% methylene blue dye buffer, gently shake the membrane at room temperature, and incubate for 30 min. Lastly, clean the membrane with dH2O until the background is clean (Figure 3C). Figure 3. Representative data of m6A level detected by dot blot. (A) Western blotting assay of METTL3 in the METTL3-KD stable cell lines in MCF-7. (B) Dot blot to detect the m6A level of mRNA isolated from total RNA of METTL3-KD MCF-7 cells. (C) Methylene blue (MB) staining served as a loading control. (D) Measurement and normalization of m6A levels in ImageJ. Data analysis Perform densitometric analysis using ImageJ (result calculation method: m6A grayscale value/MB grayscale value). Use a minimum of three biological replicates to perform statistical analysis of the m6A levels between samples (Figure 3D). For statistical analysis, use GraphPad Prism software. Notes This protocol can be modified for individual species of RNA, such as messenger RNA, tRNA, rRNA, or microRNA, by employing isolation techniques for the enrichment of the specific RNAs to be analyzed. For the preparation of wash buffer, blocking buffer, and antibody dilution buffer, the use of nuclease-free water is not necessary. Recipes 10× phosphate-buffered saline (PBS) (1,000 mL) Weigh 14.4 g of Na2HPO4, 2.4 g of KH2PO4, 2.0 g of KCl, and 80.0 g of NaCl Add 800 mL of sterile water Stir until dissolved Bring the final volume to 1,000 mL using sterile water 1× phosphate-buffered saline (PBS) (500 mL) 50 mL of 10× PBS Bring final volume to 500 mL using sterile water 1× phosphate-buffered saline/0.1% Tween-20 (PBST) (500 mL) 50 mL of 1× PBS 1 mL of 100% Tween-20 Bring final volume to 500 mL using sterile water Blocking buffer and antibody dilution buffer 50 mL of PBST 5 g non-fat milk Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (32171407) and Priority Academic Program Development of Jiangsu Higher Education Institutions. The protocol presented here was adapted from those in Terenghi (1998) and Jia et al. (2011). Competing interests The author declares there are no competing interests. References Arguello, A. E., DeLiberto, A. N. and Kleiner, R. E. (2017). RNA Chemical Proteomics Reveals the N6-Methyladenosine (m6A)-Regulated Protein-RNA Interactome. J Am Chem Soc 139(48): 17249-17252. Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., et al. (2014). m6A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15(6): 707-719. Chen, W., Feng, P., Ding, H. and Lin, H. (2016). Identifying N6-methyladenosine sites in the Arabidopsis thaliana transcriptome. Mol Genet Genomics 291(6): 2225-2229. Csepany, T., Lin, A., Baldick, C. J., Jr. and Beemon, K. (1990). Sequence specificity of mRNA N6-adenosine methyltransferase. J Biol Chem 265(33): 20117-20122. Engel, M., Eggert, C., Kaplick, P. M., Eder, M., Roh, S., Tietze, L., Namendorf, C., Arloth, J., Weber, P., Rex-Haffner, M., et al. (2018). The Role of m6A/m-RNA Methylation in Stress Response Regulation. Neuron 99(2): 389-403 e389. Harcourt, E. M., Kietrys, A. M. and Kool, E. T. (2017). Chemical and structural effects of base modifications in messenger RNA. Nature 541(7637): 339-346. Imanishi, M., Tsuji, S., Suda, A. and Futaki, S. (2017). Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease. Chem Commun (Camb) 53(96): 12930-12933. Jia, G., Fu, Y. and He, C. (2013). Reversible RNA adenosine methylation in biological regulation. Trends Genet 29(2): 108-115. Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y. G., et al. (2011). N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7(12): 885-887. Li, E., Xia, M., Du, Y., Long, K., Ji, F., Pan, F., He, L., Hu, Z. and Guo, Z. (2022). METTL3 promotes homologous recombination repair and modulates chemotherapeutic response in breast cancer by regulating the EGF/RAD51 axis. Elife 11: e75231. Li, Z., Weng, H., Su, R., Weng, X., Zuo, Z., Li, C., Huang, H., Nachtergaele, S., Dong, L., Hu, C., et al. (2017). FTO Plays an Oncogenic Role in Acute Myeloid Leukemia as a N6-Methyladenosine RNA Demethylase. Cancer Cell 31(1): 127-141. Linder, B., Grozhik, A. V., Olarerin-George, A. O., Meydan, C., Mason, C. E. and Jaffrey, S. R. (2015). Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12(8): 767-772. Liu, H., Wang, H., Wei, Z., Zhang, S., Hua, G., Zhang, S. W., Zhang, L., Gao, S. J., Meng, J., Chen, X., et al. (2018). MeT-DB V2.0: elucidating context-specific functions of N6-methyl-adenosine methyltranscriptome. Nucleic Acids Res 46(D1): D281-D287. Mishima, E., Jinno, D., Akiyama, Y., Itoh, K., Nankumo, S., Shima, H., Kikuchi, K., Takeuchi, Y., Elkordy, A., Suzuki, T., et al. (2015). Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides. PLoS One 10(11): e0143756. Nachtergaele, S. and He, C. (2018). Chemical Modifications in the Life of an mRNA Transcript. Annu Rev Genet 52: 349-372. Niu, Y., Zhao, X., Wu, Y. S., Li, M. M., Wang, X. J. and Yang, Y. G. (2013). N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function. Genomics Proteomics Bioinformatics 11(1): 8-17. Rana, A. P. and Tuck, M. T. (1990). Analysis and in vitro localization of internal methylated adenine residues in dihydrofolate reductase mRNA. Nucleic Acids Res 18(16): 4803-4808. Sun, T., Wu, R. and Ming, L. (2019). The role of m6A RNA methylation in cancer. Biomed Pharmacother 112: 108613. Terenghi, G. (1998). Detecting mRNA in tissue sections with digoxigenin-labeled probes. Methods Mol Biol 86: 137-142. Wang, Y., Li, Y., Yue, M., Wang, J., Kumar, S., Wechsler-Reya, R. J., Zhang, Z., Ogawa, Y., Kellis, M., Duester, G., et al. (2018). N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications. Nat Neurosci 21(2): 195-206. Wiedmer, L., Eberle, S. A., Bedi, R. K., Sledz, P. and Caflisch, A. (2019). A Reader-Based Assay for m6A Writers and Erasers. Anal Chem 91(4): 3078-3084. Winkler, R., Gillis, E., Lasman, L., Safra, M., Geula, S., Soyris, C., Nachshon, A., Tai-Schmiedel, J., Friedman, N., Le-Trilling, V. T. K., et al. (2019). m6A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol 20(2): 173-182. Xiang, Y., Laurent, B., Hsu, C. H., Nachtergaele, S., Lu, Z., Sheng, W., Xu, C., Chen, H., Ouyang, J., Wang, S., et al. (2017). RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543(7646): 573-576. Yin, H., Wang, H., Jiang, W., Zhou, Y. and Ai, S. (2017). Electrochemical immunosensor for N6-methyladenosine detection in human cell lines based on biotin-streptavidin system and silver-SiO2 signal amplification. Biosens Bioelectron 90: 494-500. Zhou, J., Wan, J., Gao, X., Zhang, X., Jaffrey, S. R. and Qian, S. B. (2015). Dynamic m6A mRNA methylation directs translational control of heat shock response. Nature 526(7574): 591-594. Article Information Copyright Du et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Molecular Biology > RNA > RNA detection Molecular Biology > RNA > RNA extraction Cancer Biology > General technique > Biochemical assays Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs Sajni Josson [...] Leland W.K. Chung Feb 20, 2017 10223 Views miRNA Characterization from the Extracellular Vesicles Sajni Josson [...] Leland W.K. Chung Feb 20, 2017 7589 Views Detection of Individual RNA in Fixed Cells and Tissues by Chromogenic ISH Meng Jiang [...] Rongqin Ke Feb 5, 2020 3965 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,566	https://bio-protocol.org/en/bpdetail?id=4566&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells MH Manjurul Haque * DB Dominic A. Boardman * AL Avery J. Lam KM Katherine N. MacDonald LS Lieke Sanderink QH Qing Huang VF Vivian C. W. Fung SI Sabine Ivison MM Majid Mojibian ML Megan K. Levings (*contributed equally to this work) Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4566 Views: 1329 Reviewed by: Luis Alberto Sánchez VargasTakashi Nishina Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Translational Medicine Aug 2020 Abstract Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. Graphical abstract Keywords: Regulatory T cell Graft-versus-host disease (GvHD) Hematopoietic stem cell transplantation (HSCT) Xenograft Thymic T-regulatory cells Background Hematopoietic stem cell transplantation (HSCT) is a therapeutic strategy used to treat certain immune system disorders as well as various hematological cancers such as myeloma and leukemia (Gschweng et al., 2014). In this procedure, recipients are typically conditioned with irradiation and/or chemotherapy to deplete immune cells, which are, in turn, replaced by an intravenous infusion of hematopoietic stem cells. As the hematopoietic stem cells are often derived from an allogeneic donor, a severe and potentially lethal side effect of HSCT is graft-versus-host disease (GvHD), whereby the patient’s new immune cells recognize the recipient’s cells as foreign and attack and destroy healthy tissues (Ferrara et al., 2009). This process is thought to be driven by reconstituted T cells recognizing allogeneic human leukocyte antigens (HLAs) on recipient cells (Loiseau et al., 2007). Animal models are critical for testing novel therapeutic strategies that can be used to treat GvHD. Mouse models of GvHD are well established and have significantly advanced this field of research. However, in order to examine how new therapies influence the function of human T cells, models in which mice are reconstituted with human immune cells (humanized mice) are required. Accordingly, significant work has been performed to generate immunodeficient mice that can be reconstituted with human cells from different sources. For example, NOD-scid-IL-2Rγ null (NSG) mice are immunodeficient for B, T, and natural killer (NK) cells and have defective dendritic cells and macrophages, allowing them to be efficiently engrafted with human cells/tissues (Ehx et al., 2018). However, each humanized mouse model has unique properties that must be carefully considered when attempting to answer specific scientific questions. For example, it is well established that following peripheral blood mononuclear cell (PBMC) reconstitution of NSG mice, the primary cells to engraft are T cells, making this model unsuitable for assessing humoral immune responses. Additional factors such as time and cost should also be considered when selecting an appropriate humanized mouse model. This area has been extensively reviewed in Walsh et al. (2017), Adigbli et al. (2020), and Khosravi-Maharlooei et al. (2022). For modelling the multi-system disorder known as xenoGvHD, we and others have shown that human T cells readily engraft in NSG mice upon intravenous infusion. These human T cells are stimulated in a xenogeneic manner via the recognition of mouse major histocompatibility complex molecules, and as such, the consequential pathology observed in these mice closely mimics human GvHD (King et al., 2009). Although PBMC-reconstituted xenoGvHD mouse models have their limitations, such as the lack of a complete human immune system, they can be used to generate safety data and conduct pre-clinical proof-of-concept data for T cell–directed therapies. This protocol describes a method for inducing and monitoring xenoGvHD in NSG mice. We provide details on human PBMC preparation, methods to monitor disease progression over time, and an approach to data analysis and reporting. We also provide an example of how this model can be used to demonstrate the therapeutic potential of regulatory T cells (Tregs). Materials and Reagents Materials Sterile 1.5 mL microcentrifuge tubes (Fisher Scientific, catalog number: 229442) 15 mL Falcon tubes (Corning, catalog number: 14-959-53A) 50 mL Falcon tubes (Corning, catalog number: 14-432-22) Heparinized capillaries (Fisherbrand, catalog number: 22-362-566) 2” × 2” gauze (Fisherbrand, catalog number: 22-362178) Cotton tipped applicators (Q-tips®) Petroleum jelly (i.e., Vaseline®) 27 G needles, one per animal (BD Biosciences, catalog number: 305109) Appropriate mouse restrainer for blood collection 70 µm cell strainer (Corning, catalog number: 352350) 96-well clear V-Bottom polystyrene not treated microplate (Corning, catalog number: 3896) 0.5 mL insulin syringes (BD Horizon, catalog number: 329461) 1 mL syringe (BD Horizon, catalog number: 309659) Weighing bucket (empty 1 mL tip box without the rack or any small box) Micro test tubes (Bio-Rad, catalog number: 2239391) Corning® cryogenic vials, internal thread (Corning, catalog number: CLS430488) Mice 8–16-week-old NOD/SCID/IL-2Rgammanull (NSG) mice (The Jackson Laboratory, Strain: 005557) Note: Select the sex of mice depending on the experimental question. Both commercially available and house bred mice may be used. We have used mice of either sex and have not observed any differences in the induction of xenoGvHD. Drugs Anesthetic, isoflurane (USP 250 mL) (Piramal Critical Care Inc, part number: DVM-102190) Reagents Heparin (Sigma, catalog number: H3149-10KU) 70% ethanol (VWR, catalog number: 89370-078) Fixation/permeabilization concentrate (eBioscience, catalog number: 00-5123-43) Fixation/permeabilization diluent (eBioscience, catalog number: 00-5223-56) 10× permeabilization buffer (eBioscience, catalog number: 00-8333-56) Counting beads (123count eBeadsTM, Invitrogen, catalog number: 01-1234-42) DNase I solution (1 mg/mL) (StemcellTM Technologies, catalog number: 07900) Penicillin–streptomycin (P/S) (GibcoTM, catalog number: 15140122) β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148) Dithiothreitol (DTT) (ThermoFisher, catalog number: 20290) EDTA (Sigma-Aldrich, catalog number: 03690) Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C7657) Percoll (Sigma-Aldrich, catalog number: P1644) Media and Buffers 10× RBC lysis buffer (eBioscience, catalog number: 00-4300-54) Gibco 1× Dulbecco’s phosphate buffered saline (DPBS) (ThermoFisher, catalog number: 14190) Gibco 10× Phosphate buffered saline (PBS) (ThermoFisher, catalog number: 70013032) LymphoprepTM (StemcellTM Technologies, catalog number: 07851) Immunocult-XF T-cell expansion medium (StemcellTM Technologies, catalog number: 10981) Gibco fetal bovine serum qualified (FBS) (ThermoFisher, catalog number: 12483020) Gibco RPMI 1640 medium (ThermoFisher, catalog number: 11875093) Gibco GlutaMax (ThermoFisher, catalog number: 35050061) Gibco HEPES (1 M) (ThermoFisher, catalog number: 15630080) Antibodies Mouse Fc block (BD Biosciences, catalog number: 553142) Fixable viability dye (FVD) eFluor 780 (eBioscience, catalog number: 65-0865-18) Anti-Mouse CD45 (30-F11) AF700 (BD Biosciences, catalog number: 560510) Anti-Human CD45 (HI30) V500 (BD Biosciences, catalog number: 560777) Anti-Human CD4 (SK3) BV786 (BD Biosciences, catalog number: 563877) Anti-Human CD8 (RPA-T8) BV711 (BD Biosciences, catalog number: 563677) Anti-Human CD3 (UCHT1) BB515 (BD Biosciences, catalog number: 564466) Anti-Human Foxp3 (236A/E7) PE-Cy7 (eBioscience, catalog number: 25-4777-42) Anti-Human Helios (22F6) AF488 (BioLegend, catalog number: 137223) Anti-Human HLA-A2 (BB7.2) APC (Invitrogen, catalog number: 17-9876-42) Equipment Type II biosafety cabinet (NuAire, model: LabGard ES NU-540) Microcentrifuge (Eppendorf, models: 5810R and 5452) Cell counter (e.g., Nexcelom, model: Cellometer Auto 2000 or alternative) X-ray irradiator (Rad Source-RS2000 Pro Biological Irradiator; alternate instruments including a gamma irradiator can be used) Dosimeter (Radcal-2186 Dose Meter; alternative instruments can be used) Electric shaver (Wahl Professional Animal BravMini+ Pet Trimmer, catalog number: 41590-0437) Ear notcher (Fine Science Tools, catalog number: 24214-02) Flow cytometer (BD LSRFortessaTM X-20; alternative instruments can be used) OHAUSTM NavigatorTM portable balance (Thermo Fisher, catalog number: 01-922-205) Thermo ScientificTM PrecisionTM general purpose baths (Thermo Fisher, catalog number: TSGP05) VetFloTM vaporizer single-channel anesthesia system (Kent Scientific Corporation, catalog number: VetFlo-1205SP) Software FlowJo (BD Biosciences, 10.8.1) Procedure Experimental Timeline Day -1: Prepare mice Day 0: Inject PBMCs Day 2 onwards: Monitor until endpoint Day 7 onwards: Weekly bleeding to monitor engraftment Detailed procedure Day -1: Prepare mice for infusion of PBMCs Create experimental groups Ear notch mice for identification. Randomly allocate mice to experimental groups. Mice should be housed under sterile conditions according to institutional guidelines and local ethical approvals. For reference, we house 2–4 mice per cage (UBC Animal Care Committee approval A18-0180). For the best practice, randomize mice and group them in a blinded fashion. Allocate littermates of the same cage to different experimental groups to control for cage-to-cage variation. Note: Tail marking may be used instead of ear notch, but this is short-lasting and requires repeated application. Weigh mice Wipe a weighing bucket with 70% ethanol and place on a portable balance in a biosafety cabinet. Gently transfer a mouse to the weighing bucket and record the weight. Wipe the weighing bucket and gloves with 70% ethanol between cages. OPTIONAL: Irradiate mice Keeping the mice in their cage, irradiate with 150 cGy (150 rad) X-ray irradiation (exposure time to obtain 150 cGy varies between machines and should be established in consultation with local animal facility staff). Confirm the total accumulated dose using a dosimeter. Note: Preconditioning mice with X-ray irradiation is optional. The recommended dose is sublethal and considered as mild (Gibson et al., 2015). Irradiation facilitates faster and more consistent engraftment and significantly accelerates the onset of xenoGvHD (King et al., 2009). Monitor mice Check mice for signs of stress/discomfort immediately after irradiation and 4 h later. Examples of such signs are provided in Table 1. If signs of stress/discomfort are observed, consult the veterinarian of your animal facility or euthanize the animal. Irradiation may cause minor (<10%) weight loss, which should be regained within five days. If the weight loss continues, humanely euthanize the animal and remove from the study. Table 1. Animal monitoring sheet with xenoGvHD scoring guide xenoGvHD score guide 0 1 2 3 Weight loss (%) <5% 5%–10% 10%–15% >15% Activity Bright and alert; interested in the environment; interacts with casemates; looks at observer; when nudged, moves away or sniffs observer. Less interested in the environment; interacts less with casemates; disregards observer; when nudged, reluctantly moves away or hyperactive. Isolated from casemates; sits in corner of cage; does not readily move when cage is disturbed (only moves when touched); when nudged, pauses before moving away slowly. Immobile or hyperreactive, even when nudged; cannot right itself. Hunch Normal posture. Mild or occasional hunching (round). Moderate hunched posture (easily noticed). Severe hunched posture (tip toe gait). Fur/skin Shiny, well-kept coat. Unkempt, dull, or soiled coat, ruffled fur; skin is mildly inflamed (mildly red). Fur loss (may be generalized with skin exposed by caressing or localized and presenting as alopecia—especially on face, neck, hinds); skin is moderately inflamed or affects face or footpads. Fur loss on 25% of mouse, soiled dull fur; skin is ulcerated (face, footpads). Note: conjunctivitis is also a sign of GvHD (1 = discharge, pus; 2 = closing, whitening of cornea). Pain n/a n/a Self-trauma; facial grimace: narrow or closed eyes, bulge on top of nose (mice), flattening of bridge of nose (rat), cheek convex (mice: between eye and whiskers), ears back or flat, whiskers pointing back or “standing out on end”. Other: muscle twitching or flinching, staggering, back stretch (like cat), abdominal writhing, abdominal pressing. Nothing additional, monitor as per protocol Nothing additional, monitor as per protocol Monitor mice every day Euthanize immediately Animals with a score of 2 in any one category: score every day. Experimental endpoints: cumulative score of six or seven weeks after injection of cells. Humane endpoints: score 3 in any one category. Other endpoints: Mice showing any signs of infection or severe condition such as eyelids closed, changes in respiration, loss of fur, unusually apprehensive or aggressive, scratching, biting or self-mutilation, hunched posture, aggressive vocalization, separation from group, or sunken or distended abdomen will be euthanized. Day 0: Preparation and injection of PBMCs Prepare PBMCs in a biosafety cabinet: either freshly isolated or thawed PBMCs may be used. Fresh cells: Isolate PBMCs from human peripheral blood using LymphoprepTM, according to established protocols (https://protocols.io/view/can-asc-consensus-protocol-isolation-cryopreservat-brrsm56e.pdf). PBMCs can be infused immediately after isolation or stored at 4 °C overnight and infused the following day. For the latter, cells should be stored in a 50:50 mixture of PBS and Immunocult-XF at a concentration of 10 × 106 cells/mL in a 50 mL Falcon tube laid horizontally. Frozen cells: Frozen PBMCs can be thawed on the day of infusion. Using batch frozen PBMCs allows the cells to be characterized before experimentation and can reduce experimental variability. An example of the variability that can result from using PBMCs from different donors is shown in Figure 1. An intravenous infusion of 5 × 106–10 × 106 PBMCs should achieve an engraftment level of >20% human CD45+ cells in the peripheral blood 14 days post infusion and xenoGvHD symptoms should be evident within 3–4 weeks. Using PBMCs that do not achieve these criteria may yield sub-optimal results. Note: Number of PBMCs may be a limiting factor. For large experiments, commercial leukopaks can be used as a frozen source of PBMCs. Prepare Immunocult-XF T-cell expansion media with 1% P/S. Warm up the media to 37 °C in a water bath. Add 10 mL of the media to 15 mL Falcon tubes. Prepare one 15 mL Falcon tube for each cryogenic vial. Note: We use Immunocult-XF T-cell expansion media to avoid the use of human serum, which often shows batch variations in composition. Any other T-cell expansion medium can be used here for the thawing process. Thaw cryovials in a 37 °C water bath until almost completely thawed. Add 1 mL of warm thawing media dropwise to cryotubes, then carefully transfer cells to a 15 mL Falcon tube containing 9 mL of warm media. Figure 1. PBMC donor variation in inducing xenoGvHD. Five million PBMCs from four different donors (PBMC A: male; PBMC B: female; PBMC C: male, and PBMC D: female) were tested for their ability to induce xenoGvHD. (A) % weight loss relative to day of PBMC infusion (day 0). (B) % mouse survival. (C) Cumulative GvHD score. (D) Engraftment of human (h) CD45+ cells as a proportion of total live CD45+ (mouse and human) cells. Note: For mice that were terminated before the endpoint (D42), the end date value for the calculation was carried forward in subsequent time points. Centrifuge cells at 450 × g for 5 min and discard the supernatant. To reduce cell clumping, resuspend the cells in 10 mL of Immunocult-XF + 1% P/S with DNase I at a concentration rate of 10 µg/mL, gently mix by pipetting, take a sample for counting, and incubate for 10 min at room temperature. Centrifuge cells at 450 × g for 5 min, discard the supernatant, and resuspend the PBMCs in sterile PBS at a concentration of > 100 × 106 cells/mL (approximately 120 × 106/mL). Pass cells through a 70 µm cell strainer, recount, and adjust the cell concentration to 100 × 106 cells/mL. Note: The cell concentration adjusted here is for a dose of 10 × 106 PBMCs in 100 µL injection volume. If you intend to inject a different number of cells, adjust the cell number such that 100 µL of PBS contains the desired number of cells. The number of cells required for inducing xenoGvHD varies between PBMC donors, thus a pilot study to assess the engraftment capacity of cryopreserved PBMC aliquots is strongly recommended. If additional agents or cells of interest (e.g. Tregs) are co-administered with the PBMCs, the final infusion volume should not exceed 1% of the mouse body weight (i.e., 200 µL for a 20 g mouse). Follow guidelines from your animal ethical committee for the maximum injection volume via tail vein. Just before cell injection, transfer cells to a 1.5 mL tube and store on ice until injection to minimize the time between cell storage on ice and cell injection. Note: On the day of injection the viability of PBMC should be higher than 70%. PBMC injection: Remove cells from ice 10–15 min before injection to warm up the cell suspension to room temperature. Gently mix the PBMCs by inverting the 1.5 mL collection tube. Draw 100 mL of cells into an insulin syringe and ensure no bubbles are present. Inject directly in the tail vein with the bevel pointing upwards, following the method below: Notes: 1) The temperature of circulatory blood in the mouse tail vein is approximately 37 °C. Warming up cells to 37 °C or room temperature before adoptive transfer minimizes the heat shock to cells and ensures better survival in vivo. 2) Limit drawing the cells up and down into the syringe as much as possible; instead, mix by inversion. Repeatedly drawing the cells in and out of the syringe exposes them to unnecessary sheer stress that reduces viability. If you are injecting any other cells with the PBMC, cells can be mixed together and injected as a single inoculation in a total volume of 200 µL. Vasodilate the tail vein of mice prior to intravenous injections to help with visualization. This can be performed using one of the following approaches: Place half of the cage on top of a safe heat source such as a heat pad. Only half of the cage should be on the heat source so that there is a cooler half of the cage where mice can move to in case of overheating. Apply heat directly to the tail itself using a glove filled with lukewarm water. Make sure the water is not too hot to avoid burn injury. Any of the above methods can be used for efficient vasodilation, but never leave mice unattended around heating sources. Place mice in a restrainer if injecting conscious, or in a warmed anesthetizing induction chamber if injecting anesthetized mice. Note: If injecting an anesthetized mouse, make sure to inject as quickly as possible since the anesthetic agents can cause vasoconstriction making it difficult to find the vein. Hold the warmed tail with the non-dominant hand and locate one of the two lateral tail veins. Swab the tail with 70% ethanol (avoid applying too much alcohol, which may cause vasoconstriction) to disinfect and increase the visibility of the vein. Then, insert the needle bevel up into the distal portion of the middle third of the tail with the dominant hand. Slowly press the plunger to test if you are in the vein. If the needle is in the vein there will be no resistance; however, you will feel resistance if it is not in the vein. Once confident that you are in the vein, gently inject 100 µL of cell suspension. Keep the needle in place for 2–3 s to limit backflow of the injected cells, then remove the needle and press the injection site with a sterile gauze to stop bleeding and limit backflow of cell suspension. Monitor the animals for 5–10 min for any sign of discomfort or bleeding. Note: While injecting cells in the tail vein do not pull the plunger back, as this may cause clumping of cells. We recommend using insulin syringes for injections as there is less dead space, which minimizes volume loss during injection. See Yano et al. (2020) for a detailed video protocol. Day 2 to endpoint: Monitoring Monitor body weight and health status of mice according to the approved animal use protocol. For our protocol (UBC Animal Care Committee approval A18-0180), this is three times per week. Bleed mice by puncturing the saphenous vein once every seven days post cell injection to monitor cell engraftment. Euthanize mice and end experiment according to approved animal protocol. In our case, this is when any of the following criteria are achieved: i) >15% body weight loss, ii) any health score category is ≥3, or iii) the cumulative health score is ≥6 (Table 1). Day 7 onward: Weekly bleeding to check engraftment Collect blood from the lateral or medial saphenous veins following the steps below: Prepare blood collection tubes (1.5 mL Eppendorf) by adding 3 µL of heparin (50 mg/mL) into separate tubes for each animal. Place a mouse in an appropriate restrainer with your non-dominant hand, ensuring that the animal’s leg remains extended. Locate the medial (inner) or lateral (outer) saphenous vein and remove the hair using the electric shaver. Swab the intended puncture area with a cotton tipped applicator moistened with 70% ethanol and apply a thin film of petroleum jelly. Using a sterile 27 G needle, puncture the blood vessel perpendicular to the skin at the most proximal visible area of the vein. Keep the needle in the vein for 2–3 s to allow the skin to stretch around the puncture site. Collect blood (approximately 50–70 µL) in a heparinized capillary tube by gently touching the end of the capillary tube to the blood drop. Transfer the blood from the capillary to the blood collection tubes. Store tubes at room temperature until further processing. Press the puncture site with a sterile gauze for at least 30 s or until bleeding stops. Transfer mice to cage and monitor for 10 min to ensure complete cessation of bleeding. Processing and flow staining of peripheral blood Transfer 50 µL of blood to a 96-well V-bottom plate and add 150 µL of PBS. Centrifuge the plate at 1,065 × g for 3 min and pipette off supernatant. Meanwhile, prepare 1× RBC lysis buffer in distilled water and warm up in a 37 °C water bath. First RBC lysis: Resuspend blood cell pellet in 180 µL of 1× RBC lysis buffer, mix well, incubate 3 min at room temperature, centrifuge at 1,065 × g for 3 min, and pipette off supernatant. Second RBC lysis: Resuspend the cell pellet in 180 µL of 1× RBC lysis buffer, incubate 3 min at room temperature, centrifuge at 1,065 × g for 3 min, and pipette off supernatant. Note: Cell pellet remains loosely attached during RBC lysis. To avoid cell loss, pipette off supernatant carefully. First wash: Resuspend the cell pellet in 180 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Second wash: Resuspend cell pellet again in 180 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Dilute mouse Fc Block in PBS (see Table 2), add 25 µL/well, and incubate for 10 min at room temperature. Table 2. Panel for weekly and endpoint engraftment testing Type Marker Clone Fluor Dilution (2×) Supplier Cat. No. Surface Mouse Fc Block 2.4G2 - 1: 50 BD Biosciences 553142 FVD - eF780 1: 500 eBioscience 65-0865-18 mCD45 30-F11 AF700 1: 50 BD Biosciences 560510 hCD45 HI30 V500 1: 50 BD Biosciences 560777 hCD4 SK3 BV785 1: 50 BD Biosciences 563877 hCD8 RPA-T8 BV711 1: 100 BD Biosciences 563677 hCD3 UCHT1 BB515 1: 100 BD Biosciences 564466 hHLA-A2 BB7.2 APC 1: 100 Invitrogen 17-9876-42 Intracellular hFoxp3 236A/E7 PE-Cy7 1: 50 eBioscience 25-4777-42 hHelios 22F6 AF488 1: 50 BioLegend 137223 Meanwhile, prepare the surface staining mixture in PBS as per Table 2. Top up wells with 25 µL/well of staining mix, mix well, and incubate for 20 min at room temperature in the dark. Top up each well with 150 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Note: Optional additional steps for intracellular staining, e.g. to measure FOXP3 expression: 9a. Prepare 1× fix/perm buffer (one part fixation/permeabilization concentrate, three parts fixation/permeabilization diluent). 9b. Resuspend cell pellet in 100 µL of fix/perm buffer and incubate at room temperature for 45 min in the dark. 9c. Meanwhile, prepare 1× permeabilization buffer in distilled water. 9d. Top up the well containing cell pellet with 100 µL of permeabilization buffer, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. 9e. Resuspend cell pellet in 100 µL of permeabilization buffer a second time, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. 9f. Prepare intracellular staining mix in 1× permeabilization buffer as per the dilution in Table 2. 9g. Resuspend in 25 µL of staining mix and incubate for 40 min at room temperature in the dark. 9h. Top up wells with 180 µL of permeabilization buffer, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Top up each well with 180 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Resuspend cells in 150 µL of FACS buffer (PBS + 1% FBS + 5 mM EDTA) and transfer cells to micro test tubes. Note: To obtain absolute cell concentrations, 10,000 counting beads may be added to each micro test tube at this point. Make sure to vortex beads very well as they settle quickly. Set the voltages of forward scatter (FSC) and side scatter (SSC) such that both cells and beads are visible. Beads fluoresce strongly in both FITC/PE channels. Ensure bead fluorescence in these channels are on scale before acquiring samples. Endpoint preparation of leukocytes for flow cytometry Collection of cardiac blood and spleen Prepare 1.5 mL Eppendorf tubes with 3 µL of heparin for cardiac blood and 1 mL of PBS for spleen. Put mouse into surgical plane of anesthesia using isoflurane vaporizer gas anesthesia chamber. Move mouse to nose cone, rinse a 1 mL syringe fitted with 27 G needle with heparin, perform cardiac puncture, and extract approximately 100 µL of blood. Perform cervical dislocation and harvest spleen avoiding the pancreatic tissue (pancreatic enzymes may cause damage to T cells). Processing and flow staining of cardiac blood and spleen Blood: Perform lysis and flow staining of 70 µL of blood as described in the section “Processing and flow staining of peripheral blood.” Spleen: Record the weight of the whole spleen, cut a small piece (approximately 15 mg) of spleen tissue, and mash on a pre-wet (with PBS) 70 µm cell strainer fitted on a 50 mL Falcon tube with a 1 mL syringe plunger. Rinse the filter with PBS until no visible particulate is present. Note: Alternatively, mash the whole spleen, then take a portion of the cells (i.e. mash, centrifuge cells at 450 × g for 5 min, resuspend the cells in 1 mL PBS and take 20 µL for 1/50 of the total splenocytes). Centrifuge the cell suspension at 450 × g for 5 min, discard supernatant by pouring, and transfer (approximately 200 µL) to a 96-well V-bottom plate. Centrifuge the plate at 1,065 × g for 3 min and pipette off the supernatant. Perform RBC lysis once by resuspending cell pellet in 180 µL of 1× RBC lysis buffer and incubate for 3 min at room temperature. Resuspend cell pellet in 180 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off supernatant. Resuspend cell pellet again in 180 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off and discard the supernatant. Dilute mouse Fc block in PBS (see Table 2), add 25 µL/well, and incubate for 10 min at room temperature. Meanwhile, prepare the staining mixture in PBS as per Table 2, add 25 µL of staining mix per well, and incubate for 20 min at room temperature in the dark. Top up each well with 150 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Wash cells by resuspending in 150 µL of FACS buffer, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Resuspend the cell pellet in 150 µL of FACS buffer and transfer to micro test tubes. OPTIONAL: Processing and flow staining of laminar propria lymphocytes (LPL): A major symptom of xenoGvHD is the manifestation of intestinal inflammation and colitis-like pathology. This can be further investigated by analyzing lymphocytes in the colon using the following protocol: Collect colon: Open abdomen and collect entire colon from the rectum to the cecum. Gently remove fecal material by pushing along the colon towards the rectum. Place the colon into a 50 mL Falcon tube with 15 mL of cold RPMI supplemented with 10% FBS, 2 μM Gibco GlutaMax, 1× P/S, 25 mM HEPES, and 55 μM β-mercaptoethanol and store on ice. Intestinal epithelial cell wash: Carefully discard the RPMI medium, add approximately 25 mL of PBS, and vortex for 2–3 s to wash the colon. Repeat this wash with an additional ~25 mL of PBS. Discard the PBS and add 15–20 mL of warm (37 °C) RPMI supplemented with 5% FBS, 1 mM DTT, and 1 mM EDTA. Vortex for 2–3 s and shake tubes at 37 °C for 10 min. Intraepithelial lymphocyte wash: Carefully discard the RPMI/DTT mixture and add 15–20 mL of cold (4 °C) RPMI + 2% FBS. Vortex for 2–3 s to wash the colon, discard supernatant, and repeat this wash step with an additional 15–20 mL of RPMI + 2% FBS. Discard the supernatant and add 15–20 mL of warm (37 °C) RPMI supplemented with 5% FBS, 1 mM DTT, and 1 mM EDTA. Vortex for 2–3 s and shake tubes at 37 °C for 20 min. Colon digestion: Carefully discard the RPMI/DTT mixture and add 15–20 mL of cold (4 °C) RPMI + 2% FBS. Vortex for 2–3 s to wash colon, discard supernatant, and repeat this wash step with an additional 15–20 mL of RPMI + 2% FBS. Discard supernatant and add 14 mL of warm (37 °C) RPMI supplemented with 0.5 mg/mL collagenase VIII + 20 µg/mL DNase I. Shake at 37 °C for 30 min to digest the colon. Pour the media and digested colon over a fresh 70 µm cell strainer into a fresh 50 mL Falcon tube. Mechanically disrupt the remaining colon with a syringe plunger against the cell strainer and wash the cell strainer with additional cold RPMI + 2% FBS to a final volume of 35 mL. Pellet the cells at 500 × g for 10 min. Lymphocyte enrichment: Make an isotonic Percoll solution (nine parts Percoll to one part 10× PBS) and use this to dilute with 1× PBS to yield 40% and 80% Percoll solutions. Add 3 mL of 80% Percoll to 15 mL Falcon tubes. Resuspend the cell pellet from step 5 in 4 mL of 40% Percoll and carefully overlay onto the 80% Percoll. Centrifuge at 652 × g at room temperature with brake off. Remove the lipid-like top layer to prevent contamination and collect the interphase containing LPLs (approximately 3 mL) into a fresh 15 mL tube.Top up with PBS to wash and pellet the cells at 652 × g for 5 min at 4 °C. Repeat this wash step and proceed to stain the enriched LPLs. Dilute mouse Fc block in PBS (see Table 2), add 25 µL/well, and incubate for 10 min at room temperature. Meanwhile, prepare the staining mixture in PBS as per Table 2, top up with 25 µL/well of staining mix, and incubate for 20 min at room temperature in the dark. Top up each well with 150 µL of PBS, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Wash cells by resuspending in 150 µL of FACS buffer, centrifuge at 1,065 × g for 3 min, and pipette off the supernatant. Resuspend the cell pellet in 150 µL of FACS buffer and transfer to micro test tubes. Add 10,000 counting beads if an absolute cell count is desired. Data analysis Flow cytometry data analysis Data presented here were acquired using a BD LSRFortessaTM X-20 flow cytometer and analyzed in FlowJo software. However, different flow cytometers with similar configurations can also be used. Data should be analyzed as shown in Figure 2. From total live events, human cell engraftment can be calculated as a proportion of total (human and mouse) CD45+ cells. With injection of PBMC alone, human cell engraftment is expected to range from 20% to 30% by day 21 post injection. Note: Before using PBMC for an actual experiment it is strongly recommended to pre-test their xenoGvHD-inducing capacity in a pilot study. Figure 2. Gating strategy for calculating PBMC engraftment. The voltage should be set such that the forward and side scatter (FSC-A vs. SSC-A) plot shows both total cells and beads (first plot). From total cells, gate singlets and live cells (FVD-negative fraction). From total live cells, gate mouse CD45 (AF-700-mCD45) and human CD45 (V500-hCD45) and calculate the % human CD45 cells in the upper left quadrant. The first flow-plot (SSC-A vs. FSC-A) is showing. Absolute cell count Vortex beads for 30 s and add 10,000 beads for blood and 20,000 beads for spleen samples before running sample in flow cytometer. We adjusted the bead concentration such that 10 µL contains 10,000 beads. Adjust the SSC and FSC voltage to detect both beads and cells (see Figure 2, top left). Draw a gate on the counting beads, display on a flow plot with FITC vs. PE, and gate on the double positive population (Figure 3). Use the count statistics in the data analysis software (FlowJo) to determine the cell concentration using the equation below. Note: Collect at least 7,000–8,000 bead events to obtain statistically significant results. Figure 3. Flow plot showing the gating of cells and counting beads in SSC vs. FSC and FITC+PE+ beads. Absolute cell number calculation is based upon the PE/FITC double positive event count. Thymic Tregs delay the development of xenoGvHD As an example of how this model can be used to test new therapies, in Figure 4 we show an example of Treg-mediated suppression of xenoGvHD. Preparation of thymic Tregs: Thymic Tregs were prepared according to MacDonald et al. (2019). Briefly, thymocytes were isolated by mechanical dissociation of thymic tissue followed by CD25-positive and CD8-negative selection. The protocol yields >90% pure CD4+CD25+Foxp3+ thymic Tregs. Preparation of PBMCs: Frozen PBMC were thawed following the process described in section B above. Cell injection: Resuspend both PBMCs and Tregs at a cell concentration of 70 × 106/mL in PBS and keep them separate. At the time of injection, mix 100 µL of PBMC and 100 µL of Tregs and inject 200 µL of the mixed cell suspension via the tail vein following the method described in the PBMC injection section above. Note: If Tregs are allogeneic to PBMCs, it is useful to have a known HLA-mismatch to facilitate cell tracking. We routinely phenotype PBMCs and Tregs for HLA-A2, -A3, and -A24 to facilitate this process. Allogeneic Tregs diminish the engraftment of human PBMCs whereas autologous Tregs show minimal impact on PBMC engraftment (Dawson et al., 2019). Tregs engineered with different types of receptors (e.g., chimeric antigen receptor) may have greater effects on PBMC engraftment. Figure 4. Suppression of xenoGvHD by thymic Tregs. Seven million HLA-A2+ PBMCs and seven million HLA-A2- thymic Tregs were injected into irradiated NSG mice. The infused Tregs were either freshly isolated and rested, freshly isolated and activated, or previously frozen. (A). Flow plot showing the gating strategy for detecting HLA-A2+ PBMCs and HLA-A2- thymic Tregs on day 7 post cell injection. (B). Kaplan–Meier survival curve showing that infusion of Tregs delayed the onset of xenoGvHD. OPTIONAL: Histology of organs To detect cellular infiltration, perform histology of various organs (lungs, liver, heart, intestine, kidney, skin, etc.). The onset of a higher degree of xenoGvHD shows massive infiltration of human-CD45 positive cells. For details, see Dawson et al. (2019). Acknowledgments This work was supported by funding from the Canadian Institutes of Health Research. Competing interests MKL has received research funding from Sangamo Therapeutics, Bristol-Myers Squibb, Pfizer, Takeda, and CRISPR Therapeutics for work unrelated to this study. All other authors declare no competing interests. Ethics Researchers must obtain appropriate training and ethical approval from the institutional animal ethical committee before conducting any mouse experiment. Data presented here were obtained from experiments conducted with the approval of the University of British Columbia Animal Care Committee (Protocol: A18-0180). References Adigbli, G., Menoret, S., Cross, A. R., Hester, J., Issa, F. and Anegon, I. (2020). Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine. Transplantation 104(11): 2290-2306. Dawson, N. A., Lamarche, C., Hoeppli, R. E., Bergqvist, P., Fung, V. C., McIver, E., Huang, Q., Gillies, J., Speck, M., Orban, P. C., et al. (2019). Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells. JCI Insight 4(6). e123672. Ehx, G., Somja, J., Warnatz, H. J., Ritacco, C., Hannon, M., Delens, L., Fransolet, G., Delvenne, P., Muller, J., Beguin, Y., Lehrach, H., Belle, L., Humblet-Baron, S., Baron, F. (2018). Xenogeneic Graft-Versus-Host Disease in Humanized NSG and NSG-HLA-A2/HHD Mice. Front Immunol 9:1943. Ferrara, J. L., Levine, J. E., Reddy, P. and Holler, E. (2009). Graft-versus-host disease. Lancet 373(9674): 1550-1561. Gibson, B. W., Boles, N. C., Souroullas, G. P., Herron, A. J., Fraley, J. K., Schwiebert, R. S., Sharp, J. J. and Goodell, M. A. (2015). Comparison of Cesium-137 and X-ray Irradiators by Using Bone Marrow Transplant Reconstitution in C57BL/6J Mice. Comp Med 65(3):165-72. Gschweng, E., De Oliveira, S. and Kohn, D. B. (2014). Hematopoietic stem cells for cancer immunotherapy. Immunol Rev 257(1): 237-249. Khosravi-Maharlooei, M., Madley, R., Borsotti, C., Ferreira, L. M. R., Sharp, R. C., Brehm, M. A., Greiner, D. L., Parent, A. V., Anderson, M. S., Sykes, M., et al. (2022). Modeling human T1D-associated autoimmune processes. Mol Metab 56: 101417. King, M. A., Covassin, L., Brehm, M. A., Racki, W., Pearson, T., Leif, J., Laning, J., Fodor, W., Foreman, O., Burzenski, L., et al. (2009). Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex. Clin Exp Immunol 157(1): 104-118. Loiseau, P., Busson, M., Balere, M. L., Dormoy, A., Bignon, J. D., Gagne, K., Gebuhrer, L., Dubois, V., Jollet, I., Bois, M., et al. (2007). HLA Association with hematopoietic stem cell transplantation outcome: the number of mismatches at HLA-A, -B, -C, -DRB1, or -DQB1 is strongly associated with overall survival. Biol Blood Marrow Transplant 13(8): 965-974. MacDonald, K. N., Piret, J. M. and Levings, M. K. (2019). Methods to manufacture regulatory T cells for cell therapy. Clin Exp Immunol 197(1): 52-63. Walsh, N. C., Kenney, L. L., Jangalwe, S., Aryee, K. E., Greiner, D. L., Brehm, M. A., Shultz, L. D. (2017). Humanized Mouse Models of Clinical Disease. Annu Rev Pathol 12:187-215. Yano, J., Lilly, E. A., Noverr, M. C. and Fidel, P. L. (2020). A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model. J Vis Exp(165): e61961. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Medicine > Inflammation Immunology > Host defense Cell Biology > Cell Transplantation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,567	https://bio-protocol.org/en/bpdetail?id=4567&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Measurement of Transgenes Copy Number in Wheat Plants Using Droplet Digital PCR PL Peng Liu SL Shuang Liu JL Jiajia Lei JC Jianping Chen JY Jian Yang Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4567 Views: 816 Reviewed by: Ansul LokdarshiPriyanka DuttaRaviraj Mahadeo Kalunke Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Molecular Plant Jul 2021 Abstract Genetic transformation is a powerful method for the investigation of gene function and improvement of crop plants. The transgenes copy number in the transgenic line is involved in gene expression level and phenotypes. Additionally, identification of transgene zygosity is important for quantitative assessment of phenotype and for tracking the inheritance of transgenes in progeny generations. Several methods have been developed for estimating the transgene copy number, including southern blot assay and quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization, although convincing and reliable, is a time-consuming method through which the examination of the copy number is challenging in species with large genomes like wheat plants. Although qPCR is potentially simpler to perform, its results lack accuracy and precision, especially to distinguish between one and two copy events in transgenic plants with large genomes. The droplet digital PCR (ddPCR)–based method for investigation of transgenes copy number has been widely used in an array of crops. In this method, the specific primers to amplify target transgenes and reference genes are used as a single duplexed reaction, which is divided into tens of thousands of nanodroplets. The copy number in independent transgenic lines is determined by detection and quantification of droplets using sequence-specific fluorescently labeled probes. This method offers superior accuracy and reliability with a low cost and scalability as other PCR techniques in the investigation of transgenes copy number. Graphical abstract Flow chart for the ddPCR protocol Keywords: Genetic transformation Transgene copy number Digital droplet PCR Southern blot qPCR wheat Background Plant genetic transformation has been widely used for investigating gene function in wheat as well as the production of environmentally friendly and disease-resistant wheat varieties (Vasil, 2007). During genetic transformation in wheat embryos, the delivery number of transgenes into the plant genome cannot be controlled. Moreover, the expression level of transgenes and, consequently, the phenotype can be influenced by copy numbers. Thus, the validation of the copy number of transgenes is essential for research projects involving transgenic plants (Hobbs et al., 1993; Sivamani et al., 2015). The transgenic wheat plants with a single, full-length copy of a gene at a single locus is the most desirable event because the transgenes typically segregate in a predictable Mendelian fashion (Srivastava et al., 1996). Additionally, copy number determination can also be used to track inheritance and zygosity (Wu et al., 2017). Gene copy number analysis could be determined by southern blot analysis (Southern, 1975), quantitative polymerase chain reaction (qPCR) (Higuchi et al., 2013), thermal asymmetric interlaced PCR (TAIL-PCR) (Y. G. Liu et al., 1995) and, most recently, digital droplet PCR (ddPCR). However, these methods vastly differ in precision, reproducibility, and potential to scale up. Among these, southern blot analysis was regarded as a powerful method for estimating copy number and loci complexity in transgenic plants. In this assay, genomic DNA was digested, separated by electrophoresis, blotted onto a membrane, and then detected with a radioactive, fluorescent, or chemiluminescent-labelled probe. However, it is hard to detect the copy number of transgenes using this method, which are inserted at a single locus. Moreover, the southern blot assay is impractical for examining large populations of transgenic plants because it is unable to automate and requires substantial skills, especially in species with large genomes like wheat plants. In qPCR, the concentration of the transgene in each sample is compared with either a standard curve or an endogenous gene of a known copy number, to estimate the copy number and zygosity of the transgenic allele(s) (Higuchi et al., 2013). Estimation of copy number by qPCR is very sensitive to PCR efficiency because of direct coupling between the PCR amplification and quantification. TAIL-PCR has also been used to establish the copy numbers of transgenes by amplifying the flanking sequence of the transgenic allele(s) (Y. G. Liu et al., 1995; Hanhineva and Krenlampi, 2007). The ddPCR method has been used to detect the copy number of transgenes with high accuracy (Gowacka et al., 2015). Similar to qPCR, the detection principle of ddPCR is based on a fluorescent dye or probe and requires an endogenous reference gene with a known copy number (Collier et al., 2017). In ddPCR, the PCR reaction partitions individual into separate compartments, such as oil-bounded droplets, and then detects their endpoint amplification products. Poisson probability distribution is also used to derive the template concentration. Accordingly, the ddPCR method enables a more effective calculation of gene target enumeration and accurate quantification of nucleic acid targets. In qPCR, logarithmic PCR template quantification may limit the ability to identify small copy number differences (Bubner and Baldwin, 2004). However, in ddPCR, the decoupling of amplification and quantification with the linearity of the quantification scale allows the detection of small copy differences (Hindson et al., 2013; Bharuthram et al., 2014). In recent years, ddPCR has been successfully applied for the accurate determination of transgene copy number in different plant species. Here, we summarize the protocol of ddPCR used to determine the transgene copy number in wheat plants (P. Liu et al., 2021). Materials and Reagents 2 mL safe-lock microcentrifuge tubes (Axygen, catalog number: MCT-200-C) PCR plate heat seal, foil, pierceable (Bio-Rad, catalog number: 1814040) ddPCR plates, 96-well, semi-skirted (Bio-Rad, catalog number: 12001925) DG8TM cartridge for QX200TM/QX100TM droplet generator (Bio-Rad, catalog number: 1864008) Droplet generator DG8TM gasket (Bio-Rad, catalog number: 1863009) Restriction enzyme EcoRI (ThermoFisher, catalog number: FD0274) RNase A (Sigma, CAS: 9001-99-4) Nuclease-free water (ThermoFisher, catalog number: 10977015) QubitTM dsDNA HS assay kit (ThermoFisher Scientific, catalog number: Q32851) QX200TM ddPCRTM EvaGreen supermix (Bio-Rad, catalog number: 1864033) QX200 droplet generation oil for EvaGreen® (Bio-Rad, catalog number: 1864005) 5' FAMTM (6-Carboxyfluorescein) (ThermoFisher Scientific, catalog number: C1360) 5' VICTM (6-VIC) (AAT Bioquest, catalog number: 212) Equipment QubitTM 4 fluorometer (ThermoFisher Scientific, catalog number: Q33238) QX200TM droplet generator (Bio-Rad, catalog number: 1864002) Thermal cycler (e.g., Bio-Rad C1000 Touch thermal cycler, catalog number: 1851148) Pipettes (10 µL, 20 µL, 100 µL) Heat sealer (e.g., Eppendorf S100, catalog number: 5391000036) DG8 cartridge holder (Bio-Rad, catalog number: 1863051) QX200 droplet reader (Bio-Rad, catalog number: 1864003) Droplet reader plate holder (Bio-Rad, catalog number: 12006834) Software Primer design software: Primer5 Sequence analysis software: DNAstar (https://www.dnastar.com/) QuantaSoftTM software (Bio-Rad, catalog number: 1863007) (https://www.bio-rad.com/) Software capable of reading comma-separated values (.csv) files: Microsoft Excel Droplet DigitalTM PCR Applications Guide: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf Procedure Preparation of wheat materials Grow plants in a chamber/greenhouse with the corresponding conditions. Note: Here, wheat plants were grown in a growth chamber maintained at 16 °C ± 2 °C with a 16 h light and 8 h dark photoperiod and 70% relative humidity. Collect approximately 100 mg of leaf samples from two-weeks-old wheat plants in a 2 mL safe-lock microcentrifuge tube for genomic DNA extraction. Isolate total genomic DNA from wheat plant leaves using the CTAB method (Chen et al., 2014). Digest each sample of genomic DNA using the restriction enzyme EcoRI followed by RNase A treatment. An example restriction digestion reaction is shown in Table 1. Table 1. Example reaction for restriction endonuclease digestion of genomic DNA Components Volume 10× restriction enzyme buffer 5 μL EcoRI (10–20 U/μL) 2 μL Genomic DNA 1 μg Nuclease-free water Up to 50 μL Total 50 μL Incubate the restriction reaction mixture at 37 °C for 1 h and then add 1 μL of RNase A. After incubation at 37 °C for 30 min, inactivate the mixture by heating at 95 °C for 10 min. Following inactivation, the digested genomic DNA can be used directly in the next step without further cleanup. Primers selection The wheat puroindoline-b (PINb) gene with a single copy was used as a reference gene to estimate transgene copy number (Collier et al., 2017). The primers of target genes were designed using a primer design software (e.g., Primer 5), which amplifies a 100–120 base pair region of both transgene targets. An example of target genes primers and wheat PINb gene primers are shown in Table 2. Table 2. Primers used in ddPCR Name Sequence Length Targets TaPINb-F AGTTGGCGGCTGGTACAATG 20 Wheat PINb gene TaPINb-R ACATCGCTCCATCACGTAATCC 23 TaPINb-prove FAM-TCAACAATGTCCGCAGGAGCG-VIC 23 pUbi-F GTAGATAATGCCAGCCTGTTAAAC 24 Ubi promoter pUbi-R GACGCGACGCTGCTGGTT 18 pUbi-prove FAM-CGTCGACGAGTCTAACGGACACCAAC-VIC 29 Each primer pair needs to be checked first in a test PCR reaction. Use the reaction with genomic DNA from non-transgenic wheat plants or H2O as negative control. Assemble the reaction as shown in Table 3. Table 3. PCR reaction for testing ddPCR primer pairs Components Stock concentration Final concentration Volume 2×QX200TM ddPCRTM EvaGreen® supermix 2× 1× 5 μL Forward primer 2 μM 250 nM 1.25 μL Reverse primer 2 μM 250 nM 1.25 μL Genomic DNA 50 ng Nuclease-free water Up to 10 μL Total 10 μL Label the reference gene probes with 5' FAMTM (6-Carboxyfluorescein) and the transgene probes with 5' VICTM (6-VIC). The cycling conditions of the PCR reaction are shown in Table 4. Table 4. PCR cycling conditions for testing ddPCR primer pairs Step Temperature (°C) Time Number of cycles Enzyme activation 95 5 min 1 Denaturation 95 30 s 40 Annealing 60 30 s Extension 72 30 s Signal stabilization 72 5 min 1 Hold 10 ∞ 1 Separate the PCR product by agarose gel electrophoresis. Obtain the single clear bands with the expected sizes for reference and target primers (Figure 1). Figure 1. Example of PCR product showing the expected sizes obtained for reference and target primers ddPCR setup Assemble the PCR reactions as shown in Table 5. The genomic DNA from non-transgenic wheat plants or H2O were used as template as negative controls and added into the reaction. The primers of reference gene and transgenic genes were mixed in one reaction for each sample. It is critical that the assayed ddPCR reactions are well mixed prior to proceeding to droplet generation. The concentration of each component must be the same in every droplet. Table 5. Example ddPCR reaction Components Stock concentration Final concentration Volume 2× QX200TM ddPCRTM EvaGreen® supermix 2× 1× 10 μL Forward primer of reference gene 2 μM 250 nM 2.5 μL Reverse primer of reference gene 2 μM 250 nM 2.5 μL Forward primer of reference gene 2 μM 250 nM 2.5 μL Reverse primer of reference gene 2 μM 250 nM 2.5 μL Genomic DNA 50 ng Nuclease-free water Up to 20 μL Total 20 μL Droplet generation Load the cartridge into the QX200 droplet generator. Load 20 μL of the ddPCR reaction into individual sample wells of the DG8TM droplet generator cartridge. Add 70 μL of QX200 droplet generation oil for EvaGreen® into the oil wells. Cover the cartridge with the DG8TM gasket (Figure 2). Figure 2. Workflow of droplet generation The QX200 droplet generator uses microfluidics to combine oil and aqueous samples to generate the nanoliter-sized droplets required for ddPCR analysis. It processes up to eight samples at a time in approximately 2 min. Transfer the entire volume of the droplet emulsion (typically 70 μL) to the desired wells of a semi-skirted 96-well ddPCR plate. Cover the 96-well ddPCR plate with a pierceable foil plate heat seal using a heat sealer according to the manufacturer’s instructions. Note: Take care to pipette very slowly to avoid disrupting the emulsified droplets. Place the 96-well ddPCR plate containing the PCR products into a thermal cycler and use cycling conditions as shown in Table 6. The annealing temperature was decided by the Tm value of the primers (normally < 5 °C than Tm value). Table 6. PCR cycling conditions for ddPCR. Use a 2 °C/s ramping rate for all steps. Step Temperature (°C) Time Number of cycles Enzyme activation 95 10 min 1 Denaturation 94 30 s 40 Annealing 58 1 min Signal stabilization 98 10 min 1 Hold 10 ∞ 1 Droplet counting Switch on the QX200TM droplet reader and open the QuantaSoftTM software after the PCR cycle is finished. Turn on the new experiment in the QuantaSoftTM software. Select the wells that need detection, enter the sample names, and record the well positions for target and reference genes for each sample. Select the key parameters in the wells that need detection as follows: Experiment: ABS (absolute quantitation). Supermix: QX200TM ddPCR EvaGreen supermix. Target 1 type: Ch1 unknown. Target 2 type: Ch2 unknown. Enter the sample name to distinguish transgenes and reference genes. Apply the chosen parameters to the selected wells by clicking OK. Place the 96-well PCR plate into the plate holder as follows (Figure 3): Place the 96-well PCR plate containing the amplified droplets into the base of the plate holder. Well A1 of the PCR plate must be in the top-left position. Move the release tabs of the top of the plate holder to the “up” position and place the top on the PCR plate. Firmly press down both release tabs to secure the PCR plate in the holder. Figure 3. Placing the 96-well plate into the plate holder After closing the lid, check if the indicator lights for “power,” “bottle level,” and “plate in place” are green. Click Run in the left navigation bar to start the run. In the Run Options window, select the detection chemistry. Note: If a probe supermix is selected in the well editor, the probe dye setting appears; select FAM/HEX or FAM/VIC. If an EvaGreen supermix is selected, the EvaGreen dye setting appears, and the screen confirms the number of EvaGreen wells configured on the plate. Data analysis Click the Analysis button to analyze the data after reading is complete. Click the 1D Amplitude button to analyze the data collected from each channel of selected wells. The radio button is used to select the channels and provides options for adjusting the thresholds used in assigning positives and negatives for each channel. An example distribution is shown in Figure 4. Note: A single reaction should produce 12,000–20,000 droplets. The calculation of copy number relies on the ratio of positive to negative droplets. Figure 4. Example plot showing positive droplets for six samples. Green and blue droplets indicate VIC and FAM probes. The x-axis (event number) indicates the number of droplets measured across the total experiment and the y-axis shows the amplitude of fluorescence. The image of these samples was published in a previous study (P. Liu et al., 2021). Click the 2D Amplitude button to view the channel 1 vs. channel 2 scatter plot and enable options for manually or automatically adjusting the thresholds used in assigning positives and negatives for each channel (Figure 5). Figure 5. Droplets of six examples visualized in two dimensions. The image of this sample was published in a previous study (P. Liu et al., 2021). Click the concentration button to visualize data in concentration plots. Export the concentration data as a comma-separated values (.csv) file. Note: The “copies/μL well” column displays the total amount of starting material in the ddPCR sample. Click Copy Number to view copy number for selected well/samples (Figure 6). Figure 6. Display of the calculated transgene copy number values of six examples. The image of these samples was published in a previous study (P. Liu et al., 2021). Click Ratio button to view ratio data for selected well/samples. Use the ratio buttons to select a plot of the Ratio (unknown: reference) or Fractional Abundance (% of sample). Click Events to view the number of droplet events counted for selected well/samples. Acknowledgments We acknowledge financial support from China Agriculture Research System from the Ministry of Agriculture of the P.R. China (CARS-03), National Natural Science Foundation of China (32100126, 31901954), Ningbo Science and Technology Innovation 2025 Major Project, China (Q21C140013), Natural Science Foundation of Zhejiang Province, China (LQ20C140002), and K.C. Wong Magna Funding Ningbo University. This protocol was developed to generate the transgene copy number in a previous study (P. Liu et al., 2021). Competing interests No competing interests to declare. References Bharuthram, A., Paximadis, M., Picton, A. C. P. and Tiemessen, C. T. (2014). Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes. Infect Genet Evol 25: 28-35. Bubner, B. and Baldwin, I. T. (2004). Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep 23(5): 263-271. Collier, R., Dasgupta, K., Xing, Y. P., Hernandez, B. T., Shao, M., Rohozinski, D., Kovak, E., Lin, J., de Oliveira, M. L. P., Stover, E., et al. (2017). Accurate measurement of transgene copy number in crop plants using droplet digital PCR. Plant J 90(5): 1014-1025. Chen, M., Sun, L., Wu H, Chen, J., Ma, Y., Zhang, X., Du, L., Cheng, S., Zhang, B., Ye, X., et al. (2014). Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene. Plant Biotechnol J 12(4): 447-456. Gowacka, K., Kromdijk, J., Leonelli, L., Niyogi, K. K. and Long, S. P. (2015). An evaluation of New and established methods to determine T-DNA copy number and homozygosity in transgenic plants. Plant Cell Environ 39(4): 908-917. Hanhineva, K. J. and Krenlampi, S. O. (2007). Production of transgenic strawberries by temporary immersion bioreactor system and verification by TAIL-PCR. Bmc Biotechnol 7(1): 11. Hindson, C. M., Chevillet, J. R., Briggs, H. A., Gallichotte, E. N. and Tewari, M. (2013). Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10): 1003-1005. Higuchi, R., Fockler, C., Dolinger, G. and Watson, R. (2013). Kinetic PCR: Real time monitoring of DNA ampli cation reactions. Biotechnol 11(9): 1026-1030. Hobbs, S., Warkentin, T. D. and Delong, C. (1993). Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21(1): 17-26. Liu, P., Zhang, X., Zhang, F., Xu, M., Ye, Z., Wang, K., Liu, S., Han, X., Cheng, Y. and Zhong, K. (2021). A virus-derived siRNA activates plant immunity by interfering with ROS scavenging. Mol Plant 14(7): 1088-1103. Liu, Y. G., Mitsukawa, N., Oosumi, T. and Whittier, R. F. (1995). Efficient isolation and mapping of Arabidopsis thealiana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8(3): 457-463. Sivamani, E., Li, X., Nalapalli, S., Barron, Y. and Que, Q. (2015). Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize. Transgenic Res 24(6): 1-11. Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3): 503-517. Srivastava, V., Vasil, V. and Vasil, I. K. (1996). Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.). Theor Appl Genet 92(8): 1031-1037. Wu, Y., Li, J., Li, X., Liang, J., Zeng, X. and Wu, G. (2017). Copy number and zygosity determination of transgenic rapeseed by droplet digital PCR. Oil Crop Science 2: 84-94. Vasil, I. K. (2007). Molecular genetic improvement of cereals: transgenic wheat (Triticum aestivum L.). Plant Cell Rep 26(8): 1133-1154. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant molecular biology Molecular Biology Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,568	https://bio-protocol.org/en/bpdetail?id=4568&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Conditioned Lick Suppression: Assessing Contextual, Cued, and Context-cue Compound Fear Responses Independently of Locomotor Activity in Mice YB Youcef Bouchekioua NN Naoya Nishitani YO Yu Ohmura Published: Vol 12, Iss 23, Dec 5, 2022 DOI: 10.21769/BioProtoc.4568 Views: 604 Reviewed by: Edgar Soria-GomezNanci Winke Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Translational Psychiatry Feb 2022 Abstract Pavlovian fear conditioning is a widely used procedure to assess learning and memory processes that has also been extensively used as a model of post-traumatic stress disorder (PTSD). Freezing, the absence of movement except for respiratory-related movements, is commonly used as a measure of fear response in non-human animals. However, this measure of fear responses can be affected by a different baseline of locomotor activity between groups and/or conditions. Moreover, fear conditioning procedures are usually restricted to a single conditioned stimulus (e.g., a tone cue, the context, etc.) and thus do not depict the complexity of real-life situations where traumatic memories are composed of a complex set of stimuli associated with the same aversive event. To overcome this issue, we use a conditioned lick suppression paradigm where water-deprived mice are presented with a single conditioned stimulus (CS, a tone cue or the context) previously paired with an unconditioned stimulus (US, a foot shock) while consuming water. We use the ratio of number of licks before and during the CS presentation as a fear measure, thereby neutralizing the potential effect of locomotor activity in fear responses. We further implemented the conditioned lick suppression ratio to assess the effect of cue competition using a compound of contextual and tone cue conditioned stimuli that were extinguished separately. This paradigm should prove useful in assessing potential therapeutics and/or behavioral therapies in PTSD, while neutralizing potential confounding effects between locomotor activity and fear responses on one side, and by considering potential cue-competition effects on the other side. Graphical abstract Schematic representation of the compound context-cue condition lick suppression procedure. Illustration reproduced from Bouchekioua et al. (2022). Keywords: Conditioned suppression Fear conditioning Context Cue Locomotor activity Rodents Pavlovian conditioning Background Pharmacological treatments for PTSD, such as the 5-HT2CR antagonist SB242084 (Browne et al., 2017), increase locomotor activity and could alternatively explain the reduction of fear responses when measuring the time spent in freezing. The potential locomotor activity confounding variable has thus to be neutralized before drawing any conclusion regarding the role of a treatment in fear responses. Exposure therapies developed to treat PTSD consist of gradually confronting patients to stimuli that are identified as triggers of the symptoms; yet patients do not necessarily have conscious access to these triggers. Moreover, according to associative learning theories, the intensity of fear responses to stimuli can be affected in situations where multiple conditioned stimuli (CSs), previously paired with the same aversive event, interact (Kamin, 1969; Rescorla and Wagner, 1972; Mackintosh, 1974; Pearce and Hall, 1980). It has been shown that a reduction in fear responding to a cue is associated with sustained contextual fear responses when cue-specific and contextual information are combined during conditioning, but tested separately (Grillon et al., 2002; Baas et al., 2008; Baas, 2013). Underestimating cue-competition effects may thus lead to an incomplete evaluation of potential therapeutics. To overcome these limitations and improve the screening efficiency of candidate drugs in fear conditioning, we propose a condition lick suppression procedure to assess contextual, cued, and context-cue compound fear responses. Materials and Reagents Black opaque acrylic or plastic sheet (height: 18 cm; width: 6 cm; thickness: 2 mm) Triangular-shaped ceiling made with two transparent acrylic sheets (21.5 × 13 cm, 1 mm thick) (YSTIME, catalog number: B08NPMVJGQ) Transparent acrylic hinges (IVYTOHO, catalog number: B07SMSJJK2) 0.5 mm thick high-density white polyethylene plastic sheet (Polymersan, catalog number: 0714038308181) 40 × 40 × 40 mm urethan rubber block (Tokyu Hands, catalog number: 4989606010889) Solder wire (Editone, Adafruit, catalog number: SAC305) Hook-up wire spool set, 22AWG solid core, 10 × 25 ft (Adafruit, catalog number: 3174) Momentary push button switch NO NC AC 5 A/120 V (DIYhz, catalog number: B079D923V9) DB9 D-SUB RS232 Adapter 9 pin signals terminal breakout plastic cover 2 row (male with screw) (Daier, catalog number: B01C2V2E8BU) IR break beam sensors with premium wire header ends, 3 mm LEDs (Adafruit, catalog number: 2167) MPR121 12 key capacitive touch sensor breakout (Adafruit, catalog number: B0778WYR5D) [Alternative: Adafruit 12 × capacitive touch shield for Arduino – MPR121 (Adafruit, catalog number: 2024)] Adafruit LED Sequins [luminous intensity: minimum = 250 mcd and maximum = 350 mcd with IF (Forward Current) = 20 mA; luminous intensity measurement allowance is ± 10%], warm white (Adafruit, catalog number: 1758) Speaker 8Ω8W (Akizukidenshi, catalog number: F00805) Amazon Basics USB 2.0 printer cable – A-Male to B-Male cord (Amazon Basics, catalog number: B00NH11KIK) White cotton/polyester knit gloves (Setaria Viridis, catalog number: B08QHLCZ11) Double sided tape (3M, catalog number: 0888519024546) Alligator to Dupont wire (Oiyagai, catalog number: B07CXLMBY7) Adult mice (8-week-old to 18-week-old) 70% ethanol solution Equipment Modular chamber (Lab-hacks, model: STCHK0001) Watering bottle with rubber stopper and water tube (CLEA Japan, catalog number: CL-0917) Sound attenuating cubicle for mouse (Med associates, model: MED-OFA-022) Standalone aversive stimulator/scrambler (115V/60Hz) (Med associates, model/catalog number: ENV-414S) [Alternative: Precision animal shocker (Coulbourne Instruments, Harvard Apparatus, catalog number: H13-15)] Arduino Mega2560 Rev3 (Arduino, catalog number: A000067) Raspberry Pi 3 model B+ [Raspberry Pi, catalog number: (Digi-Key)2648-RASPBERRYPI3MODELB+-ND] Digital Genuine Hakko with a standard T18-D16 tip (Hakko, catalog number: FX-888D) Software Arduino IDE (Arduino, https://www.arduino.cc/en/software) Anaconda (Anaconda, https://www.anaconda.com/) Spyder IDE (Included in Anaconda, https://www.spyder-ide.org/) Python 3 (https://www.python.org/downloads/) Python libraries: pyserial 3.5 (https://pyserial.readthedocs.io/en/latest/pyserial.html); csv, time, and os modules are part of Python’s standard libraries Excel (Microsoft, https://www.microsoft.com/fr-ww/microsoft-365/excel) Prism 9 (GraphPad Software, https://www.graphpad.com/company/) SPSS 23.0 (IBM, https://www.ibm.com/analytics/spss-statistics-software) Arduino and Python scripts (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression) Procedure Handling and water deprivation If mice are ordered from a supplier, group house same-gender mice (3–4 per cage) with unlimited access to food and water in a temperature/humidity-controlled (25 ± 2 °C, 40%–60%, respectively), 12 h reversed light/dark cycle room (light from 7 p.m. to 7 a.m.) for at least one week. If mice were born in the laboratory, make sure that they are group housed with same-gender animals (3–4 per cage), after being weaned at three weeks of age, with unlimited access to food and water in a temperature/humidity-controlled (25 ± 2 °C, 40%–60%, respectively), 12 h reversed light/dark cycle room (light from 7 p.m. to 7 a.m.). The whole procedure should be conducted during the dark phase of the light cycle (i.e., between 7 a.m. and 7 p.m.). Run mice in experiment 3 in context A during the morning and in context B during the afternoon. Run each mouse in experiments 1 and 2 within the dark cycle, around the same time of the first acclimation session (± 3 h). Single house all mice and handle them for 5 min for at least three consecutive days during the dark cycle. To habituate a mouse to handling, pick it up from the tail with one hand, and place it on the other hand while wearing cotton/polyester knit gloves. Gently keep the tail on one hand while the mouse is standing or walking on the other hand to prevent escapes. Handling is considered complete when mice stop trying to jump out of the hand, after at least three consecutive days of handling. The day following mice handling, remove water bottles from their home cage 24 h prior to the beginning of the experiment, during the dark cycle. Allocate mice (N = 7–11) to one of eight groups (Figure 1): Context-Only condition: Experimental group Context-Only condition: No-treatment Control group Context-Only condition: Unpaired Control group Cued-Only condition: Experimental group Cued-Only condition: No-treatment Control group Cued-Only condition: Unpaired Control group Compound condition: Experimental group Compound condition: No-treatment Control group Figure 1. Schematic representation of the procedure. Experimental flowchart for each condition and group. The last three columns from the left represent the three different phases of the procedure (Acclimation, Conditioning, and Test). Illustration reproduced from Bouchekioua et al. (2022). Acclimation phase (Day 1 to Day 5) Give each mouse a 30 min session of acclimation in the conditioning chamber inside a sound attenuating cubicle for five consecutive days (Figure 1). During the acclimation phase, mice have access to the water-filled lick tubes via the magazine. The number of licks and magazine entries are recorded. For the first two days of acclimation, leave two drops of water inside the magazine. Clean the chambers between animals with a 70% ethanol solution during the whole experiment. Make sure that the acclimation script “SPC_Acclimation.ino” (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression) is uploaded to the Arduino. If connected, disconnect the USB cable from the Arduino to the USB port of your PC (Mac OS or Windows) or Raspberry Pi. If you use a PC (Mac OS or Windows), open the Arduino IDE, left click Tools in the menu bar and place your mouse cursor over the Port row (Figure 2). Take note of the port names displayed, if any. Connect the Arduino USB cable to your PC and go back to the Port row of the Tools menu. Take note of the new port name that appears. It corresponds to the port name to which your Arduino is connected. Figure 2. Display of the USB port name from the Arduino IDE. (A) List of port names before connecting the Arduino to the PC. (B) List of port names after connecting the Arduino to the PC, including the port name used by the Arduino (i.e., “/dev/cu.usbserial-14110” in this case). If you use a Raspberry Pi with Linux (or any PC with a Linux OS), open the command terminal, type “ls /dev/tty”, and press the Enter key of your keyboard before connecting your Arduino to the USB port of the Raspberry Pi. Take note of the port names displayed. Connect the Arduino USB cable to your Raspberry Pi and repeat using the command “ls /dev/tty” on your command terminal. Take note of the new port name that appears. Open the Python 3 IDE (Spyder on PC and Python 3 IDLE on Raspberry Pi) and open the Python data collection script for the acclimation phase “Acclimation.py” (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression). Start running this Python script by pressing the Run File icon on Spyder (green right arrow) (Figure 3) or by left clicking Run in the menu bar and then left clicking on the Run File row on the Python3 IDLE of your Raspberry Pi. Alternatively, you can simply press the F5 key of your keyboard to start running the script. Figure 3. Menu bar of Spyder. Screenshot of the menu bar of Spyder. The Run File icon is highlighted by a red circle. Type the PC/Raspberry Pi USB port name to which your Arduino is connected and press Enter. Type a new file name and press Enter. Finally, type the time of the session in seconds and press Enter. Place the mouse in the conditioning chamber inside a sound attenuating cubicle and press the start button connected to your Arduino as soon as you release the mouse and close the front door of the chamber. Pressing the start button will turn the magazine line on. No other light is used for light housing. Use Context A for mice of the Experimental and No-treatment Control groups of the Context-Only condition (Figure 4). Figure 4. Context A during the acclimation phase. (A) Picture (from above) showing the configuration of the conditioning chamber used for Context A. The red arrow highlights a piece of transparent acrylic used as a stand and placed inside the magazine to facilitate access to the water port to mice. (B) Picture (from the inside of the chamber, facing the magazine) showing the configuration of the conditioning chamber used for Context A. The red arrows point to the magazine light, the two speakers, the water port, and the beam break sensor (the infrared light being at the opposite side of the magazine). The bottom and top of the speakers were at 3.5 and 8 cm from the grid floor, respectively. The speakers were fixed to the middle panel (i.e., the second panel from the top) of the wall (magazine side), using screws [cf., ChamberSideView (Water bottle and Speakers).jpeg, https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression/tree/main/BioProtocol%20Github]. The bottom and top of the beam break LEDs were fixed with super glue and hot glue at 1.5 and 2 cm from the grid floor, respectively. The bottom and top of the water port were at 2.5 and 3 cm from the grid floor, respectively. Holes for the beam break LEDs and the water port were drilled to the aluminum magazine with a conventional drill and drill bits. The positions of the speakers, water port, and beam break LEDs were the same for both contexts (i.e., contexts A and B). Use Context B for mice of the Unpaired Control group of the Context-Only condition, and mice of the Experimental, No-treatment Control, and Unpaired Control groups of the Cued-Only condition (Figure 5). Figure 5. Context B. Picture (from above) showing the configuration of the conditioning chamber used for Context B. The red arrows highlight the white plastic floor, the triangular-shaped ceiling (made of two 1 mm thick acrylic boards connected with two transparent acrylic hinges and forming an angle of 80°), the water port, and a white plastic board used to prevent escapes to the top of the triangular-shaped ceiling. Run all mice of the Compound condition (i.e., Experimental and No-treatment Control groups) twice a day. Use Context A in the morning and Context B in the afternoon six hours later (Figure 1). Remove the mouse from the chamber at the end of the 30 min session. Give access to water for 30 min at home cage after each session to mice in conditions Context-Only and Cued-Only. Give access to water for 30 min at home cage after the last session of the day to mice in condition Compound. Fear conditioning phase (Day 6) Place a black opaque plastic/acrylic sheet in front of the magazine to prevent magazine entries and access to the water-filled lick tube (Figure 6). Figure 6. Context A during the conditioning phase. (A) Picture (from above) showing the configuration of the conditioning chamber used for Context A during the conditioning phase. The access to the magazine is prevented by adding an opaque plastic board in front of it. (B) Picture from the inside of the chamber, facing the magazine. Upload the Conditioning Arduino script (to the Arduino) that corresponds to the condition the mouse belongs to and make sure that the foot shocker is turned on, connected to the Arduino, and set up to 0.5 mA. Upload the script “SPC_Phase_2_CS-US_30min.ino” to the Arduino for groups Experimental and No-treatment of conditions Cued-Only and Compound, and upload the script “SPC_Phase_2_US_30min.ino” to the Arduino for groups Experimental and No-treatment of conditions Context-Only (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression). Upload the script “SPC_Phase_2_US-CS_30min.ino” to the Arduino for mice of the Unpaired group in condition Cued-Only (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression). Mice of all groups and conditions, except the Unpaired group in condition Cued-Only, receive a 1 s, 0.5 mA foot shock at the following onsets: 5.15, 9.15, 14.15, 17.15, 21.15, and 25.15 min into the session. Mice of the Unpaired group in condition Cued-Only receive a 1 s, 0.5 mA foot shock at the following onsets: 8.5, 11.7, 15.9, 18.3, 22.25, and 23.8 min into the session. Mice of all groups in the Cued-Only and Compound conditions receive a 10 s, 12 kHz pure tone at the following onsets: 5, 9, 14, 17, 21, and 25 min into the session, that is, 9 s before the onset of each foot shock. Connect the Arduino to the PC/Raspberry Pi. Use Context A for mice of all groups and conditions (Figure 1). Place the mouse in the conditioning chamber inside a sound attenuating cubicle and press the start button connected to your Arduino as soon as you release the mouse. Remove the mouse from the chamber at the end of the 30 min session. Give access to water for 30 min at home cage after each session to all mice. Test phase (fear extinction: days 7–10) Give each mouse a 30 min session of fear extinction in the conditioning chamber inside a sound attenuating cubicle for only one day for mice of the Control Unpaired groups and four consecutive days for mice of all the other groups (Figure 1). During the test phase, mice have access to the water-filled lick tubes via the magazine. The number of licks and magazine entries are recorded. Follow steps B.1.a. to B.1.g except that the script for the Arduino and the Python data collection scripts should correspond to the test phase, condition, and groups. Upload the script “Acclimation.ino” to the Arduino for all mice in condition Context-Only (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression) and use the Python script “Acclimation.py” on your PC/Raspberry Pi for data collection. Test sessions of all mice in condition Context-Only are the same as acclimation sessions. Upload the script “SPC_TEST.ino” to the Arduino for all mice in condition Cued-Only and all mice in condition Compound for a.m. sessions (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression) and use the Python script “TEST.py” for data collection. Test sessions of all mice in condition Cued-Only and all mice in condition Compound for a.m. sessions consist of a tone cue delivery triggered up to five times by five cumulative seconds spent inside the magazine. The session ends if five tone cues are triggered, or after 30 min elapsed. Test sessions of all mice in condition Compound are the same as the morning acclimation sessions (i.e., step C.2.a) and the Cued-Only test sessions in the afternoon (i.e., step C.2.b). Data analysis Inclusion criteria Exclude mice that fail to drink from the water-filled lick tubes (i.e., 0 licks) on Days 1 and 2. Data processing Place the CSV files collected during the sessions you want to analyze into the same folder as the Python script for data processing corresponding to the condition and group you are analyzing. Use Python 3 for data processing. There are two Python scripts for data processing: Use the Python script “Contextual fear extinction.py” (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression) to analyze data collected from all groups of condition Context-Only and from all groups of Compound in the morning session (i.e., for contextual fear extinction). Open the file generated by the Python script whose name has “bin” added to the original CSV file name. The number of licks during the 30 min session appears under the column entitled “Lick #”. Use the other Python script “Cued fear extinction.py” (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression) to analyze data collected from all groups of condition Cued-Only and from all groups of Condition Compound in the afternoon session (i.e., for cued fear extinction). The suppression ratios for each tone triggered appear in the column entitled “Suppression ratio” of the CSV file generated by the Python script. Conditioned lick suppression ratio Baseline score For all groups of condition Contextual-Only and Compound (a.m. sessions), calculate the baseline score by averaging the rates of licks during the 30 min sessions of the two last days of the acclimation phase (i.e., Days 4 and 5). For all groups of condition Cued-Only and Compound (p.m. sessions), the baseline score is calculated by averaging the rates of licks during the 5 s period that precedes the onset of the tone cues. Performance score For all groups of conditions Contextual-Only and Compound (a.m. sessions), calculate the performance score by averaging the rates of licks during the 30 min sessions of each day of the test phase. For all groups of conditions Cued-Only and Compound (p.m. sessions), the performance score is calculated by averaging the rates of licks during the first 5 s of the tone cues. The conditioned suppression ratio for each tone triggered was already calculated by the Python script and can be found in column “Suppression ratio” of the generated file whose name has “_analyzed” added to the original CSV file name. Calculate the averaged suppression ratio for each group. Conditioned lick suppression ratio For each group calculate the averaged conditioned lick suppression ratio as follows: Conditioned Lick Suppression Ratio=(Baseline - Performance)/(Baseline + Performance) For comparison between Experimental and Control Unpaired groups of the Cued-Only and Compound conditions, use the suppression ratio of the first triggered tone of Day 7 only (Figure 7). Figure 7. Representation of the lick suppression ratios in condition Context-Only. (A) Contextual lick suppression ratios (Context-Only condition; Days 7–10) of mice from the No-treatment group (C57BL/6N wildtype male mice; N = 11) compared to that of mice from the Experimental group [5-HT2CR KO male mice (RRID:IMSR_JAX:015821); N = 11]. (B) Contextual lick suppression ratios (Context-Only condition; Day 7, Tone 1) of mice from the Control group (C57BL/6N wildtype male mice; N = 9) compared to that of mice from the Experimental group [5-HT2CR KO male mice (RRID:IMSR_JAX:015821); N = 11]. Illustration reproduced from Bouchekioua et al. (2022). Survival rate (probability of survival) For all groups of conditions Cued-Only and Compound (p.m. sessions), the survival rate corresponds to the number of tone cues triggered during a test session. The number of triggered tone cues appears in the column “Tone #” of the file generated by the Python script. The survival rate indicates the percentage of tone cues triggered within each group (Figure 8). A low number of tone cues triggered reflects a higher fear to the tone cue. Figure 8. Representation of the survival rate in condition Cued-Only. Percentage probability of survival rate (Cued-Only condition; Days 7–10) of mice from the No-treatment group (C57BL/6N wildtype male mice; N = 9) compared to that of mice from the Experimental group [5-HT2CR KO male mice (RRID:IMSR_JAX:015821); N = 9]. (A) Cued fear extinction session at day 1 (Ext D1). (B) Cued fear extinction session at day 2 (Ext D2). (C) Cued fear extinction session at day 3 (Ext D3). (D) Cued fear extinction session at day 4 (Ext D4). Illustration reproduced from Bouchekioua et al. (2022). Statistical tests Use an unpaired two-tailed Student’s t-test for independent samples, that is, for between-groups comparisons of lick suppression ratios during the first day of extinction (Day 7). As data from Experimental groups (Day 7) are used twice, use a Bonferroni correction. Use the suppression ratio of Day 7 for comparison between Experimental and No-treatment groups of the Context-Only condition. Use the suppression ratio of the first triggered tone of Day 7 only for comparison between Experimental and Control Unpaired groups, and between Experimental and No-treatment groups of the Cued-Only condition. Use the suppression ratio of the first triggered tone of Day 7 only for comparison between Experimental and No-treatment groups of the Compound condition. Use a two-way analysis of variance (ANOVA) for repeated measures, that is, for between-groups comparisons of lick suppression ratios across test sessions (fear extinction). Conduct a Bonferroni post-hoc test for multiple comparisons when applicable. If Mauchly’s sphericity test is significant, use Greenhouse-Geisser correction. When comparing Experimental and No-treatment group of the Compound condition, use a mixed-effects ANOVA if not all mice trigger the same number of tone cues. Use a log-rang (Mantel-Cox) test when comparing the survival rate performance between Experimental and No-treatment groups of the Cued-Only and Compound conditions. Notes Solder the wire connected to the MPR121 sensor directly to the water-filling lick tube (Figure 9). Figure 9. Wire soldered to the water-filling tube. Picture showing the wire [connecting the capacitive touch sensor (MPR121) to the water-filling tube] soldered directly to the water-filling tube. Use a heat shrinking tube to isolate the water-filling lick tube and cover the wire that connects the MPR121 sensor to the tube (Figure 10). When mice drink from a non-isolated water-filling lick tube, the capacitive touch sensor will only count one lick if any body parts (e.g., nose) are in contact with the tube while they drink water. By isolating the tube, the capacitive touch sensor detects only contacts between the tongue and the ball inside the tube (that prevents water leaking). The heat shrinking tube also prevents the tube to be in direct contact with the magazine. Figure 10. Heat shrinking tube. Picture showing the heat shrinking tube covering the water-filling tube and the wire connecting the capacitive touch sensor with the tube. To keep the water bottle in a fixed position with the spout of the water-filling tube being exposed inside the magazine, we super-glued a 40 × 40 × 40 mm urethane rubber block, previously drilled (10 mm of diameter) at an angle of approximately 45° [cf. “ChamberSideView (Water bottle and Speakers).jpeg”, available in our Github (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression/tree/main/BioProtocol%20Github]. Alternatively, we provide an *.STL file (“Lickometer MagAdapter.stl”) in the above-mentioned Github to 3D print a magazine adapter that supports the water bottle and includes a guide for the water-filling tube. Connect the Arduino’s ground (one of the GND pin) to the modular chamber with an alligator to Dupont wire. We found that it prevents false capacitive touch signals. Adapt the sensitivity of the sensor directly via its library (cf. “MPR121 Adafruit Doc” PDF file, p.15; https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression). Cover the top of the magazine with a plastic or acrylic board to prevent escapes from the conditioning chamber. Use a white plastic board to cover the higher part of the magazine (cf. Figure 5) to prevent mice from escaping to the top of the triangular-shaped ceiling of context B. Acknowledgments This work was supported by JSPS KAKENHI grants (numbers: JP18K07545, JP19H04976, and JP21K07473) awarded to Y.O. and a JSPS KAKENHI grant (number: JP19K23377) awarded to Y.B. This protocol was derived from Bouchekioua et al. (2022). We thank Dr. Nebuka, Dr. Sasamori, Ms. Sugiura, Dr. Sato, and Prof. Yoshioka for their support in the study Bouchekioua et al. (2022), from which this current protocol was derived. We also thank Nick Normal, Head of QC Makerspace, for having provided access to 3D printers. Competing interests The authors declare no competing interests. Ethics The treatment of animals used in Bouchekioua et al. (2022) complied with the Guidelines for the Care and Use of Laboratory Animals of the Animal Research Committee of Hokkaido University and all procedures were approved by the Animal Research Committee of Hokkaido University (approval no. 18-0070). References Baas, J. M., van Ooijen, L., Goudriaan, A. and Kenemans, J. L. (2008). Failure to condition to a cue is associated with sustained contextual fear. Acta Psychol (Amst) 127(3): 581-592. Baas, J. M. (2013). Individual differences in predicting aversive events and modulating contextual anxiety in a context and cue conditioning paradigm. Biol Psychol 92(1): 17-25. Bouchekioua, Y., Nebuka, M., Sasamori, H., Nishitani, N., Sugiura, C., Sato, M., Yoshioka, M. and Ohmura, Y. (2022). Serotonin 5-HT2C receptor knockout in mice attenuates fear responses in contextual or cued but not compound context-cue fear conditioning. Translational Psychiatry 12(1): 58. Browne, C. J., Ji, X., Higgins, G. A., Fletcher, P. J. and Harvey-Lewis, C. (2017). Pharmacological Modulation of 5-HT2C Receptor Activity Produces Bidirectional Changes in Locomotor Activity, Responding for a Conditioned Reinforcer, and Mesolimbic DA Release in C57BL/6 Mice. Neuropsychopharmacology 42(11): 2178-2187. Grillon, C. (2002). Associative learning deficits increase symptoms of anxiety in humans. Biol Psychiatry 51(11): 851-858. Kamin, L. J. (1969). In: Punishment and aversive behavior. (1st edition). Campbell, B. A. and Church, R. M. (Eds.). Ch. 9. Appleton-Century-Crofts, New York. Pearce, J. M. and Hall, l. G. (1980). A model for Pavlovian conditioning: Variations in the effectiveness of conditioned but not of unconditioned stimuli.Psychol Rev 87: 532-552. Mackintosh, N. J. (1974). The Psychology of Animal Learning. Academic Press, New York. Rescorla, R. A. and Wagner, A. R. (1972). In: Classical conditioning II: Current theory and research. Black, A. H. and Prokasy, W. F. (Eds.). Ch. 3. Appleton-Century-Crofts, New York. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Behavioral neuroscience > Learning and memory Neuroscience > Nervous system disorders > Animal model Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Protocol to Study Spatial Subgoal Learning Using Escape Behavior in Mice Philip Shamash and Tiago Branco Jun 20, 2022 1501 Views A Simplified Paradigm of Late Gestation Transient Prenatal Hypoxia to Investigate Functional and Structural Outcomes from a Developmental Hypoxic Insult Elyse C. Gadra and Ana G. Cristancho Oct 5, 2022 762 Views In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method Fayrouz Naim [...] Masaaki Murakami Apr 5, 2023 968 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,569	https://bio-protocol.org/en/bpdetail?id=4569&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Isolation and Expansion of Primary Conjunctival Stem Cells (CjSCs) from Human and Rabbit Tissues ZZ Zheng Zhong SC Shaochen Chen Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4569 Views: 991 Reviewed by: Vivien Jane Coulson-ThomasSudhir Verma Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Biomaterials Mar 2022 Abstract Conjunctival disorders are multivariate degenerative ocular surface diseases that can jeopardize ocular function and impair visual capacity in severe cases. The recent development of stem cell technologies has shed a new light on the treatment of conjunctival disorders as the regenerative medicine using endogenous stem cells becomes a potential therapeutic strategy. However, the efficient in vitro expansion of the endogenous stem cells dominating the conjunctival regeneration, the conjunctival stem cells (CjSCs), remains challenging. Existing protocols largely adopted primary culture using feeder layers, which has limited efficiency and risk of contamination. Here, we report a protocol for the isolation and expansion of primary CjSCs derived from human or animal tissues. This protocol adopts collagenase-based enzymatic digestion to release the primary cells from conjunctival tissues and utilizes a feeder-free culture strategy based on a small molecule inhibitor cocktail that stimulates the expansion of CjSCs. The CjSCs generated with this method were rapidly dividing and highly homogeneous. They also expressed characteristic stem cell markers and exhibited differentiation potency. These findings marked an important step forward in building stable CjSCs in vitro expansion, which will help researchers better understand the biology of ocular surface stem cells and develop innovative regenerative medicine approaches for ocular surface diseases. Graphical abstract Keywords: Stem cell Conjunctiva Conjunctival stem cell Endogenous stem cell Conjunctival goblet cell Stem cell culture Dual SMAD inhibition ROCK inhibition Background The conjunctiva is a significant part of the ocular surface that functions as the immune barrier and protects the integrity of the eyes (Nguyen et al., 2011; Gipson, 2016). Similar to the involuted epithelium on gastrointestinal and airway internal surfaces, it is comprised of nonkeratinized mucosal epithelium containing mucin-secreting goblet cells, which provide the fundamental support of the tear film as well as the homeostasis of the ocular surface (Barabino et al., 2012). Disorders including cicatrizing conjunctivitis, dry eye diseases, and Stevens-Johnson syndrome can disrupt the normal function of the conjunctiva, which could further damage the ocular surface and jeopardize the vision (Barabino et al., 2003; Kohanim et al., 2016). Although these conditions are threatening millions of patients worldwide, existing treatments based on pharmaceutical therapy and amniotic membrane transplantation are limited by mediocre efficacy and insufficient regeneration (Liu et al., 2010; Tseng et al., 2016). Recent advances in stem cell technology and regenerative medicine have made stem cell transplantation an alternative strategy for treating ocular surface diseases, and a huge interest has been raised in studying the endogenous stem cells residing in the conjunctiva, the conjunctival stem cells (CjSC) (Ramos et al., 2015; Nakamura et al., 2016; Williams et al., 2018; Zhong et al., 2021a). These are the bipotent progenitor that give rise to both conjunctival keratinocytes and conjunctival goblet cells, thus holding tremendous potential in conjunctival regeneration (Pellegrini et al., 1999; Majo et al., 2008; Nomi et al., 2021). However, as a critical premise in developing CjSC-based applications, the primary culture and in vitro expansion of CjSCs remains challenging. Early studies largely utilized feeder layers to support the primary culture of CjSCs, while the later ones tended to adopt the feeder-free culture system supplemented with cytokines and small molecules targeting key signaling pathways (Pellegrini et al., 1999; Stewart et al., 2015; Wu et al., 2020). The latest studies have demonstrated the efficacy of small molecule–based dual SMAD signaling inhibition (dSMADi) and ROCK signaling inhibition (ROCKi) in culturing epithelial stem cells derived from pulmonary alveoli, esophagus, and intestine (Mou et al., 2016; Zhang et al., 2018). Dual SMAD signaling (TGFβ and BMP signaling pathways) has been proved to regulate the maturation, self-renewal, and quiescence of epithelial stem cells, while ROCK signaling pathway contributes to the mechanotransduction and cell cycle progression (Kobielak et al., 2007; Amano et al., 2010; Tata et al., 2013). Therefore, we hypothesize that the combination of dSMADi and ROCKi can be applied to the CjSC culture and stimulate expansion. Here, we established an isolation and feeder-free culture method for CjSCs derived from both human and animal conjunctival tissues. The primary conjunctival epithelial cells were first isolated through enzymatic digestion and seeded on a collagen-coated surface. Then, the cells were subjected to primary culture with the conjunctival stem cell medium (CjSCM) encompassing a small molecule cocktail of dSMADi and ROCKi, which stimulated the outgrowth of the stem cell population. CjSCM outperformed the control medium in generating the cells with a higher replicative capacity and shorter doubling time. The cells cultured with CjSCM also showed upregulated expression of stemness and lineage markers while retaining the differentiation potency. This method can be applied to produce functional CjSCs and support the development of regenerative medicine and stem cell therapy for ocular surface diseases. Materials and Reagents Sterilization pouches BD syringe needle (BD, catalog number: 230-45094) 100 mm TC-treated culture dish (Corning, catalog number: 430167) Costar® 6-well plates (Corning, catalog number: 3516) Costar® 12-well plates (Corning, catalog number: 3513) FalconTM 70 μm cell strainers (Corning, catalog number: 08-771-2) Microcentrifuge tubes (ThermoFisher Scientific, catalog number: 3448PK) 15 mL conical centrifuge tubes (ThermoFisher Scientific, catalog number: 339651) 50 mL conical centrifuge tubes (ThermoFisher Scientific, catalog number: 339653) Millicell EZ SLIDE 8-well glass, sterile (Sigma-Aldrich, catalog number: PEZGS0816) Phosphate buffer solution (PBS) (ThermoFisher Scientific, catalog number: 10010023) Dulbecco's modified Eagle medium (DMEM) (ThermoFisher Scientific, catalog number: 11885084) DMEM/F12 medium (ThermoFisher Scientific, catalog number: 11330032) Penicillin-streptomycin (pen-strep) (ThermoFisher Scientific, catalog number: 15140122) 0.25% trypsin-EDTA (ThermoFisher Scientific, catalog number: 25200056) Fetal bovine serum (FBS) (ThermoFisher Scientific, catalog number: 10082147) Keratinocyte serum-free medium (KSFM) with bovine pituitary extract (BPE) (ThermoFisher Scientific, catalog number: 17005042) Collagen I, bovine (ThermoFisher Scientific, catalog number: A1064401) Insulin-transferrin-selenium (ITS-G) (ThermoFisher Scientific, catalog number: 41400045) 4% paraformaldehyde solution (ThermoFisher Scientific, catalog number: J19943.K2) Triton X-100 (ThermoFisher Scientific, catalog number: A16046.0F) Fluoromount-GTM mounting medium (ThermoFisher Scientific, catalog number: 00-4958-02) TRIzolTM reagent (ThermoFisher Scientific, catalog number: 15596026) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418) DAPI (Sigma-Aldrich, catalog number: D9542) Hydrocortisone (Sigma-Aldrich, catalog number: H0888) Cholera toxin (Sigma-Aldrich, catalog number: C8052) 3,3′,5-Triiodo-L-thyronine sodium salt (Sigma-Aldrich, catalog number: T6397) Recombinant human EGF protein (R&D Systems, catalog number: 236-EG) Recombinant human BMP-4 (BioLegend, catalog number: 795606) Recombinant human KGF (BioLegend, catalog number: 711702) Recombinant human IL-13 (BioLegend, catalog number: 571106) Recombinant human BMP-4 (BioLegend, catalog number: 795606) Y-27632 dihydrochloride (Tocris, catalog number: 1254) A83-01 (Tocris, catalog number: 2939) DMH-1 (Tocris, catalog number: 4126) Collagenase IV, powder (ThermoFisher Scientific, catalog number: 17104019) Direct-zolTM RNA purification miniprep kit (Zymo Research, catalog number: R2050) ProtoScript® II First Strand cDNA synthesis kit (New England Biolabs, catalog number: E6560L) Luna® Universal qPCR master mix (New England Biolabs, catalog number: M3003L) Conjunctival stem cell medium (CjSCM) (see Recipes) Control medium (see Recipes) Goblet cell differentiation medium (see Recipes) DMEM/F12 + pen-strep (see Recipes) DMEM/F12 + pen-strep + FBS (see Recipes) 0.5% type IV collagenase solution (see Recipes) Equipment Tweezers (Dumont, catalog number: 0203-54-PO) Surgical scissors (FST, catalog number: 15011-12) Hemocytometer (ThermoFisher Scientific, catalog number: 02-671-51B) Pipette sets (Eppendorf, catalog number: 2231300004) Centrifuge (Eppendorf, model: 5810R) Pipet-aid (Drummond Scientific, catalog number: 4-000-101) Biosafety cabinet (Labconco, model: 3460009) Orbital shaker (Benchmark, model: BT4001) Water bath (Fisher Scientific, model: 210) Cell culture incubator (VWR, model: 51014992) NanoDropTM 2000 spectrophotometer (ThermoFisher Scientific, catalog number: ND-2000) Microcentrifuge (ThermoFisher Scientific, catalog number: 75002492) StepOne M real-time PCR system (ThermoFisher Scientific, catalog number: 4376357) Confocal microscope (Leica, model: SP8) Autoclave machine (Tuttnauer, model: EZ9) Procedure Isolation of conjunctival epithelial cells from the human/rabbit conjunctival tissues The human/animal tissues used in this study were acquired from certified third-party facilities. Therefore, the procurement of eye tissues is not described in this protocol. Sterilize all forceps and tweezers in the autoclave before tissue processing. Conjunctival tissues from human donor biopsy: Transfer the human corneoscleral tissues from the preservation media to a sterile Petri dish and rinse with chilled PBS three times. Keep the tissues in chilled DMEM/F-12 with pen-strep upon dissection. Collect the conjunctival tissues from bulbar conjunctiva at 2–4 mm away from the limbus and keep the collected tissues in DMEM/F-12 with pen-strep. See Note 1. Conjunctival tissues from New Zealand white rabbit (Oryctolagus cuniculus) eyeballs: Transfer the rabbit eyeballs from the preservation media to a sterile Petri dish and rinse with chilled PBS multiple times. Clean the tissues with forceps and surgical scissors to remove the residual blood clots, eye muscles, and hairs. Immerse the cleaned eyeballs in chilled DMEM/F-12 with pen-strep upon dissection. Gently pull up the conjunctival tissue away from the sclera with a tweezer. Inject chilled DMEM/F-12 with pen-strep into the subconjunctival region of the bulbar conjunctiva that is 3–5 mm away from the limbus for the blunt separation of epithelial tissues (Figure 1). After the injection, let the eyeball stand for a couple minutes on an ice block or in the fridge (maintain moisture), which allows the solution to spread in the subconjunctival region. See Note 2. Cut and collect the conjunctival tissues with surgical scissors and keep the tissues in chilled DMEM/F-12 with pen-strep. See Note 3. Transfer the tissues to a sterile 100 mm Petri dish and add a few drops of medium to maintain the tissue moisture. Mince the dissected tissues with a surgical blade until they are fine enough to be aspirated using a T-1000 pipette. Transfer the minced tissues to a centrifuge tube (use a 15 mL or 50 mL tube depending on the number of tissues). Resuspend the minced tissues with 0.5% type IV collagenase solution. Incubate the solution at 37 °C with 5% CO2 under agitation at 150 rpm for 30–60 min. Stop the digestion when all sizable conjunctival tissues are digested (some white sclera tissues might be found in the solution; keep them in the solution and continue forward). Dilute the cell-collagenase solution with DMEM/F-12 with pen-strep (at least 1:1) to facilitate the centrifugation. Pellet the cells by centrifuging at 400 × g for 5 min. The centrifugation in this protocol was all performed at room temperature unless overwise stated. Resuspend the pellet with DMEM/F-12 with pen-strep and repeat the centrifugation (the pellet might be loose depending on the number of undigested tissue residues). Resuspend the pellet with 0.25% trypsin-EDTA and incubate the solution at 37 °C for 10 min, then quench the reaction by adding DMEM/F-12 with 10% FBS and pen-strep. Pellet the cells by centrifuging at 200 × g for 5 min. Proceed to Section B. Figure 1. Isolation of conjunctival tissues from rabbit eyeballs. Representative image showing the subconjunctival injection of DMEM/F-12 (plus phenol red) with pen-strep on the rabbit eyeball for the blunt separation (from left to right). (A) The conjunctival tissue on the injection site (red arrow) was gently pulled up with a tweezer and the injection was performed using a 30-gauge syringe needle. (B) After one injection, a bulge formed on the injection site (red arrow); the injection would be repeated on multiple sites (blue arrows indicating the potential injection sites). (C) An eyeball received complete injection and the injected solution spread in the subconjunctival region. (D) Isolated conjunctival tissues. Primary culture Collagen coating: incubate the plate/dish with a 5 µg/cm2 collagen I solution at room temperature for 1 h (the collagen-coated plate should be prepared freshly). Resuspend the pellet with 5 mL pre-warmed culture media (CjSCM or control media). Filter the cell solution with 70 μm cell strainers and measure the cell concentration with a hemocytometer (the straining can be skipped if the experiment does not require a precise seeding density; this will retain the residual microtissues in culture and allow them to grow as explants and increase the overall cell yield). Seed the cells at a density of 1 × 104 –2 × 104 cells/cm2 on a collagen-coated surface. Mark the cells as Passage 0 (P0). Perform the primary culture in the incubator at 37 °C with 5% CO2 for three days (avoid unnecessary movement). Change the culture media after three days and every other day from then on (pipette carefully and try to avoid disrupting the epithelial cell layer). See Note 4. Proceed to the Section C. Note: During the primary culture, CjSCs grow in homogeneous colonies. CjSCM can promote the formation of stem cell colonies and facilitate the outgrowth of CjSCs. The CjSC population can outgrow and dominate the total population during the primary culture phase. In vitro expansion of CjSCs Passage the P0 cells at 80%–90% confluence. Aspirate the culture supernatant and rinse three times with PBS. Add 0.25% trypsin-EDTA and incubate at 37 °C with 5% CO2 for 5 min. Stop the digestion by adding an equal amount of culture media with 10% FBS. Pellet the cells by centrifuging at 200 × g for 5 min. Resuspend the pellet with pre-warmed culture media. Filter the cell solution with 70 μm cell strainers and measure the cell concentration. Seed the cells at a density of 2 × 104 cells/cm2 on a collagen-coated surface. Mark the cells as P1. Perform medium change every other day and passage the cells at 80%–90% confluence. Mark the cells as P(n+1) after every subculture (n represents the number of passage times before the subculture). Note: The cells expanded with CjSCM are uniform in size and show compacted, cuboidal, and homogeneous morphology. In contrast, the cells cultured with the control medium that contains no dSMADi or ROCKi display an elongated spindle shape (Figure 2). Figure 2. CjSCM-expanded cells displayed uniform cell morphology. Representative bright field images of primary human conjunctival epithelial cells cultured with control medium (left) or CjSCM (right) at P3. Scale bar = 100 μm. Basic characterization of CjSCs Immunofluorescence staining and real-time quantitative PCR (qPCR) are performed to evaluate the expanded CjSCs in stemness, lineage, and proliferation by measuring corresponding markers on the transcriptional and post-transcriptional levels. Immunofluorescence staining Passage the cells at a density of 1 × 104 –2 × 104 cells/cm2 on a collagen-coated slide chamber and culture for 12–24 h (start the staining at 60%–80% confluence). See Note 5. Wash once with PBS and fix the cells with 100 μL of 4% (w/v) paraformaldehyde for 20 min at room temperature, avoiding light. Wash the samples three times with 100 μL of PBS (10 min incubation each time). Perform permeabilization and blocking by incubating the samples with 100 μL of 5% (w/v) BSA solution containing 0.3% Triton X-100 for 1 h at room temperature. Incubate the samples with the primary antibodies diluted in 100 μL of 5% (w/v) BSA solution at 4 °C overnight (antibodies are listed inTable 1). In this protocol, we adopted an epithelial stem cell marker (∆NP63), an ocular lineage marker (PAX6), and a proliferation marker (KI67) to evaluate the stem cell properties of the CjSCs. Wash the samples three times with 100 μL of PBS (10 min incubation each time). Incubate the samples with the secondary antibodies (Table 1) diluted in 100 μL of 5% (w/v) BSA solution for 1 h at room temperature, avoiding light. Wash the samples three times with 100 μL of PBS (10 min incubation each time), avoiding light. Incubate the samples with 100 μL of 1 mg/mL DAPI diluted in PBS for 10 min at room temperature, avoiding light. Make sure to wear personal protective equipment when working with DAPI. See Note 6. Remove the DAPI solution and rinse the sample with PBS. Aspirate all the solutions and disassemble the slide chamber. Air-dry the slides for 30–60 s. Mount the samples by adding Fluoromount-GTM mounting medium and seal the samples with coverslips. Proceed to imaging with the fluorescent microscope or confocal microscope. Table 1. Antibody list for immunofluorescence staining Primary Antibody Catalog Vendor Dilution Secondary Antibody Catalog Vendor Dilution Purified anti-p63 (∆N) Antibody 699501 BioLegend 1:500 Anti-rat IgG Alexa Fluor ® 647 Conjugate 4418S Cell Signaling Technologies 1:500 Purified anti-Pax-6 Antibody 901301 BioLegend 1:100 Anti-rabbit IgG Alexa Fluor ® 555 Conjugate 4413S Cell Signaling Technologies 1:500 Purified Mouse Anti-Ki-67 550609 BD Pharmagin 1:500 Anti-mouse IgG Alexa Fluor ® 488 Conjugate 4408S Cell Signaling Technologies 1:500 MUC5AC Monoclonal Antibody (45M1) 12178 ThermoFisher Scientific 1:100 Anti-mouse IgG Alexa Fluor ® 488 Conjugate 4408S Cell Signaling Technologies 1:500 Anti-MUC1 antibody [HMFG1 (aka 1.10.F3)] AB70475 Abcam 1:100 Anti-mouse IgG Alexa Fluor ® 488 Conjugate 4408S Cell Signaling Technologies 1:500 Anti-MUC16 antibody [X75] AB1107 Abcam 1:100 Anti-mouse IgG Alexa Fluor ® 488 Conjugate 4408S Cell Signaling Technologies 1:500 RNA extraction and real-time qPCR Digest the cells with 0.25% trypsin-EDTA, neutralize the digestion with culture media, and pellet the cells by centrifuging at 200 × g for 5 min. Add chilled TRIzol® reagent and lyse the cells by repeated pipetting. To ensure complete lysis, use at least 300 µL of TRIzol® per million cells. Immediately subject the lysed samples to RNA extraction or store at -80 °C. Extract the RNA using the Direct-zolTM RNA purification kit, following the manufacturer’s instructions. Measure the RNA concentration with NanoDropTM . Perform reverse transcription with ProtoScript® First Strand cDNA synthesis kit, following the manufacturer’s instructions, on the StepOneTM real-time PCR system. Perform qPCR using the Luna® Universal qPCR master mix according to the manufacturer’s instructions (primers are listed inTable 2) on the StepOneTM real-time PCR system. The PCR program was composed of a 60 s initial denaturation at 95 °C and 40 thermal cycles with a 15 s denaturation at 95 °C and a 60 s extension (signal capturing) at 60 °C. For quantitative analysis, GAPDH was used as an internal control. Table 2. Primer list for qPCR Human Gene 5'→3' KI67 Forward CTTTGGGTGCGACTTGACG Reverse GTCGACCCCGCTCCTTTT PAX6 Forward GTATTCTTGCTTCAGGTAGAT Reverse GAGGCTCAAATGCGACTTCAGCT P63 Forward CAGGAAGACAGAGTGTGCTGGT Reverse AATTGGACGGCGGTTCATCCCT VIM Forward GGACCAGCTAACCAACGACA Reverse TCCTCCTGCAATTTCTCCCG GADPH Forward CGACCACTTTGTCAAGCTCA Reverse AGGGGTCTACATGGCAACTG The immunofluorescence staining confirmed the expression of CjSC markers, highlighting the stemness, lineage, and proliferative activity of the cells cultured with CjSCM (Figure 3A). Real-time qPCR showed that the mRNA expression of epithelial stem cell marker and proliferation marker was upregulated in the cells cultured with CjSCM, while the expression of the mesenchymal marker was significantly downregulated (Figure 3B). Figure 3. Immunofluorescence and mRNA profiling of CjSCM-expanded cells. (A) Representative immunofluorescence images of ∆NP63, PAX6, and KI67 in the cells expanded in CjSCM or control medium at passage 3. Scale bar = 50 μm. (B) Real-time qPCR showing the relative mRNA expression of KI67, P63, PAX6 , and VIM in the cells expanded in CjSCM or control medium (mean ± SD, n = 4, : P < 0.05, : P < 0.001). Cell doubling quantification Cell doubling quantification is an optional experiment to measure the cell doubling time and replicative potential. We performed this experiment to validate the efficacy of the CjSCM in expanding CjSCs. The experiment was performed with fresh P0 cells that had not been subjected to any culture. Resuspend the pre-cultured P0 cells with pre-warmed media and seed the cells on a collagen-coated 12-well plate with 2 × 104 –4 × 104 cells per well (the number should be fixed among different groups for comparison). Perform medium change every other day. Passage the cells at 90% confluence. Aspirate the culture supernatant and rinse three times with PBS. Add 0.25% trypsin-EDTA and incubate at 37 °C with 5% CO2 for 5 min. Stop the digestion by adding an equal amount of culture medium with FBS. Pellet the cells by centrifuging at 200 × g for 5 min. Resuspend the pellet with a pre-warmed culture medium. Filter the cell solution with 70 μm cell strainers and measure the cell concentration. Keep the cell count as a record for later calculation. Seed the cells in a density of 1 × 105 cells per well on a collagen-coated 6-well plate. Repeat the culture until desired passage number is met. Draw the cumulative cell expansion curve by plotting cell doubling with time. Calculate the cell doubling time using the formula: DT=∆T∙ln 2/ln(Q2/Q1)(DT : doubling time; ∆T : culture time; Q1, Q2 : the cell counts of two passages). Quantification of cell doubling in long-term culture showed that the cells cultured with CjSCM exhibited faster dividing and significantly shorter doubling time compared to those cultured with the control medium (Figure 4). Figure 4. Cell doubling quantification. Representative cumulative curve of cell doublings and the average cell doubling time (P1–8) of the human primary conjunctival epithelial cells in culture with CjSCM or control medium (mean ± SD, n = 3; *: P < 0.001). Potency test: goblet cell differentiation The potency is an optional experiment to validate the goblet cell differentiation potency of the expanded CjSCs. The test should be performed with cells after P1 to ensure homogeneity. Seed the cells in a density of 2 × 104 cells/cm2 on a collagen-coated surface and culture the cells with CjSCM (the efficiency will be higher if the differentiation is performed on a 3D hydrogel matrix or collagen-coated Transwell membrane). Initiate the goblet cell differentiation when the cell confluence reaches 90%–100% by switching the culture medium to the goblet cell differentiation medium. Perform medium change every other day for 5–10 days (pipette carefully and try to avoid disrupting the epithelial cell layer). Examine the goblet cell differentiation efficiency by immunofluorescence staining of the characteristic mucins expressed in the conjunctival goblet cells. See Note 7. Note: The goblet-like cells with granules will start to appear after 3–5 days of differentiation; the number of these cells will increase over time. However, because the survival of goblet cells requires the support of surrounding cells, the number of viable goblet cells might drop in an extended differentiation after 10 days. In our test, we detected the expression of mucin proteins (MUC1, MUC5AC, and MUC16) in the cells after a seven-day differentiation, suggesting that the CjSCs expanded with CjSCM retained their differentiation potency (Figure 5). Figure 5. Goblet cell differentiation. Representative immunofluorescence staining of conjunctival goblet cell markers, MUC1, MUC5AC, and MUC16, and the corresponding bright field images of the post-differentiation cells. Scale bar = 50 μm. Notes For the purpose of growing human CjSCs, the starting materials can be whole human eyeball, ocular surface, or other type of corneoscleral tissues, as long as sufficient viable conjunctival tissues are present. Based on our experience, a minimal amount of 3–5 mm2 conjunctival tissues would be enough to establish the line but more tissue input would further ensure the success of the procedure. To ensure the viability of the isolated cells, the tissue should be processed within 72 h of the primary dissection (isolation from the donor). Using tissues with insufficient quantity or quality could significantly compromise the experiment results. Furthermore, this procedure was designed for isolating cells from normal/healthy donors. Modification of the procedure would be necessary for specific needs. The conjunctiva and limbus are adjacent anatomical structures on the ocular surface. Limbus is formed by the ring-shaped junction of corneal epithelium and conjunctival epithelium, which also separates the transparent cornea and opaque sclera (VanBuskirk, 1989). The conjunctiva is a thin mucous membrane that covers the outer surface of the sclera and the inner surface of the eyelids (Shumway et al., 2018). For the blunt dissection of the bulbar conjunctiva, the subconjunctival injection sites should be 3–5 mm away from the edge of the cornea, where the limbus is located. Our protocol mainly applies to bulbar conjunctiva, but may be modified accordingly for isolating other conjunctival regions (fornix, palpebral). The use of a dissecting microscope is recommended for the process. This step requires training in tissue processing and basic surgical skills; please get professional help if needed. The elastic nature of conjunctiva facilitates the blunt dissection. The subconjunctival injection will form bulges that separate a thin layer of the conjunctival epithelial tissues. The injection should be conducted slowly, and the syringe needle should be moved gently in the subconjunctival region to ensure a clear separation. Cut down the bulges from the bottom and ensure the collected tissues are outside of the limbus. The use of a dissecting microscope is recommended for the process. The size and shape of the collected tissues will depend on the blunt dissection and should not affect the overall yield. We tested the starting materials with only 200 viable P0 rabbit conjunctival cells and generated over a million cells in 10 days (unpublished data). In our experience, the conjunctival tissues collected from one rabbit eyeball could yield millions of cells. In most cases, attached cells started to grow into small colonies 3–5 days after the primary seeding. If residual microtissues were seeded, cells would also grow out from the tissue at the same time. The seeded cells consist of a mixed population derived from the conjunctival tissue, and CjSCM can promote the formation of compact stem cell colonies, which could rapidly dominate the total population during the primary culture. Based on our data, the ratio of KRT14-positive cells (mitotically active epithelial stem cells) was less than 2% in the cells freshly isolated from the rabbit conjunctival tissues and increased to more than 90% in the primary culture with CjSCM (unpublished data). However, the CjSC expansion efficiency can be affected by donor tissue status and isolation practice. CjSCs can grow into a compact cell sheet when they reach full confluence, which is ideal for exhibiting cell morphology. To better show cell morphology, the staining should be started with a confluence of 60%–80%. As the proliferation rate varies in cells from different donors or passages, the seeding density should be adjusted based on the cell status. Based on our experience, seeding around 10 thousand viable CjSCs per well in the Millicell EZ SLIDE 8-well glass chamber slide would result in 60%–80% confluence in less than 24 h. Direct contact with DAPI may cause eye irritation and skin irritation. DAPI is also harmful by inhalation or ingestion. Rinse immediately with water for several minutes if direct contact occurs during the practice. Move to fresh air in case of inhalation. Professional medical help can be necessary if any symptoms persist. The immunofluorescence staining of mucin follows the same procedures as described in section D, except for the permeabilization and blocking. For the staining of mucins, the fixed samples were permeabilized with PBS containing 0.2% Triton X-100 for 10 min, followed by a 1 h blocking with 5% (w/v) BSA. Recipes Conjunctival stem cell medium (CjSCM) Reagent Final concentration Amount DMEM N/A 440 mL DMEM/F-12 (1:1) N/A 440 mL FBS 10% (v/v) 100 mL Pen-strep 1% (v/v) 10 mL ITS-G 1% (v/v) 10 mL Hydrocortisone 400 ng/mL N/A Cholera toxin 0.1 nM N/A Recombinant EGF 10 ng/mL N/A 3,3′,5′-Triiodo-L-thyronine 2 nM N/A Y27632 10 μM N/A A83-01 1 μM N/A DMH1 1 μM N/A Total N/A 1,000 mL Control medium Reagent Final concentration Amount DMEM N/A 220 mL DMEM/ F-12 (1:1) N/A 220 mL FBS 10% (v/v) 50 mL Pen-strep 1% (v/v) 5 mL ITS-G 1% (v/v) 5 mL Hydrocortisone 400 ng/mL N/A Cholera toxin 0.1 nM N/A Recombinant EGF 10 ng/mL N/A 3,3′,5′-Triiodo-L-thyronine 2 nM N/A Total N/A 500 mL Goblet cell differentiation medium Reagent Final concentration Amount KSFM N/A 490 mL BPE N/A 25 mg Pen-strep 1% (v/v) 5 mL Recombinant IL-13 100 ng/mL N/A Recombinant BMP4 10 ng/mL N/A Recombinant KGF 10 ng/mL N/A Recombinant EGF 10 ng/mL N/A Total N/A 500 mL DMEM/F12 + pen-strep Reagent Final concentration Amount DMEM/ F-12 (1:1) N/A 495 mL Pen-strep 1% (v/v) 5 mL Total n/a 500 mL DMEM/F12 + pen-strep + FBS Reagent Final concentration Amount DMEM/ F-12 (1:1) N/A 445 mL FBS 10% (v/v) 50 mL Pen-strep 1% (v/v) 5 mL Total n/a 500 mL 0.5% type IV collagenase solution Reagent Final concentration Amount Type IV collagenase powder 0.5% 50 mg DMEM/ F-12 (1:1) N/A 9.9 mL Pen-strep 1% (v/v) 0.1 mL Total n/a 10 mL Acknowledgments This work was supported in part by grants from the National Institutes of Health (R21 EY031122, R01 EB021857) and National Science Foundation (1937653, 2135720). We also acknowledge the University of California San Diego School of Medicine Microscopy Core, and they were supported by National Institutes of Health grant P30 NS047101. This protocol was derived from “Rapid bioprinting of conjunctival stem cell micro-constructs for subconjunctival ocular injection” (Zhong et al., 2021b) and “Rapid 3D bioprinting of a multicellular model recapitulating pterygium microenvironment” (Zhong et al., 2022). Competing interests The authors declare no competing interests. Ethics The rabbit eyeballs were acquired from Sierra for Medical Science, Inc. (Whittier, CA) with the consent for biomedical research. The human corneoscleral tissue was provided by One Legacy or Saving Sight Eye Bank with consent for research use, and the corneoscleral tissue handling procedure was approved by the University of California, Los Angeles (UCLA) Institutional Review Boards (IRB#12-000363). The experimental work adhered to the tenets of the Declaration of Helsinki and the overall laboratory experimental procedure has been approved by the University of California, San Diego Institutional Biosafety Committee. References Amano, M., Nakayama, M. and Kaibuchi, K. (2010). Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Cytoskeleton (Hoboken) 67(9): 545-554. Barabino, S., Rolando, M., Bentivoglio, G., Mingari, C., Zanardi, S., Bellomo, R. and Calabria, G. (2003). Role of amniotic membrane transplantation for conjunctival reconstruction in ocular-cicatricial pemphigoid. Ophthalmology 110(3): 474-480. Barabino, S., Chen, Y., Chauhan, S. and Dana, R. (2012). Ocular surface immunity: homeostatic mechanisms and their disruption in dry eye disease. Prog Retin Eye Res 31(3): 271-285. Shumway, C. L., Motlagh, M. and Wade, M. (2018). Anatomy, Head and Neck, Eye Conjunctiva. StatPearls. Gipson, I. K. (2016). Goblet cells of the conjunctiva: A review of recent findings. Prog Retin Eye Res 54: 49-63. Kobielak, K., Stokes, N., De La Cruz, J., Polak, L. and Fuchs, E. (2007). Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic protein signaling. Proc Natl Acad Sci U S A 104(24): 10063-10068. Kohanim, S., Palioura, S., Saeed, H. N., Akpek, E. K., Amescua, G., Basu, S., Blomquist, P. H., Bouchard, C. S., Dart, J. K., Gai, X., et al. (2016). Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis--A Comprehensive Review and Guide to Therapy. I. Systemic Disease. Ocul Surf 14(1): 2-19. Liu, J., Sheha, H., Fu, Y., Liang, L. and Tseng, S. C. (2010). Update on amniotic membrane transplantation. Expert Rev Ophthalmol 5(5): 645-661. Majo, F., Rochat, A., Nicolas, M., Jaoude, G. A. and Barrandon, Y. (2008). Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 456(7219): 250-254. McCauley, H. A. and Guasch, G. (2015). Three cheers for the goblet cell: maintaining homeostasis in mucosal epithelia. Trends Mol Med 21(8): 492-503. Van Buskirk, E. M. (1989). The anatomy of the limbus. Eye (Lond) 3 (Pt 2): 101-8. Mou, H., Vinarsky, V., Tata, P. R., Brazauskas, K., Choi, S. H., Crooke, A. K., Zhang, B., Solomon, G. M., Turner, B., Bihler, H., et al. (2016). Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells. Cell Stem Cell 19(2): 217-231. Nakamura, T., Inatomi, T., Sotozono, C., Koizumi, N. and Kinoshita, S. (2016). Ocular surface reconstruction using stem cell and tissue engineering.Prog Retin Eye Res 51: 187-207. Nguyen, P., Khashabi, S. and C, S. (2011). Ocular Surface Reconstitution. In: Progress in Molecular and Environmental Bioengineering - From Analysis and Modeling to Technology Applications. Nomi, K., Hayashi, R., Ishikawa, Y., Kobayashi, Y., Katayama, T., Quantock, A. J. and Nishida, K. (2021). Generation of functional conjunctival epithelium, including goblet cells, from human iPSCs. Cell Rep 34(5): 108715. Pellegrini, G., Golisano, O., Paterna, P., Lambiase, A., Bonini, S., Rama, P. and De Luca, M. (1999). Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 145(4): 769-782. Ramos, T., Scott, D. and Ahmad, S. (2015). An Update on Ocular Surface Epithelial Stem Cells: Cornea and Conjunctiva. Stem Cells Int 2015: 601731. Stewart, R. M., Sheridan, C. M., Hiscott, P. S., Czanner, G. and Kaye, S. B. (2015). Human Conjunctival Stem Cells are Predominantly Located in the Medial Canthal and Inferior Forniceal Areas. Invest Ophthalmol Vis Sci 56(3): 2021-2030. Tata, P. R., Mou, H., Pardo-Saganta, A., Zhao, R., Prabhu, M., Law, B. M., Vinarsky, V., Cho, J. L., Breton, S., Sahay, A., et al. (2013). Dedifferentiation of committed epithelial cells into stem cells in vivo. Nature 503(7475): 218-223. Tseng, S. C., He, H., Zhang, S. and Chen, S. Y. (2016). Niche Regulation of Limbal Epithelial Stem Cells: Relationship between Inflammation and Regeneration. Ocul Surf 14(2): 100-112. Williams, R., Lace, R., Kennedy, S., Doherty, K. and Levis, H. (2018). Biomaterials for Regenerative Medicine Approaches for the Anterior Segment of the Eye. Adv Healthc Mater 7(10): e1701328. Wu, N., Yan, C., Chen, J., Yao, Q., Lu, Y., Yu, F., Sun, H. and Fu, Y. (2020). Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis. Stem Cells Transl Med 9(11): 1448-1461. Zhang, C., Lee, H. J., Shrivastava, A., Wang, R., McQuiston, T. J., Challberg, S. S., Pollok, B. A. and Wang, T. (2018). Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-beta Signaling. Cell Rep 25(3): 598-610 e595. Zhong, Z., Balayan, A., Tian, J., Xiang, Y., Hwang, H. H., Wu, X., Deng, X., Schimelman, J., Sun, Y., Ma, C., et al. (2021a). Bioprinting of dual ECM scaffolds encapsulating limbal stem/progenitor cells in active and quiescent statuses. Biofabrication 13(4). doi: 10.1088/1758-5090/ac1992. Zhong, Z., Deng, X., Wang, P., Yu, C., Kiratitanaporn, W., Wu, X., Schimelman, J., Tang, M., Balayan, A., Yao, E., et al. (2021b). Rapid bioprinting of conjunctival stem cell micro-constructs for subconjunctival ocular injection. Biomaterials 267: 120462. Zhong, Z., Wang, J., Tian, J., Deng, X., Balayan, A., Sun, Y., Xiang, Y., Guan, J., Schimelman, J., Hwang, H., You, S., Wu, X., Ma, C., Shi, X., Yao, E., Deng, S. X. and Chen, S. (2022). Rapid 3D bioprinting of a multicellular model recapitulating pterygium microenvironment. Biomaterials 282: 121391. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cell Biology > Cell isolation and culture Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,570	https://bio-protocol.org/en/bpdetail?id=4570&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Cell-derived Matrix Assays to Assess Extracellular Matrix Architecture and Track Cell Movement KM Kendelle J. Murphy DR Daniel A. Reed CC Cecilia R. Chambers JZ Jessie Zhu AM Astrid Magenau BP Brooke A. Pereira PT Paul Timpson DH David Herrmann Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4570 Views: 1554 Reviewed by: Yoshihiro AdachiSalah Boudjadi Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Oct 2021 Abstract The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell–ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell–ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes. Keywords: Cell-derived matrix Cancer Fibroblasts Extracellular matrix Biomechanics Cell–matrix interactions Second harmonic generation imaging Background The extracellular matrix (ECM) is the non-cellular compartment of all tissues; it is fundamental for cellular processes, providing biomechanical and biochemical cues, which can be sensed by cells activating downstream signaling pathways that can affect cell behavior (Vennin et al., 2017; Cox, 2021; Romani et al., 2021). The ECM is a highly regulated scaffold whose function is dependent on its precise molecular composition and assembly of fibrillar tissue. The highly regulated ECM is an adaptive network of fibers that respond to external forces and tensional strains, whilst also providing cells with biomechanical feedback regulating homeostasis and normal tissue function (Cox, 2021). However, in cancer, the co-option of this tightly organized infrastructure, including ECM deposition and remodeling, can trigger oncogenic signaling, leading to increased cell motility, proliferation, survival, and metastasis (Nicolle et al., 2017; Neesse et al., 2019;Cox, 2021). Although the cancer stroma was initially thought to predominantly stimulate tumor progression, recently it was shown to also restrain cancer cells (Rhim et al., 2014; Özdemir et al., 2014). Therefore, a fine-tuned approach to target the pro-tumorigenic functions of the stroma while maintaining its anti-tumorigenic roles may provide potential for anti-cancer therapies. Cancer associated fibroblasts (CAFs) are a prominent stromal cell type, which can be activated and educated by cancer cells (Pereira et al., 2019; Sahai et al., 2020). Their ability to synthesize and remodel ECM is a key driver of tumor progression. As such, there is a clear need to study the influence of the ECM on cancer cell behavior and assess the potential of ECM-targeted therapies to alter cellular locomotion and chemosensitivity. Initial methodologies and protocols to generate cell-derived matrices (CDMs) were designed to better understand the complexity of cell-matrix adhesion and cell surface structures in a 2.5D to 3D context (Cukierman et al., 2001; Cukierman, 2002; Erami et al., 2016; Franco-Barraza et al., 2016). To decipher the multifaceted influence of the ECM on cancer progression and therapeutic response, CDMs can be used to characterize the effect that genetic or pharmacological manipulation have on ECM ultrastructure. Here, we provide a step-by-step protocol for the generation of CDMs and subsequent quantification of matrix abundance, architecture, and organization (Figure 1). This protocol was utilized in our recent publication to assess fine-tuned manipulation of the matrix following focal-adhesion kinase inhibition, to reduce cancer cell locomotion and improve the efficiency of chemotherapy (Murphy et al., 2021). The protocols presented below contain steps that can be modified in a cell type–specific manner, for normal fibroblasts as well as CAFs. We also highlight the utility of CDMs to assess the potential of anti-fibrotic agents to alter ECM production and remodeling, which has recently shown promise to reduce metastasis and improve chemotherapy performance in a range of cancer types (Miller et al., 2015; Rath et al., 2018; Vennin et al., 2017, 2019; Boyle et al., 2020; Wu et al., 2020; Murphy et al., 2021). Furthermore, we provide protocols for assessing cancer cell behavior when seeded on CDMs, which can provide insight into cancer cell proliferation, survival, and single vs. collective cell migration in an ECM-rich environment (Figure 2). Here, CDMs can provide a rapid and pliable readout of stromal and cancer response to genetic and pharmacological intervention in vitro, which can be used to inform subsequent in vivo studies. Figure 1. Schematic representation of cell-derived matrix (CDM) production and cancer cell seeding on top of CDMs following fibroblast decellularization. Here, fibroblasts are seeded and allowed to grow to confluence (a.) prior to supplementation with ascorbic acid to enhance matrix production (b.). Following matrix production, fibroblasts are decellularized from the matrix (c.) and cancer cells may be seeded on top (d.). As an example timeline (e.), telomerase-immortalized fibroblasts (TIFs) are provided with ascorbic acid 24 h after cell seeding to allow matrix production for six days, prior to decellularization on day 7, followed by matrix analysis or cancer cell seeding and tracking [here shown as the example of pancreatic cancer cells isolated from the KPC ( Kras G12D/+ ; p53 R172H/+ ; Pdx-1Cre ) mouse model of pancreatic cancer (Morton et al., 2010; Murphy et al., 2021)]. Parts a-d are adapted from Murphy et al. (2021), CC BY 4.0 ( https://creativecommons.org/licenses/by/4.0/ ). Figure 2. Potential application of cell-derived matrices for analysis of matrix ultrastructure. Examples with intact fibroblasts, including picrosirius red staining (a.) imaged using bright field microscopy (top left), binary overlay (bottom left), and by polarized light (birefringence, top right) to assess collagen I and III abundance and orientation (bottom right). Second-harmonic generation (SHG) (b.) imaging (top) and analysis of fiber orientation (bottom). Applications of CDMs with fibroblast removed and cancer cells seeded on top to assess single cancer cell migration (left) and collective migration (right). Materials and Reagents For CDMs 12-well plate Cultured fibroblasts [e.g., telomerase-immortalized fibroblasts (TIFs) or CAFs] Trypsin/EDTA solution (Life Technologies, catalog number: 15400) Fibroblast growth medium [specific to fibroblast line; e.g., for TIFs: DMEM (Thermo Fisher Scientific, catalog number: 11995065) supplemented with 10% FBS] Phosphate-buffered saline (PBS) (Life Technologies, catalog number: 14190) Gelatin (Sigma-Aldrich, catalog number: G1393), store at 4 °C 10% neutral buffered formalin (Australian Biostain P/L, catalog number: ANBFC.10L) Glycine (Sigma-Aldrich, catalog number: G7126) (+)-Sodium L-ascorbate (Sigma-Aldrich, catalog number: A7631), make fresh DNase I (Roche, catalog number: 05025), aliquot and store at -20 °C (do not freeze-thaw) Fungizone (Amphotericin B, Life Technologies, catalog number: 15290) Penicillin/streptomycin (pen/strep) (Thermo Fisher Scientific, catalog number: 15070-063) 70% ethanol 0.2% sterile gelatin (see Recipes) 1 M sterile glycine (see Recipes) Ascorbic acid (1,000×), prepare fresh (see Recipes) Extraction buffer (see Recipes) Triton X-100 (Sigma-Aldrich, catalog number: 9284) Ammonium hydroxide Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750) Supplemented PBS (see Recipes) Calcium chloride (Chem Supply, catalog number: CA033) Magnesium sulfate (Chem Supply, catalog number: MA048) Supplemented PBS with antibiotics and antifungals (see Recipes) For picrosirius red staining 0.1% picrosirius red stain (Abcam, catalog number: ab150681) Phosphomolybdic acid hydrate (Sigma-Aldrich, catalog number: 221856) Glacial acetic acid (Chem Supply, catalog number: AA009-2.5L-P) Reverse Osmosis (RO) water 0.02% phosphomolybdic acid (see Recipes) 10% phosphomolybdic acid stock (see Recipes) Acidified water (see Recipes) Equipment 12-well glass bottom dishes (Celvis, catalog number: D35-20/4.5N) Sterile glass bottles (for storage of gelatin, extraction buffer, and glycine) Picrosirius red staining kit (Australian Biostain, APSRA, 500 mL) Software TWOMBLI (Wershof et al., 2021): https://github.com/wershofe/TWOMBLI FibrilTool (Boudaoud et al., 2014): https://www.quantitative-plant.org/software/fibriltool Automated ECM fiber orientation and alignment (Mayorca-Guiliani et al., 2017): https://github.com/TCox-Lab/Collagen_Orientation Automated picrosirius red and collagen birefringence analysis using polarized light (Vennin et al., 2019): https://github.com/TCox-Lab/PicRed_Biref Cell Tracker (not only) for Dummies (Piccinini et al., 2016): http://celltracker.website/about-celltracker.htmL Procedure Under sterile tissue culture conditions, prepare tissue culture glass bottom dishes Note: Although this step is not strictly necessary, it is recommended to stabilize the anchoring of the matrices to the glass surface of the dish. Coat glass bottom dishes (in a 12-well plate) with 1 mL of 0.2% sterile gelatin and incubate for 60 min at 37 °C. Wash dishes twice with warm (37 °C) PBS. Crosslink gelatin layer with 1 mL of 10% neutral buffered formalin and incubate for 30 min at room temperature. Wash twice with warm (37 °C) PBS. Quench the formalin crosslinker by the addition of 1 M sterile glycine and incubation for 20 min at room temperature. Wash twice with warm (37 °C) PBS. Add growth media to the dishes and incubate for 30 min at 37 °C. Use immediately or store in supplemented PBS with 2.5 µg/mL fungizone and 5% pen/strep sealed with parafilm to prevent evaporation for up to four weeks. After storing the coated dishes: Wash twice with warm (37 °C) PBS. Add growth media to the dishes and incubate for 30 min at 37 °C before proceeding to Procedure B. Plating fibroblasts and matrix production Note: This will vary depending on cell type. Detach and harvest fibroblasts as per established protocols for the specific cell line. Dilute fibroblasts and plate as appropriate: This will vary depending upon fibroblast type and if the cell line has overcome contact inhibition of proliferation. For lines that stop growing upon contact, such as TIFs, appropriate cell numbers should be plated to reach 90%–100% confluence in 12–24 h. For lines that continue to grow upon contact, such as pancreatic cancer CAFs (Vennin et al., 2019), appropriate cell numbers should be plated to reach 30%–40% confluence in 12–24 h. This should be determined prior to the experiment for each fibroblast cell line. For example, TIFs should be seeded at 2.5 × 105 cells per well of a 12-well plate the day prior to ascorbic acid supplementation to then obtain a CDM after six days (please also seeFigure 1e). Culture fibroblasts overnight at 37 °C. The oxygen content is dependent on the specific cell line being used. Replace media with fresh growth media supplemented with 50 µg/mL ascorbic acid and appropriate drug if applicable. Treatment timing will vary depending on fibroblast type: for lines that stop growing upon contact, wait until the cells are confluent to begin treatment. If cells continue to grow upon contact, then commence treatment when cells are 30%–40% confluent. This should be determined for each fibroblast cell line. For some fibroblast cell lines, it is beneficial to treat with 100–500 µg/mL of ascorbic acid on Day 1 of supplementation, switching to 50 µg/mL to increase matrix thickness. This should be determined individually for each cell line prior to the experiment. Change ascorbic acid media every second day. If treating cells with a pharmacological agent, refresh both drug and ascorbic acid at the same time. For cell lines that require conditioned media, remove and replenish half of the media volume with fresh media supplemented with 100 µg/mL of ascorbic acid to achieve a final concentration of 50 µg/mL. Culture for 6–10 days depending on the rate of matrix production. The longer matrices are left to develop the more likely they are to detach. The ideal culturing and matrix production should be determined for each independent fibroblast cell line prior to the experiment. Time course assays are recommended to determine this timeframe. From here, CDMs can either be used for the analysis of ECM ultrastructure (see Data analysis for further detail) or denuded to remove fibroblasts (Figures 1, 2). ECM ultrastructure analysis is discussed in step B7. We recommend leaving the fibroblasts intact to ensure that cell–matrix interactions of both fibroblasts and matrix are upheld. For analysis of the ECM by imaging or histological staining: Second-harmonic generation (SHG) imaging: perform on unfixed matrices with fibroblasts intact to visualize fibrillar collagen I. Picrosirius red staining/birefringence imaging: fix matrices with intact fibroblasts overnight in 10% neutral buffered formalin, then change to 70% ethanol prior to picrosirius red staining (as per manufacturer’s instructions, see steps i.–v. below) and imaging. CDMs should be imaged immediately. Incubate CDMS in 0.02% phosphomolybdic acid for 2 min. Rinse three times with Reverse Osmosis (RO) water. Stain CDMs with 0.1% picrosirius red solution for 1 h. Rinse twice with acidified water (10 s for wash 1 and 2 min for wash 2). Rinse three times with 70% ethanol (store in 70% ethanol for imaging and do not let the CDMs dry out). For analysis of cancer cell interactions with CDMs, proceed to Procedure C and D . Denuding fibroblasts Aspirate media and wash cells twice with PBS. Gently add pre-warmed extraction buffer, sufficient to cover the cell/matrix layer. Lyse for roughly 2 min. Cell lysis is virtually instantaneous but can vary depending on matrix thickness. Leave the extraction buffer on until no intact cells are visible by transmitted light microscopy. It is not recommended to lyse for more than 5 min as the CDM is delicate and extended incubation time in extraction buffer can cause detachment. From this point onwards, it is also recommended not to use suction for media and buffer changes anymore. Aspirate extraction buffer and wash twice with supplemented PBS (see Recipes). Incubate in supplemented PBS with 10 mg/mL DNase I to minimize DNA debris in the CDMs. Aspirate DNase and wash twice with supplemented PBS. Use immediately or store at 4 °C in supplemented PBS with antibiotics and antifungals (pen/strep 5% and 0.25 µg/mL fungizone) sealed with parafilm to prevent evaporation for up to two weeks. Preparation of denuded matrix for plating of cancer cells If matrices have been stored, allow them to acclimatize to room temperature before washing twice with supplemented PBS. Otherwise, continue directly to step D3. As per instructions from Franco-Barraza et al. (2016), if you are performing antibody staining following the seeding of cancer cells, it is recommended to block the matrices with BSA. Otherwise, continue to step D3. Incubate matrices in appropriate growth media for the cancer cells for 30 min at 37 °C. Remove media and seed cancer cells at the appropriate density: For example, using KPC pancreatic cancer cells from the KPC (Kras G12D/+; p53 R172H/+; Pdx-1Cre) mouse model of pancreatic cancer, seed at a density of 2.5 × 104 for migration assays or 1 × 105 for biochemical assays per well of a 12-well plate. For analysis of cell migration, seeding density may have to be further optimized to track single cell or collective cell migration; we recommend tracking cells for 72 h. Data analysis For analysis of the ECM by imaging or histological staining: SHG imaging to visualize fibrillar collagen I (Figure 2b): Perform SHG imaging using a multiphoton microscope [excitation tuned to a wavelength of 880 nm with detection at 440 nm, as previously performed (Vennin et al., 2017;Wu et al., 2020;Murphy et al., 2021)] equipped with precise z-control capacity, ensuring to acquire: z-stacks (we recommend 2.52 µm steps, with 512 µm × 512 µm representative fields of view, acquired at 25× magnification and a line average of 4) for intensity-based analysis; High-resolution single plane images for analysis of the matrix ultrastructure (1,024 µm × 1,024 µm representative fields of view, acquired at 25× magnification and a line average of 64). Analyze SHG intensity of z-stacks to determine the amount of cross-linked collagen I present in the samples over depth, with z-stack imaging allowing the maximum SHG signal to be obtained. High-resolution images can then be processed through: TWOMBLI, as per protocol instructions (Wershof et al., 2021, see Software). Automated ECM fiber orientation and alignment analysis, to assess the distribution and orientation of collagen fibers (Mayorca-Guiliani et al., 2017). CDMs can then be formalin-fixed and stained with picrosirius red. Picrosirius red staining (Figure 2a; this can be performed on CDMs with intact fibroblasts or denuded to reduce background): Perform polarized light imaging at a high resolution (we recommend 1,024 × 1,024) at 20× zoom (brightfield images can also be acquired but polarized light reduces background noise and provides fibrillar images for further analysis). Analyze images using: TWOMBLI, as per protocol instructions (Wershof et al., 2021, see Software). Potential readouts: High-density matrix, endpoints, curvature, fractal dimension, and branchpoints. Automated ECM fiber orientation and alignment (Mayorca-Guiliani et al., 2017). Potential readouts: Fiber orientation and alignment. Automated picrosirius red and birefringence analysis of collagen I/III using polarized light (Vennin et al., 2019). Potential readouts: Collagen deposition and maturity of fibrillar bundles; further analysis can be performed using TWOMBLI. FibrilTool plug-in on ImageJ (Boudaoud et al., 2014). For this tool, it is best to segment the image into separate regions of interest and analyze each region, followed by averaging the results for each image. Potential readouts: Image anisotropy (organization) and analysis of collagen fibers. For analysis of cancer cell migration: Tracking of single cancer cell migration: Following denuding of the CDM and seeding of cancer cells, collect videos of cellular migratory patterns over time, imaged at 15 min intervals (Video 1). For tracking single cancer cell migration, we recommend having a low density of cells (starting at roughly 5% confluence to ensure single cells can be identified and tracked over time). Analyze single cell migration using Cell Tracker (not only for) Dummies (Piccinini et al., 2016) and plot polar plots of cell migration (we recommend using the in-built Matlab application to pertain readouts of cell speed, direction, persistence, and total distance). Video 1. KPC cell migration across TIF-generated CDMs Tracking of collective cancer cell migration: Following denuding of the CDM and seeding of cancer cells, collect videos of cellular migratory patterns over time at 2 h intervals (Video 2). We recommend having a medium density of cells (starting at roughly 30% confluence to ensure that collective cellular patterns can be identified). Analyze collective cell migration by converting greyscale images to binary images in ImageJ. Analyze binary images using FibrilTool (Boudaoud et al., 2014). For this tool, it is best to segment the image into separate regions of interest and analyze each individual region, followed by averaging the results for each image to provide an overall analysis of collective cell migration. Video 2. KPC cell streaming across TIF-generated CDMs Notes CDMs are best used immediately rather than after long-term storage: in our hands, SHG signal intensity is stable for up to three days and then reduces over time. Continued cell proliferation during CDM production can result in the CDM detaching from the surface. For CDM production by fibroblasts that have not overcome contact inhibition, it is recommended to start ascorbic acid supplementation at 30%–40% confluence. When seeding fibroblasts onto the gelatin-coated dishes, some primary fibroblast lines are sensitive to the gelatin coating process. Here, we recommend an extended incubation at step A7 with twice the normal concentration of FBS to ensure that cells can adhere properly to the base. For further analysis and methodologies, such as immunofluorescence, please also refer to Franco-Barraza et al. (2016). Recipes 0.2% sterile gelatin Reagent Final concentration Amount Gelatin 0.2% (w/v) 8 mL PBS n/a 72 mL Total n/a 80 mL Filter-sterilize prior to use 1 M sterile glycine Reagent Final concentration Amount Glycine 1 M 6 g PBS n/a 80 mL Total n/a 80 mL Filter-sterilize prior to use Ascorbic acid (1,000×): prepare fresh Reagent Final concentration Amount (+)-Sodium L-ascorbate 50 mg/mL 0.05 mg PBS n/a 1 mL Total n/a 1 mL Filter-sterilize prior to use Extraction buffer Reagent Final concentration Amount Triton X-100 0.5% (v/v) 400 µL NH4 OH 20 mM 1.6 mL Sodium deoxycholate 1% (v/v) 800 µL PBS n/a 77.2 mL Total n/a 80 mL Filter-sterilize prior to use Supplemented PBS Reagent Final concentration Amount Calcium chloride (CaCL2 ) 1 mM 500 µL Magnesium sulfate (MgSO4 ) 1 mM 500 µL PBS n/a 499 mL Total n/a 500 mL Filter-sterilize prior to use Supplemented PBS with antibiotics and antifungals Reagent Final concentration Amount Pen/strep 100 U/100 µg/mL 5 mL Fungizone 0.25 µg/mL PBS n/a 495 mL Total n/a 500 mL Filter-sterilize prior to use 10% phosphomolybdic acid stock Reagent Final concentration Amount Phosphomolybdic acid hydrate 10% 10 g Distilled water n/a 100 mL Total n/a 100 mL 0.02% phosphomolybdic acid stock Reagent Final concentration Amount Phosphomolybdic acid 10% stock 0.02% 4 mL Distilled water n/a 196 mL Total n/a 200 mL Acidified water Reagent Final concentration Amount Glacial acetic acid (17.4 M) 87.1 mM/0.5% 5 mL Distilled water n/a 995 mL Total n/a 1,000 mL Acknowledgments This study was supported by the National Health and Medical Research Council (NHMRC), Australian Research Council (ARC), Cancer Council NSW, Cancer Institute NSW (CINSW), Cancer Australia, Tour de Cure, St. Vincent’s Clinic Foundation, Australian Government Research Training Program Stipend, Baxter Family Postgraduate Scholarship and Suttons. This work was made possible by an Avner Pancreatic Cancer Foundation (now PanKind) Grant and the ACRF INCITe Centre. P.T. is supported by the Len Ainsworth Fellowship in Pancreatic Cancer Research and is a National Health and Medical Research Council (NHMRC). K.J.M, B.A.P, and D.H. are supported by CINSW Early Career Research Fellowship. This protocol is largely derived from the original works (Cukierman et al., 2001; Franco-Barraza et al., 2016; Murphy et al., 2021). Competing interests P.T. receives reagents from Kadmon Inc., InxMed (consultant), Redx Pharma, Équilibre Biopharmaceuticals, and Amplia Therapeutics. Under a licensing agreement between Amplia Therapeutics and Garvan Institute of Medical Research, K.J.M., D.H., and P.T. (consultant) are entitled to milestone payments. References Boudaoud, A., Burian, A., Borowska-Wykret, D., Uyttewaal, M., Wrzalik, R., Kwiatkowska, D. and Hamant, O. (2014). FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nat Protoc 9(2): 457-463. Boyle, S. T., Poltavets, V., Kular, J., Pyne, N. T., Sandow, J. J., Lewis, A. C., Murphy, K. J., Kolesnikoff, N., Moretti, P. A. B., Tea, M. N., et al. (2020). ROCK-mediated selective activation of PERK signalling causes fibroblast reprogramming and tumour progression through a CRELD2-dependent mechanism. Nat Cell Biol 22(7): 882-895. Cox, T. R. (2021). The matrix in cancer. Nat Rev Cancer 21(4): 217-238. Cukierman, E. (2002). Preparation of Extracellular Matrices Produced by Cultured Fibroblasts. Curr Protoc Cell Biol 16(1): 10.19.11-10.19.15. Erami, Z., Herrmann, D., Warren, S. C., Nobis, M., McGhee, E. J., Lucas, M. C., Leung, W., Reischmann, N., Mrowinska, A., Schwarz, J. P., et al. (2016). Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue. Cell Rep 14(1): 152-167. Franco-Barraza, J., Beacham, D. A., Amatangelo, M. D. and Cukierman, E. (2016). Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts. Curr Protoc Cell Biol 71(1): 10.19.11-10.19.34. Mayorca-Guiliani, A. E., Madsen, C. D., Cox, T. R., Horton, E. R., Venning, F. A. and Erler, J. T. (2017). ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix. Nat Med 23(7): 890-898. Miller, B. W., Morton, J. P., Pinese, M., Saturno, G., Jamieson, N. B., McGhee, E., Timpson, P., Leach, J., McGarry, L., Shanks, E., et al. (2015). Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. EMBO Mol Med 7(8): 1063-1076. Morton, J. P., Timpson, P., Karim, S. A., Ridgway, R. A., Athineos, D., Doyle, B., Jamieson, N. B., Oien, K. A., Lowy, A. M., Brunton, V. G., et al. (2010). Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc Natl Acad Sci U S A 107(1): 246-51. Murphy, K. J., Reed, D. A., Vennin, C., Conway, J. R. W., Nobis, M., Yin, J. X., Chambers, C. R., Pereira, B. A., Lee, V., Filipe, E. C., et al. (2021). Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status. Sci Adv 7(40): eabh0363. Neesse, A., Bauer, C. A., Ohlund, D., Lauth, M., Buchholz, M., Michl, P., Tuveson, D. A. and Gress, T. M. (2019). Stromal biology and therapy in pancreatic cancer: ready for clinical translation? Gut 68(1): 159-171. Nicolle, R., Blum, Y., Marisa, L., Loncle, C., Gayet, O., Moutardier, V., Turrini, O., Giovannini, M., Bian, B., Bigonnet, M., et al. (2017). Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived Xenografts. Cell Rep 21(9): 2458-2470. Özdemir, B. C., Pentcheva-Hoang, T., Carstens, J. L., Zheng, X., Wu, C. C., Simpson, T. R., Laklai, H., Sugimoto, H., Kahlert, C., Novitskiy, S. V., et al. (2014). Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25(6): 719-734. Cukierman, E., Pankov, R., Stevens, D. R. and Yamada, K. M. (2001). Taking cell-matrix adhesions to the third dimension. Science 294(5547): 1708-1712. Pereira, B. A., Vennin, C., Papanicolaou, M., Chambers, C. R., Herrmann, D., Morton, J. P., Cox, T. R. and Timpson, P. (2019). CAF Subpopulations: A New Reservoir of Stromal Targets in Pancreatic Cancer. Trends Cancer 5(11): 724-741. Piccinini, F., Kiss, A. and Horvath, P. (2016). CellTracker (not only) for dummies. Bioinformatics 32(6): 955-957. Rath, N., Munro, J., Cutiongco, M. F., Jagiello, A., Gadegaard, N., McGarry, L., Unbekandt, M., Michalopoulou, E., Kamphorst, J. J., Sumpton, D., et al. (2018). Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth. Cancer Res 78(12): 3321-3336. Rhim, A. D., Oberstein, P. E., Thomas, D. H., Mirek, E. T., Palermo, C. F., Sastra, S. A., Dekleva, E. N., Saunders, T., Becerra, C. P., Tattersall, I. W., et al. (2014). Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell 25(6): 735-747. Romani, P., Valcarcel-Jimenez, L., Frezza, C. and Dupont, S. (2021). Crosstalk between mechanotransduction and metabolism. Nat Rev Mol Cell Biol 22(1): 22-38. Sahai, E., Astsaturov, I., Cukierman, E., DeNardo, D. G., Egeblad, M., Evans, R. M., Fearon, D., Greten, F. R., Hingorani, S. R., Hunter, T., et al. (2020). A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer 20(3): 174-186. Vennin, C., Chin, V. T., Warren, S. C., Lucas, M. C., Herrmann, D., Magenau, A., Melenec, P., Walters, S. N., Del Monte-Nieto, G., Conway, J. R., et al. (2017). Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis. Sci Transl Med 9(384). Vennin, C., Melenec, P., Rouet, R., Nobis, M., Cazet, A. S., Murphy, K. J., Herrmann, D., Reed, D. A., Lucas, M. C., Warren, S. C., et al. (2019). CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan. Nat Commun 10(1): 3637. Wershof, E., Park, D., Barry, D. J., Jenkins, R. P., Rullan, A., Wilkins, A., Schlegelmilch, K., Roxanis, I., Anderson, K. I., Bates, P. A. and Sahai, E. (2021). A FIJI macro for quantifying pattern in extracellular matrix. Life Sci Alliance 4(3). Wu, S. Z., Roden, D. L., Wang, C., Holliday, H., Harvey, K., Cazet, A. S., Murphy, K. J., Pereira, B., Al-Eryani, G., Bartonicek, N., et al. (2020). Stromal cell diversity associated with immune evasion in human triple-negative breast cancer. EMBO J 39(19): e104063. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > Microenvironment Cancer Biology > Invasion & metastasis > Tumor microenvironment Cell Biology > Cell movement > Cell migration Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Generation of Human Blood Vessel and Vascularized Cerebral Organoids Xin-Yao Sun [...] Zhen-Ge Luo Nov 5, 2023 1659 Views Iterative Immunostaining and NEDD Denoising for Improved Signal-To-Noise Ratio in ExM-LSCM Lucio Azzari [...] Elina Mäntylä Sep 20, 2024 570 Views An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene Srisathya Srinivasan and Hsin-Yi Henry Ho Jan 20, 2025 980 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,571	https://bio-protocol.org/en/bpdetail?id=4571&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Stimulation of Human Periodontal Ligament Fibroblasts Using Purified Dentilisin Extracted from Treponema denticola SG Sean Ganther JF J. Christopher Fenno YK Yvonne L. Kapila Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4571 Views: 490 Reviewed by: Kristin L. ShinglerBruno Lima Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in PLOS Pathogens Jul 2021 Abstract Periodontal disease is a chronic multifactorial disease triggered by a complex of bacterial species. These interact with host tissues to cause the release of a broad array of pro-inflammatory cytokines, chemokines, and tissue remodelers, such as matrix metalloproteinases (MMPs), which lead to the destruction of periodontal tissues. Patients with severe forms of periodontitis are left with a persistent pro-inflammatory transcriptional profile throughout the periodontium, even after clinical intervention, leading to the destruction of teeth-supporting tissues. The oral spirochete,Treponema denticola, is consistently found at significantly elevated levels at sites with advanced periodontal disease. Of allT. denticolavirulence factors that have been described, its chymotrypsin-like protease complex, also called dentilisin, has demonstrated a multitude of cytopathic effects consistent with periodontal disease pathogenesis, including alterations in cellular adhesion activity, degradation of various endogenous extracellular matrix–substrates, degradation of host chemokines and cytokines, and ectopic activation of host MMPs. Thus, the following model ofT. denticola–human periodontal ligament cell interactions may provide new knowledge about the mechanisms that drive the chronicity of periodontal disease at the protein, transcriptional, and epigenetic levels, which could afford new putative therapeutic targets. Keywords: Periodontal disease Host–microbe interactions Periodontal ligament cells Treponema denticola Dentilisin Background The human periodontal ligament serves as a connector between the tooth and the surrounding alveolar bone proper. Its main role is to convert mechanical forces into chemical signals, which largely mediate tissue turnover through the expression of various proteases, primarily matrix metalloproteinases (MMPs) (Ho et al., 2007; Sokos et al., 2015; Takimoto et al., 2015; Jiang et al., 2016; Lin et al., 2017; Kim et al., 2020). Periodontal diseases are caused by bacterially derived factors, leading to a chronic infectious inflammatory disease that affects the periodontium and gradually destroys the tooth-supporting alveolar bone (Sela et al., 1997; Asai et al., 2003; Hayashi et al., 2010; Trindade et al., 2014; Cecil et al., 2017; Deng et al., 2017; Gao et al., 2020) (Figure 1). As periodontal disease progresses, periodontopathogenic bacteria invade deeper into the subgingival space, compromising the periodontal ligament (PDL) function and contributing to tooth loss. Figure 1. Pathophysiology of periodontal disease Among over 500 species of bacterium, the oral spirochete, Treponema denticola, is consistently found at significantly elevated levels in advanced lesions (Ateia et al., 2018; Solbiati and Frias-Lopez, 2018). Additionally, elevated T. denticola biofilm levels, combined with elevated MMP levels in host tissue, display robust combinatorial characteristics in predicting advanced periodontal disease severity. Thus, clinical data regarding the increased presence of T. denticola in periodontal lesions, together with basic research results involving the role of T. denticola products, suggest that it plays a pivotal role in driving periodontal disease progression. Therefore, delineation of causative mechanisms that T. denticola uses to drive host modulation may help to identify novel targets for better or alternative treatments for this chronic disease. Conserved lipid moieties of the protease complex recognized by host receptor complexes may contribute to the activation of innate immune responses (Schenk et al., 2009). Because predominant host responses to lipoproteins are believed to be to their lipid moieties, most studies have focused on diacylated lipopeptide, Pam2CSK4, and triacylated lipopeptide, Pam3CSK4, which mimic bacterial lipoproteins for their potent immunostimulatory and osteoclastogenic activities (Schenk et al., 2009; Kim et al., 2013; Wilson and Bernstein, 2016). Recent studies have demonstrated that synthetic di- and tri-acylated lipopeptides, which preferentially activate TLR2/6 and TLR2/1-dependent pathways respectively, are sufficient to induce alveolar bone loss in mice (Kim et al., 2013; Souza et al., 2020), broadening the avenues of investigation into the role of lipoproteins underpinning the pathogenesis of periodontal disease. However, studies that utilize endogenously expressed bacterial lipopeptides are lacking. Several proteinases and peptidases secreted by T. denticola have been identified as causative factors that likely contribute to periodontal disease pathogenesis, due to their roles in processing host tissue proteins and peptides to fulfill the nutritional requirements of these highly motile and invasive organisms (Veith et al., 2009; Ellis and Kuehn, 2010; Visser et al., 2011; Asai et al., 2003; Cecil et al., 2016). Of all T. denticola surface-expressed proteins that have been described, a chymotrypsin-like protease complex, also called dentilisin, has demonstrated a multitude of cytopathic effects consistent with periodontal disease pathogenesis, including changes in cellular adhesion activity (Bamford et al., 2007; Sano et al., 2014), degradation of various endogenous extracellular matrix–substrates (Bamford et al., 2007; Miao et al., 2011; Inagaki et al., 2016), and degradation of host chemokines and cytokines (Miyamoto et al., 2006; McDowell et al., 2012). Despite the many studies demonstrating its role at the protein level, few direct links have been reported between the activity of T. denticola ’s protease and the cellular and tissue processes driving periodontal tissue destruction. The following protocol delineates the experimental setup to study the interactions between T. denticola’s dentilisin protease and human periodontal ligament cells. Materials and Reagents 6-well clear multi-well plate (Fisher Scientific, catalog number: 25373-187) 10 cm Falcon tissue culture plate (The Lab Depot, catalog number: 25373-10) Minimal essential medium-α (MEM-α) (ThermoFisher Scientific, catalog number: 12571063) Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14190-094) Penicillin/streptomycin (P/S) (Thermo Fisher Scientific, Gibco, catalog number: 15140122) Amphotericin B (Thermo Fisher Scientific, Gibco, catalog number: 15290018) 0.25% trypsin with phenol red (Thermo Fisher, catalog number: 25200-05 Heat-inactivated fetal bovine serum (FBS) (Gibco, catalog number: 10-438-026) BCA protein assay kit (Millipore Sigma, catalog number: 71285-3) Equipment 96-well SpectraMax Plus microplate reader (VWR, catalog number: 89212-396) Labomed Lx400 phase contrast HD digital microscope (Microscope Central, catalog number: 9126017T-HDS) SteriCycle 370 CO2 incubator (Thermo Fisher Scientific, catalog number: TH-370N) NanoDrop One Microvolume UV-Vis spectrophotometer (Fisher Scientific, catalog number: 13-400-519) Bright line hemacytometer chamber (Carolina, catalog number: 700722) Thermo Sorvall ST16R refrigerated centrifuge (Thermo Fisher Scientific, catalog number: 75-004-240) Procedure Human periodontal ligament cell cultures (hPDL) As described previously (Scanlon et al., 2011), prepare the primary culture of human periodontal ligament cells via the direct cell outgrowth method, by isolating cells from the periodontal ligament tissue around the middle third of healthy human teeth extracted. Maintain cells in approximately 10 mL of MEM-α augmented with 10% FBS, 1% P/S, and 1% amphotericin B in 10 cm Falcon tissue culture plates in a humid atmosphere with 95% air and 5% CO2 at 37 °C. Passage cell outgrowths when they reach approximately 90% confluency using 0.25% trypsin and a Thermo Sorvall ST16R refrigerated centrifuge. Although variation will be observed from patient to patient, cells usually take approximately three to four days to become confluent after seeding cells at approximately 70%. Use cells passaged three to six times for experimentation. Validate human periodontal ligament cell parameters by 1) measuring mRNA expression of a specific isoform of the POSTN gene, which is exclusively expressed by periodontal ligament cells, 2) evaluating cell morphology, and 3) examining expression of other confident cell biomarkers, such as vimentin (general fibroblast marker) and CD45 , which ensure cultures are free of macrophages (Marchesan et al., 2011). If cultures are contaminated with other cell types, hPDL cells can be further purified via FACS sorting using the biomarkers reported above (Basu et al., 2010). Seed approximately 9 × 105 cells into 6-well clear multi-well plates and allow them to adhere in the presence of MEM-α with 10% FBS, 1% amphotericin B, and 1% P/S for approximately 24 h before stimulating or challenging the cells. Stimulation of human periodontal ligament cells using purified dentilisin Use a BCA protein assay kit according to the manufacturer’s recommendations to determine purified dentilisin (UniProt Accession #: P96091) sample concentrations Scan the plates using a 96-well SpectraMax Plus Microplate Reader. Determine enzymatic activity using gelatin zymography as described previously (Cathcart, 2016). Wash cells with PBS 2–3 times. Add the purified dentilisin/PBS solution to MEM-α media with no phenol red, no serum, and no antibiotics to a final concentration of 1 μg/mL. Add this dentilisin solution to healthy human periodontal ligament cell cultures and incubate in a humid atmosphere containing 95% air and 5% CO2 at 37 °C for 2 h. Stimulation with purified dentilisin will cause significant changes to the cells’ morphology (Figure 2). Gently wash cells with PBS twice and incubate in a humid atmosphere containing 95% air and 5% CO2 at 37 °C for an additional 22 h in MEM-α with no FBS, P/S, or amphotericin B. Data analysis Figure 2. Morphological Change of hPDL Cells Following Purified Dentilisin Stimulation. Healthy human periodontal ligament fibroblasts were challenged with purified dentilisin at a final concentration of 1 μg/mL for 2 h in MEM-α without serum or antibiotics, followed by a 22 h incubation in a humid atmosphere containing 95% air and 5% CO2 at 37 °C in MEM-α media with no supplementation. Following this experimental setup, harvest cells for analysis of various parameters. In our studies, we harvested cells or conditioned media to evaluate MMP enzymatic activity (gelatin zymography) and protein and RNA expression (Western Blot and qRT-PCR), and for cell imaging (immunofluorescence) (Ganther et al., 2021). Notes If collecting conditioned media samples, it is highly recommended to use media without phenol red, serum, or antibiotics as dentilisin’s protease activity can cleave numerous substates and may interfere with protein concentration measurements. Acknowledgments We thank Dr. Christopher Fenno for his donation of purified dentilisin samples and isogenic mutants. These studies were supported by funding from the NIH (R01 DE025225) to YLK (https://www.nih.gov/) and to SG by a Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant (T32DE007306) (https://www.nih.gov/). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript. Competing interests The authors declare no conflict of interest. Ethics Approval to conduct human subjects’ research was obtained from the University of California San Francisco Institutional Review Board (# 16-20204; reference #227030). References Asai, Y., Jinno, T. and Ogawa, T. (2003). Oral Treponemes and Their Outer Membrane Extracts Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2. Infect Immun 71(2): 717-725. Ateia, I. M., Sutthiboonyapan, P., Kamarajan, P., Jin, T., Godovikova, V., Kapila, Y. L. and Fenno, J. C. (2018). Treponema denticola increases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications. Cell Microbiol 20(4): 10.1111/cmi.12815. Bamford, C. V., Fenno, J. C., Jenkinson, H. F. and Dymock, D. (2007). The chymotrypsin-like protease complex of Treponema denticola ATCC 35405 mediates fibrinogen adherence and degradation. Infect Immun 75(9): 4364-4372. Basu, S., Campbell, H. M., Dittel, B. N. and Ray, A. (2010). Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp(41): 1546. Cathcart, J. (2016). Assessment of Matrix Metalloproteinases by Gelatin Zymography. Methods Mol Biol 1406: 151-159. Cecil, J. D., O'Brien-Simpson, N. M., Lenzo, J. C., Holden, J. A., Singleton, W., Perez-Gonzalez, A., Mansell, A. and Reynolds, E. C. (2017). Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo. Front Immunol 8: 1017. Cecil, J. D., O'Brien-Simpson, N. M., Lenzo, J. C., Holden, J. A., Chen, Y. Y., Singleton, W., Gause, K. T., Yan, Y., Caruso, F. and Reynolds, E. C. (2016). Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens. PLoS One 11(4): e0151967. Deng, Z. L., Szafranski, S. P., Jarek, M., Bhuju, S. and Wagner-Dobler, I. (2017). Dysbiosis in chronic periodontitis: Key microbial players and interactions with the human host. Sci Rep 7(1): 3703. Ellis, T. N. and Kuehn, M. J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 74(1): 81-94. Ganther, S., Radaic, A., Malone, E., Kamarajan, P., Chang, N. N., Tafolla, C., Zhan, L., Fenno, J. C. and Kapila, Y. L. (2021). Treponema denticola dentilisin triggered TLR2/MyD88 activation upregulates a tissue destructive program involving MMPs via Sp1 in human oral cells. PLoS Pathog 17(7): e1009311. Gao, L., Kang, M., Zhang, M. J., Reza Sailani, M., Kuraji, R., Martinez, A., Ye, C., Kamarajan, P., Le, C., Zhan, L., et al. (2020). Polymicrobial periodontal disease triggers a wide radius of effect and unique virome. NPJ Biofilms Microbiomes 6(1): 10. Hayashi, C., Gudino, C. V., Gibson, F. C. 3rd, Genco, C. A. (2010). Review: Pathogen-induced inflammation at sites distant from oral infection: bacterial persistence and induction of cell-specific innate immune inflammatory pathways. Mol Oral Microbiol 25(5): 305-16. Ho, S. P., Marshall, S. J., Ryder, M. I. and Marshall, G. W. (2007). The tooth attachment mechanism defined by structure, chemical composition and mechanical properties of collagen fibers in the periodontium. Biomaterials 28(35): 5238-5245. Inagaki, S., Kimizuka, R., Kokubu, E., Saito, A. and Ishihara, K. (2016). Treponema denticola invasion into human gingival epithelial cells.Microb Pathog 94: 104-111. Jiang, N., Guo, W., Chen, M., Zheng, Y., Zhou, J., Kim, S. G., Embree, M. C., Songhee Song, K., Marao, H. F. and Mao, J. J. (2016). Periodontal Ligament and Alveolar Bone in Health and Adaptation: Tooth Movement. Front Oral Biol 18: 1-8. Kim, J., Yang, J., Park, O. J., Kang, S. S., Kim, W. S., Kurokawa, K., Yun, C. H., Kim, H. H., Lee, B. L. and Han, S. H. (2013). Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation. J Bone Miner Res 28(11): 2381-2391. Kim, K., Kang, H. E., Yook, J. I., Yu, H. S., Kim, E., Cha, J. Y. and Choi, Y. J. (2020). Transcriptional Expression in Human Periodontal Ligament Cells Subjected to Orthodontic Force: An RNA-Sequencing Study. J Clin Med 9(2): 358. Lin, J. D., Jang, A. T., Kurylo, M. P., Hurng, J., Yang, F., Yang, L., Pal, A., Chen, L. and Ho, S. P. (2017). Periodontal ligament entheses and their adaptive role in the context of dentoalveolar joint function. Dent Mater 33(6): 650-666. Marchesan, J. T., Scanlon, C. S., Soehren, S., Matsuo, M. and Kapila, Y. L. (2011). Implications of cultured periodontal ligament cells for the clinical and experimental setting: a review. Arch Oral Biol 56(10): 933-943. McDowell, J. V., Miller, D. P., Mallory, K. L. and Marconi, R. T. (2012). The Pathogenic Spirochetes: strategies for evasion of host immunity and persistence. Embers, M. E. (Ed.): 43-62. Springer. Miao, D., Fenno, J. C., Timm, J. C., Joo, N. E. and Kapila, Y. L. (2011). The Treponema denticola chymotrypsin-like protease dentilisin induces matrix metalloproteinase-2-dependent fibronectin fragmentation in periodontal ligament cells. Infect Immun 79(2): 806-811. Miyamoto, M., Ishihara, K. and Okuda, K. (2006). The Treponema denticola surface protease dentilisin degrades interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha. Infect Immun 74(4): 2462-2467. Sano, Y., Okamoto-Shibayama, K., Tanaka, K., Ito, R., Shintani, S., Yakushiji, M. and Ishihara, K. (2014). Dentilisin involvement in coaggregation between Treponema denticola and Tannerella forsythia. Anaerobe 30: 45-50. Scanlon, C., Marchesan, J., Soehren, S., Matsuo, M. and Kapila, Y. (2011). Capturing the regenerative potential of periodontal ligament fibroblasts. J Stem Cells Regen Med 7(1): 54-56. Schenk, M., Belisle, J. T. and Modlin, R. L. (2009). TLR2 Looks at Lipoproteins. Immunity 31: 847-849. Sela, M. N., Bolotin, A., Naor, R., Weinberg, A. and Rosen, G. (1997). Lipoproteins of Treponemadenticola: their effect on human polymorphonuclear neutrophils. J Periodontal Res 32(5): 455-466. Sokos, D., Everts, V. and de Vries, T. J. (2015). Role of periodontal ligament fibroblasts in osteoclastogenesis: a review. J Periodontal Res 50(2): 152-159. Solbiati, J. and Frias-Lopez, J. (2018). Metatranscriptome of the Oral Microbiome in Health and Disease. J Dent Res 97(5): 492-500. Souza, J. A. C., Magalhaes, F. A. C., Oliveira, G., RS, D. E. M., Zuanon, J. A. and Souza, P. P. C. (2020). Pam2CSK4 (TLR2 agonist) induces periodontal destruction in mice. Braz Oral Res 34: e012. Takimoto, A., Kawatsu, M., Yoshimoto, Y., Kawamoto, T., Seiryu, M., Takano-Yamamoto, T., Hiraki, Y. and Shukunami, C. (2015). Scleraxis and osterix antagonistically regulate tensile force-responsive remodeling of the periodontal ligament and alveolar bone. Development 142(4): 787-796. Trindade, F., Oppenheim, F. G., Helmerhorst, E. J., Amado, F., Gomes, P. S. and Vitorino, R. (2014). Uncovering the molecular networks in periodontitis. Proteomics Clin Appl 8(9-10): 748-761. Veith, P. D., Dashper, S. G., O'Brien-Simpson, N. M., Paolini, R. A., Orth, R., Walsh, K. A. and Reynolds, E. C. (2009). Major proteins and antigens of Treponema denticola. Biochim Biophys Acta 1794(10): 1421-1432. Visser, M. B., Koh, A., Glogauer, M. and Ellen, R. P. (2011). Treponema denticola major outer sheath protein induces actin assembly at free barbed ends by a PIP2-dependent uncapping mechanism in fibroblasts. PLoS One 6(8): e23736. Wilson, M. M. and Bernstein, H. D. (2016). Surface-Exposed Lipoproteins: An Emerging Secretion Phenomenon in Gram-Negative Bacteria. Trends Microbiol 24(3): 198-208. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > Microbe-host interactions > Bacterium Medicine Cell Biology > Cell signaling > Intracellular Signaling Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Bacterial Pathogen-mediated Suppression of Host Trafficking to Lysosomes: Fluorescence Microscopy-based DQ-Red BSA Analysis Mădălina Mocăniță [...] Vanessa M. D'Costa Mar 5, 2024 394 Views Purification of Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography Pachiyappan Kamarajan [...] Yvonne L. Kapila Apr 5, 2024 379 Views Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry Hui Jing Lim and Hamish E. G. McWilliam May 5, 2024 650 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,572	https://bio-protocol.org/en/bpdetail?id=4572&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Single Oligonucleotide Capture of RNA And Temperature Elution Series (SOCRATES) for Identification of RNA-binding Proteins AY Allen T. Yu DA Disha Aggarwal DP Darryl Pappin David L. Spector Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4572 Views: 1506 Reviewed by: Julie WeidnerRakesh ChatrikhiWeiyan Jia Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE May 2021 Abstract The importance of studying the mechanistic aspects of long non-coding RNAs is being increasingly emphasized as more and more regulatory RNAs are being discovered. Non-coding RNA sequences directly associate with generic RNA-binding proteins as well as specific proteins, which cooperate in the downstream functions of the RNA and can also be dysregulated in various physiologic states and/or diseases. While current methods exist for identifying RNA–protein interactions, these methods require high quantities of input cells or use pooled capture reagents that may increase non-specific binding. We have developed a method to efficiently capture specific RNAs using less than one million input cells. One single oligonucleotide is used to pull down the target RNA of choice and oligonucleotide selection is driven by sequence accessibility. We perform thermal elution to specifically elute the target RNA and its associated proteins, which are identified by mass spectrometry. Ultimately, two target and control oligonucleotides are used to create an enrichment map of interacting proteins of interest. Graphical abstract Schematic representation of the SOCRATES workflow. SOCRATES utilizes a single 20-mer oligonucleotide for RNA pull down followed by a temperature elution series and liquid chromatography–mass spectrometry (LC-MS)/MS to identify specific RNA–protein interactions. Keywords: Pull down Mass spectrometry RNA–protein interactions Temperature elution Non-coding RNA Background The interplay between an organism’s two essential macromolecules, RNA and proteins, dictate much of the cell’s functions (Zhu, 2010). Messenger RNAs contain instructions for protein synthesis and may contain regulatory sequences that affect the levels of the encoded protein (Mayr, 2017). As a complement, proteins can bind to RNA and modulate expression, function, and stability (Moore, 2005). Perturbation or loss of physical interactions can lead to loss of genomic stability, aberrant cellular signaling, or global changes in gene expression (Allerson et al., 1999; Batista and Chang, 2013; Lee et al., 2016). It has been shown that at least 5% of the human proteome is capable of binding to RNA, and our catalogue of RNA binding domains is incredibly incomplete (Castello et al., 2012). Of particular recent interest are a class of non-protein-coding RNA transcripts called long non-coding RNAs (lncRNAs) that have been shown to hold regulatory roles in embryonic stem cell (ESC) differentiation and can be dysregulated in cancer (Guttman et al., 2011; Bergmann et al., 2015; He et al., 2017). In addition, lncRNAs have also been shown to serve as therapeutic molecular targets, and the ease of designing antisense oligonucleotides to target them for degradation or inhibition is a large untapped field of therapeutics (Liang et al., 2017; Crooke et al., 2017; Zong et al., 2015; Arun et al., 2016, 2018; Chang et al., 2020; Yu et al., 2021). However, as many more functional lncRNAs are being discovered, their molecular mechanisms of action are largely unknown. Elucidation of RNA–protein interactions have not progressed as quickly, due to the lack of streamlined and efficient methods. Current methods include crosslinking cells, needing an incredibly high quantity of starting material, or requiring expensive and long modified capture reagents Hacisuleyman et al., 2014; Chen et al., 2016; Chu et al., 2011). Multiple oligonucleotides used for antisense capture also can result in possible off-target effects (Chu et al., 2015). In addition, the final elution conditions may not necessarily be compatible with mass spectrometry (MS). Because the ultimate goal is to identify proteins, the final elution buffer used must be compatible with downstream applications in order to avoid additional buffer exchange or purification steps, which could lead to loss of material and cause potential biases. We have developed a method to efficiently capture RNAs without the need to use a high quantity of input or pooled capture reagents to identify the associated proteins. One single oligonucleotide is used to pull down the target RNA of choice and oligonucleotide selection is driven by sequence accessibility. Because the pull down is mediated by one single oligonucleotide, temperature can be used as an option for elution. The melting point of the oligonucleotide-to-RNA interaction can be empirically determined and thus an optimal elution temperature can be found. This allows for a very high degree of selectivity and discrimination, as tighter interactors that elute at higher temperatures are more likely to be specific. An additional step of quality control is possible with this method as well, as the full elution of the target RNA can be quantified using qRT-PCR on the eluate and the beads. In addition, elution can be performed in a buffer that is completely compatible with any downstream application, such as digestion or peptide-labelling for MS. Elution of the RNA–protein complex from the capture oligonucleotide is thermally controlled and measurable by qRT-PCR, thus allowing for quality control and specificity of protein hits. We call this method S ingle O ligonucleotide C apture of R NA A nd T emperature E lution S eries, or SOCRATES . Materials and Reagents 10 cm sterile cell culture dish (e.g., Corning, Falcon® , catalog number: 353003) 15 mL and 50 mL conical tubes (e.g., Crystalgen, catalog number: 23-2265; Corning, Falcon® , catalog number: 352098) Sterile pipette tips (e.g., Corning) 0.2 mL PCR strip tubes (e.g., Corning, Axygen®, catalog number: PCR-0208-CP-C) Low adhesion microcentrifuge (Eppendorf) tube (e.g., USA Scientific, catalog number: 1415-2690) 96-well reaction plates (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4346907) Optical adhesive film (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4311971) 200 proof ethyl alcohol (e.g., UltraPure, catalog number: 200CSPTP) Sterile water Ice 1× DPBS (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 14190250) DynabeadsTM MyOneTM streptavidin C1 (Invitrogen, catalog number: 65001) IP lysis buffer (e.g., Pierce IP Lysis Buffer; Thermo Fisher Scientific, catalog number: 87787) cOmpleteTM mini protease inhibitor cocktail (Roche, catalog number: 11836153001) SUPERase-In RNase inhibitor (Invitrogen, catalog number: AM2694) Magnetic racks (e.g., Invitrogen DynaMag, catalog number: 12321D for 1.5–2 mL Eppendorf tubes, 12331D for 0.2 mL PCR strip tubes) BCA assay kit (e.g., Pierce BCA protein assay kit; Thermo Fisher Scientific, catalog number: 23227) Triethylammonium bicarbonate (TEAB) 1 M for mass spectrometry (Thermo Scientific, catalog number: 90114) TRIzol (Thermo Fisher Scientific, Invitrogen, catalog number: 15596018) GlycoBlue (Thermo Fisher Scientific, Invitrogen, catalog number: AM9516) DNase I, amplification grade, for cDNA synthesis (e.g., Thermo Fisher Scientific, Invitrogen, catalog number: 18068015) Ethylenediaminetetraacetate acid disodium salt (EDTA) (included in DNase I kit; Thermo Fisher Scientific, catalog number: 18068015) Nuclease-free water (e.g., Thermo Fisher Scientific, Invitrogen, catalog number: AM9937) TaqMan reverse transcription kit (Thermo Fisher Scientific, Invitrogen, catalog number: N8080234) PowerUp SYBR green master mix (Thermo Fischer Scientific, Applied Biosystems, catalog number: A25743) Chloroform, purified (e.g., Avantor Performance Materials, MACRON, catalog number: 4432-10) Isopropanol, molecular biology grade (e.g., Fisher Scientific, catalog number: BP2618500) Cell lysis buffer (see Recipes) cDNA master mix per reaction (see Recipes) qPCR master mix (see Recipes) Equipment Biological safety cabinet for tissue culture (e.g., NuAire) Cell culture incubator (e.g., Heracell-I Copper CO2 incubator, Thermo Fischer Scientific, Thermo ScientificTM, model: HeracellTM 150i and 240i, catalog number: 50116050) Gentle rotating mixer for Eppendorf tubes (end-over-end rotation e.g., VWR, catalog number: 10136-084) Centrifuge for 15 and 50 mL tubes (e.g., Eppendorf, model: 5804) Centrifuge for 1.5 mL reaction tubes with cooling function (e.g., Eppendorf, model: 5427 R) Vacuum suction Micro-pipettes (e.g., Gilson, catalog number: F167300) NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000, catalog number: ND-2000) PCR machine (e.g., Applied Biosystems Proflex Thermocycler, Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4484073) qPCR machine (e.g., Applied Biosystems StepOne Plus Real-Time PCR system, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4376600) -80 °C freezer (e.g., VWR, catalog number: 10160-728) Refrigerator (4 °C) Procedure Design of antisense oligonucleotides (ASOs) for RNA capture Choose a target gene for pull down. The protocol described here is equally suitable for long non-coding RNAs and protein-coding transcripts. Design 20-mers complementary to exons of the RNA sequence. Here, 100% sequence identity is required; design at least two ASOs for the target sequence as well as a negative control [e.g., peptidylprolyl isomerase B (PPIB)]. It is recommended to start with up to ten individual ASOs for new targets tiling the entire length of the mature transcript, as not every ASO will result in an efficient pull down. Use Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure the ASOs are specific to the intended target only. Stringency is important at this step—even one or two mismatches can result in unwanted off-target pull downs. ASOs used in pull-down experiments carry a modified 3’ end with biotin to assist in capture. In the United States, 3’ biotin modifications can be ordered, for example, from LGC Biosearch Technologies. We generally prefer a 50 nmol scale with a 3’ biotin, six carbon linker, and reverse-phase cartridge purification. Upon arrival, resuspend lyophilized ASOs in sterile DPBS to a working stock concentration of 200 µM. Store at 4 °C for short-term storage (up to one week) or at -20 °C for long-term storage. Aliquot to avoid repeated freeze-thaw cycles. Preparation of cell lysate Prepare a master mix of cell lysis buffer (see Recipe 1). The volume of each reaction is 500 μL, which can be scaled up if needed. Obtain a 10 cm plate at 70%–80% confluence of the cell lines of interest. Place on ice and aspirate the medium. Wash twice with 1× DPBS. Leave the plate tilted for a minute to remove all of the residual PBS. This is to prevent dilution of the lysis buffer. Lyse the cells on the plate using approximately 1 mL of cell lysis buffer per plate on ice for 10 min; then, harvest cells by scraping and transfer to low adhesion microcentrifuge tubes. Spin down lysate at 13,000 × g for 10 min at 4 °C to pellet debris. If harvesting from multiple plates and using multiple Eppendorf tubes, combine and mix supernatant lysate in a 15 mL conical tube before proceeding to the capture step. (Highly recommended) Perform a BCA assay on the cell lysate. Adjust the cell lysate to a concentration of 0.3 mg/mL using cell lysis buffer. Note: While this step is optional, we highly recommend for it to be performed. We have found that in general, a 0.3 mg/mL protein concentration provides the optimum balance between pull-down efficiency and protein yield for peptide detection, as the cell input for this protocol is very low compared to previously published protocols (seeFigure 1below). As lysate protein concentration increases, we observe that yield decreases as seen inFigure 2below. Figure 1. SOCRATES requires 150,000 cells as input. SOCRATES input cell number requirement is much lower than current methods (West et al., 2014; Chu et al., 2015; McHugh et al., 2015). CHART: Capture Hybridization Analysis for RNA Targets; ChIRP: Chromatin isolation by RNA Purification; RAP-MS: RNA Antisense Purification with Mass Spectrometry. Figure 2. Lysate protein concentration affects pull-down efficiency. In this case, a protein concentration of 0.1 mg/mL would be optimal to pull down RMRP, a 286 nt transcript. Anti-sense capture with ASOs and magnetic beads From the lysate stock, aliquot 50 μL for 10% RNA input and 50 μL for 10% protein input, if needed. Leave the tubes on ice for the whole duration of the pull down. Do not add TRIzol to the input until the pull-down samples are in TRIzol at the end of this protocol. Aliquot 500 μL of lysate stock per sample and add 100 pmol of oligonucleotide to make a final concentration of 200 nM. This is generally 0.5 μL of the 200 µM stock. Do not vortex. Note: Extra lysate can be labelled and stored at -80 °C for future validation experiments such as western blot. Incubate at room temperature for 1 h with gentle agitation on the rotator. Note: As a general note for mass spectrometry and pull-down experiments, it is very important to adhere to the same protocol for each replicate. Any deviation should be recorded in the event of inconsistent results. In addition, fluctuations in room temperature (e.g., 20–28 °C) may also be a source of error that may be compounded throughout the experiment. Take care not to vortex the sample prior to capture with the magnetic beads. While the samples are incubating, prepare the Dynabeads by aliquoting 100 μL per reaction into a fresh Eppendorf tube for each reaction and place on the magnetic stand for 2 min to capture the beads. Remove the Dynabeads storage buffer gently and wash twice with 100 µL of lysis buffer, capturing the beads for 2 min in between washes. Basic lysis buffer without protease inhibitor cocktail and RNase inhibitor can be used here as well. Remove the lysis wash buffer and remove the beads from the magnet. Place on ice until ready to use. Add samples to the washed Dynabeads and incubate at room temperature for 30 min with the same gentle agitation. Do not vortex. Once the incubation is done, place the samples on a magnetic stand on ice to capture the beads for 2 min. Wash the beads three times with 800 µL of basic lysis buffer. Allow 2 min between washes for bead capture on the stand. Take care to remove all buffer, as any remaining will interfere with the elution process. Elution of RNA and protein from capture ASOs Preheat a thermocycler to 40 °C. Very carefully resuspend beads in 90 µL of ice cold 100 mM TEAB by pipetting up and down and transfer to a 0.2 mL PCR tube or a PCR tube compatible with the thermocycler. Incubate the sample at 40 °C for exactly 10 min and immediately place on ice afterwards. Note: In our experience, the vast majority of the oligonucleotides that we have tested achieve >95% elution at 40 °C. However, if elution is not fully achieved at that temperature, another oligonucleotide should be chosen or a temperature curve can be performed in order to identify the optimum elution temperature, as inFigure 3below. Figure 3. Temperature elution curve for PHAROH . PHAROH ’s optimal elution temperature is 40 °C; >95% has been eluted off the beads and only <5% remained on the beads. Allow the samples to cool on ice for 15 s. Perform a quick spin and place the sample on a magnetic stand on ice compatible with PCR tubes. Leave the samples in the magnetic stand for 60 s to capture the beads. Very carefully remove the eluate and transfer to a new Eppendorf tube. Note: Removing the eluate from the beads is a critical step. After performing temperature elution, do not wait longer than one minute to transfer the eluate. The RNA will rebind to the capture oligonucleotide over time and efficiencies will not be accurate. Process in batches if handling many samples. Immediately aliquot 30 µL for mass spectrometry, 30 µL for RNA, and the remaining for immunoblots. As TEAB is a volatile compound, take care when pipetting. Add 1 mL of TRIzol to the RNA aliquot as well as the input that was set aside in the beginning of the experiment. Store protein samples at -20 °C until ready to process. Proceed to cDNA synthesis and qPCR analysis with the RNA samples. Note: Ideally, the samples should be processed within 24 h for consistency, but they are stable in TRIzol for up to one month. Care must be taken in order to have consistent results across replicates. cDNA synthesis and qPCR to analyze pull-down efficiency Isolate RNA from pull-down samples according to the manufacturer’s instructions. Addition of glycogen (GlycoBlue) is necessary in order to ensure complete extraction of RNA. Allow the RNA pellet to air dry for 5 min. Resuspend in 8 µL of nuclease-free water. Perform DNase digestion to remove any potentially co-purified genomic DNA by adding 1 µL of 10× DNase reaction buffer and 1 µL (1 U/µL) of DNase I to the RNA sample (total volume: 10 µL). Mix reaction by gently pipetting up and down. Spin briefly in a microcentrifuge. Incubate reaction at 25 °C in a PCR machine for 15 min. Add 1 µL of 25 mM EDTA. Mix reaction by gently pipetting up and down. Spin briefly in a microcentrifuge. Inactivate reaction at 65 °C in a PCR cycler for 10 min. Transfer samples on ice immediately after the 10 min incubation is completed. Do not leave samples in the PCR machine while ramping down to room temperature. Prepare cDNA master mix (see Recipe 2). Add 39 µL of cDNA master mix to samples on ice. Mix reaction by gently pipetting up and down. Spin briefly in a microcentrifuge. Perform the following incubation in a PCR machine for cDNA synthesis: 25 °C for 10 min 48 °C for 30 min 95 °C for 5 min cDNA can be stored short-term at 4 °C or long-term at -20 °C. Prepare qPCR master mix (see Recipe 3) containing primers for the target gene as well as qPCR master mix containing primers for an internal control (housekeeping) gene. When calculating the quantity of master mix needed, consider that samples will be pipetted in triplicates. Note: For optimal results, primers for qPCR should not overlap with the capture oligonucleotide binding site, and the capture oligonucleotide should not bind within the qPCR amplified region. Commonly used genes for internal controls include beta-actin, GAPDH , or ribosomal protein genes. Internal controls should be chosen carefully; their expression level should be consistent and independent of experimental conditions such as ASO treatments. The expression level of an internal control gene should be in the same range as the target gene. General rules regarding qPCR experiments and qPCR primer design can be found in Bustin et al. (2009). Prepare a 96-well plate on ice. Add 15 µL of master mix containing either the target gene or internal control gene primers to the wells. Dilute the prepared cDNA 1:3 by adding 100 µL of nuclease-free water to each reaction. Pipette 5 µL of diluted cDNA into one well of the 96-well plate. Each sample will be pipetted in triplicate for both the target and the housekeeping gene (= six wells containing 5 µL of cDNA per sample). Seal the 96-well plate with an optical adhesive film. Avoid touching the film with your gloves, handling carefully by the edges. Spin down the plate at 100 × g for 1 min at room temperature. Place the plate in the qPCR machine and run the following program: 95 °C for 10 min 40 cycles of 95 °C for 15 s 60 °C for 60 s Followed by Melt curve 95 °C→60 °C, 1 °C/min After pull-down analysis, choose the best two oligonucleotides with the highest pull-down efficiency (ideally >70%) for the target gene and control gene and submit the eluate for digestion, iTRAQ 4-plex labelling, and sequencing at the mass spectrometry facility. Note: Submission for digestion and labelling should occur within 24 h of obtaining the eluate if possible. The samples can be kept at -20 °C until the pull-down efficiency for the batch is confirmed via qRT-PCR. Lastly, TEAB is highly volatile, so care must be taken when handling this reagent. Data analysis Pull-down efficiency Export Ct values from the qPCR machine and analyze using the 2-ΔΔCt Method (Livak and Schmittgen, 2001). Input should be adjusted to 100% depending on how much was taken from the initial reaction. Pull down should range from 10%–90%, which can vary based on the oligonucleotide position along the RNA. Poor efficiencies may be due to saturated lysate or incorrect temperature for elution. Adherence to the protocol has a large impact on result consistency. Note: We hypothesize that pull-down efficiency generally correlates with accessibility to the binding site. We have tiled the lncRNA NORAD with 32 oligonucleotides and found that areas of decreased binding correlated with published sites protein occupancy (see Figure 4). Regarding RNA length and pull-down efficiency, we have been successful in pull downs of RNAs that range from 286 nt (RMRP) to 5,300 nt (NORAD), with no fragmentation of the RNA detected (see Figure 5). Figure 4. Pull-down profile maps accurately to published data. Smoothed moving average pull-down profile of NORAD and occupancy PAR-CLIP data taken from West et al. (2014), Chu et al. (2015), and Lee et al. (2016); CLIP-seq data taken from Munschauer et al. (2018). The blue bars indicate areas of low pull-down efficiency, which correspond well with published occupancy data. Figure 5. Full-length NORAD can be pulled down using single oligonucleotides. Two primers spanning different regions of NORAD that are approximately 2 kb apart yield similar pull-down efficiencies, assayed by primer pairs on the 5’ and 3’ end, implying that NORAD is not fragmented. N = 3, error bars represent standard error of mean. The two oligonucleotides for the target and control gene with the highest efficiency should be chosen for downstream mass spectrometry processing. Mass spectrometry and ranked target acquisition To identify proteins that bind to the target RNA, calculate the log2 enrichment ratio of target RNA to control RNA for each oligonucleotide. If using two control oligonucleotides, take the average enrichment of the two control oligonucleotides and use the resulting number as the denominator for the enrichment ratio. Plot the log2 enrichment ratio as a scatterplot with oligonucleotide 1 and oligonucleotide 2 as the x and y coordinates (see Figure 6 adapted from Yu et al., 2021). Quadrant I will contain proteins that bind to both oligonucleotides against the target RNA and quadrant III will be enriched for proteins that bind specifically to the control RNA. Averaging the two enrichment ratios will provide a ranked list of the top protein hits that bind to the target RNA. Proceed to confirmation via immunoblot. Figure 6. Mass spectrometry target scatterplot. Log2 enrichment ratios plotted by oligonucleotide reveal the interacting proteins with the highest enrichments (adapted from Yu et al., 2021). Recipes Cell lysis buffer Pierce IP buffer base containing 150 mM NaCl, 25 mM Tris-Cl pH 7.4, 1% NP-40, 1 mM EDTA, and 5% glycerol 1× cOmplete mini protease inhibitor cocktail: one tablet in 10 mL of lysis buffer 100 U/mL SUPERase-In (Invitrogen) Note: We have tested the protocol without SUPERase-In as well and found little change to the RNA integrity number. cDNA master mix per reaction Note: All components, except for nuclease-free H2O, are included in the TaqMan reverse transcription kit. 10× TaqMan RT buffer 5 µL Random hexamers (50 µM) 2.5 µL MgCl2 (25 mM) 11 µL dNTPs (10 mM) 10 µL RNase inhibitor (20 U/µL) 1 µL RTase (50 U/µL) 1.25 µL Nuclease-free H2O 8.25 µL Total volume 39 µL Use immediately after preparation. qPCR master mix PowerUp SYBR green master mix 10 µL Nuclease-free H2O4 µL Primer mix 1 µL Total 15 µL Primer mix consists of forward and reverse primers, each at 10 µM, final concentration 500 nM per well Use immediately after preparation. Acknowledgments We would like to acknowledge the following funding sources: NIGMS 5R35GM131833 (D.L.S.), NCI 5P01CA013106-Project 3 (D.L.S.), NCI 2P3OCA45508, and NCI 5F31CA220997 (A.T.Y.). The original research paper in which this method was used is Yu et al. (2021). Competing interests The authors of this manuscript declare that there are no conflicts of interest or competing interests. References Allerson, C. R., Cazzola, M. and Rouault, T. A. (1999). Clinical severity and thermodynamic effects of iron-responsive element mutations in hereditary hyperferritinemia-cataract syndrome. J Biol Chem 274(37): 26439-26447. Arun, G., Diermeier, S., Akerman, M., Chang, K. C., Wilkinson, J. E., Hearn, S., Kim, Y., MacLeod, A. R., Krainer, A. R., Norton, L., et al. (2016). Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes Dev 30(1): 34-51. Arun, G., Diermeier, S. D. and Spector, D. L. (2018). Therapeutic Targeting of Long Non-Coding RNAs in Cancer. Trends Mol Med 24(3): 257-277. Batista, P. J. and Chang, H. Y. (2013). Long noncoding RNAs: cellular address codes in development and disease. Cell 152(6): 1298-1307. Bergmann, J. H., Li, J., Eckersley-Maslin, M. A., Rigo, F., Freier, S. M. and Spector, D. L. (2015). Regulation of the ESC transcriptome by nuclear long noncoding RNAs. Genome Res 25(9): 1336-1346. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., et al. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4): 611-622. Castello, A., Fischer, B., Eichelbaum, K., Horos, R., Beckmann, B. M., Strein, C., Davey, N. E., Humphreys, D. T., Preiss, T., Steinmetz, L. M., et al. (2012). Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149(6): 1393-1406. Chang, K. C., Diermeier, S. D., Yu, A. T., Brine, L. D., Russo, S., Bhatia, S., Alsudani, H., Kostroff, K., Bhuiya, T., Brogi, E., et al. (2020). MaTAR25 lncRNA regulates the Tensin1 gene to impact breast cancer progression. Nat Commun 11(1): 6438. Chen, C. K., Blanco, M., Jackson, C., Aznauryan, E., Ollikainen, N., Surka, C., Chow, A., Cerase, A., McDonel, P. and Guttman, M. (2016). Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science 354(6311): 468-472. Chu, C., Qu, K., Zhong, F. L., Artandi, S. E. and Chang, H. Y. (2011). Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. Mol Cell 44(4): 667-678. Chu, C., Zhang, Q. C., da Rocha, S. T., Flynn, R. A., Bharadwaj, M., Calabrese, J. M., Magnuson, T., Heard, E. and Chang, H. Y. (2015). Systematic discovery of Xist RNA binding proteins. Cell 161(2): 404-416. Crooke, S. T., Baker, B. F., Witztum, J. L., Kwoh, T. J., Pham, N. C., Salgado, N., McEvoy, B. W., Cheng, W., Hughes, S. G., Bhanot, S., et al. (2017). The Effects of 2'-O-Methoxyethyl Containing Antisense Oligonucleotides on Platelets in Human Clinical Trials. Nucleic Acid Ther 27(3): 121-129. Guttman, M., Donaghey, J., Carey, B. W., Garber, M., Grenier, J. K., Munson, G., Young, G., Lucas, A. B., Ach, R., Bruhn, L., et al. (2011). lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477(7364): 295-300. Hacisuleyman, E., Goff, L. A., Trapnell, C., Williams, A., Henao-Mejia, J., Sun, L., McClanahan, P., Hendrickson, D. G., Sauvageau, M., Kelley, D. R., et al. (2014). Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat Struct Mol Biol 21(2): 198-206. He, H., Wang, N., Yi, X., Tang, C. and Wang, D. (2017). Long non-coding RNA H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in clear cell renal cell carcinoma. Cell Biosci 7: 65. Lee, S., Kopp, F., Chang, T. C., Sataluri, A., Chen, B., Sivakumar, S., Yu, H., Xie, Y. and Mendell, J. T. (2016). Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins. Cell 164(1-2): 69-80. Liang, X. H., Sun, H., Nichols, J. G. and Crooke, S. T. (2017). RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus. Mol Ther 25(9): 2075-2092. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408. Mayr, C. (2017). Regulation by 3'-Untranslated Regions. Annu Rev Genet 51: 171-194. McHugh, C. A., Chen, C. K., Chow, A., Surka, C. F., Tran, C., McDonel, P., Pandya-Jones, A., Blanco, M., Burghard, C., Moradian, A., et al. (2015). The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521(7551): 232-236. Moore, M. J. (2005). From birth to death: the complex lives of eukaryotic mRNAs. Science 309(5740): 1514-1518. Munschauer, M., Nguyen, C. T., Sirokman, K., Hartigan, C. R., Hogstrom, L., Engreitz, J. M., Ulirsch, J. C., Fulco, C. P., Subramanian, V., Chen, J., et al. (2018). The NORAD lncRNA assembles a topoisomerase complex critical for genome stability. Nature 561(7721): 132-136. West, J. A., Davis, C. P., Sunwoo, H., Simon, M. D., Sadreyev, R. I., Wang, P. I., Tolstorukov, M. Y. and Kingston, R. E. (2014). The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol Cell 55(5): 791-802. Yu, A. T., Berasain, C., Bhatia, S., Rivera, K., Liu, B., Rigo, F., Pappin, D. J. and Spector, D. L. (2021). PHAROH lncRNA regulates Myc translation in hepatocellular carcinoma via sequestering TIAR. Elife 10: e68263. Zhu, X. (2010). Seeing the yin and yang in cell biology. Mol Biol Cell 21(22): 3827-3828. Zong, X., Huang, L., Tripathi, V., Peralta, R., Freier, S. M., Guo, S. and Prasanth, K. V. (2015). Knockdown of nuclear-retained long noncoding RNAs using modified DNA antisense oligonucleotides. Methods Mol Biol 1262: 321-331. Article Information Copyright Yu et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Biochemistry > RNA > RNA-protein interaction Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP) Marisa Müller [...] Peter B. Becker Mar 5, 2021 2979 Views Identification of Protein-RNA Interactions in Mouse Testis Tissue Using fRIP Alexis S. Bailey [...] Margaret T. Fuller Jan 5, 2022 2571 Views Revised iCLIP-seq Protocol for Profiling RNA–protein Interaction Sites at Individual Nucleotide Resolution in Living Cells Syed Nabeel-Shah and Jack F. Greenblatt Jun 5, 2023 2780 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,573	https://bio-protocol.org/en/bpdetail?id=4573&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Direct in vitro Fatty Acylation Assay for Hedgehog Acyltransferase AS Adina R. Schonbrun MR Marilyn D. Resh Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4573 Views: 431 Reviewed by: Gal HaimovichQin Tang Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Oct 2022 Abstract Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity. Our assay utilizes purified Hhat, a fluorescently labeled fatty acyl-CoA donor substrate, and a Sonic hedgehog (Shh) peptide recipient substrate sufficient for fatty acylation. The protocol is a straightforward process that yields direct readout of fatty acylated Shh peptide via fluorescence detection of the transferred fatty acyl group. Graphical abstract Graphical abstract adapted from Schonbrun and Resh (2022) Keywords: Hedgehog (Hh) Sonic hedgehog (Shh) Hedgehog acyltransferase (Hhat) Nitrobenzoxadiazole (NBD) Fatty acyl-CoA Background The Hedgehog (Hh) pathway plays an essential role in embryonic development and in multiple cancers in adults. Signaling is mediated by the secretion of Hh proteins, e.g., Sonic hedgehog (Shh), ultimately resulting in the activation of GLI transcription factors and expression of Hh pathway target genes. An essential component of the signaling pathway is the enzyme Hedgehog acyltransferase (Hhat), which catalyzes the transfer of palmitate as well as other fatty acyl groups to the N-terminal cysteine of Hh proteins (Schonbrun and Resh, 2022). This post-translational modification has been shown to be required for Hh signaling (Micchelli et al., 2002;Chen et al., 2004). Hhat is a member of the membrane-bound O-acyltransferase enzyme family and is localized in the membrane of the endoplasmic reticulum. To date, several assays have been reported to measure the catalytic activity of Hhat in vitro. However, some of these assays rely on the use of radioactive substrates (Buglino and Resh, 2008; Jiang et al., 2021), which have safety hazards and require specialized waste disposal. Others require specialized equipment (Lanyon-Hogg et al., 2017) or multiple steps (Lanyon-Hogg et al., 2015) to achieve product readout, or measure a change in fluorescence anisotropy conferred by fatty acylation of a fluorescently conjugated Shh peptide (Lanyon-Hogg et al., 2019; Andrei et al., 2022). We set out to develop a straightforward, fluorescence-based assay to quantify the catalytic activity of purified Hhat in vitro by directly monitoring the transfer of a fluorescently labeled fatty acid to Shh. This protocol has the advantage of allowing for unambiguous competition analysis with other fatty acyl-CoAs (in addition to competition analysis with alternative forms of Shh substrate), by including these unlabeled substrates in the reaction. The two labeled substrates, biotinylated Shh peptide and NBD-labeled fatty acyl-CoAs (as well as the corresponding, unlabeled compounds for competition analysis) are readily obtained from commercial sources. Our assay is sensitive and specific and can potentially be adapted to a high-throughput format using a streptavidin-coated multi-well plate to capture the biotinylated Shh substrate. Materials and Reagents 0.5 mL tubes (RPI, catalog number: 145505) Black side, transparent bottom 96-well plate (Thermo Scientific, catalog number: 265301) Biotinylated Shh peptide [CGPGRGFGKR-(PEG2)-K(Biotin)-NH2] [Anaspec and Peptide 2.0, custom synthesis, PEG2 = Fmoc-NH-PEG2-CH2-COOH; M.W. (theoretical) 1532.82 g/mol, order at least 5 mg, free N-terminus, NH2 at C-terminus, trifluoroacetate salt, QC analysis by MS and >95% purity by peak area on HPLC (220 nm, C18 column, Buffer A: 0.05% TFA in H2O, Buffer B: 0.05% TFA in 90% CH3CN, linear gradient 10%–35% B in 25 min] (store at a minimum of -20 °C) Nitrobenzoxadiazole (NBD)-fatty acyl-CoAs (Avanti, catalog numbers: 810229P and 810705P) (store aliquots at a minimum of -20 °C) n-Dodecyl-β-D-maltopyranoside (DDM) (Anatrace, catalog number: D310) (store at -20 °C) 3× FLAG® peptide (Millipore Sigma, catalog number: F4799) (store at -20 °C) n-Octyl-β-D-glucopyranoside (OG) (EMD Millipore, catalog number: 494459) (store at room temperature) Triton X-100 (Fisher Scientific, catalog number: BP151-500) (store at room temperature) MES (Fisher Scientific, catalog number: BP300-100) (store at room temperature) DTT (Promega, catalog number: V3151) (store aliquots at -20 °C) PierceTM high-capacity streptavidin beads (Thermo Scientific, catalog number: 20361) (store at 4 °C) Purified Hhat and mock purified eluate or elution buffer (see Recipes) (Schonbrun and Resh, 2022) (store at -80 °C) Reaction buffer (see Recipes) Wash buffer (see Recipes) Equipment Fluorescence plate reader (BioTek Synergy H1) Refrigerated tabletop microcentrifuge (Eppendorf Centrifuge, model: 5417R) PicoFuge (Stratagene) Vacuum aspirator with attached 26½ G needle (Becton Dickinson, catalog number: 305111) Repeat or regular pipettors Software Gen5 (BioTek) Excel (Microsoft) Prism (GraphPad) Procedure Assay setup Set up the appropriate number of 0.5 mL plastic tubes in a rack at room temperature. Reactions should be performed in at least duplicate. Prepare fresh reaction buffer (room temperature) by adding DTT prior to the start of the assay. Prepare a suspension of streptavidin-coated beads in wash buffer. Transfer a total volume of slurry corresponding to 15 µL of slurry per reaction into a tube. Example: In an assay with eight reactions to be assayed in duplicate, 240 µL [(8 × 2) × 15 µL] of slurry should be prepared along with an additional 60 µL (4 × 15 µL) for the standards, to make a total of 300 µL slurry (240 µL + 60 µL). The combined slurry should be resuspended with 1,000 µL [(16 + 4) × 50 µL] wash buffer after washing. Be sure to include in the calculation for the beads the additional beads needed for NBD standards at the end of the assay (see step C5). Wash the beads once by adding a total volume of wash buffer corresponding to 50 µL per reaction to the tube and inverting the tube several times. Centrifuge the beads at 2,700 × g and 4 °C for 1 min in a tabletop microcentrifuge and aspirate the supernatant using a 26½ G needle. Resuspend the beads with a total volume of wash buffer corresponding to 50 µL of wash buffer per reaction. Keep tube on ice. Aliquot reaction buffer and the desired concentrations of NBD-fatty acyl-CoA (we typically use 10 μM) and Shh peptide (we typically use 10 μM) into each tube. For inhibition or substrate competition assays, include drug/inhibitor [e.g., RU-SKI 201 (Cayman, catalog number: 21101)] or unlabeled substrates (e.g., non-fluorescent fatty acyl-CoA or non-biotinylated Shh protein/peptide) at desired concentrations. Include appropriate control reactions. Note: The final volume in each tube should be 100 µL, and importantly, should include a final concentration of 0.1% DDM (detergent component of the purified Hhat eluate). Final concentrations of MES, OG (w/v), and DTT from the reaction buffer should be in the range of 125 mM, 0.06%, and 750 µM, respectively. If desired, a master mix of reaction buffer and substrates can be prepared. Reaction Initiate the reaction by adding purified Hhat enzyme (or equivalent volume of mock purified eluate or elution buffer as a negative control) (we typically use 130 nM Hhat). Briefly vortex and pulse-spin in a PicoFuge to collect the sample at the bottom of the tube. Immediately incubate the reactions in the dark at 37 °C for the desired amount of time. Note: For kinetic studies, a time course must be performed first to determine the linear range of the reaction. Suggested time points for initial kinetic studies are 0, 10, 20, 40, 60, and 90 min. Quench the reactions by placing the tubes on ice and adding 60 µL of ice-cold bead suspension. Isolation and quantification of reaction products Incubate tubes in the dark with rocking at 4 °C for 1 h. Centrifuge the beads at 2,700 × g and 4 °C for 1 min in a tabletop microcentrifuge and carefully aspirate the supernatant using a 26½ G needle. Wash the beads three times with 100 µL of wash buffer. Between each wash, spin tubes and aspirate supernatant as above. Tip for washing: place the tubes in a single rack, cover the top of the rack, and invert the rack approximately five times to wash. After the final wash, carefully aspirate the supernatant, resuspend the beads in each tube with 60 µL of wash buffer, and carefully transfer the beads from each tube to individual wells of a black side, transparent bottom 96-well plate. Use a fresh pipette tip for each tube. Note: To maximize bead transfer, after the first transfer of beads into the plate, use the pipette tip to push residual beads from the sides down to the bottom of the tube where some suspension remains and transfer this remaining suspension into the same well. To obtain a conversion factor of NBD fluorescence (RFU) to pmol of NBD-labeled product for data quantification (see next section), load standards [known amounts (e.g., 500–2,000 pmol)] of NBD-acyl-CoA in bead suspension (60 µL, as in step C4) into separate wells of the plate. Read NBD fluorescence (RFU) in a plate reader at excitation/emission wavelengths of 465/535 nm. Data analysis Export RFU readings from Gen5 software directly to an Excel spreadsheet. Quantify NBD-acylated Shh product using the RFU-to-pmol conversion factor obtained from the standards. To obtain the conversion factor, divide the RFU output value by the known number of pmol of NBD donor loaded. For calculation of the initial rate, choose a time point at which the reaction is linear (we typically use 10 min). Divide pmol of NBD-acylated Shh produced at that time point by the number of minutes, then divide by the number of pmol of Hhat to give pmol NBD-acylated Shh per min per pmol Hhat in units of min-1 . Data can be plotted in Excel or GraphPad Prism with a standard bar or line graph, or in GraphPad Prism with best-fit kinetic models, as appropriate (e.g., Figure 1). Note: For kinetic models, data should be presented as an initial rate (pmol product per min or pmol product per min per amount of enzyme). Figure 1. Hhat enzymatic activity as a function of NBD-palmitoyl-CoA concentration. Increasing concentrations of NBD-palmitoyl-CoA were incubated with 25 µM wild-type Shh peptide and purified Hhat for 10 min at 37°C. The initial rate of Hhat-mediated NBD-palmitoylation of Shh was quantified; Michaelis–Menten fit of the data (produced in GraphPad Prism) is shown with apparent kinetic parameters as insets (Schonbrun and Resh, 2022). Recipes Elution buffer 20 mM HEPES, pH 7.5 350 mM NaCl 1% (v/v) glycerol 1% (w/v) DDM 150 µg/mL 3× FLAG® peptide Reaction buffer 167 mM MES, pH 6.5 0.083% (w/v) OG 1 mM DTT (added fresh) Wash buffer 167 mM MES, pH 6.5 0.083% (v/v) Triton X-100 Acknowledgments This research was supported by NIH Grant GM116860, by a grant from the Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research and The Center for Experimental Therapeutics at Memorial Sloan Kettering Cancer Center, by Cancer Center Core Support Grant P30 CA008748 from the National Institutes of Health to Memorial Sloan Kettering Cancer Center, and by an American Heart Association Predoctoral Fellowship #20PRE35040000 (2020) to ARS. Competing interests The authors declare they have no competing interests with the contents of this article. References Andrei, S. A., Tate, E. W. and Lanyon-Hogg, T. (2022). Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay. Methods Mol Biol 2374: 13-26. Buglino, J. A. and Resh, M. D. (2008). Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog. J Biol Chem 283(32): 22076-22088. Chen, M. H., Li, Y. J., Kawakami, T., Xu, S. M. and Chuang, P. T. (2004). Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev 18(6): 641-659. Jiang, Y., Benz, T. L. and Long, S. B. (2021). Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT. Science 372(6547): 1215-1219. Lanyon-Hogg, T., Masumoto, N., Bodakh, G., Konitsiotis, A. D., Thinon, E., Rodgers, U. R., Owens, R. J., Magee, A. I. and Tate, E. W. (2015). Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase. Anal Biochem 490: 66-72. Lanyon-Hogg, T., Patel, N. V., Ritzefeld, M., Boxall, K. J., Burke, R., Blagg, J., Magee, A. I. and Tate, E. W. (2017). Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation. SLAS Discov 22(4): 418-424. Lanyon-Hogg, T., Ritzefeld, M., Sefer, L., Bickel, J. K., Rudolf, A. F., Panyain, N., Bineva-Todd, G., Ocasio, C. A., O'Reilly, N., Siebold, C., Magee, A. I. and Tate, E. W. (2019). Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes. Chem Sci 10(39): 8995-9000. Micchelli, C. A., The, I., Selva, E., Mogila, V. and Perrimon, N. (2002). Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development 129(4): 843-851. Schonbrun, A. R. and Resh, M. D. (2022). Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates. J Biol Chem 289(10): 102422. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > Cancer biochemistry > Protein Biochemistry > Protein > Activity Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays Tobias Schachinger [...] Peter L. Turecek Sep 5, 2024 340 Views Measurement of the Activity of Wildtype and Disease-Causing ALPK1 Mutants in Transfected Cells With a 96-Well Format NF-κB/AP-1 Reporter Assay Tom Snelling Nov 20, 2024 272 Views Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays Tom Snelling Nov 20, 2024 257 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,574	https://bio-protocol.org/en/bpdetail?id=4574&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Synthesis of Novel MicroRNA-30c Analogs to Reduce Apolipoprotein B Secretion in Human Hepatoma Cells YZ Ya Ying Zheng PH Phensinee Haruehanroengra PY Pradeep Kumar Yadav SI Sarah Irani SM Song Mao TW Ting Wang MH M. Mahmood Hussain JS Jia Sheng Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4574 Views: 589 Reviewed by: Chiara AmbrogioMatthew Swire Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Apr 2022 Abstract Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2’-O-methyl (2’OMe), 2’-fluoro (2’F), pseudouridine (ᴪ), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases. Keywords: Lipids Lipoproteins Microsomal triglyceride transfer protein Modified microRNAs GalNAc Background Atherosclerosis, the aggregation of fibrofatty plaques in the artery wall, has been implicated in the pathogenesis of cardiovascular diseases with high morbidity and mortality rates in the United States and worldwide. High levels of plasma cholesterol is a risk factor leading to myocardial infarctions and strokes. Cholesterol is insoluble and, therefore, is packaged into lipoproteins for transport and delivery. As the major carriers, lipoproteins consist of fatty components such as free cholesterol, triglycerides, and phospholipids, enclosed by apolipoproteins (Apo) on the surface (Jialal and BartonDuell, 2016). These serum lipoproteins, synthesized in the liver, mainly include apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB). Aside from endogenous lipids production, the liver is also a key player in lipid metabolism (Yan et al., 2019). Abnormal lipid metabolism is one of the most important indications of cancer cell formation (Liu et al., 2017). Hepatocellular carcinoma (HCC), the most common form of liver cancer, accounts for approximately 80%–90% of all liver cancer types diagnosed worldwide. Studies have found that the ApoA1 level was substantially higher in curative resection HCC patients with overall improved survival. This suggested that ApoA1 can inhibit tumor proliferation and induce apoptosis, possibly by the mitogen-activated protein kinase (MAPK) pathway (Ma et al., 2016). Moreover, another study noticed that elevated ApoB is an alarming sign of tumor size greater than 5 cm at diagnosis and is associated with poor prognosis in HCC patients (Yan et al., 2019). Taken together, these findings indicate that both ApoA1 and ApoB are potential biomarkers for prognosis in post-surgery HCC patients. Additionally, ApoB is the main driver in atherogenesis, as it is the primary transporter for low density lipoprotein (LDL) (Libby et al., 2019). On the other hand, ApoA1 is a constituent of high density lipoprotein, known as the good cholesterol, playing a role in the elimination of excess cholesterol from peripheral tissues by the process of reverse cholesterol transport to the liver (Frank and Marcel, 2000). Deregulation in LDL cholesterol levels has also been linked to risk of myocardial infarction and cardiovascular disease (Mortensen and Nordestgaard, 2020). Statins and proprotein convertase subtilis/kexin type 9 inhibitors (PCSK9) lower the plasma levels of low-density lipoprotein cholesterol (LDL-C) in hyperlipidemia and hypercholesterolemia patients (Blumenthal, 2000; Stoekenbroek et al., 2015); however, roughly 1–2 out of 10 patients develop statin intolerance with adverse symptoms (Ahmad, 2014). Moreover, cholesterol absorption inhibitors are ineffective in patients with genetic conditions caused by loss-of-function mutations in the LDL receptor or gain-of-function mutations in PCSK9, making homozygous familial hypercholesterolemia (FH) especially challenging to treat (Rader and Kastelein, 2014). Therefore, there is a need for more robust therapies that would encompass FH patients with lifelong drug durability. Additional approaches could involve reducing assembly and secretion of ApoB-containing lipoproteins. Biosynthesis of lipoproteins requires two components: a chaperone protein, the microsomal triglyceride transfer protein (MTP), and a structural protein, the ApoB. Both of these proteins are target candidates for therapeutic intervention in treating premature coronary diseases (Hussain and Bakillah, 2008). Lomitapide, a MTP inhibitor, is the current available therapy for patients with FH; however, this drug is known to cause hepatic steatosis and increase plasma transaminases, markers of liver injury (Rizzo and Wierzbicki, 2011). Hence, new avenues must be considered to reduce plasma LDL-C levels. MicroRNAs (miRs) are short, non-coding RNA species, approximately 22–23 nucleotides in length that regulate gene expression post transcriptionally (Bartel, 2009). miRs are also known to be involved in various biological processes of cancerous cells, such as cell cycle and apoptosis, and were therefore further defined as either proto-oncogenes or tumor suppressors (Gong et al., 2015). The overexpression of tumor suppressor miRs can be used as a gene silencing tool that inhibits cell proliferation and metastasis, which ultimately leads to tumor cells arrest (Khare et al., 2013). MiRs interact with the 3′-untranslated regions of target mRNAs by base pair complementarity to signal for their degradation and/or translational repression (Iwakawa and Tomari, 2015). Since their discovery, miR therapeutics have been an area of active research for their potential in treating different diseases. However, these developments have encountered difficulties. Chemical modifications result in loss of potency. Safe and targeted delivery is also challenging (Rupaimoole and Slack, 2017). To address these shortcomings, we synthesized an azido-modified N-acetylgalactosamine (GalNAc) moiety (GalNAcαProN3) that can be attached to any position of the RNA oligonucleotide modified with an alkyne group. In addition, phosphorothioate linkages were also introduced for the best action. Of course, this design could be extended to other miR systems, such as miR-122, which has been shown to inhibit growth and proliferation of HepG2 cells (Li et al., 2018). In our study, miR-30c was the best candidate in lowering plasma lipids and testing the effect on cell penetrating capacity when modifying the passenger strand. MiR-30c is a short, double-stranded non-coding RNA that belongs to the miR-30 family (miR-30a-e), highly conserved in the seed sequence (Irani and Hussain, 2015). We have previously reported that miR-30c interacts with the 3′-untranslated region of the MTP mRNA, leading to mRNA degradation and thereby reducing the secretion of apolipoprotein B by liver cells (JamesSoh, 2018. The successful synthesis of modified miR-30c passenger analogs with increased duplex stability and enhanced uptake by hepatoma cells, resulting in significant ApoB reductions without affecting the level of ApoA1(Yadav et al., 2022), have proven the feasibility of this approach. By introducing GalNAc residues at either end, we eliminated the need for lipid emulsion delivery as it might be more cost effective. Here, we detail the synthesis of modified miR-30c analogs. We envision this approach to be versatile as it can be applied to any tumor suppressor miR systems, to facilitate the development of their analogs as possible therapeutic drugs for lipid-lowering therapies and for the treatment of other diseases. Materials and Reagents U-shape cell culture flask, canted neck, 75 cm2 (Corning, catalog number: 430641U) 96-well ELISA plates (Fisher Scientific, Corning, catalog number: 07-200-640) 6-well plate (Fisher Scientific, Corning, catalog number: 07-200-80) PrecisionGlideTM 25G needle (BD, catalog number: 305125) Thin layer chromatography (TLC) plates pre-coated with silica gel F254 (Sigma-Aldrich, catalog number: 717185) WatersTM Corp Sep-Pak C18 3 cc Vac cartridge (Fisher Scientific, catalog number: 50785823) Protected RNA phosphoramidites [ChemGenes, catalog numbers: ANP-5671 (rA-CE), ANP-5673 (rG-CE), ANP-6676 (Ac-rC-CE), and ANP-5674 (rU-CE)] Oligonucleotide synthesizer empty column (Alibaba, catalog number: HJ-H-818) CPG-500 Å solid support (ChemGenes, catalog number: N-6103-05) Anhydrous acetonitrile or ACN (CH3CN), HPLC grade (Sigma-Aldrich, catalog number: 34851-4L) 3% trichloroacetic acid in dichloromethane (ChemGenes, catalog number: RN-1462) 5-Ethylthio-1H-tetrazole (ETT) in acetonitrile solution (0.25 M) (ChemGenes, catalog number: RN-1466) Tetrahydrofuran (THF) (Sigma-Aldrich, catalog number: 186562-1L) CapA (80% THF/10% acetic anhydride/10% 2,6-lutidine) (ChemGenes, catalog number: RN-1458) CapB (16% N-methylimidazole in THF) (ChemGenes, catalog number: RN-7776) 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione) (DDTT) (ChemGenes, catalog number: RN-1588) Ammonium hydroxide (Sigma-Aldrich, catalog number: 221228-500mL) Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418-100mL) Triethylamine trihydrogen fluoride (Et3N·3HF) (Sigma-Aldrich, catalog number: 344648-25g) Sodium acetate (Invitrogen, catalog number: AM9740) N-Acetyl-D-galactosamine (Thermo Fisher Scientific, catalog number: J66095.06) Acetyl chloride (Thermo Fisher Scientific, catalog number: 219472500) Sodium azide (NaN3) (Thermo Fisher Scientific, catalog number: J21610.A1) Sodium iodide (NaI) (Thermo Fisher Scientific, catalog number: 203182500) 2'-O-propargyl adenosine (n-bz) CED phosphoramidite (ChemGenes, catalog number: ANP-7751) 2'-O-propargyl cytidine (n-bz) CED phosphoramidite (ChemGenes, catalog number: ANP-7752) 2'-O-propargyl guanosine (n-ibu) CED phosphoramidite (ChemGenes, catalog number: ANP-7753) 2'-O-propargyl uridine CED phosphoramidite (ChemGenes, catalog number: ANP-7754) Copper(I) bromide (CuBr) (Thermo Fisher Scientific, catalog number: 047211.30) Tert-butanol (tBuOH) (Sigma-Aldrich, catalog number: 471712) Sodium phosphate, monobasic (NaH2PO4) (Fisher Scientific, catalog number: 567545500GM) Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: 7558794) Sodium chloride (NaCl) (Fisher Scientific, catalog number: AC447300010) Modified Eagle’s medium (DMEM) (Fisher Scientific, catalog number: MT10027CV) Fetal bovine serum (FBS) (Fisher Scientific, catalog number: MT35010CV) L-glutamine (Fisher Scientific, catalog number: AAJ60573A1) LipofectamineTM RNAiMAX transfection reagent (Fisher Scientific, Invitrogen, catalog number: 13778075) Human ApoB ELISA development kit (HRP) (MABTECH lnc., catalog number: 3715-1H-6) 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific, catalog number: J61325.EQE) Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific, catalog number: 23200) Human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems, catalog number: DY3664) Substrate reagent pack (R&D Systems, catalog number: DY999) Stop solution 2 N sulfuric acid (R&D Systems, catalog number: DY994) Protease inhibitor cocktail (Fisher Scientific, catalog number: PIA32965) Anti-MTTP/MTP antibody (Abcam, catalog number: ab63467) β-Actin (D6A8) rabbit mAb (Cell Signaling, catalog number: 8457) Anti-rabbit IgG, HRP-linked antibody (Cell Signaling, catalog number: 7074) Phosphate buffer saline (PBA 10×) (Thermo Fisher Scientific, catalog number: AAJ62851AP) Oxidation solution (0.02 M iodine in THF/pyridine/water, 7:2:1 v/v) (ChemGenes Corporation, catalog number: RN14552L) Screw cap tube (Thermo Scientific, catalog number: AB1389500) Solid phase synthesis column frit (BioAutomation, catalog number: FR-1502-1) Molecular Biology Grade (200 Proof) Ethanol (Fisher Scientific, catalog number: BP2818100) RNase/DNase free water (MP BiomedicalsTM, catalog number: MP112450204) 3-chloroproanol (Thermo ScientificTM, catalog number: C0270100G) TEAAC buffer (see section B, #14 for detailed preparation) 8 M urea (see section B, #15 for detailed preparation) 30% acrylamide/bis stock (Thermo ScientificTM, catalog number: HBGR329500) 10× TBE running buffer (Invitrogen, catalog number: AM9864) TEMED (Thermo ScientificTM, catalog number: 17919) Ammonium Persulfate (APS) (Thermo ScientificTM, catalog number: 17874) Loading buffer (Thermo ScientificTM, catalog number: J62832S0) Ethidium Bromide 10mg/ml (InvitrogenTM, catalog number: 15585011) Tm Buffer or sodium phosphate buffer pH 6.5 (see Recipes) Buffer K containing one tablet of protease inhibitor cocktail (see Recipes) Equipment Nuclear magnetic resonance NMR (Brucker, Ascend 500 MHz spectrometer) Automated solid-phase oligonucleotide synthesizer (BIOSSET Ltd, ASM800) DNA/RNA speed-vac concentrator (Thermo Electron Corporation, Savant DNA 120 SpeedVac Concentrator) Mass spectrometer (Agilent Technologies, Accurate-Mass Q-TOF LC/MS 6530) UV-visible spectrometer (Perkin Elmer, Lambda 35 UV/VIS Spectrometer) Circular Dichromism (CD) Spectropolarimeter (JASCO, J-815 spectropolarimeter) ChemiDocTM touch imaging system (Bio-Rad, 1708370) Eppendorf centrifuge (Eppendorf, 5424) UV lamp (MilliporeSigmaTM SupelcoTM, Z16964114EA) Tube revolver rotator (Thermo ScientificTM, 88881001) Heating Blot (Thermo SchientificTM, 88870003) Procedure Synthesis of chemically modified novel miR-30c-3p analogs (Figure 1) Note: Standard phosphoramidites and reagents were available from ChemGenes. Modified antisense miR-30c oligonucleotides were synthesized at one micromole scale using Oligo-800 DNA/RNA solid-phase synthesizer. The system is protected under helium gas. The synthesis proceeds in 3′ to 5′ direction with four main steps per synthesis cycle: detritylation, coupling, capping, and oxidation that adds one nucleotide at the completion of one cycle. All synthesis steps are performed on control-pore glass (CPG-500) that has the first nucleotide base immobilized through succinate linker inside the column. Figure 1. Preparation for RNA synthesis. (A) Solid phase synthesis empty columns with frits. (B) Column assembled with tow frits that hold the CPG-500 beads in the center, where the synthesis takes place. (C) Securely mound the columns onto the system. (D) Screw on each nucleotide AUCG reservoir, measured and dissolved in anhydrous acetonitrile (ACN). Dissolve the modified and native RNA phosphoramidites separately in anhydrous acetonitrile to a concentration of 0.07 M. Note: 0.07 M is the final concentration for each type of phosphoramidite (modified and native); the volume of anhydrous acetonitrile is determined by the length, sequence, and molecular weight of commercial AUCG phosphoramidites. Assemble the column with two frits that hold the CPG-500 Å beads in the center, where the synthesis takes place. Employ the Oligo-800 synthesizer in DMTr-off mode to perform automated solid-phase oligonucleotide synthesis of modified miR-30c analogs on a one micromole scale. All the necessary phosphoramidite solutions, including A, U, C, and G, are pre-filled in the synthesis lines and pressure-checked before the synthesis can begin. Note: This is an automatic system; the input sequence is the final synthesized product. The synthesis adds one nucleotide per synthesis cycle, beginning with detritylation and ending with oxidation. The average time for eight -mers of RNA is 4 h under working pressure of 1.5 bars. Detritylation is the removal of the 5′ DMT-protecting group on the 3′-end of the oligonucleotide, using 3% trichloroacetic acid in dichloromethane. Coupling is carried out with ETT (0.25 M in acetonitrile) for 12 min. Capping of the 5′-OH is performed using CapA (80% THF/10% acetic anhydride/10% 2,6-lutidine) and CapB (16% N-methylimidazole in THF) solutions Phosphate backbone oxidation is performed with 20 mM iodine solution in pyridine/THF/water; the synthesis of phosphorothioate backbones is performed with the sulfurization reagent, DDTT. After the completion of the synthesis cycle, transfer the RNA product into a screw cap tube. Cleave the RNAs from the solid support and deprotect using 800 µL of concentrated aqueous ammonium hydroxide at room temperature for 18 h. Dry the resulting RNA solution in a speed vacuum concentrator (approximately 3–4 h). The Savant DNA/RNA 120 speed-vac concentrator operates at three drying rates—low, medium, and high heat—with temperature ranging from approximately 43°C to 65°C, a displacement capacity of 31 L/min at 50 Hz and 36 L/min at 60 Hz, and maximum vacuum at 7 Torr or 9 mbar. Low or medium heat is preferred for drying to prevent sample denaturing. Redissolve the solid residue with 100 µL of DMSO by vortexing, and desilylate by incubating with 125 µL of Et3N·3HF solution at 65 °C for 2.5 h using a heating block. Desilylation is the removal of silyl moiety. Precipitate RNAs by adding 25 µL of 3 M sodium acetate solution and 1 mL of 100% ethanol and keep at -80 °C for at least 3 h. Centrifuge at 15,000 × g and 4 °C for 45 min to recover the RNA. Completely dry the RNA under vacuum. Dissolve the RNA in 500 µL of RNase-free water and assess its integrity if necessary. Perform desalting twice prior to storage at -20 °C. Synthesis of GalNAcαProN3 3 (Figure 1) and 2′-GalNAc-modified RNA strands (Figure 2) Figure 2. Synthesis of 3-chloropropyl GalNAc 2 and GalNAcαProN3 Dissolve 330 mg (1.5 mmol) of N-acetyl-D-galactosamine in 5 mL of 3-chloropropanol. Add 0.13 mL (1.8 mmol) of acetyl chloride to the above solution while on ice (approximately 0 °C). Heat the resulting solution at 70 °C for 15 h using a hot plate. Concentrate the solution by speed vacuum using medium heat and purify the residue by silica gel chromatography to get 3-chloropropyl GalNAc 2 . Typical yield of approximately 45% for 200 mg white solid with the following parameters: TLC Rf = 0.5 (20% MeOH in CH2Cl2) 1H NMR (500 MHz, D2O) δ 4.92 (d, J = 4.0 Hz, 1H), 4.15 (dd, J = 4.4, 12.8 Hz, 1H), 4.00–3.84 (m, 4H), 3.77–3.72 (m, 4H), 3.62–3.56 (m, 1H), 2.10–2.02 (m, 5H) Dissolve 200 mg (0.671 mmol) of 3-chloropropyl GalNAc 2 in 6 mL of CH3CN by heating the solution. Add 436 mg (6.71 mmol) of NaN3 and 101 mg (0.671 mmol) of NaI to the solution and stir at 60 °C for 15 h. Concentrate the solution by speed vacuum and purify the residue by silica gel chromatography to obtain GalNAcαProN3 3. Approximately 54% yield for approximately 110 mg white solid with the following parameters: TLC Rf = 0.4 (20% MeOH in CH2Cl2) 1H NMR (500 MHz, D2O) δ 4.94 (m, 1H), 4.21–4.18 (m, 1H), 4.03-3.94 (m, 3H), 3.85–3.78 (m, 3H), 3.59–3.47 (m, 3H), 2.08 (d, 3H), 1.95–1.91 (m, 2H). Note: The synthesis of GalNAc-modified RNA strands begins with synthesizing propargyl-modified RNA oligonucleotides, following protocol A, with commercially available 2′-(O-propargyl)-phosphoramidite building blocks from ChemGenes. The chemically synthesized azido-GalNAc moiety is conjugated to the propargyl RNA via click chemistry with copper as catalyst. Figure 3. Synthesis of GalNAc-modified RNA strands through Cu(I)-catalyzed click reaction To make GalNAc-modified RNA strands (sequences shown in Tables 1 and 2), add 100 equivalents of azido-modified GalNAc (GalNAcαProN3) to the freshly synthesized propargyl RNA (1 equivalent) in a 1.5 mL Eppendorf tube (i.e., for every microgram of propargyl RNA use 100 µg of azido-modified GalNAc (GalNAcαProN3) to react) (Yadav et al., 2022). Table 1. MiR-30c analogs and sequences containing GalNAc modification Compound Short form Strand Sequence (5’ to 3’) miR30c-B1 B1 miR-30c-1-3p (pC)UgGgAgAgGgUuGuUuAcUcC miR30c-B2 B2 miR-30c-1-3p CuGgGaGaGgGuUgUuUaCuC(pC)u miR30c-B3 B3 miR-30c-1-3p (pC)uGgGaGaGgGuUgUuUaCuC(pC)u miR30c-B4 B4 miR-30c-2-3p (pC)UgGgAgAaGgCuGuUuAcUcU miR30c-B5 B5 miR-30c-1-3p CuGgGaGaGgGuUgUuUa(pC)U(pC)(pC)u miR30c-B6 B6 miR-30c-1-3p (pC)UgGgAgAgGgUuGuUuA(pC)U(pC)(pC)u miR30c-B7 B7 miR-30c-2-3p CuGgGaGaAgG(pC)uGuUuA(pC)u(pC)U miR30c-B8 B8 miR-30c-2-3p (pC)UgGgAgAaGg(pC)UgUuUa(pC)U(pC)u miR30c-B9 B9 miR-30c-1-3p (pC)(pC)(pC)UgGgAgAgGgUuGuUuAcUcC miR30c-B10 B10 miR-30c-2-3p (pC)(pC)(pC)UgGgAgAaGgCuGuUuAcUcU miR30c-B11 B11 miR-30c-2-3p (pC)(pC)(pC)ugggagaaggcuguuuacucu Upper case letters: 2′-deoxy-2′-fluoro (2′-F) ribosugar-modified nucleosides; lower case letters: 2′-O-methyl (2′-OMe) ribosugar-modified nucleosides; (pC): 2′-GalNAc clicked cytidines. Table 2. MiR-30c analogs and sequences containing GalNAc and phosphorothioate linkages Compound Short form Strand Sequence (5' to 3') miR30c-C1 C1 miR-30c-1-3p (pC)•U•gGgAgAgGgUuGuUuAcUc•C miR30c-C2 C2 miR-30c-1-3p (pC)•UgGgAgAgGgUuGuUuAc•U•c•C miR30c-C3 C3 miR-30c-1-3p (pC)•U•gGgAgAgGgUuGuUuAc•U•c•C miR30c-C4 C4 miR-30c-1-3p GgGaGaGgGuUgUuUaCuC(pC)•u miR30c-C5 C5 miR-30c-1-3p GgGaGaGgGuUgUuUaCu•C•(pC)•u miR30c-C6 C6 miR-30c-1-3p C•u•GgGaGaGgGuUgUuUaCuC(pC)u Upper case letters: 2′-deoxy-2′-fluoro (2′-F) ribosugar modifications; lower case letters: 2′-O-methyl (2′-OMe) ribosugar modifications; (pC): 2′-GalNAc clicked cytidine; •: phosphorothioate linkages In a separate tube, dissolve 22 equivalents of CuBr (100 mM in 25% tBuOH/75% DMSO) in 20% acetonitrile and mix well. Transfer the mixture to the RNA solution and rotate at approximately 20 rpm using the tube revolver rotator at room temperature for 12 h. Precipitate the RNA with 3 M sodium acetate and 1 mL of cold ethanol and store at -80 °C for at least 3 h. Centrifuge the RNA at 18,400 × g for 15 min. Resuspend the RNA pellet in 500 µL of RNase-free water. Desalt the RNA solution using Sep-Pak C18 cartridges. First, condition the cartridge with 1 mL of ACN and equilibrate with 1 mL of RNase free water and 1 mL of 2 M TEAAC buffer. Discard the flow through each time. Load RNA samples dropwise and allow for RNA to bind. Perform three washes with RNase free water, 1 mL each wash, and elute the desalted RNA with 1 mL of 50% ACN. Note: 2 M TEAAC pH 8: in the fume hood, prepare 1 L of stock with 619 mL of distilled water, 268 mL of triethylamine, and 114 mL of glacial acetic acid. Recipe is adopted from the Cold Spring Harbor laboratory. Concentrate the eluted RNA using the speed vac and check the resulting products by analytical gel electrophoresis with 15% polyacrylamide containing 8 M urea. Make the 15% polyacrylamide gel containing 8 M urea stock solution (500 mL) with 50 mL of 10× TBE, 250 mL of 30% acrylamide/bis stock, and 240 g of 8 M urea. A small analytical gel is made with 6.5 mL of 15% 8 M urea stock solution, 37 µL of 10% APS, and 6.8 µL of TEMED. Heat the samples at 95 °C for 5 min and cool down on ice for another 5 min. Use 10 µM sample as final concentration after adding loading buffer. Perform the gel electrophoresis under 1× TEB as running buffer at 250 V for approximately 30 min depending on the percentage of the gel. Following electrophoresis, stain the gel with EthBr for 15 min prior to imaging. Cell culture studies with the focus on the effects of GalNAc-modified miR-30c analogs on ApoB, ApoA1, and MTP in human hepatoma Huh-7 cell line Maintain the human hepatoma Huh-7 cells in DMEM containing 10% FBS and 1% L-glutamine in a 75 cm2 culture flask with vent cap at 37 °C and 5% CO2 in a humidified incubator. Seed the Huh-7 cells in a 6-well plate with 2 mL of aforementioned media at the concentration of approximately 100,000 cells/well. Mix the non-GalNAc-modified microRNAs with RNAiMAX at a ratio of 3:1 and incubate for 30 min at room temperature prior to cell transfection. Add GalNAc-modified microRNAs directly into the cells without the need for liposome-assisted delivery. Change into 1 mL of fresh DMEM (10% FBS) media 72 h post transfection and incubate overnight. Collect media for ApoB and ApoA1 measurements. Wash and harvest the cells in the presence of protease inhibitor cocktail for MTP protein level measurements. Determine the ApoB and ApoA1 levels in the collected media using specific human ApoB/ApoA1 ELISA development kit with 3,3′,5,5′ tetramethylbenzidine substrate. Later, stop solutions in 96-well ELISA plates. Calculate each concentration using ApoB/ApoA1 standard curve prepared in parallel with the experimental samples using the standard provided by the manufacturer. Normalize ApoB/ApoA1 levels to total protein in the respective wells, i.e., set the control microRNA–transfected cells to 100% and modified miR-30c normalized to this value. Determine the MTP protein levels within the cells, wash the transfected Huh-7 cells with ice-cold PBS, and scrap off the wells in ice-cold buffer K (see Recipes). Lyse the cells by passing them through a PrecisionGlideTM 25G needle. Measure the total protein concentration using the Coomassie (Bradford) protein assay kit prior to MTP detection on western blot. Data analysis UV thermal denaturation and circular dichroism (CD) spectroscopy studies with 2′-OMe-modified miR-30c strands Note: To study the biophysical properties, the synthesized 2’-OMe-modified miR-30c-1-3p and miR-30c-2p antisense strands were annealed with sense unmodified native miR-30c-5p in sodium phosphate buffer (10 mM, pH 6.5) containing 100 mM NaCl. Heat the solution at 95 °C for 3 min and slowly cool down at the rate of 1 °C /min at room temperature for approximately 2 h. Store at 4 °C overnight prior to running on the UV-visible spectrometer. For the UV melting study, collect all data points at 260 nm with two cycles of heating and cooling from 5 °C to 85 °C (4 ramps total) at the rate of 0.5 °C/min. Record the CD spectra over a wavelength range of 200–300 nm using a 1 cm path length quartz cuvette. Collect all data points with a scanning speed of 100 nm/min, bandwidth of 1.0 nm, and integration time of 1.0 s. Figure 4. CD spectra of modified miR-30c duplexes. Compared to the native miR-30c-1-3p annealed with miR-30c-5p, the modified duplexes showed an overall increase in thermal stability by approximately 7 °C, indicating enhanced duplex stability (A). The CD spectra showed similar conformation indicating that the modification of miR-30c-1-3p does not impair the interaction with the miR-30c-5p (B) (Yadav et al., 2022). Confirmation check for the synthesized compounds Figure 5. 1H NMR spectra of 3-chloropropyl GalNAc 2 and GalNAcαProN3 3, and analysis of antisense-oligonucleotide in 15% PAGE gel. Correct synthesis of both compounds 3-Chloropropyl GalNAc 2 (A) and GalNAcαProN3 3 (B) was confirmed by 1H NMR spectroscopy and (C) 15% polyacrylamide analytical 8 M urea gel electrophoresis. Lane 1: antisense-oligonucleotide (ASO), lane 2: post-clicked GalNAc-ASO, and lane 3: reference GalNAc-ASO (Yadav et al., 2022). Effect of GalNAc-modified miR-30c analogs on ApoB and ApoA1 secretion and cellular MTP protein levels in human hepatoma Huh-7 cells Figure 6. Effects of various GalNAc-modified miR-30c analogs on apoB secretion in Huh-7 human hepatoma cells. All analogs from Table 1 were transfected into cells using lipofectamine RNAiMAX. All potently inhibited ApoB secretion (A) without affecting ApoA1 secretion (B). However, when these analogs were given to cells without lipofectamine RNAiMAX, only analogs B5 and B8 had no effect on ApoB, while all other analogs reduced secretion, ranging from 40% to 80% (C). Any of the analogs had an effect on ApoA1 (D). These results indicate that analogs B1 and B2 containing one copy of GalNAc have better biological activity compared to analogs with multiple GalNAc-modified nucleotides B5 and B8. The level of MTP protein is detected by western blot resolved on an SDS-PAGE (10%) gel. Briefly, 50 µg of total proteins were used in this experiment. A polyclonal rabbit primary antibody to human MTP and a monoclonal rabbit antibody to β-actin were used at 1:1,000 dilution. Anti-rabbit IgG and HRP-linked secondary antibody were used at 1:2,000 dilution. The blots were imaged using ChemiDocTM touch imaging system. Analysis on western blot also showed a decrease in MTP protein expression (Yadav et al., 2022). Figure 7. Effects of various phosphorothioate-linked miR-30c analogs on apoB secretion in Huh-7 human hepatoma cells. Since B1 and B2 analogs have shown potent inhibition towards ApoB secretion, we further modified the analogs (Table 2) by introducing phosphorothioate linkages, which have been suggested to improve specificity and silencing activity. The analogs synthesized with phosphorothioate linkages showed potent inhibition towards ApoB secretion (A) without having an effect on ApoA1 when cells were transfected with RNAiMAX. More importantly, all the analogs with phosphorothiorate linkages reduced ApoB secretion (C) but had no effect on ApoA1 (D) when cells were transfected without the lipofectamine-assisted delivery (Yadav et al., 2022). Recipes Tm Buffer or sodium phosphate buffer pH 6.5 Reagent Final concentration Amount NaCl 100 mM 294.4 mg Na2HPO4 10 mM 70.95 mg NaH2PO4 10 mM 59.99 mg Dissolve in total of 50 mL of Milli-Q H2O Buffer K contains 1 tablet of protease inhibitor cocktail Reagent Final concentration Amount Tris-HCl 1 mM 7.88 mg EGTA 1 mM 19.02 mg MgCl2 1 mM 4.76 mg Dissolve in a total of 50 mL of Milli-Q H2O Acknowledgments We thank U.S. National Institutes of Health (NIH) DK121490, HL137202, and HD094778 and VA Merit Award BX004113 to MMH, NSF CHE1845486 to JS, and MRI grant CHE1726724 for the financial support. The content of this protocol is based on the published work (Yadav et al., 2022). Competing interests The authors declare no conflict of interests. Ethics No human and/or animal subjects were used in this study. References Ahmad, Z. (2014). Statin intolerance. Am J Cardiol 113(10): 1765-1771. Bartel, D. P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136(2): 215-233. Blumenthal, R. S. (2000). Statins: effective antiatherosclerotic therapy. Am Heart 139(4): 577-583. Frank, P. G. and Marcel, Y. L. (2000). Apolipoprotein A-I: structure-function relationships. J Lipid Res 41(6): 853-872. Gong, J., He, X. X. and Tian, A. (2015). Emerging role of microRNA in hepatocellular carcinoma (Review). Oncol Lett 9(3): 1027-1033. Hussain, M. M. and Bakillah, A. (2008). New approaches to target microsomal triglyceride transfer protein. Curr Opin Lipidol 19(6): 572-578. Irani, S. and Hussain, M. M. (2015). Role of microRNA-30c in lipid metabolism, adipogenesis, cardiac remodeling and cancer. Curr Opin Lipidol 26(2): 139-146. Iwakawa, H. O. and Tomari, Y. (2015). The Functions of MicroRNAs: mRNA Decay and Translational Repression. Trends Cell Biol 25(11): 651-665. James Soh, J., Iqbal, J., Queiroz, J., Fernandez-Hernando, C. and Hussain, M. M. (2018). MicroRNA-30c reduces hyperlipidemia and atherosclesrosis by decreasing lipid synthesis and lipoprotein secretion. Nat Med 19: 892-900. Jialal, I. and Barton Duell, P. (2016). Diagnosis of Familial Hypercholesterolemia. Am J Clin Pathol 145(4): 437-439. Khare, S., Zhang, Q. and Ibdah, J. A. (2013). Epigenetics of hepatocellular carcinoma: role of microRNA. World J Gastroenterol 19(33): 5439-5445. Li, S., Yao, J., Xie, M., Liu, Y. and Zheng, M. (2018). Exosomal miRNAs in hepatocellular carcinoma development and clinical responses. J Hematol Oncol 11(1): 54. Libby, P., Buring, J. E., Badimon, L., Hansson, G. K., Deanfield, J., Bittencourt, M. S., Tokgozoglu, L. and Lewis, E. F. (2019). Atherosclerosis. Nat Rev Dis Primers 5(1): 56. Liu, Q., Luo, Q., Halim, A. and Song, G. (2017). Targeting lipid metabolism of cancer cells: A promising therapeutic strategy for cancer. Cancer Lett 401: 39-45. Ma, X. L., Gao, X. H., Gong, Z. J., Wu, J., Tian, L., Zhang, C. Y., Zhou, Y., Sun, Y. F., Hu, B., Qiu, S. J., et al. (2016). Apolipoprotein A1: a novel serum biomarker for predicting the prognosis of hepatocellular carcinoma after curative resection. Oncotarget 7(43): 70654-70668. Mortensen, M. B. and Nordestgaard, B. G. (2020). Elevated LDL cholesterol and increased risk of myocardial infarction and atherosclerotic cardiovascular disease in individuals aged 70-100 years: a contemporary primary prevention cohort. Lancet 396(10263): 1644-1652. Rader, D. J. and Kastelein, J. J. (2014). Lomitapide and mipomersen: two first-in-class drugs for reducing low-density lipoprotein cholesterol in patients with homozygous familial hypercholesterolemia. Circulation 129(9): 1022-1032. Rizzo, M. and Wierzbicki, A. S. (2011). New lipid modulating drugs: the role of microsomal transport protein inhibitors. Curr Pharm Des 17(9): 943-949. Rupaimoole, R. and Slack, F. J. (2017). MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov 16(3): 203-222. Stoekenbroek, R. M., Kastelein, J. J. and Huijgen, R. (2015). PCSK9 inhibition: the way forward in the treatment of dyslipidemia. BMC Med 13: 258. Yadav, P. K., Haruehanroengra, P., Irani, S., Wang, T., Ansari, A., Sheng, J. and Hussain, M. M. (2022). Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells. J Biol Chem 298(4): 101813. Yan, X., Yao, M., Wen, X., Zhu, Y., Zhao, E., Qian, X., Chen, X., Lu, W., Lv, Q., Zhang, L., et al. (2019). Elevated apolipoprotein B predicts poor postsurgery prognosis in patients with hepatocellular carcinoma. Onco Targets Ther 12: 1957-1964. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Drug Discovery > Drug Design Medicine > Cardiovascular system Biochemistry > RNA > miRNA targeting Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq Sungchul Kim and Kwangseog Ahn Apr 20, 2016 11037 Views Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets Pornima Phatak and James M Donahue May 5, 2017 18436 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,575	https://bio-protocol.org/en/bpdetail?id=4575&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Profiling of Single-cell-type-specific MicroRNAs in Arabidopsis Roots by Immunoprecipitation of Root Cell-layer-specific GFP-AGO1 LF Lusheng Fan BG Bin Gao XC Xuemei Chen Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4575 Views: 723 Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Developmental Cell Apr 2022 Abstract MicroRNAs (miRNA) are small (21–24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription ofMIRNA (MIR) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis, some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves. Graphical abstract Keywords: AGO1 Small RNA Root cell-layer-specific promoters Immunoprecipitation Small RNA-seq Background MicroRNAs (MiRNAs) play an important role in many cellular growth and development processes. A fantastic feature of miRNAs is the non-cell autonomous action, in which miRNA moves from cell to cell or over long distances (Melnyk et al., 2011), serving as a signal molecule that functions locally or systemically. Previous studies have shown that the long distance or systemic movement of miRNA is mainly through the phloem, while cell-to-cell movement is through plasmodesmata (Melnyk et al., 2011). Mobile miRNAs, such as miR394 and miR165/6, have been shown to serve as morphogens, which shape the cell pattern formation during development (Breakfield et al., 2012; Carlsbecker et al., 2010; Knauer et al., 2013). Root cell layer-specific promoter-driven expression of AGO1, the core protein in the miRNA-induced silencing complex (miRISC), shows that AGO1 is cell autonomous (Brosnan et al., 2019; Fan et al., 2022). Microtubules have been shown to specifically regulate the association of miRNA165/6 with AGO1 in the cytoplasm and play a vital role in miRNA cell-to-cell movement (Fan et al., 2022). Therefore, characterization of miRNA distribution at a high spatial resolution is critical for understanding their functions in gene regulation. Here, we describe a protocol to profile cell-type-specific miRNA by immunoprecipitation of root cell layer-specific green fluorescent protein (GFP)-AGO1, followed by small RNAseq, that provides a new tool to deepen our understanding of the regulation of miRNA functions. Materials and Reagents RNase-free pipette tips [Genesee Scientific, catalog numbers: 23-130R (10 µL), 23-150R (200 µL), 23-165R (1,250 µL)] 1.5 mL centrifuge tubes (Genesee Scientific, catalog number: 22-282) 50 mL centrifuge tubes (Genesee Scientific, catalog number: 21-108) 15 mL centrifuge tubes (Genesee Scientific, catalog number: 21-103) Petri dishes (Genesee Scientific, catalog number: 32-106) Nylon mesh (mesh opening sizes 100 µm) (NITEX, catalog number: NTX100-098) Liquid nitrogen Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, catalog number: F-530XL) In-Fusion HD cloning plus (Takara, catalog number: 638911) Murashige and Skoog medium (MS) (PhytoTech Lab, catalog number: M524) IGEPAL CA-630 (MilliporeSigma, catalog number: I8896) GFP-Trap magnetic agarose (ChromoTek, gtd-20) Sodium dodecyl sulfate (SDS) (MilliporeSigma, catalog number: L3771-500G) β-mercaptoethanol (MilliporeSigma, M3148-250ML) Glycerol (Thermo Fisher Scientific, catalog number: J16374.K2) Glycogen RNA grade (Thermo Fisher Scientific, catalog number: R0551) NEBNext multiplex small RNA library prep set for Illumina (New England Biolabs, catalog number: E7300) 10 bp O'RangeRuler DNA ladder (Thermo Fisher Scientific, catalog number: SM1313) Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: AAJ2161836) DEPC-treated water (Thermo Fisher Scientific, catalog number: 4387937) Trizma base (MilliporeSigma, catalog number: T1503) TRI reagent (MRC, catalog number: TR118) Clorox germicidal bleach (Thermo Fisher Scientific, catalog number: Essendant CLO30966EA) Triton X-100 solution (MilliporeSigma, catalog number: 93443) Bromophenol blue (MilliporeSigma, catalog number: B0126) cOmplete, EDTA-free protease inhibitor cocktail (MilliporeSigma, catalog number: 11836170001) GFP antibody (MilliporeSigma, catalog number: 11814460001) Chloroform (Thermo Fisher Scientific, catalog number: 423550040) Isopropanol (Thermo Fisher Scientific, catalog number: 423835000) 6× DNA loading dye (Thermo Fisher Scientific, catalog number: R0611) Ethidium bromide (Thermo Fisher Scientific, catalog number: J67270.14) Sodium acetate (Thermo Fisher Scientific, catalog number: A13184.30) Seed sterilization solution (see Recipes) 2× protein SDS loading buffer (see Recipes) IP lysis buffer (see Recipes) Washing buffer (see Recipes) 5× TBE buffer (see Recipes) 12% polyacrylamide gel (see Recipes) 8% SDS-PAGE resolving gel (see Recipes) SDS-PAGE stacking gel (see Recipes) 3 M sodium acetate (pH 5.2) (see Recipes) Equipment Plant growth chamber (Percival, model: CU36L5) Microcentrifuge (Eppendorf, model: 5424R) High-speed centrifuge (Beckman Counter, model: Avanti J-E Series) DynaMag-2 magnet (Invitrogen, catalog number: 12321D) H5600 revolver tube mixer (VWR, catalog number: H5600-15) Thermocyclers (Bio-Rad, catalog number: 1851148) Blade Thermomixer C (Eppendorf, catalog number: 5382000015) Spatula Mortar and pestle Mini-PROTEAN tetra vertical electrophoresis cell (Bio-Rad, catalog number: 1658001FC) Procedure Plasmid construction and plant transformation Select root cell type-specific promoters [Endodermis 7 (EN7) for endodermis and Acaulis5 (ACL5) for metaxylem/procambium were used in this study] and amplify them from Arabidopsis genomic DNA. Amplify the GFP-coding sequence from pMDC107 plasmid and the AGO1 genomic sequence containing the coding region and 3’ UTR from Arabidopsis genomic DNA using PCR (Phusion high-fidelity DNA polymerase). Assemble them with a root cell layer-specific promoter into pCambia1300 using In-Fusion HD cloning plus, according to the manual of the kit. A linker sequence of five amino acids (GGGGA) was inserted between GFP and AGO1 . The recombinant constructs were transformed into E. coli DH5α competent cell for verification (Renzette, 2011). The constructs were transformed into Agrobacterium tumefaciens GV3101 by electroporation. Transgenic lines were generated by A. tumefaciens -mediated ﬂoral dip transformation (Clough and Bent, 1998). Homozygous single insert transformants were selected by growing the transgenic lines on ½ MS (Half strength Murashige and Skoog Medium) containing antibiotics and used for the subsequent experiment. Plant material preparation and collection Measure 200 µL of transgenic seeds and sterilize with seed sterilization solution for 10 min by gently rotating at 18 rpm on a H5600 revolver tube mixer. Briefly spin at 2,000 × g for 20 s to pellet the seeds and discard the bleach solution. Resuspend the seeds with sterile water and gently rotate at 18 rpm for 1 min on a H5600 revolver tube mixer to wash the seeds. Repeat five times. Cut the nylon mesh into 9 × 9 cm squares to fit inside 150 mm Petri dishes. Autoclave them for 20 min. Transfer the nylon mesh onto the top of ½ MS Petri dishes in the sterile hood. Sow 500 seeds per Petri dish in a line on top of the nylon mesh. Grow the plants vertically at 16 h light and 8 h dark and 22 °C for nine days. Note: 200 μL transgenic seeds can be sowed into 10 plates and produce 2–3 g root tissues. Harvest the roots by cutting them off with a blade at approximately 2 cm above the root tip. Note: Change the blade regularly to avoid damaging the roots when slicing. GFP-AGO1 immunoprecipitation Grind 2 g of root tissue into fine powder in liquid nitrogen and add 4 mL of freshly prepared IP lysis buffer. Incubate at 4 °C with gentle rotation at 18 rpm for 30 min. Centrifuge at 16,000 × g for 20 min at 4 °C to remove the insoluble debris and repeat this step one more time. Transfer 100 µL of supernatant to a new tube. Add 100 µL of 2× protein SDS loading buffer and heat at 95 °C for 5 min. Store the sample at -20 °C for western blotting. Prewash 50 µL of GFP-Trap magnetic agarose beads with 1 mL of IP lysis buffer. Separate the beads with a magnetic stand and discard the supernatant. Repeat this step two more times. Transfer the remaining supernatant from step C3 into a new tube and add the prewashed GFP-trap magnetic agarose beads. Incubate on a rotator at 4 °C for 2 h. Remove the supernatant by placing the tube on a magnetic stand. Wash the beads with 1 mL of washing buffer by rotating the tube at 4 °C for 5 min. Use the magnetic stand to separate the beads from the supernatant and discard the supernatant. Repeat this step four more times. Resuspend the beads in 200 µL of washing buffer and transfer 20 µL (10% of the sample) into a new tube for western blot analysis. The remainder of the sample should be kept for RNA extraction and is referred to as the RNA sample. Use a magnetic stand to separate the beads from the supernatant in the western blot sample and discard the supernatant. Resuspend the beads with 20 µL of 1× SDS loading buffer and heat the sample at 95 °C for 5 min. Separate the beads from the supernatant using the magnetic stand and transfer the supernatant to a new tube for western blot analysis. Separate the beads from the supernatant in the RNA sample using the magnetic stand and discard the supernatant. Add 500 µL of TRI reagent to the beads to resuspend the beads. Add 100 µL of chloroform to the tube, shake vigorously, and allow sample to stand at room temperature for 5 min. Centrifuge the mixture at 10,000 rpm and 4 °C for 15 min. Transfer aqueous phase to a new tube and add 20 µg of glycogen. Add an equal volume of isopropanol into the aqueous liquid, invert several times to mix and incubate at -20 °C for 60 min. Centrifuge for 15 min at 10,000 rpm and 4 °C. Discard the supernatant and wash the RNA pellets with 70% ethanol. Remove the supernatant and air-dry the pellet at room temperature. Dissolve the RNA in 7 µL of nuclease-free water. The RNA can be stored at -80 °C for small RNA library construction. Western blot analysis for immunoprecipitation quality control Load 20 µL per sample of the crude extract from step C4 and the GFP-AGO1 immunoprecipitate from step C9 on an 8% SDS-PAGE gel. Follow the protocols for SDS-PAGE electrophoresis and western blotting to detect GFP-AGO1 using GFP antibody (Mahmood and Yang, 2012). The size of GFP-AGO1 is approximately 157 kDa. Genome wide detection of root cell layer-specific AGO1-bound small RNAs Recovered RNAs were subjected to library construction using NEBNext Small RNA Library Prep Set for Illumina according to the manual (https://www.neb.com/protocols/2018/03/27/protocol-for-use-with-nebnext-small-rna-library-prep-set-for-illumina-e7300-e7580-e7560-e7330). sRNA library recovery for sequencing Make a 12% polyacrylamide gel. Add 6× DNA loading dye into the library, mix well by pipetting, and load the sample into the gel. Load 10 µL of 10 bp O'RangeRuler DNA ladder into the same gel. Perform gel electrophoresis in 0.5× TBE buffer at 150 V for 90 min. Stain the gel with ethidium bromide in 0.5× TBE buffer for 5 min. Cut out the bands at 140–150 bp. Place the gel slice into a 1.5 mL centrifuge tube and smash the gel into small pieces using pipette tips. Add 500 µL of 0.4 M NaCl into the tube and elute the sRNA library DNA from the gel by rotating the sample for 6 h or overnight at 4 °C. Centrifuge the tube at 15,000 × g and room temperature for 1 min and transfer the supernatant into a new tube. Repeat this step two times; avoid transferring any gel into the tube. Add 1/10 volume of 3 M sodium acetate, two volumes of 100% ethanol, and 20 µg of glycogen, mix the solution, and keep at -20 °C for 6 h. Centrifuge at 16,000 × g for 30 min at 4 °C to pellet the library. Discard the supernatant and wash the pellet with 70% ethanol. Centrifuge at 16,000 × g for 10 min at 4 °C, discard the supernatant, and air-dry the pellet. Dissolve the library DNA in 30 µl of nuclease-free water. Process the small RNAseq data using home-made pipeline pRNASeqTools (https://github.com/grubbybio/pRNASeqTools). Data analysis Small RNAseq data were analyzed by pRNASeqTools pipeline. Briefly, the adaptor sequence was trimmed by cutadapt 3.0. The trimmed reads were mapped to the genome Araport 11 using Shortstack. Mapped reads were normalized by calculating RPM (reads per million mapped reads) value. Recipes Seed sterilization solution 50% Clorox germicidal bleach 0.05% Triton X-100 2× protein SDS loading buffer 100 mM Tris-Cl (pH 6.8) 4% (w/v) SDS (sodium dodecyl sulfate; electrophoresis grade) 0.2% (w/v) bromophenol blue 20% (v/v) glycerol 200 mM β-mercaptoethanol IP lysis buffer 50 mM Tris (pH 7.5) 150 mM NaCl 4 mM MgCl2 2 mM DTT 10% glycerol 0.1% IGEPAL CA-630 1× EDTA-free protease inhibitor cocktail Store at 4 °C. Washing buffer 50 mM Tris (pH 7.5) 150 mM NaCl 4 mM MgCl2 2 mM DTT 10% glycerol 0.5% IGEPAL CA-630 1× EDTA-free protease inhibitor cocktail 5× TBE buffer 450 mM Tris base 450 mM boric acid 10 mM EDTA pH 8.3 12% polyacrylamide gel 8.85 mL of ddH2O 1.5 mL of 5× TBE buffer 4.5 mL of 40% acrylamide/Bis solution (29:1) 105 µL 10% APS 5.25 µL of TEMED 8% SDS-PAGE resolving gel 2.65 mL of ddH2O 1 mL of 40% acrylamide/Bis solution (29:1) 1.3 mL of 1.5 M Tris (pH 8.8) 50 µL of 10% SDS 50 µL of 10% APS 2 µL of TEMED SDS-PAGE stacking gel 1.51 mL of ddH2O 0.20 mL of 40% acrylamide/Bis solution (29:1) 0.25 mL of 1.5 M Tris (pH 8.8) 20 µL of 10% SDS 20 µL of 10% APS 2 µL of TEMED 3 M sodium acetate (pH 5.2) 3 M sodium acetate Adjust pH to 5.2 using glacier acid Acknowledgments This protocol was adapted from our previous work (Fan et al., 2022). This work was supported by a grant from National Institutes of Health (GM129373) to Xuemei Chen. Competing interests The authors declare no conflicts of interests. References Breakfield, N. W., Corcoran, D. L., Petricka, J. J., Shen, J., Sae-Seaw, J., Rubio-Somoza, I., Weigel, D., Ohler, U. and Benfey, P. N. (2012). High-resolution experimental and computational profiling of tissue-specific known and novel miRNAs in Arabidopsis. Genome Res 22(1): 163-176. Brosnan, C. A., Sarazin, A., Lim, P., Bologna, N. G., Hirsch-Hoffmann, M. and Voinnet, O. (2019). Genome-scale, single-cell-type resolution of microRNA activities within a whole plant organ. EMBO J 38(13): e100754. Carlsbecker, A., Lee, J. Y., Roberts, C. J., Dettmer, J., Lehesranta, S., Zhou, J., Lindgren, O., Moreno-Risueno, M. A., Vaten, A., Thitamadee, S., et al. (2010). Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465(7296): 316-321. Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6): 735-743. Fan, L., Zhang, C., Gao, B., Zhang, Y., Stewart, E., Jez, J., Nakajima, K. and Chen, X. (2022). Microtubules promote the non-cell autonomous action of microRNAs by inhibiting their cytoplasmic loading onto ARGONAUTE1 in Arabidopsis. Dev Cell 57(8): 995-1008 e5. Knauer, S., Holt, A. L., Rubio-Somoza, I., Tucker, E. J., Hinze, A., Pisch, M., Javelle, M., Timmermans, M. C., Tucker, M. R. and Laux, T. (2013). A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem. Dev Cell 24(2): 125-132. Mahmood, T. and Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4(9): 429-434. Melnyk, C. W., Molnar, A. and Baulcombe, D. C. (2011). Intercellular and systemic movement of RNA silencing signals. EMBO J 30(17): 3553-3563. Renzette, N. (2011). Generation of transformation competent E. coli. Curr Protoc Microbiol 22(1): A.3L.1-A.3L.5. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant molecular biology > RNA Plant Science > Plant cell biology > Intercellular communication Molecular Biology > RNA > miRNA interference Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Micrografting in Arabidopsis Using a Silicone Chip Hiroki Tsutsui [...] Michitaka Notaguchi Jun 20, 2021 5559 Views A Novel Method to Map Small RNAs with High Resolution Kun Huang [...] Jeffrey L. Caplan Aug 20, 2021 2892 Views Quantitative Analysis of RNA Editing at Specific Sites in Plant Mitochondria or Chloroplasts Using DNA Sequencing Yang Yang and Weixing Shan Sep 20, 2021 1904 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,576	https://bio-protocol.org/en/bpdetail?id=4576&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Faster Bacterial Gene Cloning Using the Brick into the Gateway (BiG) Protocol FP Flaviani G. Pierdoná * YC Yajahaira Carbajal * MV Mateus H. Vicente * LF Letícia F. Ferigolo FN Fabio T. S. Nogueira (*contributed equally to this work) Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4576 Views: 1127 Reviewed by: Alba BlesaDemosthenis ChronisChangyi Zhang Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plasmid May 2022 Abstract Cloning systems like Gateway and Golden Gate/Braid are known because of their efficiency and accuracy. While the main drawback of Gateway is the expensive cost of the enzymes used in its two-step (LR and BP) reaction, Golden Gate requires non-reusable components due to their specific restriction sites. We present the Brick into the Gateway (BiG) protocol as a new cloning strategy, faster and more economic method that combines (i) reusable modules or bricks assembled by the GoldenBraid approach, and (ii) Gateway LR reactions [recombination of attachment sites: attL (L from left) and attR (R from right)] avoiding the BP reaction [recombination of attachment sites: attP (P from phage) and attB (B from bacteria)] usually necessary in the Gateway cloning. The starting point is to perform a PCR reaction to add type IIS restriction sites into DNA fragments generating specific fusion sites. Then, this PCR product is used to design GoldenBraid bricks, including the attL Gateway recombination sites. Using the Golden Gate method, these bricks are assembled to produce an attL1–gene of interest–attL2 fragment, which is integrated into a compatible vector producing a Gateway entry vector. Finally, the fragment containing the target gene is recombined by LR reaction into the Gateway destination vector. Graphical abstract Keywords: DNA cloning GoldenBraid Gateway Golden Gate brick assembly LR reaction Background DNA cloning tools have been improved to optimize the efficiency of cloning systems. New cloning methods were designed, such as (i) Gibson assembly, which integrates a number of DNA fragments in a single reaction using a mix of the three enzymes exonuclease, DNA polymerase, and DNA ligase (Gibson et al., 2009), (ii) Gateway, which requires specific recombination sites (atts) in both the DNA fragment and plasmids used in the two reactions steps: BP reaction—in which an entry vector, harboring a DNA fragment flanked by two attL sites, is created; and LR reaction—wherein an expression vector containing a DNA fragment flanked by attB sites is generated (Karimi et al., 2007; Invitrogen, USA); (iii) Golden Gate cloning, a method based on the use of type IIS endonucleases that have cut sites distal from their recognition sites producing overhangs; the complementary overhangs are traditionally ligated by T4 DNA ligase (Engler et al., 2008); (iv) USER cloning (Uracil-Specific Excision Reagent), which employs primers containing uracil to perform PCR reactions; the PCR product is incubated with a commercial deoxyuridine-excision enzyme to eliminate the uracil and generate 3' overhangs that allow the direct cloning of PCR fragments into a USER-compatible vector (Geu-Flores et al., 2007; Hansen et al., 2014); and (v) In-Fusion, in which four or more DNA fragments containing an overlapping of 15 bp at both ends are combined by a reaction equivalent to recombination; the ends overlap is achieved by including the 15 bp in the primers used for DNA amplification (Zhu et al., 2007). Gateway and Golden Gate are the most used cloning systems. Both have high efficiency and accuracy. However, the drawback of Gateway is the dependency on specific commercial enzymes used for the two clonase reactions, BP and LR, which makes this technique more expensive. On the other hand, Golden Gate requires specific restriction sites in the vector as well as in the DNA fragments, which could be a limiting factor when designing constructs for a large number of genes, as the components of this method are generally not reusable (Marillonnet and Grützner, 2020). Thus, modular assembly systems have been developed to standardize the parts, or bricks (DNA fragments of interest), which is crucial for cloning construction. These modular systems facilitate cloning assembly, making it simpler, more versatile, and autonomous (Patron et al., 2015). Here, we provide a new cloning method, Brick into the Gateway (BiG), that uses the GoldenBraid/Gate methods to assemble the bricks and the LR reaction from the Gateway system to transfer DNA fragments into any destination vector. Using the GoldenBraid system, we generated standard bricks of the attL sites into an acceptor plasmid pUPD2, which can be combined with other bricks harboring any DNA fragments through a Golden Gate reaction. After this reaction, an entry vector containing the DNA of interest flanked by attL sites is generated, allowing its transfer to a destination vector using Gateway LR clonase. BiG is a low-cost method with a short processing time and directional cloning, whose efficiency was successfully demonstrated (Ferigolo et al., 2022). Most importantly, BiG allows the use of assembled bricks in different studies due to its common syntax (Ferigolo et al., 2022). Materials and Reagents Enzymes High-fidelity DNA polymerase with proper buffer (e.g., platinum SuperFi II polymerase, InvitrogenTM, catalog number: 12361010) Restriction enzyme cloning BsmBI-v2 with proper buffer (New England Biolabs Inc., catalog number: R0580) Restriction enzyme cloning BsaI-HFv2 with proper buffer (New England Biolabs Inc., catalog number: R3733) T4 DNA ligase with proper buffer (e.g., Thermo Fisher ScientificTM, catalog number: EL0014) Gateway LR clonase II enzyme mix with proper buffer (InvitrogenTM, catalog number: 11791100) GoTaq green master mix (PromegaTM, catalog number: M7122) Cloning vectors Vector harboring attL sites (e.g., pENTRTM/D-TOPOTM) LM22_attL1 (Addgene, catalog number: 184008) LM22_attL2 (Addgene, catalog number: 184010) Universal GoldenBraid acceptor (e.g., pUPD2) Golden Gate acceptor vector (e.g., pICSL86900OD) Gateway expression vector of choice Kits Gel extraction kit (e.g., QIAEX II, QIAGENTM, catalog number: 20021) Plasmid Miniprep kit (e.g., GeneJET, Thermo Fisher ScientificTM, catalog number: K0702) Others dNTPs, 10 mM (Thermo Fisher ScientificTM, catalog number: R0192) PCR primers DNase- and RNase-free water PCR tubes, 0.2 mL (AxygenTM, catalog number: 14-222-262) 6×loading dye (New England Biolabs Inc., catalog number: B7024S) 1 Kb plus DNA ladder (e.g., InvitrogenTM, catalog number: 10787018) Agarose 1.5% gel (BioBasic, catalog number: AB0014) SYBRTM Safe DNA gel stain (InvitrogenTM, catalog number: S33102) Escherichia coli DH10α chemically competent cells (Thermo Fisher Scientific, catalog number: 18265017) LB plates with the appropriate antibiotic, IPTG (Fermentas, catalog number: R0392), and X-gal substrate (Fermentas, catalog number: R0401) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) Esp31 (Thermo Fisher ScientificTM, catalog number: ER0451) Eco31I (Thermo Fisher ScientificTM, catalog number: ER0291) GoldenBraid (GB) reaction (TV = 10 µL) (see Recipes) LB medium (see Recipes) Golden Gate reactions (TV = 10 µL) (see Recipes) LR reaction (TV = 5 µL) (see Recipes) Equipment PCR cycler (Mastercycler® nexus, catalog number: 6330000021) Microwave oven Gel electrophoresis chamber Blue-Light transilluminator (Safe ImagerTM 2.0, catalog number: G6600) NanoDropTM OneC Microvolume UV-Vis spectrophotometer (Thermo Fisher ScientificTM, catalog number: 13-400-519) Heat block at 42 °C ColiRollersTM plating beads, Novagen® (Sigma-AldrichTM, catalog number: 71013-3) Incubators at 37 °C Software Benchling R&D Cloud (https://www.benchling.com/) Procedure Primers design Primers should be designed according to the GoldenBraid syntax, including BsmBI (Esp3I) restriction sites followed by pUPD2 and GoldenBraid fusion sites in both extremities (Figure 1A). In case the sequence from the gene or DNA fragment of interest presents BsmBI (Esp31) or BsaI restriction sites, the fragment should be divided into sub-fragments, inserting synonymous mutations in the primers to silence the restriction site (Patron et al., 2015). Alternatively, the target gene can be amplified using primers with the BsaI sites instead of BsmBI sites, with the forward overhang GGGGTCTCAAATG and reverse overhang GGGGTCTCAAAGC (Figure 1B). The PCR products can be used directly in the Golden Gate reaction without the pUPD2 cloning step. Figure 1. Primer design to clone attL sequences and the target gene into GoldenBraid bricks, according to Vazquez-Vilar et al. (2017). Primers are designed to use the standard (A) or alternative (B) BiG protocol. Green box: BsmBI recognition site with the pUPD2 fusion sites (underlined); Orange box: fusion site to follow the Golden Gate assembly syntax; Purple box: specific sequence to amplify attL or target gene; Blue box: BsaI recognition site; RC: reverse complement. GoldenBraid bricks assembly into pUPD2 vector Individually amplify attL1 and attL2 fragments, as well as the target gene, by touchdown PCR using a high-fidelity DNA polymerase such as the platinum SuperFi II polymerase. Use a pENTRTM/D-TOPOTM vector as a DNA template to amplify attL1 and attL2 sequences. Perform the PCR reaction in 50 μL of final volume according to the enzyme manufacturer's instructions. Touchdown PCR steps: 98 °C for 1 min, followed by nine cycles of 98 °C for 30 s, proper melting temperature for 30 s, and 65 °C for 1 min, starting with 66 °C of melting temperature and decreasing 1 °C each cycle, plus 30 cycles of 98 °C for 30 s, 54 °C for 30 s, and 65 °C for 1 min, and ending with 65 °C for 3 min. Add loading dye to the PCR samples to the final concentration of 1× and perform the electrophoresis using the total PCR reaction volume and the 1 Kb plus DNA ladder in 1.5% agarose with 0.002% of SYBRTM Safe DNA gel stain. Confirm the PCR amplification and the amplicon size, 150 pb, by visualizing in the Blue-Light transilluminator. Cut the DNA band corresponding to attLs or the target gene fragments and purify it from the agarose using the gel extraction kit. Note: Purification can be performed directly from the PRC products. Quantify DNA concentration using the spectrophotometer. Clone the attL1, attL2, and target gene fragments into the GoldenBraid acceptor vector (e.g., pUPD2) by an assembly reaction using BsmBI restriction enzyme and T4 DNA ligase (Figure 2). Set up the GoldenBraid reactions according to Recipe 1 (see Recipes). Thermal cycler steps: 37 °C for 10 min followed by 30 cycles of 37 °C for 3 min and 25 °C for 4 min, ending with 37 °C for 10 min and 80 °C for 5 min. Figure 2. Schematic GoldenBraid bricks assembly. attL s and target gene individual PCR fragments (top) flanked by the BsmBI (dark green box), pUPD2 fusion sites (light green box), and Golden Gate fusion sites (light blue box) along with the pUPD2 vector (middle), were submitted to the GoldenBraid reaction to obtain the first bricks (bottom). All fusion sites are in the sense direction. Transform E. coli DH10α chemically competent cells with the total assembling reaction volume by heat shock. Plate the cells using plating beads on LB (see Recipe 2) with chloramphenicol (25 μL/mL), IPTG (1 mM), and X-gal (200 μg/mL) and incubate cell plates at 37 °C overnight. Select from two to five isolated white colonies and pick them to perform PCR colony using GoTaq green master mix to confirm the assembly construction. Perform PCR colony using specific primers for each cloned DNA sequence. Inoculate a single positive colony in liquid LB medium with chloramphenicol (25 μL/mL) and incubate shaking at 37 °C overnight. Extract the plasmid DNA from the liquid culture using plasmid Miniprep kit and quantify DNA concentration with the spectrophotometer. Golden Gate assembly Perform the Golden Gate (GG) assembly with attL1-pUPD2, attL2-pUPD2, target gene-pUPD2, and a GG acceptor vector (e.g., pICSL86900OD) using BsaI restriction enzyme and T4 DNA ligase (Figure 3). Set up the Golden Gate reactions according to Recipe 3 (see Recipes). Thermal cycler steps: 37 °C for 10 min followed by 30 cycles of 37 °C for 3 min and 25 °C for 4 min, ending with 37 °C for 10 min and 80 °C for 5 min. Figure 3. Schematic Golden Gate assembly to produce the BiG entry vector. Individual bricks harboring attL s and the target gene were submitted to the Golden Gate reaction with the pICSL86900OD vector. Transform E. coli DH10α chemically competent cells with the total assembly reaction volume and plate on selective LB medium with the proper antibiotic as described previously. Select from two to five isolated white colonies by performing PCR colony. Then, one colony can be used to cultivate cells in a liquid LB medium following the plasmidial DNA extraction. Perform PCR colony using specific primers for each cloned DNA sequence. LR reaction Perform LR reaction using the GoldenGate entry vector containing the gene of interest flanked by attL sites and the Gateway expression vector of choice. To reduce costs, we performed the LR reaction with half the volume indicated by the manufacturer’s protocol in an overnight incubation at room temperature (see Recipe 4). Transform E. coli DH10α chemically competent cells with the LR reaction and plate in selective LB medium with the proper antibiotic as described previously. Plasmidial DNA extraction from positive colonies selected by PCR colony can be used in further experiments. Data analysis To confirm the nucleotide sequence and the correct assembly of the DNA constructions, miniprep samples can be sent for Sanger sequencing. Notes E. coli liquid culture expressing the attL-pUPD2 GoldenBraid bricks can be used to prepare a glycerol stock and stored at -80 °C for future assemblies with different target genes. Furthermore, these vectors are available in the plasmid repository Addgene as LM22_attL1 (catalog number: 184008) and LM22_attL2 (catalog number: 184010). BsmBI restriction enzyme can be substituted for Esp31 (Thermo Fisher ScientificTM, catalog number: ER0451). BsaI restriction enzyme can be substituted for Eco31I (Thermo Fisher ScientificTM, catalog number: ER0291). The Golden Gate fusion sites were defined according to the GoldenBraid Standard (Vazquez-Vilar et al., 2017). However, different fusion sites can be used. Recipes GoldenBraid (GB) reaction (TV = 10 µL) Reagent Final concentration Amount T4 DNA ligase buffer (10×) 1× 1.0 µL Restriction enzyme-specific buffer (10×) 1× 1.0 µL T4 DNA ligase -- 1.0 µL BsmBI restriction enzyme -- 1.0 µL GB acceptor vector (e.g., pUPD2) 10 ng µL-1 100 ng DNA fragment 5–10 ng µL-1 50–100 ng DNase- and RNase-free water -- q.s. 10 µL LB medium (TV = 1L) Reagent Final concentration Amount Tryptone -- 10 g Yeast extract -- 5 g NaCl 1 M 5 g For a solid medium add agar at a final concentration of 8%. Golden Gate (GG) reactions (TV = 10 µL) Reagent Final concentration Amount T4 DNA ligase buffer (10×) 1× 1.0 µL Restriction enzyme–specific buffer (10×) 1× 1.0 µL T4 DNA ligase -- 1.0 µL BsaI restriction enzyme -- 1.0 µL GG acceptor vector (e.g., pICSL86900OD) 15 ng µL-1 150 ng attL1-pUPD2 5–10 ng µL-1 50–100 ng attL2-pUPD2 5–10 ng µL-1 50–100 ng target gene-pUPD2 5–10 ng µL-1 50–100 ng DNase- and RNase-free water -- q.s. 10 µL LR reaction (TV = 5 µL) Reagent Final concentration Amount LR clonase II Mix (5×) 1× 1.0 µL Gateway expression vector 15 ng µL-1 150 ng BIG entry vector 15 ng µL-1 150 ng Tris-EDTA (TE) buffer pH 8.0 -- q.s. 5 µL 1Overnight incubation at room temperature. Acknowledgments This work was supported by The Sao Paulo Research Foundation – FAPESP (2018/17441–3, 2019/20157–8, 2020/12940-1, 2021/14640-8), and the Coordination for the Improvement of Higher Education Personnel – CAPES (88882.461731/2019-01; 88887.309513/2018-00, and 88887.613665/2021-00). This protocol was adapted from our recent work (Ferigolo et al., 2022). Competing interests The authors declare non-financial competing interests. References Engler, C., Kandzia, R. and Marillonnet, S. (2008). A one pot, one step, precision cloning method with high throughput capability. PLoS One 3(11): e3647. Ferigolo, L. F., Vicente, M. H. and Nogueira, F. T. S. (2022). Brick into the Gateway (BiG): A novel approach for faster cloning combining Golden Gate and Gateway methods. Plasmid 121: 102630. Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd and Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5): 343-345. Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T. and Halkier, B. A. (2007). USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35(7): e55. Hansen, N. B., Lübeck, M. and Lübeck, P. S. (2014). Advancing USER cloning into simple USER and nicking cloning. J Microbiol Methods 96: 42-49. Karimi, M., Depicker, A. and Hilson, P. (2007). Recombinational cloning with plant gateway vectors. Plant Physiol 145(4): 1144-1154. Marillonnet, S. and Grützner, R. (2020). Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline. Curr Protoc in Mol Biol 130(1). https://doi.org/10.1002/cpmb.115. Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., Raitskin, O., Leveau, A., Farre, G., Rogers, C., et al. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol 208(1): 13-19. Vazquez-Vilar, M., Quijano-Rubio, A., Fernandez-del-Carmen, A., Sarrion-Perdigones, A., Ochoa-Fernandez, R., Ziarsolo, P., Blanca, J., Granell, A. and Orzaez, D. (2017). GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. Nucleic Acids Res 45(4): 2196-2209. Zhu, B., Cai, G., Hall, E. O. and Freeman, G. J. (2007). In-FusionTM assembly: Seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43(3): 354-359. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > DNA > DNA cloning Plant Science > Plant molecular biology > DNA Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions Amit K. Tripathi [...] Sneh Lata Singla-Pareek Aug 20, 2017 13563 Views Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers Gozde S. Demirer and Markita P. Landry Jan 5, 2021 4802 Views Autolysin Production from Chlamydomonas reinhardtii Justin Findinier Jul 5, 2023 529 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,577	https://bio-protocol.org/en/bpdetail?id=4577&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Arrayed CRISPR/Cas9 Screening for the Functional Validation of Cancer Genetic Dependencies LP Ludovica Proietti GM Gabriele Manhart EH Elizabeth Heyes ST Selina Troester FG Florian Grebien Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4577 Views: 1736 Reviewed by: Rene WinklerSalim Gasmi Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Leukemia Sep 2021 Abstract CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50–300 gene knockouts on various cellular parameters within eight weeks. Graphical abstract Keywords: Cancer genetic dependencies CRISPR/Cas9 Guide RNAs Lentivirus Flow cytometry Cell fitness Background The experimental identification of genes that are essential for cancer cell proliferation and survival has radically changed over the last decade. The CRISPR/Cas9 technology is a powerful tool for the assessment of cancer-specific vulnerabilities (u and Yusa, 2019; Behan et al., 2019). It has been successfully employed in various cancer models and led to the identification of multiple genetic dependencies, such as WRN1 in cancers with microsatellite instability, YTHDF2 in triple-negative breast cancer, TRIM8 in Ewing sarcoma, or STAUFEN2 and ERG in acute myeloid leukemia (Lin and Sheltzer, 2020; Chan et al., 2019; Bajaj et al., 2020; Einstein et al., 2021; Seong et al., 2021; Schmoellerl et al., 2022). Indeed, pooled genome-wide loss-of-function screens to identify genetic dependencies have become common practice in cancer biology (Behan et al., 2019; Bajaj et al., 2020) as well as in many other fields of the molecular life sciences, such as stem cell biology, immunology, or cell and developmental biology (Arroyo et al., 2016; Y. Zhu, et al., 2021; Dong et al., 2022; Zhang et al., 2022). In parallel, pooled focused screens covering smaller sets of genes representing specific pathways or protein families are often employed (X. G. Zhu et al., 2021; Cao et al., 2021). Furthermore, next-generation sequencing–based methods, such as RNA-seq, ATAC-seq, or Cut&Run, as well as mass spectrometry–based proteomics are increasingly used to characterize molecular alterations in cancer cells in an unbiased global fashion. However, all these experimental approaches yield large amounts of data and high numbers of candidate genes, ranging from a few hundred to several thousand. Consequently, streamlined strategies for efficient functional genomic screening and validation of candidate genes downstream of pooled screens need to be developed. Here, we describe how to design and perform arrayed CRISPR/Cas9 screening experiments of candidate sets ranging from 50 to 300 gene knockouts for the validation and identification of functional genetic dependencies of cancer cells using flow cytometry that can serve as starting points for detailed molecular analyses. Materials and Reagents Cloning Scalpel Pipette tips, filtered, sterile, 0.5–1,250 µL (Biozym Surphob) 1.5 mL tubes (Eppendorf, catalog number: 0030121872) 96-well PCR plates (Biozym Scientific, catalog number: 710880) 96-well deep well plates, 2.2 mL, V-bottom (Biozym Scientific, catalog number: 710850) LentiGuide-Puro-P2A-EGFP (Addgene, Plasmid #137729) (Panda et al., 2020) BsmBI-v2 (New England Biolabs, catalog number: R0739L) NEBuffer r3.1 (New England Biolabs, catalog number: B6003S) Double-distilled water (ddH2O) Antarctic phosphatase (New England Biolabs, catalog number: M0289L) Antarctic phosphatase reaction buffer (New England Biolabs, catalog number: B0289S) Agarose (Sigma Aldrich, catalog number: A9539) Monarch® DNA Gel Extraction kit (New England Biolabs, catalog number: T1020L) T4 DNA ligase (New England Biolabs, catalog number: M0202L) T4 DNA ligase reaction buffer (New England Biolabs, catalog number: B0202S) T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201L) NEB stable competent E. coli (New England Biolabs, catalog number: C3040H) LB medium (Carl Roth, catalog number: X964.4) LB agar (Carl Roth, catalog number: 6671.2) Carbenicillin (Carl Roth, catalog number: 6344) Petri dish, square, 12 × 12 cm (Greiner Bio-One, catalog number: 688161) Inoculation loop (Sarstedt, catalog number: 86.1562.050) Monarch® Plasmid Miniprep kit (New England Biolabs, catalog number: T1010L) Cell culture Sterile flat-bottom 96-well plates (Greiner, catalog number: 655180) Round bottom 96-well plates (Greiner, catalog number: 650188) 10 cm2 dishes (Greiner, catalog number: 655160) 24-well cell culture plates (Stölzle Oberglas, catalog number: GR-662160) Syringe filter 0.45 µm, 33 mm diameter (M&B Stricker, catalog number: 99745) Sterile distilled water (Ecotainer, Braun, catalog number: 82479E-E) DMEM high glucose, no glutamine, 10 × 500 mL (Life Tech, catalog number: 11960085) Fetal bovine serum (FBS), heat inactivated (Sigma-Aldrich, catalog number: F9665-500ML) L-glutamine 200 mM (100×), liquid (Gibco, catalog number: 25030024) Penicillin-streptomycin, 10,000 U/mL (Life Tech, catalog number: 15140122) Phosphate buffered saline (PBS), 10×, pH 7.2–10 (Life Tech, catalog number: 70013065) Trypsin, 0.25% (1×) with EDTA 4Na, liquid (Gibco, catalog number: 25200056) Lenti-X 293T cells (Takara bio, catalog number: 632180) psPAX2 (packaging plasmid) (Addgene, catalog number: 12260) pMD2.G (packaging plasmid) (Addgene, catalog number: 12259) Polyethylenimine HCl MAX, linear, MW 40,000 (PEI) (Polysciences Europe, catalog number: 24765-1) PEI stock: 1 mg/mL in water according to the supplier’s protocol Polybrene transfection reagent (Merck, catalog number: TR-1003-G) Oligonucleotides DNA oligonucleotide primers from Integrated DNA Technologies (IDT) in 96-well format, premixed hU6 prom_fwd GACTATCATATGCTTACCGT Polybrene stock: 10 mg/mL in sterile water Equipment Single-channel pipettes, 0.5–1000 µL (Eppendorf Research) Multi-channel pipettes, 1–200 µL (Thermo Scientific Finnpette F2) Thermomixer (Eppendorf, catalog number: 5355) Thermal cycler S1000 (Bio-Rad, catalog number: 1852196) Incubator (Thermo Scientific, catalog number: 51029323) Shaker (GFL, catalog number: 3015) Tabletop microcentrifuge (Eppendorf 5424) Vortex (Scientific Industries, model: Vortex Genie 2) Spectrophotometer (Spark, Tecan) Incubator (Thermo Scientific Heraeus, BBD 6220) Incubator settings: 37 °C, 5 % CO2 , 95 % relative humidity Centrifuge (Eppendorf, 5810, equipped with aerosol-tight buckets located in a BSL2 laboratory) Fully-equipped biosafety level 2 laboratory authorized for work with virus particles (BSL 2) IQue Screener Plus [Intellicyt, equipped with plate reader, blue (488 nm) and red (640 nm) laser] Software Geneious Prime 2022.0.2 Forecyt standard Edition 7.0 (R2) (7.0.7035) Microsoft Excel R studio version: 1.3.1056 Procedure Guide RNA (gRNA) design Use the user-friendly VBC Score online tool (https://www.vbc-score.org/) (Michlits et al., 2020) to select gRNAs targeting the genes of interest. It is recommended to select two to three gRNAs per target gene. A high VBC score indicates high targeting efficiency and low off-target effects. The complete oligonucleotide sequences needed for cloning can be found in columns “GeCKO_cloning_FW” and “GeCKO_cloning_RV” in the Microsoft Excel files provided by the tool. The oligonucleotide sequences are as follows: Oligo forward: 5’-CACCG NNNNNNNNNNNNNNNNNNNN-3’ Oligo reverse: 3’-C NNNNNNNNNNNNNNNNNNNN CAAA-5’ Note: The PAM sequence is not included in the primers but is specified in the “sgRNA + NGG” column. Order pre-mixed gRNA (forward and reverse) oligonucleotides in 96-well plate format from IDT (https://eu.idtdna.com) as follows: Plate Specifications Scale: 25 nmol DNA Purification: Standard de-salted Plate type: PCR Loading Scheme Forward/Reverse mixed in wells Shipping: Normalization: nmol/well Amount per well: 4 nm If wet: Final concentration 40 micromolar Final volume 100 microliters/well Buffer: IDTE buffer pH 8.0 Preparation of gRNA expression vector Digest 5,000 ng of the LentiGuide-Puro-P2A-EGFP backbone (Figure 1) for 2 h at 55 °C with the restriction enzyme BsmBI-v2 (Figure 2A). Component Volume/Amount LentiGuide-Puro.P2A-EGFP 5,000 ng BsmBI-v2 3 µL NEBuffer 3.1 5 µL ddH2O up to 50 µL (= final volume) Figure 1. LentiGuide-Puro-P2A-EGFP from Addgene (Plasmid #137729) For dephosphorylation, add the following reagents to the digestion reaction from step B1 and incubate for 1 h at 37 °C. Component Volume Antarctic phosphatase 1 µL Antarctic phosphatase buffer 6 µL ddH2O 3 µL Total 60 µL Separate the backbone (approximately 9,000 bp) from the filler piece (1,885 bp) on a 0.7% agarose gel. Add an aliquot of undigested backbone to a separate gel pocket for comparison and to verify successful digestion of the backbone. Excise the band at approximately 9 kb with a scalpel and purify with a gel extraction kit. Elute DNA in 30–50 µL ddH2O. Note: As a considerable fraction of DNA is usually lost during gel purification, we recommend performing three to four digestion reactions in parallel and purify DNA from all bands using a single purification column. This will ensure obtaining sufficient quantities of backbone DNA. Measure the concentration of the eluted DNA and dilute to 100 ng/µL with ddH2O. gRNA cloning in 96-well plate format Figure 2. Workflow for gRNA cloning in 96-well format. Created with BioRender.com. Figure 3. Overview of plates A-E and the corresponding final concentration of oligos Thaw pre-mixed gRNA forward and reverse primers from IDT at room temperature (RT) (Plate A). For the phosphorylation and annealing of oligonucleotides (Figure 2B): Prepare the following master mix: Component Volume (×1) Volume (×96) T4 DNA ligase reaction buffer 0.4 µL 38.4 µL T4 PNK 0.2 µL 19.2 µL ddH2O 1.4 µL 134.4 µL Master mix 2 µL 192 µL Add 2 µL per well of the master mix to a new 96-well plate (plate B). Add 2 µL per well of oligonucleotides from plate A according to plate layout (final concentration: 20 µM) (Figure 3). Seal plate B and transfer to a thermocycler and apply the following conditions: Temperature Time 37 °C 30 min 95 °C 30 s Ramp down to 25 °C at 5 °C/min Dilute phosphorylated and annealed oligonucleotides in a new 96-well plate (plate C) by adding 2 µL of oligos to 198 µL of ddH2O (dilution factor: 100; final concentration: 200 nM) (Figure 3). Dilute plate C further in a new 96-well plate (plate D) by adding 5 µL of diluted oligonucleotides from plate C to 95 µL of ddH2O (dilution factor: 20; total dilution: 1:2,000; final concentration: 10 nM) (Figure 3). Set up the ligation reaction: Prepare the following master mix: Component Volume (×1) Volume (×96) Digested backbone 100 ng/µL 0.4 µL 38.4 µL T4 DNA ligase reaction buffer 0.4 µL 38.4 µL T4 DNA ligase 0.2 µL 19.2 µL ddH2O 1 µL 96 µL Total 2 µL 192 µL Add 2 µL per well of master mix to a new plate (plate E). Add 2 µL per well of annealed oligos from plate D (final concentration: 5 nM) (Figure 3). Let plate E stand for 20 min at RT to allow the ligation reaction to occur (Figure 2C). Bacterial transformation Dilute ligation reaction by adding 6 µL of ddH2O to each well of plate E. Thaw competent E. coli on ice. Seed 10 µL of bacteria per well in a 96-well plate on ice. Add 2 µL of ligation reaction to bacteria and pipette up and down two to three times. Incubate for 5 min on ice. Perform a heat shock at 42 °C for 45 s using a thermocycler. Incubate for 1 min on ice. Add 180 µL of LB medium to each well. Loosely cover the plate with aluminum foil and incubate at 37 °C for 30 min. Seed 5 µL of recovered bacteria on 12 × 12 cm petri dishes with LB medium + carbenicillin (Figure 2D). Note: Distribute 5 µL bacteria with a multichannel pipette and streak them out using an inoculation loop. Incubate overnight at 37 °C. Isolation of plasmids (backbone + gRNA) Add 2 mL of LB medium + carbenicillin into each well of a 96-deep-well plate. Pick single colonies using an inoculation loop or a pipette tip. Incubate overnight at 37 °C on a shaker. On the next day, isolate plasmid DNA using Monarch® Plasmid Miniprep kit according to the manufacturer’s instructions. Measure plasmid DNA concentration using a spectrophotometer. Submit plasmid DNA to Sanger Sequencing using hU6 prom_fwd sequencing primer (GACTATCATATGCTTACCGT). Check sequencing results using appropriate software (e.g., Geneious Prime) (Figure 4). Figure 4. Example of sequence analysis using Geneious Prime Cell culture Notes: For the arrayed CRISPR/Cas9 screen (Figure 5), we recommend the generation of a functional SpCas9-expressing clone from your cell line of interest. For validation, conduct a competition assay as described below using control gRNAs. For instance, in human cell lines, gRNAs targeting AAVS1 should be used as negative control, as the introduction of indels into this locus is not expected to have any effect on cellular fitness. gRNAs targeting RPL17 can be used as positive controls, as this gene is essential in most human cells. It is recommended to perform all functional experiments in at least three biological replicates. Transfection of Lenti-X cells with lentiviral constructs Figure 5. Workflow for virus production, transduction of target cells in 96-well plate format, and functional analysis of gene knockouts using a flow cytometry–based competition assay. Created with BioRender.com. Day 1: Determine the concentration of all gRNA constructs and dilute to 20 ng/µL in ddH2O in a 96-well plate. This plate can be stored at -20 °C for later use. Culture Lenti-X 293T (Lenti-X) in 10 cm2 dishes in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Make sure cultures do not grow beyond confluency (≥60%–80%). For the transfection, remove the supernatant, wash once with 5–10 mL of PBS and detach cells by incubating them in 1 mL of trypsin solution for 3–5 min in the incubator. After detachment, resuspend cells in 10 mL of fresh medium. Dilute the cells to 1.4 × 105 cells/mL and transfer 100 µL per well to a flat-bottom 96-well plate. Day 2: Prepare the transfection master mix as follows: Component Volume (×1) Volume (×96) psPAX2 100 ng 9600 ng pMD2.G 10 ng 960 ng DMEM (plain) 25 µL 2.4 mL Add 25 µL of master mix and 10 µL of each construct [20 ng/µL] to each well of a 96-well plate. Dilute PEI 1:10 in plain medium, add 12.6 µL per well, and mix thoroughly. Incubate for 25 min at RT. Carefully add 30 µL of each mix to the pre-seeded Lenti-X cells and incubate overnight. Note: From this step onwards, a BSL2 environment is required for work with virus particles. Day 3: Carefully remove the supernatant and replace with 170 µL per well of fresh medium. Note: Choose the fresh medium according to the growth medium of the cell line to be transduced. Since the chosen vector contains a fluorescent reporter, such as GFP, you can check the success of the transfection with a fluorescence microscope after changing the medium (Figure 6). Figure 6. Bright field (left) and fluorescence (right) microscope picture of Lenti-X cells 24 h post transfection (10×), showing expression of GFP Day 4/5: To harvest virus particles, carefully transfer 150 µL of supernatant from Lenti-X cells to a 96-well plate, store at 4 °C, and add 170 µL per well of the same medium to Lenti-X cells for an optional second harvest on the next day. Pool the second with the first harvest and freeze viruses in 100 µL of aliquots at -80 °C. Note: The infectious capacity of lentiviral particles may be reduced upon freezing. However, this step efficiently eliminates Lenti-X cells, which might eventually be present in the supernatant and contaminate target cells with Lenti-X cells during the transduction process. Transduction of target suspension cells and setup of the flow cytometry–based competition assay Count target cells and collect the required number of cells by centrifugation. Note: The supernatant obtained after centrifugation and filtering with a 0.45 µm filter can be kept at 4 °C and used as conditioned medium to supplement cells after the transduction. This is recommended for cells that are particularly susceptible to stress upon external perturbations, such as lentiviral infection. Optionally, the medium can be supplemented with 20% FBS during the transduction process to increase cell viability. Resuspend cell pellet in medium to reach a concentration of 2 × 106 cells/mL and transfer 100 µL per well to a flat-bottom 96-well plate (= 2 × 105 cells/well). Pre-dilute polybrene to 100 µg/mL in the growth medium of target cells, add 8 µL to 100 µL of virus, and add the mix (virus + polybrene) to each 100 µL of cells (polybrene final = 4 µg/mL). Centrifuge the plate in an aerosol-tight bucket at 1,000 × g for 90 min. After the spinoculation, transfer the plate to an incubator. Notes: For an optional second spinoculation, carefully remove 100 µL per well of supernatant, defrost new 100 µL aliquots of virus, add fresh pre-diluted polybrene, add the mix to cells, and centrifuge again. For adherent cells, the addition of polybrene and the spinoculation may be omitted. Remove the virus 6–24 h after the last spinoculation. Centrifuge the plate (300 × g, 5 min) and carefully remove most of the supernatant. Optionally wash cells with PBS before resuspending them in fresh growth medium. Culture the cells as usual. Note: Cells may show reduced growth and increased cell death for a few days post transduction. After three days, determine the percentage of transduced cells per well by flow cytometry, e.g., via a fluorescent reporter protein such as GFP. After successful transduction, measure the percentage of GFP-positive cells per well every two to three days and split cultures as required. Note: In addition to monitoring GFP signal, the analysis can be extended to any other parameter that is accessible with flow-based measurements, such as differentiation, apoptosis, and cell cycle analysis to acquire deeper insights into the cellular effects of gene knockout. The typical monitoring time is two to four weeks post transduction but will depend on the growth characteristics of the target cell line and the effect of the knockout. We recommend transferring the cells to 24-well plates for culturing and monitoring the percentage of GFP-positive cells. Cultures can be efficiently split using a 12-channel pipette using every second tip only. During each splitting event, transfer an aliquot of cells to a 96-well plate and analyze on the IQue Screener Plus flow cytometer or a similar device. Notes: Transduction rates between 20%–80% are recommended; the optimum is 50%. Test the procedure with only a few constructs (e.g., a set of controls) and adjust if required. Optional changes include: Adjust concentration of Lenti-X cells (e.g., 1 × 104 –2 × 104 cells per well). Adjust concentration of target cells (e.g., scale down to 1 × 105 cells per well). Adjust dilutions of virus (dilute virus more or less to decrease or increase transduction rates, respectively). Increase/decrease number of spinoculations. Scale up to a 24-well format for transfection and/or transduction. In general, every flow cytometer equipped with the required laser and detector system to measure the reporter protein can be used for monitoring the percentage of transduced cells. We recommend a device with a plate loader module to most efficiently analyze high numbers of samples that are usually present in an arrayed CRISPR/Cas9 screen. Analysis of the competition assay In the Forecyt software of the IQue Screener Plus, use the gating strategy shown in Figure 7 to identify the number of transduced cells as “percentage of GFP-positive cells of singlets.” Figure 7. Gating strategy for identifying GFP-positive cells as the percentage of all live singlets per well Generate a heat map of each measurement plate with GFP-positive cells as percentage of all singlet cells for each well. These values can be exported via the metrics function in a .csv format and further analyzed in Microsoft Excel. In Microsoft Excel, normalize all data to the first measurement, e.g., at day 3 post-transduction (Day3pt) and to a negative control (sgAAVS1). Note: It can happen that the percentage of GFP-positive cells still rises after Day3pt. If this is the case, normalize to a later time point, e.g., Day5pt or Day7pt. Normalization to measurement start Day3pt (normDay3): Formula: %GFPnormDay3= %GFPDayXpt/%GFPDay3pt 100 Example calculation in Microsoft Excel: Normalization to control sgAAVS1 (normAAVS1): Formula: %GFP(Sample, normDay3 + normAAVS1)= %GFP(Sample,normDay3)/%GFP(sgAAVS1,normDay3) 100 Example calculation in Microsoft Excel: Data from a limited number of samples can be presented in bar charts (Figure 8). Targeting a gene essential for cell proliferation will result in a decrease of the percentage of GFP-positive cells over time. Targeting of a non-essential gene will result in unchanged percentages of GFP-positive cells. Figure 8. Bar chart diagram of a) raw data of % GFP-positive cells over time and b) normalized to Day3 post-transduction and to sgAAVS1 (negative control) A heat map format can be used to present the results of a big data set in a comprehensible way (Figure 9a). To further condense the results, it is possible to calculate the area under the curve (AUC) for each gRNA using the following R script. Create an excel file containing percentages of GFP values normalized to the measurement start point and a negative control (e.g., sgAAVS1) with the following structure and save it as a .csv file. In R studio, apply the following code to calculate AUC values for each gene/target. Adjust file name, day numbers, and column numbers according to your data. Example of a heat map with AUC values is shown inFigure 9b. Figure 9. Heat maps representing a) %GFP normalized to sgAAVS1 and Day3pt for each target and b) area under the curve (AUC) values for each target Acknowledgments This protocol was adapted from previous work (Schmidt et al., 2019; Heyes et al., 2021). We thank all members of the Grebien laboratory for discussions. This project has received funding from the European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant agreement No 813091), the Austrian Science Fund (projects P-35628 and TAI-490 to FG), and a grant from the Fellinger Krebsforschungsverein. ST is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. Competing interests The authors declare no competing interests. References Arroyo, J. D., Jourdain, A. A., Calvo, S. E., Ballarano, C. A., Doench, J. G., Root, D. E., Mootha, V. K. (2016). A genome-wide CRISPR death screen identifies genes essential for oxidative phosphorylation. Cell Metab 24(6):875-885. Bajaj, J., Hamilton, M., Shima, Y., Chambers, K., Spinler, K., Van Nostrand, E. L., Yee, B. A., Blue, S. M., Chen, M., Rizzeri, D., et al. (2020). An in vivo genome-wide CRISPR screen identifies the RNA-binding protein Staufen2 as a key regulator of myeloid leukemia. Nat Cancer 1(4): 410-422. Behan, F. M., Iorio, F., Picco, G., Gonçalves, E., Beaver, C. M., Migliardi, G., Santos, R., Rao, Y., Sassi, F., Pinnelli, M., et al. (2019). Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens. Nature 568(7753): 511-516. Cao, Z., Budinich, K. A., Huang, H., Ren, D., Lu, B., Zhang, Z., Chen, Q., Zhou, Y., Huang, Y. H., Alikarami, F., et al. (2021). ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency. Mol Cell 81(17): 3604-3622 e3610. Chan, E. M. , Shibue, T., McFarland, J. M., Gaeta, B., Ghandi, M., Dumont, N., Gonzalez, A., McPartlan, J. S., Li, T., Zhang, Y., et al. (2019). WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568(7753): 551-556. Dong, C., Fu, S., Karvas, R. M., Chew, B., Fischer, L. A., Xing, X., Harrison, J. K., Popli, P., Kommagani, R., Wang, T., et al. (2022). A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells. Nat Commun 13(1): 2548. Einstein, J. M., Perelis, M., Chaim, I. A., Meena, J. K., Nussbacher, J. K., Tankka, A. T., Yee, B. A., Li, H., Madrigal, A. A., Neill, N. J., et al. (2021) Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer. Mol Cell 81(15):3048-3064.e9. Heyes, E., Schmidt, L., Manhart, G., Eder, T., Proietti, L. and Grebien, F. (2021). Identification of gene targets of mutant C/EBPalpha reveals a critical role for MSI2 in CEBPA-mutated AML. Leukemia 35(9): 2526-2538. Lin, A. and Sheltzer, J. M. (2020). Discovering and validating cancer genetic dependencies: approaches and pitfalls. Nat Rev Genet 21(11): 671-682. Michlits, G., Jude, J., Hinterndorfer, M., de Almeida, M., Vainorius, G., Hubmann, M., Neumann, T., Schleiffer, A., Burkard, T. R., Fellner, M., et al. (2020). Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles. Nat Methods 17(7): 708-716. Panda, S. K., Wigerblad, G., Jiang, L., Jimenez-Andrade, Y., Iyer, V. S., Shen, Y., Boddul, S. V., Guerreiro-Cacais, A. O., Raposo, B., Kasza, Z., et al. (2020). IL-4 controls activated neutrophil FcgammaR2b expression and migration into inflamed joints. Proc Natl Acad Sci U S A 117(6): 3103-3113. Schmidt, L., Heyes, E., Scheiblecker, L., Eder, T., Volpe, G., Frampton, J., Nerlov, C., Valent, P., Grembecka, J. and Grebien, F. (2019). CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex. Leukemia 33(7): 1608-1619. Schmoellerl, J., Barbosa, I. A. M., Minnich, M., Andersch, F., Smeenk, L., Havermans, M., Eder, T., Neumann, T., Jude, J., Fellner, M., et al. (2022). EVI1 drives leukemogenesis through aberrant ERG activation. Blood. doi: 10.1182/blood.2022016592. Seong, B. K. A., Dharia, N. V., Lin, S., Donovan, K. A., Chong, S., Robichaud, A., Conway, A., Hamze, A., Ross, L., Alexe, G., et al. (2021). TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma. Cancer Cell 39(9): 1262-1278.e7. Yu, J. S. L. and Yusa, K. (2019). Genome-wide CRISPR-Cas9 screening in mammalian cells. Methods 164-165: 29-35. Zhang, Y., Wang, J., Ruan, Y., Yang, Y., Cheng, Y., Wang, F., Zhang, C., Xu, Y., Liu, L., Yu, M., et al. (2022). Genome-Wide CRISPR Screen Identifies Puf60 as a Novel Stemness Gene of Mouse Embryonic Stem Cells. Stem Cells Dev 31(5-6): 132-142. Zhu, X. G., Chudnovskiy, A., Baudrier, L., Prizer, B., Liu, Y., Ostendorf, B. N., Yamaguchi, N., Arab, A., Tavora, B., Timson, R., et al. (2021). Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells. Cell Metab 33(1): 211-221 e216. Zhu, Y., Feng, F., Hu, G., Wang, Y., Yu, Y., Zhu, Y., Xu, W., Cai, X., Sun, Z., Han, W., Ye, R., Qu, D., Ding, Q., Huang, X., Chen, H., Xu, W., Xie, Y., Cai, Q., Yuan, Z. and Zhang, R. (2021). A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry. Nat Commun 12(1): 961. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cancer Biology > General technique > Molecular biology technique Computational Biology and Bioinformatics Molecular Biology > DNA > DNA cloning Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols 3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines Kari-Anne M. Frikstad [...] Sebastian Patzke Mar 20, 2022 1998 Views In-Cell Western Protocol for Semi-High-Throughput Screening of Single Clones Arpita S. Pal [...] Andrea L. Kasinski Aug 20, 2022 2195 Views Efficient Large DNA Fragment Knock-in by Long dsDNA with 3′-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells Wenjie Han [...] Jianqiang Bao Oct 20, 2023 1161 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,578	https://bio-protocol.org/en/bpdetail?id=4578&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Monitoring Mitochondrial Protein Import Using Mitochondrial Targeting Sequence (MTS)-eGFP JM Jonas B. Michaelis * SB Süleyman Bozkurt * JS Jasmin A. Schäfer * CM Christian Münch (*contributed equally to this work) Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4578 Views: 1634 Reviewed by: Julie WeidnerOlli MatilainenAlberto Rissone Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Molecular Cell Jan 2022 Abstract Mitochondria are cellular organelles essential for the function and survival of eukaryotic cells. Nearly all mitochondrial proteins are nuclear-encoded and require mitochondrial import upon their synthesis in the cytosol. Various approaches have been described to study mitochondrial protein import, such as monitoring the entry of radiolabeled proteins into purified mitochondria or quantifying newly synthesized proteins within mitochondria by proteomics. Here, we provide a detailed protocol for a commonly used and straightforward assay that quantitatively examines mitochondrial protein import by monitoring the co-localization of mitochondrially targeted enhanced green fluorescent protein (eGFP) with the mitochondrial fluorescence dye MitoTracker TM Deep Red FM by live cell imaging. We describe the preparation and use of a stable mammalian cell line inducibly expressing a mitochondrial targeting sequence (MTS)-eGFP, followed by quantitative image analysis using an open-source ImageJ-based plugin. This inducible expression system avoids the need for transient transfection while enabling titration of MTS-eGFP expression and thereby avoiding protein folding stress. Overall, the assay provides a simple and robust approach to assess mitochondrial import capacity of cells in various disease-related settings. Graphical abstract Keywords: Mitochondrial protein import Microscopy Mitochondria Protein translocation Live cell imaging Background Mitochondria are crucial for cellular survival. Almost all mitochondrial proteins are encoded in the nuclear genome and proper mitochondrial function and biogenesis rely on mitochondrial protein import (Dimogkioka et al., 2021). Multiple mitochondrial protein import machineries exist; each mediates protein translocation into a distinct mitochondrial sub-compartment (Schmidt et al., 2010). Stress conditions, such as the loss of mitochondrial membrane potential, are known to disturb mitochondrial protein import, and defective import can lead to a series of pathologies, such as neurodegenerative and cardiovascular diseases and cancer (Geissler et al., 2000; Palmer et al., 2021). To investigate mitochondrial protein import, several methods have been established. Radiolabeling of individual mitochondrial proteins followed by incubation with purified mitochondria allows following mitochondrial import of the labeled protein (Murschall et al., 2021; Poveda-Huertes et al., 2021). Besides the technical (e.g., purification of fully active mitochondria) and bureaucratic requirements of performing radioactive lab work, this in vitro approach lacks cellular context and thus does not account for potential contribution of cytosolic factors on protein import. Recently developed proteomics methods allow monitoring the import of newly synthesized proteins into mitochondria on the global level (Schafer et al., 2022). This method is carried out within the cellular context and allows monitoring hundreds of mitochondrial proteins; however, it is a complex proteomics method that requires a mass spectrometry infrastructure not available in most laboratories. Here, we describe a fluorescence microscopy–based approach that allows monitoring mitochondrial import of the previously established mitochondrial targeting sequence (MTS)–enhanced green fluorescent protein (eGFP) reporter protein by live cell imaging (Chen et al., 2003). The reporter consists of eGFP that is selectively targeted to the mitochondrial matrix by means of a N-terminal MTS, which consists of the first 69 amino acids of subunit 9 of the F0-ATPase. Transcription of the reporter gene can be induced with the Tet-On system by treating cells with doxycycline, which allows for a controlled and titratable MTS-eGFP expression. Defective import of the reporter is assessed by its mislocalization to the cytosol, which is monitored by co-staining of mitochondria with MitoTrackerTM Deep Red FM. Subsequent co-localization analysis with the ImageJ-implemented Coloc2 plugin allows assessing mitochondrial protein import defects in a quantitative manner. Materials and Reagents Cultivating cell lines 10 cm tissue culture dish (Sarstedt, catalog number: 83.3902) 6-well cell culture plate (Sarstedt, catalog number: 83.3920.005) Cell counting slides (dual-chamber) (Bio-Rad, catalog number: 1450015) 0.45 µm filters (Whatman, catalog number: 10462100) HeLa FlpIn TRex cell line (Invitrogen, catalog number: R71407) Note: This protocol was established for HeLa FlpIn TRex cell lines. It can be transferred to other cell lines that are suitable for microscopy. HEK 293T cells (human embryonic kidney 293T) (ATCC, catalog number: CRL-3216) RPMI 1640 medium (+L-Glutamine) (Thermo Fisher Scientific, catalog number: 21875-034) DMEM medium (+L-Glutamine) (Thermo Fisher Scientific, catalog number 41966029) Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 10270-106) Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, catalog number: 25200056) Dulbecco's phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14190-169) Trypan blue solution (Sigma-Aldrich, catalog number: T8154) Stable and inducible MTS-eGFP cell line generation 15 mL tube (Greiner, catalog number: 188271-N) Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, catalog number: 11668027) Puromycin (InvivoGen, catalog number: ant-pr-1) Opti-MEM I (Thermo Fisher Scientific, catalog number: 31985-047) Polybrene (Sigma-Aldrich, catalog number: TR-1003-G) pHDM-VSV-G (Addgene, catalog number: 164440); encodes VSV-G protein pHDM-Hgpm2 (Addgene, catalog number: 164441); encodes codon-optimized HIV gag-pol pHDM-tatIB (Addgene, catalog number: 164442); encodes HIV-1 Tat accessory protein pRC-CMV-revIB (Addgene, catalog number: 164443); encodes HIV-1 Rev accessory protein pLD-puro-MTS-EGFP (Addgene, catalog number: 190270); contains tetracycline/doxycycline-inducible MTS-eGFP and lentiviral packaging signal MTS-eGFP mitochondrial import assay Cell culture microplate, 96-well, black (Greiner, catalog number: 655090) MitoTrackerTM Deep Red FM (Thermo Fisher Scientific, catalog number: M22426) Doxycycline (0.25 µg/mL stock in RNAse-free H2O) (Sigma-Aldrich, catalog number: D9891-10G) Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759) Note: CCCP is toxic if swallowed. Avoid skin contact and wash hands thoroughly after handling. Dimethyl sulfoxide for cell culture (DMSO) (VWR, catalog number: A3672.0100) Gamitrinib-triphenylphosphonium (GTPP) (custom synthesized) Equipment -80 °C freezer (Ewald, catalog number: V86-520.1) TC20 automated cell counter (Bio-Rad, catalog number: 1450102) CQ-1 confocal quantitative (Yokogawa) or other live cell microscope with 60× magnification (488 nm, 640 nm laser) Emission filters 525/50 nm for eGFP (CQ-1, Yokogawa) Emission filters 685/40 nm for MitoTrackerTM Deep Red FM (CQ-1, Yokogawa) Incubator for mammalian cell culture (Thermo Fisher Scientific, catalog number: 16416639) Software ImageJ (NIH, version: 1.53e) Procedure Generation of a stable and inducible MTS-eGFP cell line with lentiviral particles Note: All steps described in section A have to be conducted in a laboratory approved for lentiviral work. Seed 3.5 × 106 HEK 293T cells in a 10 cm tissue culture dish containing DMEM (v/v) 10% FBS medium to obtain a density of 80% confluence the next day. Exchange medium with 9 mL of pre-warmed DMEM (v/v) 1% FBS the day after seeding. Mix plasmids with corresponding ratio (5 μg total DNA for 10 cm culture dish) of pHDM-VSV-G (ratio: 2, 0.4 µg), pHDM-Hgpm2 (ratio: 1, 0.2 µg), pHDM-tatIB (ratio: 1, 0.2 µg), pRC-CMV-revIB (ratio: 1, 0.2 µg), and pLD-puro-MTS-EGFP (ratio: 20, 4 µg) with 500 µL of Opti-MEM I in tube number 1. Mix 15 µL of lipofectamine 2000 transfection reagent with 500 µL of Opti-MEM I in tube number 2. Note: Instead of Lipofectamine 2000, other transfection reagents can be used. Add DNA mixture from tube number 1 to tube number 2, mix, and incubate for 10 min at room temperature. Add mix to the cells in a dropwise manner to reach 10 mL in total. Exchange medium with 10 mL of pre-warmed DMEM (v/v) 10% FBS after 6 h. Harvest lentiviral particles after 48 h by collecting medium. Spin the collected supernatant at 2000 × g for 3 min to pellet transferred HEK 293T cells. Transfer the supernatant containing lentiviral particles to a new tube. Note: Alternatively, 0.45 µm filters can be used to remove transferred HEK 293T cells. Store the lentiviral particle containing supernatant at 4 °C for up to one week or at -80 °C for longer storage. Seed 2 × 106 HeLa FlpIn TRex cells in a 10 cm tissue culture dish containing RPMI (v/v) 10% FBS medium to obtain a density of 60% confluence the next day. Add 0.8–1 mL of supernatant containing lentiviral particles obtained in step A9 per 10 cm dish with polybrene (final concentration of 8 µg/mL) the day after seeding. Note: Do not remove the culture medium before adding the supernatant containing lentiviral particles. Also, it is not necessary to determine the concentration of lentiviral particles in the supernatant. Incubate cells with lentiviral particles for 48 h. Note: Exchange the medium with fresh, pre-warmed RPMI (v/v) 10% FBS medium during the incubation period if the cells show signs of stress. Start selection of lentivirus-infected HeLa FlpIn TRex cells by puromycin addition (final concentration of 1 µg/mL) to the RPMI (v/v) 10% FBS medium, 48 h after transduction. Select for lentivirus-infected cells for a total of 11 days and by a minimum of three passages in RPMI (v/v) 10% FBS medium containing 1 µg/mL puromycin. Note: The generated cell line is stable, can be frozen in RPMI (v/v) 10% FBS medium containing 10% DMSO (v/v) at -80 °C and stored ≤ -150 °C. Cells can be cultured in regular RPMI (v/v) 10% FBS medium after completing puromycin selection for 11 days. Cell seeding Aspirate medium from a 10 cm tissue culture dish containing HeLa FlpIn TRex cells carrying the genomically integrated MTS-eGFP reporter grown in RPMI (v/v) 10% FBS at 60%–80% confluence. Note: This step is performed with cells obtained in the previous step, i.e., after 11 days of puromycin selection. Wash cell layer once with 5 mL of room temperature PBS and aspirate. Add 1 mL of 0.25% trypsin to cell layer and incubate the plate for 5 min in the incubator. Add 9 mL of RPMI (v/v) 10% FBS to stop tryptic reaction and resuspend cells by pipetting up and down repeatedly. Collect cell suspension in a 15 mL tube and pellet cells by centrifugation at 800 × g for 3 min. Aspirate supernatant and resuspend cells in 5 mL of RPMI (v/v) 10% FBS by pipetting up and down repeatedly. Transfer 5 µL of cell suspension to a 1.5 mL tube and mix with 5 µL of trypan blue solution. Transfer 8.5 µL of stained cell suspension to a counting slide and count viable cells. Seed 10–20,000 viable cells per well of a black 96-well cell culture microplate in RPMI (v/v) 10% FBS and grow cells overnight in the incubator. Treatment, induction of MTS-eGFP expression, and mitochondrial staining Prepare treatment solution containing the compound or vehicle at the appropriate concentration and 0.25 µg/mL doxycycline to induce MTS-eGFP expression in pre-warmed RPMI (v/v) 10% FBS. Note: According to Moullan et al. (2015), the used doxycycline concentration should not affect mitochondrial function in HeLa cells. Start treatment and doxycycline induction by aspirating the growth medium and adding the treatment medium to the cells in the 96-well plate. Note: The protocol was tested for 6 h of treatment with 10 µM CCCP, a mitochondrial uncoupler, and 10 µM GTPP, a mitochondrial chaperone inhibitor, both of which rapidly reduce mitochondrial protein import and might be used as positive controls (Michaelis et al., 2022). DMSO was used as vehicle control. Incubate cells in the incubator during treatment. Aspirate treatment medium and stain cells with 50 nM MitoTrackerTM Deep Red FM in pre-warmed RPMI (v/v) 10% FBS for 20 min in the incubator. Aspirate medium, wash cells once with PBS, and add fresh RPMI (v/v) 10% FBS. Live cell imaging Transfer the 96-well plate with cells to the Yokogawa CQ-1 microscope chamber pre-equilibrated to 37 °C, 5% CO2 . Acquire live cell images of eGFP (488 nm excitation, 525/50 nm emission) and MitoTrackerTM Deep Red FM (640 nm excitation, 685/40 nm emission) of at least 100 cells per replicate at 60× magnification with automated focus (example images shown in Figure 1). Notes: Adjust the laser power and exposure times (maximum 500 ms) to obtain non-saturated images. It is recommended to acquire z-stacks to obtain in-focus images of as many cells as possible per field of view. Z-stacks are typically acquired with 6–10 slides within 3–5 µm radius around the central focus. Figure 1. Live cell images of MTS-eGFP and MitoTrackerTM Deep Red FM upon DMSO and CCCP treatment. HeLa FlpIn TRex cells carrying MTS-eGFP were treated for 6 h with doxycycline and 10 µM CCCP or 1 µL/mL DMSO as vehicle control (left). Scale bar represents 25 µm. Results of correlation-based co-localization analysis using ImageJ Coloc2 are shown (right). Data analysis Use ImageJ (Schneider et al., 2012) and the implemented Coloc2 plugin for co-localization analysis. Open images of eGFP and MitoTrackerTM Deep Red FM of the same field of view in ImageJ and start Coloc2, following Analyze – Colocalization – Coloc2. Select the images to be analyzed in Channel 1 and 2; select Bisection as Threshold regression and Manders’ correlation in the Coloc2 selection window (Figure 2). Note: Coloc2 also calculates the Pearson’s coefficient, which is based on another correlation-based co-localization method. The coefficients range from -1 to +1 for Pearson’s and from 0 to 1 for Manders’. The values correlate with the confidence of co-localization. For visualization purposes, perform background subtraction on individual image files using the background subtraction feature in ImageJ and adjust brightness of images to comparable intensities (Figure 3). Figure 2. Coloc2 selection window. Images for co-localization analysis are selected in Channels 1 and 2. The parameters used for co-localization analysis are displayed. Figure 3. Image adjustment by background subtraction, brightness, and contrast adjustment in ImageJ. This figure demonstrates how to access the implemented features for figure preparation. Notes This assay can also be combined with siRNA knock downs. For this, MTS-eGFP expression is induced by doxycycline treatment after 2–4 days of siRNA transfection. Acknowledgments The MTS-eGFP protein import assay was adapted from Chen et al. (2003). Plasmid lentiviral destination (pLD) was established as described previously by Mak et al. (2010). C.M. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG) SFB 1177 (subproject D08) Project-ID 259130777, Emmy Noether Programm Project ID 390339347, and Excellence Strategy Program of the DFG (Exc 2026), and the European Research Council (ERC) Starting Grant 803565. The protocol was established as part of the following research papers: Schafer et al. (2022) and Michaelis et al. (2022). The graphical abstract was created with BioRender.com. Competing interests The authors declare that they have no competing interests. References Chen, H., Detmer, S. A., Ewald, A. J., Griffin, E. E., Fraser, S. E. and Chan, D. C. (2003). Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol 160(2): 189-200. Dimogkioka, A. R., Lees, J., Lacko, E. and Tokatlidis, K. (2021). Protein import in mitochondria biogenesis: guided by targeting signals and sustained by dedicated chaperones. RSC Adv 11(51): 32476-32493. Geissler, A., Krimmer, T., Bomer, U., Guiard, B., Rassow, J. and Pfanner, N. (2000). Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence. Mol Biol Cell 11(11): 3977-3991. Mak, A. B., Ni, Z., Hewel, J. A., Chen, G. I., Zhong, G., Karamboulas, K., Blakely, K., Smiley, S., Marcon, E., Roudeva, D., et al. (2010). A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency. Mol Cell Proteomics 9(5): 811-823. Michaelis, J. B., Brunstein, M. E., Bozkurt, S., Alves, L., Wegner, M., Kaulich, M., Pohl, C. and Munch, C. (2022). Protein import motor complex reacts to mitochondrial misfolding by reducing protein import and activating mitophagy. Nat Commun 13(1): 5164. Moullan, N., Mouchiroud, L., Wang, X., Ryu, D., Williams, E. G., Mottis, A., Jovaisaite, V., Frochaux, M. V., Quiros, P. M., Deplancke, B., et al. (2015). Tetracyclines Disturb Mitochondrial Function across Eukaryotic Models: A Call for Caution in Biomedical Research. Cell Rep 10(10): 1681-1691. Murschall, L. M., Peker, E., MacVicar, T., Langer, T. and Riemer, J. (2021). Protein Import Assay into Mitochondria Isolated from Human Cells. Bio Protoc 11(12): e4057. Palmer, C. S., Anderson, A. J. and Stojanovski, D. (2021). Mitochondrial protein import dysfunction: mitochondrial disease, neurodegenerative disease and cancer. FEBS Lett 595(8): 1107-1131. Poveda-Huertes, D., Taskin, A. A., Dhaouadi, I., Myketin, L., Marada, A., Habernig, L., Buttner, S. and Vogtle, F. N. (2021). Increased mitochondrial protein import and cardiolipin remodelling upon early mtUPR. PLoS Genet 17(7): e1009664. Schafer, J. A., Bozkurt, S., Michaelis, J. B., Klann, K. and Munch, C. (2022). Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery. Mol Cell 82(2): 435-446 e437. Schmidt, O., Pfanner, N. and Meisinger, C. (2010). Mitochondrial protein import: from proteomics to functional mechanisms. Nat Rev Mol Cell Biol 11(9): 655-667. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cell Biology > Cell-based analysis Cell Biology > Cell imaging > Fluorescence Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy Stephan Tetenborg [...] John O`Brien Jul 20, 2024 341 Views Calibrating Fluorescence Microscopy With 3D-Speckler (3D Fluorescence Speckle Analyzer) Chieh-Chang Lin and Aussie Suzuki Aug 20, 2024 404 Views Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca2+ Imaging Sergei G. Gaidin [...] Sultan T. Tuleukhanov Feb 5, 2025 46 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,579	https://bio-protocol.org/en/bpdetail?id=4579&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Safety Profiling of Tumor-targeted T Cell–Bispecific Antibodies with Alveolus Lung- and Colon-on-Chip SK S. Jordan Kerns * CB Chaitra Belgur * MK Marianne Kanellias DM Dimitris V. Manatakis DP Debora Petropolis RB Riccardo Barrile WT Will Tien-Street LE Lorna Ewart NG Nikolche Gjorevski LC Lauriane Cabon (contributed equally to this work) Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4579 Views: 1582 Reviewed by: Gal HaimovichTakashi NishinaMarieta Ruseva Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Aug 2021 Abstract Traditional drug safety assessments often fail to predict complications in humans, especially when the drug targets the immune system. Rodent-based preclinical animal models are often ill-suited for predicting immunotherapy-mediated adverse events in humans, in part because of the fundamental differences in immunological responses between species and the human relevant expression profile of the target antigen, if it is expected to be present in normal, healthy tissue. While human-relevant cell-based models of tissues and organs promise to bridge this gap, conventional in vitro two-dimensional models fail to provide the complexity required to model the biological mechanisms of immunotherapeutic effects. Also, like animal models, they fail to recapitulate physiologically relevant levels and patterns of organ-specific proteins, crucial for capturing pharmacology and safety liabilities. Organ-on-Chip models aim to overcome these limitations by combining micro-engineering with cultured primary human cells to recreate the complex multifactorial microenvironment and functions of native tissues and organs. In this protocol, we show the unprecedented capability of two human Organs-on-Chip models to evaluate the safety profile of T cell–bispecific antibodies (TCBs) targeting tumor antigens. These novel tools broaden the research options available for a mechanistic understanding of engineered therapeutic antibodies and for assessing safety in tissues susceptible to adverse events. Graphical abstract Figure 1. Graphical representation of the major steps in target-dependent T cell–bispecific antibodies engagement and immunomodulation, as performed in the Colon Intestine-Chip Keywords: Cancer Immunotherapy Organs-on-Chip Organoids Safety Cancer biology Intestinal biology Alveolar biology Preclinical safety T Cell bispecifics Primary cell models Background Cancer immunotherapies are treatments that promise delivering durable treatment by harnessing the cytotoxic potential of the immune system against tumor cells (Yang, 2015; Gong et al., 2018; Waldman et al., 2020). Although impressive improvement in long-term survival has been reported (Hodi et al., 2010; Schadendorf et al., 2015; Wolchok et al., 2017), systemic immunomodulation mediated by these drugs often elicits immune-related adverse events, limiting their broad clinical application in battling cancer (Naidoo et al., 2015; Champiat et al., 2017; Kennedy and Salama, 2020). T cell–engaging bispecific antibodies (TCBs) are a novel class of cancer immunotherapeutic agents that have the potential to improve on the clinical efficacy and safety of traditional immunotherapy (Clynes and Desjarlais, 2019; Labrijn et al., 2019). TCBs exert their anti-tumor activity by simultaneously binding to a cancer surface antigen and to the CD3 T-cell receptor, thereby both activating the T cell and physically crosslinking it to target cells (Bacac et al., 2016). This synthetic immunity approach is particularly favorable for targeting less immunogenic, neo-antigen-lacking tumors, as T cells can be recruited and activated independently of their T-cell receptor specificity. This strictly tumor–targeted immunomodulation is also expected to reduce the systemic inflammatory toxicities associated with traditional immunotherapies (Milling et al., 2017). The therapeutic potential of TCBs is exemplified by the large number of molecules targeting solid and blood tumors, which are currently in various stages of clinical evaluation (Ishiguro et al., 2017; Goebeler and Bargou, 2020). Although TCBs hold promise for a safer therapeutic option, they are not risk-free. The antigens targeted are rarely exclusive to the tumor, but are also often expressed, albeit at lower levels, in normal tissues, rendering TCBs subject to on-target, off-tumor safety liabilities. This is particularly true for epithelial tumor antigens, as they are frequently targeted in solid tumor indications. For example, a bispecific T-cell engager (BiTE) targeted to the epidermal growth factor receptor (EGFR) produced severe liver and kidney toxicities in non-human primates, in line with EGFR expression in these organs, and led to the termination of the animals (Lutterbuese et al., 2010; Klinger et al., 2016). Clinical adverse events were reported in a recent Phase I study evaluating an epithelial cell adhesion molecule (EpCAM)–targeted BiTE as a therapy for a variety of epithelial carcinomas. Consistent with the expression of EpCAM in the gastrointestinal tract, the molecule triggered severe diarrhea and ultimately prevented escalation to efficacious doses and the identification of a therapeutic window (Kebenko et al., 2018; Trabolsi et al., 2019). Reliable human TCB safety evaluation at the preclinical stage is therefore of vital importance to ensure that well-tolerated and efficacious therapeutics reach patients. Traditional animal-based preclinical models are often ill-suited for predicting some cancer immunotherapy-mediated adverse events in humans, in part because of the fundamental differences in the immunological responses between the species (Bjornson-Hooper et al., 2019). In the EpCAM example mentioned above, the severity of the diarrhea elicited by the treatment was not predicted by preclinical studies in mice (Brischwein et al., 2006). Moreover, an increasing number of TCBs target human-specific antigens that lack expression in animals, rendering preclinical animal studies uninformative for safety and efficacy assessment (Bacac et al., 2016). Indeed, the development of preclinical models that better translate to human immunity is regarded as one of the top current challenges of cancer immunotherapy (Hegde and Chen, 2020). While human-relevant cell-based models of tissues and organs promise to bridge this gap, conventional in vitro two-dimensional models fail to provide the complexity required to model the biological mechanisms of immunotherapeutic effects. Furthermore, their reductive microenvironment, devoid of essential cellular, biochemical, and biophysical factors found in the native organ, precludes the expression of TCB targets at physiologically relevant levels and patterns, crucial for capturing TCB pharmacology and safety liabilities. Organ-on-Chip models aim to overcome these limitations by combining micro-engineering with cultured primary human cells to recreate the complex multifactorial microenvironment and functions of native tissues and organs (Huh et al., 2010). The tissue microenvironment in vivo provides the external signals that help driving cellular differentiation toward mature phenotypes. The key functional aspects of the Organs-on-Chip model regarding tissue-level physiology, such as epithelial and microvascular tissue–tissue interfaces and physiologically relevant mechanical forces, have been shown to capture in vivo relevant phenotypes more accurately (Gayer and Basson, 2009; Kasendra et al., 2018, 2020). The enhanced tissue maturation promoted by Organs-on-Chip could help ensure organ-specific expression of TCB targets, while the modularity of these devices and the possibility for controlled circulation of molecules and immune cells could better capture the functional interactions between TCBs, immune cells, and target-expressing cells that occur in patients. Motivated by these advantages, we set out to evaluate Alveolus Lung- and Colon Intestine-Chip as platforms for the assessment of on-target, off-tumor TCB safety risks in human organs, using a panel of targeting and non-targeting molecules, and leveraging in vivo target expression and toxicity data (Figure 1). We found that these systems could reproduce and predict target-dependent TCB safety liabilities, showing sensitivity to key determinants thereof, such as target expression and antibody affinity (Kerns et al., 2021). Briefly, the protocol can be outlined broadly as a four-step process. In the first step, Organ-Chips are washed and activated to facilitate the covalent attachment of extracellular matrix proteins (ECM) to the surface of the chip. Appropriate deposition of ECM to the surface is critical to enable productive and physiological downstream cellular attachment. In the second step, cells are harvested from various primary stocks (e.g., frozen vials, organoids, or tissue culture flasks) for seeding onto the prepared chip surfaces. Key considerations in the seeding process are the density of cells and the incubation time needed for cells to adhere to the coated chip surface. After the attachment period, the chips are then connected to the pod reservoir system, which provides media and interface with the Zoë unit, necessary to establish media flow throughout the duration of the experiment. In the third step, the connected chip/pod unit is equilibrated with the Zoë to support continuous flow for the entire experimental period. This period includes an equilibration and maturation window that allows for the seeded epithelium and endothelial layers to mature and develop, according to standard biomarker assessments experimental interrogation. A critical step during this period is the introduction of microenvironmental cues, such as stretch motions or an air–liquid interface, which are necessary for tissue-specific differentiation and maturation. Once the Organ-Chips have achieved the appropriate characteristics for tissue maturation (e.g., barrier function and morphology), the experimental phase of the protocol begins. In this stage, chips are dosed with immune cell populations preincubated with test article solutions and endpoints are collected according to the desired biomarkers panel and experimental timepoints. At the terminal timepoint, the final endpoint specimens are collected, including the harvesting of tissues, and the pod reservoir units are discarded. Chips intended for downstream imaging or other applications can be stored in sterile solutions of balanced salt solutions at 4 °C. The expected result of the above protocol is an assessment of the dose- and time-dependent levels of TCB-mediated T cell activation and killing of the respective alveolar lung or colonic epithelial tissues. Materials and Reagents Organ-Chip materials Emulate Basic Research kit 12 pack (Emulate, catalog number: BRK-WER-12). Kit components: Chip-S1® stretchable chips, Pod® portable modules, ER-1® /ER-2® chip activation reagents, Steriflip® filter. Storage: ER-1® and ER-2® reagents: 2–8 °C; other kit components: ambient temperature (15–25 °C) Pod imaging adapter kit (Emulate, catalog number: POD-IMG) Fixed chip imaging adaptor kit (Emulate, catalog number CHIP-IMG) Emulate colon intestine Bio Kit 12-pack (Emulate, catalog number: BIO-CH1-12). Kit components: Chip-S1® stretchable chips, Pod® portable modules, ER-1® /ER-2® chip activation reagents, Steriflip® Filter. Qualified, biopsy-derived human colonic organoids and primary colonic microvascular colonic endothelial cells (HIMEC). Storage: ER-1® and ER-2® reagents: 2–8 °C; Cells: store in liquid nitrogen; other kit components: ambient temperature (15–25 °C) Cells and Reagents Note: Please refer to the Notes section below for more information regarding the specification of the carcinoembryonic antigen (CEA) and folate receptor 1 (FLOR1) targeting reagents below. Steriflip filters (EMD Millipore, catalog number: SE1M003M00) T-25 flasks T-75 flasks T-150 flasks 15 mL conical tubes 50 mL conical tubes 96-well V-bottom plates 24-well plates 15 mL conical tubes, Protein LoBind® tubes (Eppendorf, catalog number: 0030122216) 50 mL conical tubes, Protein LoBind® tubes (Eppendorf, catalog number: 0030122240) 1.5 mL Eppendorf tubes, Protein LoBind® (Eppendorf, catalog number: 022431081) 1.5 mL Eppendorf tubes P200 pipette tips with filter (Labcon, catalog number: 1179-965-008-9, or equivalent) P1000 pipette tips with filter (Labcon, catalog number: 1177-965-008-9, or equivalent) Square cell culture dish (VWR, catalog number: 82051-068) Trypan blue (Sigma, catalog number: 93595) Hemocytometer (SKC Inc., catalog number: DHCN015) Human primary alveolar epithelial cells (HPAEC): Human pulmonary alveolar epithelial cells (Accegen, catalog number: ABC-TC3770), or Human primary alveolar epithelial cells (CellBiologics, catalog number: H-6053) Human lung microvascular endothelial cells (HMVEC-L) (Lonza, catalog number: CC-2527) SABM basal medium (Lonza, catalog number: CC-3119), store at 4 °C SAGM SingleQuots supplement pack (Lonza, catalog number: CC-4124), store at -20 °C Fetal bovine serum (FBS) (Sigma, catalog number: F4135 or F8317), store at -20 °C Bovine serum albumin (BSA) (Sigma, catalog number: A9576), store at 4 °C Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D2650), store at room temperature until expiration Dexamethasone (Sigma, catalog number: D4902), store at 2-8 °C Keratinocyte growth factor (KGF) (Thermo Fisher, catalog number: PHG0094) 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt (cAMP) (Sigma, catalog number: B7880) Isobutyl methylxanthine (IBMX) (Sigma, catalog number: I7018) EBM-2 basal medium (Lonza, catalog number: CC-3156), store at 4 °C EGM-2MV SingleQuots supplement pack (Lonza, catalog number: CC-4147), store at -20 °C Medium 199 (Thermo Fisher, catalog number: 11043023), store at 4 °C Epidermal growth factor (EGF) (PromoCell, catalog number: C-60170) Basic human fibroblast growth factor (FGF) (PromoCell, catalog number: C-60243) Vascular endothelial growth factor (VEGF) (PromoCell, catalog number: C-64420) Hydrocortisone (Sigma, catalog number: H0135) Heparin (Sigma, catalog number: H3149) Gelatin solution (ATCC, catalog number: PCS-999-027) Accutase (Stemcell Technologies, catalog number: 07920) Penicillin-Streptomycin (Pen-Strep) (Sigma, catalog number: P4333), store at -20 °C Easy 50 EasySep magnet (Stemcell Technologies, catalog number: 18002) Direct human PBMC isolation kit (Stemcell Technologies, catalog number: 19654), store at 4 °C Dulbecco’s PBS (DPBS) without Ca2+ , Mg2+ (Gibco, catalog number: 21-031-CV), store at room temperature Placental collagen type IV (Sigma, catalog number: C5533), store at -20 °C Human plasma fibronectin (Corning, catalog number: 356008), store at 2–8 °C (lyophilized) for three months or at -20 °C for two weeks Laminin (Sigma, catalog number: 6274) L-Glutamax (Thermo Fisher, catalog number: 35050-061), store at 4 °C Advanced DMEM/F12 (Thermo Fisher, catalog number: 12634028), store at 4 °C IntestiCult (Stem Cell Technologies, catalog number: 06010), store media components at -20 °C Y-27632 (Stem Cell Technologies, catalog number: 72304), store at -20 °C desiccated, protect from light CHIR99021 (Reprocell, catalog number: 04-0004-10), store powder at 4 °C protected from light, store aliquots at -20 °C Primocin (InvivoGen, catalog number: ANT-PM-1), store at -20 °C Attachment factor (Cell Systems, catalog number: 4Z0-210), store at 4 °C Matrigel-growth factor reduced (Corning, catalog number: 356231), store at -20 °C TrypLE Express (Gibco, catalog number: 12604013) Cell recovery solution (Corning, catalog number: 354253) Mini cell scraper (Biotinium, catalog number: 22003) RPMI-1640 (Gibco, catalog number: 11875093), store at 4 °C Dextran Cascade Blue 3000 MW (Invitrogen, catalog number: D7132) CMFDA cell tracker green (Thermo Fisher, catalog number: C7025), store in freezer -5 °C to -30 °C, protect from light NucView 405 (Biotium, catalog number: 10407) 4% paraformaldehyde in aqueous solution (PFA) (VWR, catalog number: 102091-904), store at room temperature 1× PBS with 0.05% sodium azide (Teknova, catalog number: P0202) BD perm/wash buffer (BD BioSciences, catalog number: 554723, store at room temperature Cell staining buffer (BioLegend, catalog number: 420201), store at 4 °C Anti-Human FOLR1 (R&D Systems, catalog number: MAB5646) Mouse IgG1 isotype control (R&D Systems, catalog number: MAB002) QIFIKIT® (Agilent Technologies, Inc., catalog number: K007811-8), store at 4 °C Anti-human CD3 HIT3a Alexa Flour 700 (BioLegend, catalog number: 300324), store at 4 °C Anti-human CD4 OKT4 BV785 (BioLegend, catalog number: 317442), store at 4 °C Anti-human CD69 FN50 BV650 (BioLegend, catalog number: 310934), store at 4 °C Anti-human CD3 HIT3a APC-Cy7 (BioLegend, catalog number: 300318), store at 4 °C Anti-human CD8 SK1 PE/Dazzle-594 (BioLegend, catalog number: 344744), store at 4 °C Anti-human CD25 BC96 PerCP-Cy5.5 (BioLegend, catalog number: 302625), store at 4 °C Anti-human CD69 FN50 APC (BioLegend, catalog number: 310910), store at 4 °C NucBlue fixed cell ready probes reagent (DAPI) (ThermoFisher, catalog number: R37606) Mouse anti-human FOLR1 (L.S Bio, catalog number: LS-C125620) Rabbit polyclonal anti-E-cadherin (Abcam, catalog number: ab15148) 7.5% BSA (Thermo Fisher, catalog number: 15260037) Normal donkey serum (Abcam, catalog number: ab7475) Anti-CEACAM5 antibody (CI-P83-1) (Santa Cruz Biotechnology, catalog number: sc-23928) Purified mouse IgG2a kappa isotype Ctrl (MOPC-173) (BioLegend, catalog number: 400202) Recombinant rabbit anti-CEA (Abcam, catalog number: ab133633), store at 4 °C short term (1–2 weeks), upon delivery aliquot and store at -20 °C Monoclonal rat anti-CD45 (Invitrogen, catalog number: MA5-17687), store at 4 °C short term (1–2 weeks), upon delivery aliquot and store at -20 °C DRAQ5TM fluorescent probe solution (5 mM) (Thermo Fisher, catalog number: 62251), store at 4 °C, protect from light DyLightTM 405 AffiniPure donkey anti-rat IgG (H+L) (Jackson ImmunoResearch, catalog number: 712-475-153), store at 4 °C short term (1–2 weeks). Upon delivery, aliquot and store at -20 °C, protect from light Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa FluorTM 555 (Thermo Fisher, catalog number: A-31572), store at 4 °C, protect from light Goat anti-human IgG (H+L) secondary antibody, Alexa FluorTM 555 (Thermo Fisher, catalog number: A-21433), store at 4 °C, protect from light CellEventTM Caspase-3/7 green detection reagent (Thermo Fisher, catalog number: C10423), store at ≤ -20 °C protected from light LIVE/DEADTM fixable yellow dead cell stain kit (Thermo Fisher, catalog number: L34959), store at ≤ -20 °C protected from light Customized ProcartaPlex multiplex immunoassay (Invitrogen, catalog number: PPX-12-MXNKRV6). Panel including human targets: IFNγ, TNFα, Granzyme-B, IL-2, IL-4, and IL-8 ER-1 (Emulate, catalog number: BRK-WER-12) ER-2 (Emulate, catalog number: BRK-WER-12) PBMC culture media (see Recipes) ER-1 solution (see Recipes) Alveolus ECM stock solutions (see Recipes) Alveolus ECM working solutions (see Recipes) HPAEC culture medium or complete SAGM culture medium (see Recipes) HPAEC maintenance media (see Recipes) HMVEC-L culture medium or complete EGM-2MV culture medium (see Recipes) ALI culture medium (see Recipes) CMFDA cell tracker green (see Recipes) PCMC dosing media (see Recipes) FACs buffer (see Recipes) 1% PFA solution (see Recipes) Y-27632 (ROCK inhibitor) (see Recipes) CHIR99021 (GSK-3 inhibitor) (see Recipes) Matrigel-growth factor reduced (see Recipes) Collagen IV (see Recipes) Fibronectin (see Recipes) 3 KDa Dextran Cascade Blue (see Recipes) Colon ECM stock solutions (see Recipes) Colon ECM working solutions (see Recipes) Colonoid thawing media (see Recipes) IntestiCult expansion media (see Recipes) IntestiCult maintenance media (see Recipes) Colonoid dissociation solution (see Recipes) Cell counting solution (see Recipes) HIMEC culture medium (see Recipes) Live/dead fixable yellow dead stain (see Recipes) PBMC dosing media (see Recipes) Equipment Zoë culture module (Emulate, catalog number: ZOE-CM2) Orb hub (Emulate, catalog number: Orb-HM1) Centrifuge (Sorvall, Thermo Fisher, model number: 75-200-395) UV light box (Emulate, Inc.) Inverted phase contrast microscope with 10× and 20× objectives (Echo Revolve Microscopes) Inverted fluorescent microscope with 10× and 20× objectives (Echo Revolve and Olympus Microscopes) Biosafety cabinet BD FACSCelestaTM flow cytometer (BD Biosciences, model: 660344) ProcartaPlex multiplex immunoassays (Invitrogen PPX-12-MXNKRV6) BioPlex-200 (Bio-Rad) Software ICY software (BioImage Analysis Lab, Institut Pasteur, https://icy.bioimageanalysis.org/) FACSDiva (BD Bioscience, https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software) FIJI (Schindelin et al., 2012) ( https://imagej.net/software/fiji/) Bio-Formats plugin for ImageJ (The Open Microscopy Environment, https://www.openmicroscopy.org/bio-formats/downloads/) Zen (blue edition) (Carl Zeiss AG, https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html) FlowJo (FlowJo LLC, https://www.flowjo.com/) GraphPad Prism (GraphPad, https://www.graphpad.com/) BioPlex Manager (Bio-Rad, https://www.bio-rad.com/en-us/product/bio-plex-manager-software-standard-edition?ID=5846e84e-03a7-4599-a8ae-7ba5dd2c7684) Part I: Culture of the Alveolar Lung-Chip for TCB-mediated T cell activation and killing Procedure Figure 2. Experimental timeline for the culture of the human Alveolar Lung-Chip Isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) Note: Please refer to the Notes section below for a further discussion on why PBMCs are the recommended cell source used for the TCB-mediated T cell activation and killing assessment. Refer to Alveolar Lung-Chip experimental timeline (Figure 2). Prepare PBMC culture media (see Recipes). Isolate PBMCs from fresh human buffy coat using the direct human PBMC isolation kit following manufacturer’s instructions. Approximately 10 million viable PBMCs per chip will be needed to complete the T cell activation and killing assessment. Cryo-preserve PBMC in FBS supplemented with 10% DMSO. Thawing human primary alveolar epithelial cells (HPAEC) (Day -1) Heat 50 mL of complete SAGM culture medium to 37 °C. Coat a T-25 flask with gelatin solution by adding 3 mL of 0.1% gelatin solution. Incubate the flask at 37 °C and 5% CO2 for 5–10 min. Thaw the vial(s) of cells by immersing in a 37 °C water bath. Immediately transfer the contents of the vial into 3 mL of warm complete SAGM in a sterile 15 mL conical tube. Rinse the vial with 1 mL of complete SAGM and collect in the 15 mL tube. Bring the volume to 15 mL with complete SAGM culture medium. Centrifuge at 200 × g for 5 min at room temperature. Aspirate and discard supernatant, leaving approximately 100 µL of medium covering the cell pellet. Resuspend cells in 7 mL of complete SAGM culture medium. Add the HPAEC suspension to the T-25 flask. Incubate overnight at 37 °C and 5% CO2 . Exchange with freshly warm complete SAGM once a day until use for seeding in the chip. Thawing human lung microvascular endothelial cells, HMVEC-L (Day -1) Heat 50 mL of complete EGM-2MV culture medium to 37 °C. Thaw the vial(s) of cells by immersing in a 37 °C water bath. Immediately transfer the contents of the vial into 3 mL of warm complete EGM2-MV culture medium in a sterile 15 mL conical tube. Rinse the vial with 1 mL of complete EGM-2MV culture medium and collect in the 15 mL tube. Bring the volume to 15 mL with complete EGM2-MV culture medium. Add the HMVEC-L suspension to the T75 flask. Incubate for 6 h (until 60%–80% cells have adhered) at 37 °C and 5% CO2 . Aspirate the culture medium and add complete EGM-2MV culture medium to remove traces of DMSO. Incubate overnight at 37 °C and 5% CO2 . Exchange with freshly warm complete EGM-2MV culture medium every other day until use for seeding in the chip. Chip activation and preparation (Day -1) ReviewVideo 1: Chip activation. Video 1. Chip activation Open the chip cradle sterile packaging and place the cradle into the 120 mm square dish, making sure the chip cradle is oriented properly with the corners facing up. Open the chip packaging carefully and place the first chip into the chip cradle by sliding the back of the carrier under the tabs on the cradle. Prepare the ER-1 solution (see Recipes) Note: All solutions in these steps are used at room temperature. Using a P200 pipette and a sterile 200 μL filtered pipette tip, take up 200 μL of ER-1 solution. Note: 200 μL of ER-1 solution will fill approximately three chips. Also, filtered tips help to maintain sterile conditions during chip preparation steps. Carefully introduce approximately 20 μL of ER-1 solution through the bottom channel inlet, pipetting until the solution begins to exit the bottom channel outlet. Without releasing the pipetting plunger, take the pipette out from the bottom channel inlet and move the pipette containing the remaining ER-1 solution to the top channel inlet. Introduce approximately 50 μL of ER-1 solution to the top channel inlet, pipetting until the solution begins to exit the top channel outlet. Remove all excess ER-1 solution from the surface of the chip by gentle aspiration. Be sure only to remove ER-1 solution from the chip surface—do not aspirate ER-1 from the channels. Repeat steps D1–D5 for each chip. Inspect the channels for bubbles prior to UV activation. If bubbles are present, dislodge by washing the channel with ER-1 solution until all bubbles have been removed. If bubbles persist, it may be helpful to aspirate the channel dry and slowly reintroduce ER-1 solution. Bring the ER-1-coated chips to the UV light box. Before placing the chips into the UV light box, make sure to remove the cover from the 120 mm square dish. Note: If the cover is not removed prior to placing the dish in the UV light box, the chips will not activate properly, which could result in poor cell attachment. Set the switch at the back of the UV light box to the “Constant” setting. Turn on the “Power” and press the “On” button to begin UV activation. Allow the chips to activate under UV light for 10 min. After UV treatment, bring the chips back to the biosafety cabinet (BSC). Aspirate activated ER-1 from all channels and refill channels with fresh ER-1 as before. Bring the chips back to the UV light box and activate under UV light for an additional 5 min. While the chips are being treated, prepare the top and bottom channel alveolus ECM working solution (see Recipes). After UV treatment, bring chips back to the BSC. Note: The light may be on in the BSC from this point forward. Fully aspirate the ER-1 solution from both channels for approximately 1–2 s. Wash each channel with 200 μL of ER-2 solution. Fully aspirate the ER-2 from the channels for approximately 1–2 s. Wash each channel with 200 μL of sterile DPBS. Leave DPBS inside the channels. Chip coating with ECM (Day -1) Review Video 2. Activation and coating of chips with extracellular matrix proteins. Video 2. Activation and coating of chips with extracellular matrix proteins Fully aspirate cold DPBS from both channels. Using a P200 pipette, take up 200 μL of alveolus or colon ECM working solution. Carefully introduce alveolus or colon ECM working solution through the bottom channel inlet until a small ECM droplet forms on the outlet. Without releasing the pipetting plunger, take the pipette out from the bottom channel inlet and move to the next chip, repeating steps E2–E4 for each chip. Using a P200 pipette, take up 200 μL alveolus or colon ECM working solution. Carefully introduce alveolus or colon ECM working solution through the top channel inlet until a small ECM droplet forms on the outlet. Without releasing the pipetting plunger, take the pipette out from the top channel inlet and move to the next chip, repeating steps E5–E7 for each chip. Inspect channels to ensure that no bubbles are present. If bubbles are present, dislodge by washing the channel with the respective top or bottom channel ECM solutions until all bubbles have been removed. To prevent evaporation during incubation, fill central reservoir of the chip cradle with 0.75 mL of DPBS (Figure 3), place the lid onto a 120 mm square dish, and incubate overnight at 37 °C and 5% CO2 . Note for Alveolar Lung-Chip: If desired, HPAEC can be seeded the same day as chip activation and ECM coating, though incubation overnight is preferred for best result. Chips can be ready for same-day seeding of HPAEC 4 h after adding the ECM solutions and incubating chips at 37 °C and 5% CO2 . If chips will be stored longer than overnight before seeding, store the chips at 4 °C for up to two days. Figure 3. Fill central reservoir of chip cradle Prepare chips for seeding (Day 0) Gently wash each channel of the chip with 200 μL of warm complete SAGM culture medium. Aspirate the medium outflow at the surface of the chips, leaving the medium in the channels. Repeat the wash with an additional 200 μL of complete SAGM culture medium per channel, leaving the excess medium outflow covering the inlet and outlet ports. Cover the 120 mm dish and place the chips in the incubator until the cells are ready for seeding. Harvest of HPAECs (Day 0) Bring the T-25 flask containing HPAECs from the incubator into the BSC. Aspirate culture media from a T-25 flask of confluent HPAECs. Add 5 mL of DPBS preincubated to 37 °C to wash the culture surface and aspirate the DPBS wash. Add 3 mL of TrypLE Express to the flask and incubate for 10–15 min at 37 °C and 5% CO2 . Tap the side of the flask gently and inspect the culture under the microscope to assess complete detachment of cells from the flask surface. Add 7 mL of warm complete SAGM culture medium to the flask and pipette gently to mix, while collecting all cells from the flask surface. Transfer the contents of the flask into a sterile 15 mL conical tube. Centrifuge HPAECs at 200 × g for 5 min at room temperature. Carefully aspirate the supernatant, leaving approximately 100 μL of medium above the cell pellet. Note: The cell pellet will be very small. Aspirate carefully. Loosen the cell pellet by flicking the tube gently. Using a P1000 pipette, gently resuspend the cells by adding 500 μL of warm complete SAGM culture medium. Pipette gently to create a homogeneous mixture and transfer 10 μL of the cell suspension to the cell counting solution of 80 μL SAGM culture medium + 10 μL trypan blue, making a 1:10 dilution. Mix the counting solution thoroughly and count cells using a manual hemocytometer. Count both viable and non-viable cells. Calculate percent viability of the cell solution. The expected viability of the HPAECs should be greater than 80%. Calculate viable cell concentration. Calculate viable cell yield, or total number of viable cells. Dilute with warm complete SAGM culture medium to the required final cell density of 1 × 106 cells/mL viable HPAECs. Seed HPAECs to top channel (Day 0) Bring the 120 mm dish containing the prepared chips to the BSC. Avoiding contact with the inlet and outlet ports, carefully aspirate excess medium droplets from the surface of one chip. Very gently agitate cell suspension before seeding each chip to ensure a homogeneous cell suspension. Quickly and steadily pipette 35–50 μL of the cell suspension (at 1 × 106 cells/mL) into the top channel inlet port, while aspirating the outflow fluid from the chip surface. Avoid direct contact with the outlet port. Cover the dish and transfer to the microscope to check the seeding density within the chip. Note: At this stage, optimal seeding density should form an even layer with cell-to-cell spacing of approximately half the radius of a single cell. If seeding density is not optimal, return the chips to the BSC and wash the channel with 200 μL of fresh medium twice. Adjust cell density accordingly and repeat steps H3–H5 until the correct density is achieved within the channel. After confirming the correct cell density, seed cells in the remaining chips. Note: Minimize the amount of time the cells are outside the incubator by seeding no more than six chips at a time and immediately placing them in the incubator at 37 °C after seeding each batch. Place the chips with DPBS in the central reservoir of the chip cradle (Figure 3) at 37 °C for at least 2 h or until cells have attached (Figure 4). Note: Correct seeding density is essential for success of Alveolar-Lung Chips. Figure 4. Representative brightfield images of epithelial (HPAEC) cell seeding density. (A) Optimal seeding density immediately after seeding and (B) 2 h post-seeding at 1 × 106 per mL. Wash chips (Days 1 and 2) Note: A gentle wash is performed 2 h post-seeding once the cells have attached, to ensure that nutrients are replenished and the channels do not dry out. If cells are not attached, incubate overnight and then wash. Gently pipette 200 μL of warm complete SAGM culture medium to the top and bottom channels of each chip to wash. Aspirate the outflow, leaving media in the channel. Place additional droplets of media to fully cover all inlet and outlet ports to prevent evaporation from the ports (Figure 5). Incubate chips overnight at 37 °C. Repeat steps I2 and I3 once daily with HPAEC maintenance media (see Recipes) for the next two days. Figure 5. Chip with medium drops covering the inlet and outlet ports Prepare chips (Day 3) Gently pipette 200 μL of warm HPAEC maintenance medium to the top channel of each chip to wash. Aspirate the outflow, leaving medium in the channel. Pipette 200 μL of warm complete EGM-2MV culture medium to the bottom channel of each chip. Aspirate the outflow, leaving medium in the channel. Return chips to the incubator until HMVEC-Ls are ready for seeding. Harvest HMVEC-Ls (Day 3) Aspirate culture medium and add 15 mL of 1× DPBS to wash the culture surface. Aspirate the DPBS wash. Add 5 mL of TrypLE Express to the flask. Incubate for 5–10 min at 37 °C. Tap the side of the flask gently and inspect the culture under the microscope to assess complete detachment of cells from the culture surface. Add 7 mL of warm complete EGM-2MV culture medium to the flask and pipette gently to mix, while collecting all cells from the culture surface. Transfer the contents of the flask (12 mL) into a sterile 15 mL conical tube. Centrifuge HMVEC-Ls at 200 × g for 5 min at room temperature. Carefully aspirate the supernatant, leaving approximately 50–100 μL of medium above the cell pellet. Note: The cell pellet will be small. Aspirate carefully. Loosen the cell pellet by flicking the tube gently. Using a P1000 pipette, gently resuspend the cells by adding 500 μL of complete EGM-2MV culture medium. Pipette gently to create a homogeneous mixture and transfer 10 μL of the cell suspension to the cell counting solution of 80 µL SAGM and 10 µL trypan blue (this will make a 1:10 dilution). Mix the counting solution thoroughly and count cells using a manual hemocytometer. Count both viable and non-viable cells. Calculate percent viability of the cell solution. The expected viability of the HMVEC-Ls should be greater than 80%. Calculate viable cell concentration. Calculate viable cell yield. Dilute to 5 × 106 cells/mL viable HMVEC-Ls in complete EGM-2MV culture medium. Seed HMVEC-Ls to bottom channel (Day 3) Bring the 120 mm dish containing the prepared chips to the BSC. Avoiding contact with the ports, aspirate DPBS from cradle reservoir and carefully aspirate excess medium droplets from the surface of one chip. Gently agitate cell suspension before seeding each chip to ensure a homogeneous cell suspension. Seed 15–20 μL of the endothelial cell suspension into the bottom channel of one chip first, while aspirating the outflow. Cover the dish and transfer to the microscope to check the seeding density within the chip (see Figure 6 for reference). Figure 6. Endothelial (HMVEC-L) optimum cell seeding density. (A) Optimal seeding density under brightfield microscopy, immediately after seeding into the bottom channel at 5 × 106 per mL. (B) HMVEC-Ls attached on chip 1.5 h post-seeding. If seeding density is not optimal, return the chip to the BSC and wash the channel twice with 200 μL of fresh endothelial cell culture medium. Do not aspirate the medium from the channel prior to washing. Adjust the volume of the cell suspension as needed to obtain correct seeding density and repeat steps L3–L5 until the correct density is achieved within the channel. After confirming the correct cell density, seed the remaining chips in the chip cradle. Note: Minimize the amount of time the cells are outside the incubator by seeding no more than six chips at a time and immediately placing them in the incubator at 37 °C after seeding. Once all six chips have been seeded place each in the cradle, cover the dish and carefully invert it (Figure 7). Figure 7. Inverting chips during endothelial attachment To prevent evaporation during incubation, refill central reservoir with 0.75 mL of DPBS and place cover onto square dish. Proceed with remaining chips until all have been seeded. Incubate at 37 °C for 2 h, or until cells in the bottom channel have attached. Once endothelial cells have attached (approximately 2 h post-seeding), aspirate DPBS from the central reservoir and flip the dish back so the chips are in an upright position in the chip cradle. Note: It is recommended to always seed any remaining cells into a plate as control for cell quality. Wash with tips (Day 3) Once HMVEC-Ls have attached, flip the chips back to an upright position. Note: Remove the chip cradle, wipe with 70% ethanol to clean, and autoclave for use in next experiment. A wash with 200 μL of HPAEC maintenance medium for the top channel and complete EGM-2MV culture medium for the bottom channel per chip will provide nutrients to cells. Since there are two different media being used, these must be separated by keeping them in filtered tips instead of drops (Figure 8). Figure 8. Chip with filtered tips inserted into ports with respective media Return chips with pipette tips inserted in each inlet and outlet port to the incubator overnight. Maintain cells in static culture in chips until connecting to pods and Zoë the next day. Note: If desired, chips can be connected at least 2 h post-attachment. Gas equilibration of media (Day 4) Note: The media equilibration step is important for the success of Organ-Chip culture. Omission of this step will cause the eventual formation of bubbles in the chip, the pod, or both, which will in turn cause irregular flow and negatively impact cell viability. Minimize the time media is outside of a warmed environment to no more than 10 min, as gas equilibrium can become compromised when media is allowed to cool. Place at least 3 mL of HPAEC maintenance medium for each chip in a 50 mL conical tube. Place at least 3 mL of complete EGM-2MV culture medium for each chip in a separate 50 mL conical tube. Heat both 50 mL conical tubes of media at 37 °C in a water or bead bath for at least 1 h. Immediately connect the 50 mL tube containing each warmed medium to a Steriflip® unit. Attach each conical tube containing warmed medium to a Steriflip® unit. With the unit right-side up (medium in the bottom conical tube), apply vacuum for 10 s. Invert the Steriflip® -connected tubes and check that the medium begins to pass from the top conical tube to the lower tube. Note: The vacuum source must operate at least at -70 kPa. At this correct pressure, it should take approximately 2 s for every 10 mL of medium to flow through the filter. If it takes longer, stop and see the troubleshooting protocol, as this indicates the medium is not equilibrated properly. Leave the filtered medium under vacuum for 5 min. Remove the vacuum tubing from the Steriflip® units. Separate the conical tubes containing media from the Steriflip® unit and immediately place them in the incubator with the caps loose. Media priming of pods (Day 4) Open the pod package and place the pods into the trays. Orient the pods with the reservoirs toward the back of the tray (Figure 9A). Pipette 2 mL of pre-equilibrated warm media to each inlet reservoir. In the top channel inlet reservoir add HPAEC maintenance medium; in the bottom channel inlet reservoir add complete EGM-2MV culture medium (Figure 9C). Pipette 300 μL of pre-equilibrated warm media to each outlet reservoir, directly over each outlet via (Figure 9C). Bring trays containing pods to the incubator and slide completely into Zoë with the tray handle facing outward. Run the prime cycle on Zoë. Use the rotary dial to highlight “Prime” on the display. Press the rotary dial to select “Prime.” Rotate the dial clockwise to highlight “Start.” Press the dial again to select “Start” and begin. Note: Once “Start” is selected, there will be an audible sound as Zoë engages the pods. Close the incubator door and allow Zoë to prime the pods; this process takes approximately 1 min. Note: When the status bar reads “Ready,” the “Prime” cycle is complete. Remove the tray from Zoë and bring to the BSC. Verify that the pods were successfully primed (Figure 9B). This is important for success. Inspect the underside of each pod—look for the presence of small droplets at all four fluidic ports. Droplets will vary in size from a small meniscus to larger droplets; often, droplets on the outlet ports will be larger. If any pod does not show droplets, rerun the “Prime” cycle on those Pods. If any media dripped onto the tray (this may occur more often by the outlet ports), clean tray with a wipe sprayed with 70% ethanol. Once it is confirmed that all pod ports are wet with droplets, put the tray of pods to the side in the BSC. Figure 9. Illustration of chip & pod handling. (A) Illustration of chip tray and pods. (B) Illustration demonstrating pod handling and description of the visual inspection necessary to observe droplet formation after priming step. (C) Schematic representation of the pod reservoirs indicating the respective top and bottom channels and inlet and outlet ports. Wash chips (Day 4) Transfer the seeded chips in a 150 mm dish from the incubator to the BSC. Remove the pipette tips from the chip inlet and outlet ports. Gently wash the top channel of each chip with warm equilibrated HPAEC maintenance medium to remove any possible bubbles in the channel. Place small droplets of equilibrated HPAEC maintenance medium on the top of each top channel inlet and outlet port of each chip. Gently wash the bottom channel of each chip with warm equilibrated complete EGM-2MV culture medium to remove any possible bubbles in the channel. Place small droplets of equilibrated complete EGM-2MV culture medium on the top of each bottom channel inlet and outlet ports of each chip. Chips-to-Pods (Day 4) Holding one chip (while it remains in the chip carrier) with the dominant hand and one pod with the non-dominant hand, slide the chip carrier into the tracks on the underside of the pod until the chip carrier has seated fully. Place the thumb on the chip carrier tab and gently but firmly depress the tab in and up to engage the tab of the chip carrier with the pod. Aspirate any excess medium on the chip surface from the pod window. Place the pod with the connected chip onto the tray. Repeat steps Q1–Q4 for each pod and chip carrier. Confirm that there is sufficient media in each pod inlet and outlet reservoirs and that the pod lids are flat and secure. Pods-to-Zoë (Day 4) Place trays that are holding pods and chips immediately into Zoë to prevent media from cooling and losing its gas equilibration. Set top channel flow rate to 0 μL/h and bottom channel flow rate to 30 μL/h. Run the regulate cycle. Using the rotary dial, highlight the “Regulate” field. Press the dial to select “Regulate” and rotate the dial clockwise to “Start.” Press the dial again to select “Start” and begin the regulate cycle. Note: Once “Start” is selected, there will be an audible sound as Zoë engages the pods. At this point the “Activation” button will glow blue. The regulate cycle lasts 2 h. After the cycle has finished, Zoë will begin flow at the preset Organ-Chip culture conditions. To cancel the regulate cycle (only if needed) on Zoë, select the “Regulate” field with the dial and press the button to select. Rotate the dial counterclockwise to select “Cancel.” Press the dial once more and wait 1 min for the cycle to end, after which the tray can be removed. If canceled, always rerun a complete regulate cycle before proceeding. Via wash and the regulate cycle (Day 5) ReviewVideo 3: Pod priming and regulate cycle. The day after connecting chips and pods to Zoë, which begins the process of Organ-Chip culture, pause the Zoë by pressing the silver “Activation” button located above the tray bays. This stops flow and releases the pods. Slide the tray out of the bay and transfer to the BSC. Remove the pod lids. Using a 200 μL pipette, perform a via wash on each pod inlet and outlet reservoir: Using media within the pod reservoir, pipette 200 μL of media directly over the top of the via to dislodge any bubbles that may be present. Repeat this wash step for each of the four pod reservoirs. Replace pod lids and return the trays to Zoë. Video 3. Pod priming and regulate cycle Establishing air–liquid interface (ALI) and maintenance (Day 5) Note: Only establish ALI 12 or more hours after running regulate. Do not rerun regulate cycle once ALI is established. Pause Zoë by pressing the silver “Activation” button and move pods into the BSC. Using a complete aspiration technique, aspirate all media from both inlet and outlet reservoirs along all four edges of the reservoir for the top channel for all pods that require culture at ALI. Transfer all trays and pods back into Zoë. Set top channel flow rate to 1,000 μL/h and bottom channel to 0 μL/h. Start flow and allow to run for 1 min. This step gently pushes any remaining media from the top channel and collects it in the top channel outlet reservoir. Pause Zoë as previously described and move pods into the BSC. Immediately aspirate all remaining media from top channel outlet reservoir to avoid media backflow from the outlet reservoir into the channel. Using the microscope, check to confirm that the top channel does not contain medium. If channels are not completely free of medium, place back in Zoë and run for another 1 min at 1,000 μL/h. Aspirate all medium from bottom channel inlet and outlet reservoirs, leaving a small liquid layer over the bottom inlet reservoir via. This will prevent introducing unwanted bubbles during flow. Add 2–4 mL of warm ALI culture medium to the bottom channel inlet reservoirs. Pipette 1 mL of media in the air channel pod inlet reservoir first, then immediately pipette 1 mL of media in the air channel pod outlet reservoir. Note: This equal media distribution in the pod reservoirs is required to maintain static pressure in the air channel. Transfer all trays and pods back to Zoë. Set top channel to “Air.” Set bottom channel flow rate to 30 μL/h. Press the activation button to resume Zoë operation. Refresh medium in bottom channel inlet reservoir every other day or as needed. Sampling and media replenishment (Day 6+) Pause Zoë by pressing the silver “Activation” button. Remove the trays and place in the BSC. Visually inspect each chip for bubbles. Using a microscope, inspect cells in the chips to assess morphology and viability. Capture representative images at 10× or 20× magnification at the following locations (Figure 10): Inlet junction. Center of channel. Outlet junction. Remove the pod lids and collect effluent medium from pod outlet reservoirs for analysis. Collect effluent from the indicated regions, avoiding disturbing the pod reservoir vias. Gently aspirate any medium not collected for analysis, ensuring that a thin liquid film still covers the reservoir vias so that no air is introduced into the vias. Refill the pod media reservoirs with fresh complete ALI medium and perform a via wash: using medium within the pod reservoir, pipette 1 mL of medium directly over the top of the via to dislodge any bubbles that may be present. Replace the pod lids and return trays to Zoë. Press the silver “Activation” button to resume pre-set Organ-Chip culture conditions. Zoë will engage when the “Activation” button glows blue. Figure 10. Representative schematic of image collection performed on chip. (A) Image capture of regions along the length of the chip. (B) Representative images of colonic epithelial cells and human microvascular endothelial cells seeded in the top and bottom chip channels, respectively. Pre-dosing media condition change and PBMC thawing (Day 8) Aspirate media from bottom channel inlet and outlet reservoirs. Replace bottom channel media with ALI culture medium without hydrocortisone. Note: Hydrocortisone is an immunosuppressant and needs to be removed a day before dosing. Check for bubbles in the channel and vias, following standard troubleshooting procedures. Maintain ALI setting. PBMC thawing protocol (Day 8) Heat PBMC culture medium to 37 °C in a water bath. Thaw frozen vials, only three vials at a time, in a 37 °C water bath. When cells are nearly completely thawed, carry the vials to the hood and swipe them with 70% ethanol. Take out PBMCs (by very slow and gentle pipetting) and transfer the cells into a 50 mL Falcon tube. Dilution of DMSO: add 10 mL of warm complete medium dropwise, i.e., one drop per second, while gently mixing the cells. Use 1 mL to rinse out the vials. Wash 1: spin the cells at 300 × g for 8 min at room temperature. Remove supernatant by tube inversion. Wash 2: suspend the cell pellet in 1 mL of medium and add 9 mL of complete medium. Spin the cells at 300 × g for 8 min at room temperature. Count cells and determine viability: add 1 mL of complete medium, slowly suspend the cell pellet using the 1 mL pipette, and add more medium to a cell concentration of approximately 3 × 106 –4 × 106 cells/mL (e.g., if the vial contained 20 × 106 fresh cells, add approximately 4 mL of medium since cell loss is expected after thawing). Perform the counting on 10 μL aliquots in duplicate with ½ trypan blue dilution. Just prior to pipetting out the cells for counting, gently invert the tube in order to homogenize the cell suspension. Resting: adjust cell concentration to 2 × 106 cells/mL in complete medium, transfer cells in a 25 or 75 cm2 flask, and let them rest for 4–16 h before performing the experiments. The flask should be kept standing or slightly bended, not flat in the incubator. PBMC staining for live imaging (Day 9) Transfer the PBMCs from the flask to a 50 mL conical tube and centrifuge at 300 × g for 8 min. Resuspend the PBMC pellet with 10 mL of RPMI + 5% FBS and count the cell suspension. Stain PBMCs with the CMFDA cell tracker green at a final concentration of 5 μM for 20 min at 37 °C and 5% CO2 . Add 10 mL of media after the incubation time and centrifuge at 300 × g for 8 min. PBMC pretreatment with TCBs (Day 9) Resuspend PBMC cells with PBMC dosing medium (see Recipes) at a working concentration of 8 × 106 cells/mL, or 2-fold higher than the final PBMC density. Prepare TCBs in PBMC dosing media at working concentrations 2-fold higher than the final concentration specified in the experimental design. Combine the 2-fold working concentrations of PBMCs with the corresponding TCBs. Preincubate the dosing suspension for 1 h at 37 °C and 5% CO2 . Reintroduction of liquid–liquid interface (LLI) (Day 9) Add 500 μL of complete ALI medium (without hydrocortisone) in the top inlet reservoirs of all chips. Make sure the bottom channel reservoirs have enough media. If not, add complete ALI medium (without hydrocortisone) to the bottom channel inlets. Run a flush cycle to reintroduce liquid–liquid interface: Set top channel flow rate to 1,000 μL/h and bottom channel flow rate to 0 μL/h. Start flow and allow to run for 2 min. Note: This step gently pushes media to the top channel and collects it in the top channel outlet reservoir. Pause Zoë as previously described and move pods into the BSC. Aspirate all media from the top channel outlet reservoir. Use the microscope to confirm that the top channel is submerged with medium. If the top channel is not filled with medium, place back in the Zoë and run for another 1 min at 1,000 μL/h. Repeat steps Z3c–Z3d as needed. Set bottom and top channel flow rates to 30 μL/h and activate the Zoë. PBMC-TCB introduction to the Alveolus Lung-Chip epithelium (Day 9) Add 500 µL of the PBMC dosing medium to the top channel inlets in appropriate chips. Run a flush cycle for the top channel: Set top channel flow rate to 1,000 μL/h and bottom channel flow rate to 0 μL/h. Start flow and allow to run for 3 min. Pause Zoë as previously described and move pods into the BSC. Aspirate all media from the top channel outlet reservoir. Disconnect the chips from Zoë and check under the microscope, making sure the PBMCs are distributed throughout the top channel (Figure 11). Note: PBMCs might not be evenly distributed throughout the top channel after the flush cycle but will settle down evenly after the static period. Place the chips back in the Zoë and leave the top channel static for 2.5 h to allow the PBMCs to settle. Aspirate top channel inlets and outlets and refresh the top channel inlet reservoir with fresh dosing media. Aspirate the bottom channel inlets and outlets and add fresh complete ALI medium (without hydrocortisone). Connect the chips to flow and set the top and bottom channel flow rates to 30 µL/h. Note: Record the time of PBMC-TCB dosing and the static incubation time. Figure 11. Representative brightfield micrographs of healthy alveolar epithelial cells on the top channel of the Alveolar Lung-Chip in the absence and presence of well-distributed PBMCs in co-culture Timepoint collection and endpoints (Days 10 and 11) Collect around 350 µL of effluent from the top and bottom outlet reservoirs and store at -80 °C immediately after collection. Live imaging of apoptotic cells (Days 10 and 11) Dilute NucView 405 at 1:500 with PBMC dosing medium. Add 500 µL of this NucView medium solution to the top inlets of each chip. Run flush cycle. Set top channel flow rate at 1,000 µL/h and bottom channel at 0 µL/h for 5 min. Stop flow and set the flow rate back to 30 µL/h for both channels for 30 min. Aspirate top inlet and outlet reservoirs and replace with fresh complete ALI medium (without hydrocortisone). Run flush cycle again to wash excess dye off the chip. Set top channel flow rate at 1,000 µL/h and bottom channel at 0 µL/h for 5 min. Image using fluorescence microscope. Take nine images per chip at a 10× magnification focused on the top channel from inlet to outlet. PBMCs: GFP or green channel. Apoptotic cells: DAPI channel. Phase contrast: bright field. Note: NucView 405 requires an exposure time of 500 ms. Save images as .vsi or .tiff files for quantification of apoptotic epithelial cells. Connect chips to Zoë and flow overnight at a flow rate of 30 µL/h in top and bottom channels. An example of live imaging of apoptotic cells is shown in Figure 12. Figure 12. Representative brightfield (top) and immunofluorescent images (bottom) of Alveolar Lung-Chip epithelium (nuclei, blue) 48 h after addition of PBMC (cyan). The control group did not have PBMC administered. The FOLR1(Hi) group showed higher levels of PBMC attachment and caspase-3/7-positive, apoptotic cells (magenta). PBMC collection for flow cytometry analysis (Day 11) Disconnect chips from Zoë and pods. Plug the bottom channel with tips on both ends and the top channel inlet. Take 100 µL of complete ALI medium (without hydrocortisone) and triturate using a pipette, to collect as many PBMCs as possible from the top channel from each chip in separate tubes or wells of a V-bottom, 96-well plate. Collect all remaining media from the top channel of chips as well in corresponding tubes or wells. Add fresh complete ALI medium (without hydrocortisone) to the top channel of each chip. Collect fluorescent images in the GFP channel of the top channel of chips after PBMC collection to enable downstream quantification of TCB-mediated PBMC attachment. Fix chips for downstream immunofluorescent endpoints by applying 4% PFA solution for 20 min in both channels. Wash both chip channels with DPBS and store for up to one week at 4 °C. Centrifuge PBMCs collected in tubes or V-bottom, 96-well plates at 300 × g for 5 min. Collect supernatant for downstream bioanalyte analysis. Resuspend the PBMC pellets in 100 µL of FACs buffer (see Recipes). Centrifuge at 300 × g for 5 min. Discard supernatant and resuspend the pellets in each well with 50 µL of a master staining mix: 1 µL Anti-human CD3 HIT3a Alexa Flour 700. 1 µL Anti-human CD4 OKT4 BV785. 1 µL Anti-human CD69 FN50 BV650. Remainder FACs buffer. Incubate in the dark at 4 °C for 20 min. Add 120 µL of FACS buffer to each well and centrifuge at 300 × g for 5 min. Discard supernatants and resuspend the PBMC pellets in 200 µL of FACS buffer. Centrifuge at 300 × g for 5 min. Resuspend the pellet in 1% PFA solution for 15 min at room temperature. Add 100 µL of FACS buffer and centrifuge at 300 × g for 5 min. Discard the supernatant and resuspend the pellets in 200 µL of FACS buffer. Cover with foil and store at 4 °C for up to one week for flow cytometry analysis. QIFIKIT single-cell quantification of target antigen Dissociation of epithelial cells from Alveolus Lung-Chip: Note: It is recommended to have samples in triplicate (separate Organ-Chip per well), including an unstained control. Wash chip channels twice with DPBS (1×). Add tips to outlets of both channels with P200 pipette tips. Do not fully block the flow. Add 100 µL of accutase to top and bottom channels. Keep tips inside the top and bottom channel inlet ports and transfer chips to incubator (37 °C) for 5 min. Check the dissociation of cells from the top channel by triturating the top channel epithelium using a P200 and checking under a microscope. If more dissociation is required, repeat steps EE5–EE6 until cells are detached. Note: This process usually takes from 15 to 20 min. Collect the contents of the top channel only into Eppendorf tubes. Break up the cells further by titrating with a P200 pipette. Add 500 µL of medium 199 to quench the accutase digestion and pipette to mix. Centrifuge at 350 × g for 5 min. Resuspend pellet in 200 µL FACS buffer to wash. Take a small volume (approximately 5 µL) of the sample of cell suspension and count using the hemocytometer. Indirect immunofluorescence staining of cell surface antigens: Centrifuge tubes at 350 × g for 5 min. Resuspend the pellet in FACS buffer to adjust the cell concentration to 0.5 × 106 cells/mL. Transfer 100 µL (approximately 50,000 cells) of the target cell suspension into each well of V-bottom, 96-well plate, as indicated. Centrifuge the plate and discard supernatant. Add 50 µL of FACS buffer containing indicated amounts of primary antibody dilutions: Anti-human FOLR1 diluted to 10 µg/mL. Ensure that the primary antibody is used at saturating concentration. Mouse IgG1 isotype control diluted to 10 µg/mL. Note: Allocate one extra well of epithelial cells as an unstained control. Perform primary staining for 30 min at 4 °C in the dark. Transfer 100 µL of vial 1 (setup beads) and vial 2 (calibration bead solution) (QIFIKIT® ) to two wells of a 96-well plate. Next, wash cells (and beads) twice with FACS buffer. Dilute QIFIKIT® anti-mouse IgG (FITC) 50× in FACS buffer. Resuspend wells in 25 µL of FACS buffer containing anti-mouse IgG. Stain cells and beads for 30 min at 4 °C in the dark. Wash wells once with FACS buffer. Fix with FACS buffer + 1% PFA solution for 20 min at 4 °C. Wash cells with FACS buffer and resuspended in 100 µL FACS buffer. Store in the dark at 4 °C until measurement. Note: If taking multiple timepoints, samples can be kept in the dark at 4 °C for up to one week. Calibration and setup should be generated each time for staining controls. Collection and analysis of QIFIKIT® data Run samples using FACSDiva flow cytometer or equivalent system, using the setup beads to inform voltage settings. Using FlowJo software, open the setup bead sample data and view in histogram plot with intensity of FITC-A as x-axis. Gate the positive population as “FITC+” and calculate the geometric mean intensity. Repeat for all experiment samples (using the unstained control and setup beads as getting reference) (Figure 13). Figure 13. Example QIFIKIT® analysis. (A) Histogram of FITC signal for setup beads, with FITC+ signal selected. (B) Overlay of unstained control (orange), sample 1 (red), and sample 2 (blue) on FITC signal histogram to determine geometric mean of FITC+ signal. Calculate the standard curve and the number of antigenic sites present on the cell surface: Open the calibration beads sample in histogram plot with five separate FITC intensity peaks. Using the range gate tool, gate the separate peaks and obtain their geometric mean intensity of FITC-A signal. Using the instructions provided in the QIFIKIT® kit, calculate the standard curve and apply to samples to report the number of antigenic target sites (Figure 14). Figure 14. QIFIKIT® calibration beads in FlowJo. (A) Histogram of FITC signal showing five gated peaks of calibration beads. (B) For each peak, the geometric mean of FITC+ intensity is measured in order to produce the standard curve. Endpoint fixation with PFA Note: Prepare the workspace of the chemical fume hood prior to beginning your work, ensuring that the space within the hood is organized and free from clutter and that the path of airflow is not blocked. Ensure all chip carriers are labeled and identify the different conditions clearly. Detach chips from PodTM modules and organize them in petri dishes for handling. Gently wash each channel with 200 μL of DPBS once. Place 200 μL tips gently in the outlets of both channels. We recommend using filtered tips for this step. Be careful to not push the tips too hard against the bottom of the chip channel, as this could seal off the outlet and prevent reagents from going through the channel and outlet. Add 100 μL of 4% PFA (in PBS, pH 7.4) to each channel from the inlet, leaving the tips inserted into the inlet as shown inFigure 7. Incubate for 20 min at room temperature. After incubation, remove all four pipette tips and wash each channel with 200 μL of DPBS three times. Optional: Add 200 μL of 100 mM glycine to each channel and incubate at room temperature for 30 min [PFA tends to increase the sample auto-fluorescence in the green (FITC, 488) wavelength and glycine can be used to quench the autofluorescence]. Note: Fixed chips can be stored at 4 °C for up to one week in DPBS with 0.05% sodium azide. To ensure channels do not dry up during this period, it is recommended that DPBS is added with 200 μL tips as described in steps FF3 and FF4 above. Then, place the chips with tips in the ports inside plastic containers sealed with parafilm. We recommended using empty 200 μL tip boxes for storage. Ensure that the tips remain snug in the ports during transport and storage to avoid drying of the channels. Permeabilization and blocking Use PBS to prepare a blocking/permeabilizing solution containing: 0.1% Triton X-100 (it is recommended that permeabilization be skipped for surface markers). 1% BSA and 5% normal donkey serum (in DPBS) obtained from the same animal where secondaries antibodies have been raised in (species-matched serum). Incubate the samples at room temperature for 30 min in the blocking/permeabilizing solution. Wash samples with 200 μL of PBS three times. Immunofluorescent staining on fixed samples Prepare primary antibody staining solution(s) in 2% BSA/DPBS or 10% serum/DPBS: Anti-human FOLR1 IgG1 1:50 (v/v). Anti E-cadherin polyclonal rabbit (1:100). After preparing the primary antibody solution(s), add 100 μL to the top and bottom channels, leaving pipette tips inserted in the inlet ports. Incubate chips overnight at 4 °C. After incubation remove all pipette tips and wash each channel with 200 μL of DPBS three times. Prepare secondary antibody solution(s) in 2% (v/v) BSA in DPBS: Donkey anti-mouse Alexa Flour 568. Donkey anti-rabbit Alexa Flour 488. Add 100 μL of the secondary antibody solution to the top and bottom channels, leaving pipette tips inserted in the ports as described in steps HH3 and HH4. If you are staining just one channel, add DPBS to the opposite channel. Incubate chips for 2 h at room temperature taking care to protect them from light. After incubation, remove all pipette tips and wash each channel with 200 μL of DPBS three times. Prepare NucBlue solution: 2 drops of NucBlue solution in 1 mL of DPBS to stain cell nuclei. Add 100 μL of this solution and incubate for 10 min. Wash three times with DPBS. Image the samples (Figure 15) or store at 4 °C. Figure 15. Example of immunofluorescent staining of FOLR1 target expression (blue) and E-cadherin (red) in epithelium of chips administered with FOLR1(Hi)-treated PBMC (green) Data analysis Alveolus Lung-Chip PBMC attachment and epithelial cell apoptosis quantification The images are saved as .vsi files; these need to be converted to .tiff before analyzing on ICY or ImageJ. Convert .vsi to .tiff image type using FIJI: Download FIJI software and install the Bio-Formats plugin (version 6.9.0 or higher) using the instructions provided here: https://docs.openmicroscopy.org/bio- formats/5.8.2/users/imagej/installing.html. Open FIJI software. Navigate to Process > Batch > Convert. Choose Input and Output directory. Select “Read Images Using BioFormats” box. Select Convert. These images can now be inputted in ICY image analysis software using the apoptosis protocol . Open five random images on ICY to set the HK means and threshold for the entire set of images for both GFP (PBMC marker) and DAPI (apoptotic cells) channels to get optimal ROIs to run the entire protocol (in the example below, channel 1 is PBMCs and channel 2 is apoptotic cells). Using co-localization tools, quantify the number of apoptotic epithelial-positive cells (Caspase-3+ NucView405+) that are also PBMC-cell-tracker-negative (GFP) and set different object sizes for alveolar epithelial cells (~900–3,000 pixels) and PBMCs (~200–600 pixels) in the ICY image analysis software. Choose a directory to input images to be analyzed on ICY. Input file name for analyzed images in the output. Run the protocol. The results are displayed in Excel format. A screenshot of the protocol is displayed below (Figure 16). An example of a graph generated for apoptotic cells and PBMC attachment is shown in Figure 17. Figure 16. Schematic representation of the ICY image analysis protocol used to quantify immune cell attachment and epithelial cell apoptosis Figure 17. Example of quantification of apoptotic caspase-3/7-positive cells and PBMC attachment from live chips (n = 4). One-way ANOVA, Tukey’s multiple comparison test, p < 0.05. Cytokine analysis Collect affluents from Alveolus Lung-Chip pod outlets at 24 and 48 h after PBMC-TCB administration. Immediately freeze effluents at -80 °C until measurement. Measure cytokines for Alveolus Lung-Chip (GranzymeB, IFNg, IL-2, IL-6, IL-8, IL-10, IL-13, IL1RA, TNFa, MIP-1b, G-CSF, and GM-CSF) using the customized ProcartaPlex multiplex immunoassays. ProcartaPlex multiplex immunoassays kit contains a black 96-well plate (flat bottom), antibody-coated beads, detection antibody, streptavidin-R-phycoerythrin (SAPE), reading buffer, and universal assay buffer. In addition, standards with known concentration are provided to prepare a standard curve. According to the Invitrogen publication number MAN0017081 [Revision B.0 (33)], the assay workflow is as follows: After adding the beads into the flat bottom plate, wash the beads using a flat magnet and an automated plate washer (405TS microplate washer from Bioteck). Then, add standards and samples diluted with a universal buffer into the plate and incubate for 2 h. After a second wash, add detection antibodies. After 30 min incubation and a wash, add SAPE. Finally, after 30 min incubation and a final wash, resuspend the beads in the reading buffer. The plates are ready for analysis. Acquire the data with a BioPlex-200 system. Using the certificate of analysis provided with the kit, enter the bead region and standard concentration value S1 for each analyte of the current lot in the software BioPlex Manager. Plotting the expected concentration of the standards against the mean fluorescent intensity generated by each standard, the software generates the best curve fit and calculates the concentrations of the unknown samples (in pg/mL). The data is then exported in Excel and plotted in GraphPad Prism. Flow cytometry List of flow cytometry panel for PBMC: PBMC: FITC CD3: Anti-human CD3 HIT3a Alexa Flour 700 CD4: Anti-human CD4 OKT4 BV785 CD69: Anti-human CD69 FN50 BV650 CD25: Anti-human CD25 BC96 PerCP-Cy5.5 Apoptotic cells: Pacific blue Acquire sample data using BD FACSCelestaTM flow cytometer and analyze data using FlowJo V10 software. Open all samples in FlowJo software and copy single stain control samples into compensation group. Use FlowJo compensation window to calculate a new compensation matrix and apply to all samples. Apply gating strategy to samples, using the unstained and compensation control samples for guidance: Open the unstained control sample and view in SSC-FSC dot plot. Place a polygon gate to encircle the lymphocyte population. Within lymphocyte population, determine the PBMC (FITC) population for live cells. In the lymphocyte+/PBMC+ population, plot CD3 (Alexa Flour 700) vs. CD4 (BV786) population and gate CD3+ CD4- population to isolate CD8+ T cells. Within the CD8+ gate, create a gate for CD69+ (BV650) to assess the activation of CD8+ T cells. Report statistics of frequency of parent to Excel for plotting: % lymphocyte+ % lymphocyte+/PBMC-FITC % lymphocyte+/PBMC-FITC/CD3+ CD4- % lymphocyte+/PBMC-FITC/CD3+ CD4-/CD69+ Note: Figure 18 shows an example of FACS panel for Colon-Chip using live/dead (BV605) to determine the live PBMC population. For the Alveolus-Chip experiments, PBMC cell tracker green was used instead to determine the live PBMC population. Either version can be used for FACS analysis.Figure 19shows a representative example of the relative populations of activated CD8+ T cells. Figure 18. Example of flow cytometry analysis in FlowJo. (A) SSC-FSC dot plot of unstained control sample to select lymphocyte+/PBMC+ population. (B) Histogram of live/dead stain to gate on live cells. (C) Quadrant dot plot of CD3 and CD4 signal to select CD8+ population. (D) Histogram of CD69 signal to gate on CD69+ activated CD8 T cells. Figure 19. Example of flow cytometry analysis of PBMC harvested from chips for percentage of live, CD69+ activated CD8+ T cells (n = 4, approximately 10,000 cells per chip) after 48 h Part II: Colon-Chip Procedure Figure 20. Experimental timeline for the culture of the human Colon Intestine-Chip Isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) Follow the instructions outlined in section A of Part I: Alveolar Lung-Chip protocol. Thawing of human intestinal microvasculature endothelial cells (HIMECs) (Day -3) Note: HIMECs are included in the Emulate Basic Research kit. Heat 50 mL of HIMEC culture medium to 37 °C. Add 5 mL of attachment factor onto the entire growth surface of a T-75 flask and incubate at 37 °C and 5% CO2 for 5 min. Discard excess attachment factor. Add 15 mL of HIMEC culture medium to the flask and leave it in a 37 °C incubator until ready for plating. Thaw the vial(s) of cells by immersing in a 37 °C water bath. Immediately transfer the contents of the vial using a P1000 pipette into the prepared T-75 flask containing warm HIMEC culture medium. Incubate the flask at 37 °C and 5% CO2 for 6 h. Aspirate medium and carefully add 15 mL of fresh HIMEC culture medium. Return the flask back to the incubator at 37 °C and 5% CO2 overnight. Exchange medium in flask with fresh HIMEC culture medium every other day until use for chip seeding. Chip activation and preparation (Day -1) Follow instructions outlined in section D of Part I: Alveolar Lung-Chip protocol. An overview of the timeline for the Colon Intestine-Chip experimental timeline is shown in Figure 20. Chip coating with ECM (Day -1) Follow procedure outlined in section E of Part I: Alveolar Lung-Chip protocol, with the following alterations: In steps 2–4, use colon ECM working solution for bottom channel. In steps 5 and 6, use colon ECM working solution for top channel. Prepare chips for seeding (Day 0) Gently wash each channel of the chip with 200 μL of warm complete HIMEC culture medium. Aspirate the medium outflow at the surface of the chips, leaving the medium in the channels. Repeat the wash with an additional 200 μL of complete HIMEC culture medium per channel, leaving the excess medium outflow covering the inlet and outlet ports. Cover the 150 mm dish and place the chips in the incubator until the cells are ready for seeding. Harvest of human intestine microvascular endothelial cells (HIMECs) (Day 0) Aspirate culture media from a T-75 flask of confluent HIMECs. Add 15 mL of DPBS to wash the culture surface and aspirate the DPBS wash. Add 3 mL of TrypLE Express to the flask and incubate for 2–3 min at 37 °C and 5% CO2 . Tap the side of the flask gently and inspect the culture under the microscope to assess complete detachment of cells from the flask surface. Add 3 mL of warm complete HIMEC culture medium to the flask and pipette gently to mix, while collecting all cells from the flask surface. Transfer the contents of the flask into a sterile 15 mL conical tube. Pipette gently to create a homogeneous mixture and transfer 20 μL of the cell suspension to a 1.5 mL tube containing the cell counting solution of 20 μL of HIMEC culture medium + 20 μL of trypan blue, making a 1:3 dilution. Mix the counting solution thoroughly and count cells using a manual hemocytometer. Count both viable and non-viable cells. Calculate percent viability of the cell solution. The expected viability of the HIMECs should be greater than 80%. Calculate viable cell concentration. Discard cell counting suspension and calculate viable cell yield. Centrifuge HIMEC suspension at 150 × g for 5 min at room temperature. Carefully aspirate the supernatant, leaving approximately 100 μL of medium above the cell pellet. Note: The cell pellet will be very small. Aspirate carefully. Loosen the cell pellet by flicking the tube gently. Using a P1000 pipette, gently resuspend the cells by adding 200 μL of warm complete HIMEC culture medium. Dilute with warm complete HIMEC culture medium to the required final cell density of 8 × 106 cells/mL viable HIMECs. Seed HIMECs to bottom channel (Day 0) Bring the 120 mm dish containing the prepared chips to the BSC. Avoiding contact with the inlet and outlet ports, carefully aspirate excess medium droplets from the surface of one chip. Very gently agitate cell suspension before seeding each chip to ensure a homogeneous cell suspension. Using a pipette, pipette out the medium from the bottom channel, leaving it empty. Quickly and steadily pipette 10–15 μL of the HIMEC cell suspension into the bottom channel inlet port, while aspirating the outflow fluid from the chip surface. Avoid direct contact with the outlet port. Cover the dish and transfer to the microscope to check the seeding density within the chip (see Figure 21 for reference). Note: At this stage, optimal seeding density should form an even layer with cell-to-cell spacing of approximately half the radius of a single cell. Figure 21. Endothelial (HIMEC) optimum cell seeding density. (A) Optimal seeding density under brightfield microscopy immediately after seeding into the bottom channel. (B) HIMECs attached on chip two hours post-seeding. If seeding density is not optimal, return the chips to the BSC and wash the channel with 200 μL of fresh medium two times. Do not aspirate the medium from the channel. Adjust cell density accordingly and repeat steps F3–F5 until the correct density is achieved within the channel. After confirming the correct cell density, seed cells in the remaining chips, invert each chip, and rest the edge of the chip carrier on the chip cradle. Note: Each chip cradle can support up to six chips inside a square cell culture dish (Figure 7). Place DPBS at the cradle to provide humidity for the cells. Replace dish lid. Note: Minimize the amount of time the cells are outside the incubator by seeding no more than 12 chips at a time and immediately placing them in the incubator at 37 °C and 5% CO2 after seeding each batch. Place the chips still in the dish in the incubator at 37 °C and 5% CO2 for approximately 30 min–1 h, or until cells in the bottom channel have attached. Once HIMECs have attached (approximately 1 h post-seeding), flip the chips back to an upright position. Wash chips (Days 1 and 2) Note: A gentle wash is performed 30 min–1 h post-seeding, once the cells have attached, to ensure that nutrients are replenished and that channels do not dry out. If cells are not attached, incubate overnight and then wash. Gently pipette 200 μL of warm complete HIMEC culture medium to the top and bottom channels of each chip to wash. Aspirate the outflow, leaving media in the channel. Repeat the step using complete IntestiCult expansion media to wash the top channel. Harvest of colonoids (Day 0) Note: Due to the donor-dependent protocol variation, this protocol assumes the user has in-culture expanded colonic organoid material from a primary, healthy donor. For more information on colonic organoid expansion and culture please see Sato et al. (2011) and Sontheimer-Phelps et al. (2020). Carefully aspirate medium from each colonoid without disturbing the matrigel dome. Gently add 500 μL of cell recovery solution to each well. Scrape the matrigel using a cell scraper. Using a P1000 pipette, collect contents of each well and transfer to a cold, LoBind 15 mL conical tube. Incubate conical tube on ice for 45 min, inverting the tube every 2–5 min during this time. While cells are incubating on ice, ensure the centrifuge is cooled down to 4 °C. Centrifuge the organoid suspension at 300 × g for 5 min at 4 °C. After centrifugation, observe the tube to confirm complete disappearance of matrigel and clear formation of a cell pellet. Note: If a thin layer of matrigel is present, remove the supernatant carefully using a P1000 pipette without disrupting the pellet and add 5 mL of new cell recovery solution. Incubate for 5 min on ice and repeat centrifugation in Step H8. If no clear pellet is formed, repeat step H8. Once a defined cell pellet is observed, aspirate the supernatant, gently flick the tube to disrupt the pellet, and add the prepared dissociation solution, 2 mL for every 24-well plate. Incubate the conical tube in the water bath at 37 °C for 1–2 min to dissociate the organoids into fragments. Note: Incubation time will vary based on the size of organoids; however, do not incubate for longer than 2 min, as this may result in dissociation of organoids into single cells, leading to decreased seeding efficiency. Dilute at least two times with advanced DMEM/F12 medium to wash. Centrifuge to pellet the dissociated organoids at 300 × g for 5 min at 4 °C. Aspirate the supernatant and adjust seeding density by suspending the pellet in IntestiCult expansion media. Adjust cell density (Day 0) The seeding density depends on the size and density of organoids cultured on the 24-well plate. Use of two to three wells of organoids per chip has been recommended (Figure 12). To calculate volume for suspension of dissociated organoids: Chip seeding volume = 30 μL. Number of chips = 6. Volume of media required to resuspend dissociated organoids = 30 μL × 6 chips = 180 μL. Dilute the organoids with warm IntestiCult expansion media to the required final cell density (see Figure 22). Figure 22. Optimal single and fragments organoids density.(A) Low magnification; (B) Higher magnification. Note that the porous membrane is completely covered by the cells. Seed colonoids to top channel (Day 0) Note: Work with one chip at a time. After seeding the first chip, assess cell density within the channel through the microscope, adjusting the density of the cell suspension accordingly for the subsequent chips if necessary. Pipette out the medium from the top channel, leaving it empty. Very gently, agitate the cell suspension before seeding each chip to ensure a homogeneous mixture for even seeding. Quickly and steadily, pipette 30 μL of the cell suspension into the top channel inlet. Avoid direct contact between the aspirator tip and the outlet port. Cover the dish and transfer to the microscope to check the seeding density within the chip. Note: At the optimal seeding density, the organoid fragments will form an even cell layer on the top channel of the chip, covering the whole chip membrane (Figure 11). If seeding density is not optimal, return the chips to the BSC and wash the channel with 200 μL of fresh medium two times. Do not aspirate the medium from the channel. Adjust cell density accordingly and repeat steps J3–J5 until the correct density is achieved within the channel. To avoid density gradient between chips, once the cells density is confirmed, transfer approximately 300 μL aliquots of the cell suspension to 1.5 mL low protein binding tubes. Seed cells in the remaining chips. Note: Minimize the amount of time the cells are outside the incubator by seeding no more than 12 chips at a time, immediately incubating them at 37 °C and 5% CO2 after seeding each batch. Place the dish of chips with a filled DPBS reservoir at 37 °C and 5% CO2 and incubate undisturbed overnight. Gas equilibration of media (Day 1) Note: The media equilibration step is important for the success of Organ-Chip culture. Omission of this step will cause the eventual formation of bubbles in the chip, the pod, or both, which will in turn cause irregular flow and negatively impact cell viability. Minimize the time media is outside of a warmed environment to no more than 10 min, as gas equilibrium can become compromised when media is allowed to cool. Prepare at least 3 mL of HIMEC culture medium for each chip in a 50 mL conical tube. Prepare at least 3 mL of IntestiCult expansion media for each chip in a separate 50 mL conical tube with the addition of 10 μM Y-27632, 5 μM CHIR99021, and a tracer of your choice to check the permeability. The most common one is the 3 KDa Dextran Cascade Blue at final concentration in medium of 100 µg/mL. The tracer can be kept in culture medium throughout the course of the experiment to ensure a tighter barrier function. The permeability assay section will provide further instructions. Note: Approximately 2 mL of medium will be used per reservoir of the chip. For 12 chips, prepare approximately 30 mL of medium for the top channel and 30 mL of medium for the bottom channel. As noted above, minimize the time media is outside of the incubator during pod preparation to maintain temperature. This is a critical step to ensure success of the chips. Follow steps 3–10 in section N of Part I: Alveolar Lung-Chip protocol [Gas equilibration of media (Day 4)] to complete gas equilibration of media. Media priming of pods (Day 1) Open the pod package and place the pods into the trays. Orient the pods with the reservoirs toward the back of the tray (Figure 9A). Pipette 2 mL of pre-equilibrated, warm media to each inlet reservoir. In the top channel inlet reservoir, add complete IntestiCult expansion medium; in the bottom channel inlet reservoir, add Complete HIMEC culture medium (Figure 9C). Pipette 300 μL of pre-equilibrated, warm media to each outlet reservoir, directly over each outlet via (Figure 9C). Follow steps 4–16 in section O of Part I: Alveolar Lung-Chip protocol [Media priming of pods (Day 4)]. Wash chips (Day 1) Transfer the seeded chips in a 120 mm square dish from the incubator to the BSC. Wash twice the top channel of each chip with 200 μL warm equilibrated IntestiCult expansion media, aspirating the outflow from the chip surface. Wash the bottom channel of each chip with warm, equilibrated HIMEC culture medium, aspirating outflow from the chip surface. Place small droplets of equilibrated IntestiCult expansion media on all inlet and outlet ports of the top channel and HIMEC culture medium on the inlet and outlet of the bottom channel. Repeat steps M2–M4 for each chip. Chips-to-Pods (Day 1) Follow procedure outlined in section Q of Part I: Alveolar Lung-Chip protocol [Chips-to-Pods (Day 4)]. Pods-to-Zoë (Day 1) Follow procedure outlined in section R of Part I: Alveolar Lung-Chip protocol [Pods-to-Pods (Day 4)]. Alevolar-Chip via wash and the regulate cycle (Day 2) Follow procedure outlined in section S of Part I: Alveolar Lung-Chip protocol [Via wash and the regulate cycle (Day 5)]. Colon-Chip via wash and the regulate cycle (Day 2) Replenish media in the top inlet reservoir with IntestiCult maintenance media (without the addition of Y-27632 and CHIR99021 inhibitors). Add HIMEC culture medium to the bottom inlet reservoir. Replace pod lids. Inspect the chip for bubbles. Initiating mechanical stretch (Day 3) Pause Zoë by pressing the silver “Activation” button. Using the rotary dial, highlight the “Stretch” field. Press the dial to select “Stretch” and rotate the dial clockwise to increase stretch to “2%.” Press the dial to select “Freq.” and rotate the dial clockwise to increase stretch to “0.15 Hz.” Sampling and maintenance (Day 4) Follow protocol outlined in section V of Part I: Alveolar Lung-Chip protocol (Sampling and media replenishment). Permeability assay (Day 2+) Notes: This protocol is to assess the permeability of an Organ-Chip's endothelial-epithelial barrier. Apparent permeability (Papp) of tracer molecules is determined by dosing the inlet of the top channel, collecting the effluent of both top and bottom channels, and calculating the amount of compound that crossed through the membrane over time. A recommended tracer molecule is Dextran Cascade Blue 3000MW (Thermo Fisher, catalog number: D7132). It is recommended to prepare and analyze the permeability assay samples fresh for each timepoint day. Prepare standard curves of the fluorescent tracer molecule (i.e., Dextran Cascade Blue) used in your permeability assay in both the dosing channel media as well as receiving channel media. This is required to quantify and further analyze barrier integrity on the Organ-Chip. Start by preparing at least 500 μL of a solution of fluorescent tracer dissolved in the dosing media at the same concentration that is added to the dosing channel reservoir. (You can prepare a larger volume if needed to accommodate a greater dilution of the tracer.) Label this as solution 1 . Label seven other tubes 2–8 and add 200 μL of the respective media (dosing or receiving channel media) to each of the tubes. Take 200 μL solution from tube 1 and add to 2, mixing evenly by pipetting up and down a few times. Then collect 200 μL from 2 and add to 3, continuing this way until you have prepared a serial dilution until tube 7. Add media without compound to tube 8, which will be used as the blank. Repeat this serial dilution for receiving channel media but start with a concentration of the fluorescent molecule that is 25% of the concentration added to the donor channel reservoir. (This is done to align the standard curve with the expected recovery concentrations.) Label tubes 9–16. Load a 96-well plate with either 50 or 100 μL (as per the requirements of your plate reader) of each of the solutions from tubes 1–16 (standards), as well as samples collected while running the barrier function assay. Load your plate and read on a plate reader. Using the standard curves generated by the plate reader, quantify the concentration of each sample, making sure to use the curve used for the media type being analyzed (e.g., top or bottom channel). A linear relationship is expected between concentration and plate reader output—check to ensure that the data is indeed linear in the concentration range being measured from the samples, especially the receiving channels, which is expected to be a much lower concentration than the donor channel. Perform a linear regression on the standard curve data to determine the equation of the form Y = m*X + b, which is needed to correlate plate reader data to concentrations. Correlate plate readings to data using the regression equation. The Emulate Standard Curve Calculator (EC003, https://emulatebio.com/protocol-archive/ep187-v1-0) automatically generates the standard curves and converts inputted data based on the curve generated. This calculator performs a log-log linear regression analysis, which minimizes the percent error across the full range of the standard curve data. Once all the concentrations have been calculated, Papp can be calculated using the following equation, which accounts for any loss of compound by assuming first-order loss dynamics along the length of the chip: where, Papp is the apparent permeability in units of cm/s; SA is the surface area of sections of the channels that overlap (0.17 cm2); QR and QD are the fluid flow rates in the dosing and receiving channels, respectively, in units of cm3/s; CR,0 and CD,0 are the recovered concentrations in the dosing and receiving channels, respectively, in any consistent units. The Emulate Papp Calculator (EC004) automatically performs this calculation (https://emulatebio.com/protocol-archive/ep187- v1-0). Sample data is shown below in Figure 23 to illustrate expected Papp over time for healthy Colon Intestine-Chips (“Control”) compared to chips with barrier disrupted by cytokine IFNγ. Seeded Colon Intestine-Chip typically forms an intact barrier around day 3–5, corresponding to apparent permeability below 1 × 10-6 cm/s (https://emulatebio.com/support/ep203-v1-0/). Figure 23. Sample Papp of Colon Intestine-Chip. Control Colon Intestine-Chip forms an intact barrier around experimental days 3–5, depending on seeding density. Intact barrier threshold marked by dotted line at y = 1 × 10-6 cm/s, which should be used as a guideline. Compared to healthy control (“Control”, blue line), cytokine treatment by IFNγ (pink line) replicates the damage of unhealthy permeable barrier (note: IFNγ treatment not included in this protocol). Increase stretch to 10% (Day 4) Pause Zoë by pressing the silver “Activation” button. Using the rotary dial, highlight the “Stretch” field. Press the dial to select “Stretch” and rotate the dial clockwise to increase stretch to “10.0%.” Press the dial to select “Freq.” and rotate the dial clockwise to increase stretch to 10% and the frequency to 0.15 Hz. Press the “Activation” button. PBMC thawing protocol (Day 4) Follow protocol outlined in section X of Part I: Alveolar Lung-Chip protocol [PBMC thawing protocol (Day 9)]. PBMC staining for live imaging (Day 5) Transfer the PBMCs to be stained from flask to a 50 mL conical tube and centrifuge at 300 × g for 8 min. Resuspend the PBMC pellet with 10 mL of RPMI + 5% FBS and count the cell suspension. Stain PBMCs with CMFDA cell tracker green at a final concentration of 5 μM for 20 min at 37 °C and 5% CO2 . Add 10 mL of PBMC culture media after the incubation time and centrifuge at 300 × g for 8 min. Follow protocol outlined in section Y of Part I: Alveolar Lung-Chip protocol [PBMC staining for live imaging (Day 10)]. PBMC pretreatment with TCBs (Day 5) On the day of PBMC administration to chip, collect the PBMCs from culture flasks into 50 mL conical tubes. Rinse flask with 10 mL of DPBS to collect as many PBMCs as possible. Centrifuge the PBMCs for 8 min at 300 × g . Resuspend the PBMCs in IntestiCult maintenance media at 8 × 106 cells/mL, or 2-fold higher than the final PBMC density. Prepare TCBs in PBMC dosing media at working concentrations 2-fold higher than the final concentration specified in the experimental design. Combine the 2-fold working concentrations of PBMCs with the corresponding TCBs. Divide the suspension into a 6-well plate (Falcon), separated by treatment condition. Account for at least 500 µL of suspension for each chip. Allow the PBMC to incubate at 37 °C and 5% CO2 with TCB antibodies for at least 1 h. PBMC-TCB introduction to the Colon-Chip epithelium (Day 5) Aspirate the pod inlets: Pause Zoë by pressing the silver “Activation” button. Remove the trays and place in the BSC. Visually inspect each chip for bubbles. Using a microscope, inspect cells in the chips to assess morphology and viability. Capture representative images at 10× or 20× magnification. Remove the pod lids and collect effluent medium from pod outlet reservoirs for analysis. Collect effluent from the indicated regions, avoiding disturbing the pod reservoir vias. Collect effluents for downstream endpoint analysis. Gently aspirate any medium not collected for analysis, ensuring that a thin liquid film still covers the reservoir vias so that no air is introduced into the vias. Add 500 µL of the dosing solution to the top channel inlets in appropriate chips. Mix the PBMC-TCB suspensions well with a P1000 pipette before administering. Note: For appropriate PBMC-free controls, add only IntestiCult maintenance media. To the endothelial channel pod inlet reservoir, add 500 µL of EGM2-MV complete medium. Place pods in Zoë culture module. Flow at 1,000 µL/h at 10% stretch (0.15 Hz) for 10 min. Disconnect from Zoë and check under the microscope making sure the PBMCs are distributed throughout the top channel. Aspirate all media from top channel outlet reservoir. Resume flow at 30 μL/h overnight for top and bottom channels. Note: PBMCs might not be evenly distributed throughout the top channel after the flush cycle but will settle down evenly after the static period. Connect the chips to flow and set the top and bottom channel flow rates to 30 µL/h. Note: Record the time of PBMC-TCB dosing and the static incubation time. Timepoint collection and endpoints (Days 5, 6, 7, 8+) Collect approximately 350 µL of effluent from the top and bottom outlet reservoirs and store at -80 °C immediately after collection. Live imaging of apoptotic cells (Days 5, 6, 7, 8+) Follow procedure outlined in section DD of Part I: Alveolar Lung-Chip protocol [Live imaging of apoptotic cells (Day 11 and 12)]. PBMC collection for flow cytometry analysis (Days 6, 7, 8+) Disconnect chips from Zoë and pods. Plug the bottom channel with tips on both ends and the top channel inlet. Take 100 µL of IntestiCult maintenance media and triturate using a pipette to collect as many PBMCs from the top channel of each chip as possible in separate tubes or wells of a V-bottom, 96-well plate. Collect all remaining media from the top channel of chips as well in corresponding tubes or wells. Add fresh IntestiCult maintenance media to the top channel of each chip. Collect fluorescent images in the GFP channel of the top channel of chips after PBMC collection to enable downstream quantification of TCB-mediated PBMC attachment. Fix chips for downstream immunofluorescent endpoints by applying 4% PFA solution for 20 min in both channels. Wash both chip channels with DPBS and store for up to one week at 4 °C. Centrifuge PBMCs collected in tubes or V-bottom 96-well plates at 300 × g for 5 min. Collect supernatant for downstream bioanalyte analysis. Resuspend the PBMC pellets in 100 µL of FACs buffer. Add 2 mM live/dead fixable yellow dead stain (see preparation) and incubate for 30 min at 37 °C. Wash cells with 100 µL of DPBS. Centrifuge at 300 × g for 5 min. Discard supernatant and resuspend the pellets in each well with 50 µL of a master staining mix: 1 µL of Anti-human CD3 HIT3a APC-Cy7. 1 µL of Anti-human CD4 OKT4 BV785. 1 µL of Anti-human CD8 SK1 PE/Dazzle-594. 1 µL of Anti-human CD69 FN50 APC. 1 µL of Anti-human CD25 BC96 PerCP-Cy5.5. Remainder FACs buffer. Incubate in the dark at 4 °C for 20 min. Follow steps 14–21 in section EE of Part I: Alveolar Lung-Chip protocol [PBMC collection for flow cytometry analysis (Day 12)] to complete flow cytometry workflow. QIFIKIT® single-cell quantification of target antigen Dissociation of epithelial cells from Colon Intestine-Chip: Note: It is recommended to have samples in triplicate (separate Organ-Chip per well), including an unstained control. Wash chip channels twice with DPBS (1×). Add tips to outlets of both channels with P200 pipette tips and do not fully block flow. Add 100 µL of TrypLE Express to top and bottom channels. Keep tips inside top and bottom channel inlet ports and transfer chips to incubator (37 °C) for 2–3 min. Check the dissociation of cells from the top channel by triturating the top channel epithelium using a P200 and checking under a microscope. If more dissociation is required, repeat steps CC5–CC6 until cells are detached. Note: This process takes usually from 15 to 20 min. Avoid excessive TrypLE incubation as this could damage surface epitopes. Collect the contents of the top channel only into Eppendorf tubes. Break up the cells further by titrating with a P200 pipette. Add 500 μL of serum-free advanced DMEM/F12 (+1% Pen-Strep) to quench the TrypLE digestion. Pipette to mix. Centrifuge at 350 × g for 5 min. Resuspend pellet in 200 µL of FACS buffer to wash. Take a small sample volume (approximately 5 µL) of cell suspension and count using hemocytometer. Indirect immunofluorescence staining of cell surface antigens: Centrifuge tubes at 350 × g for 5 min. Resuspend the pellet in FACS buffer to adjust the cell concentration to 0.5 × 106 cells/mL. Transfer 100 µL (approximately 50,000 cells) of the target cell suspension into each well of V-bottom, 96 well plate, as indicated. Centrifuge the plate and discard supernatant Add 50 µL of FACS buffer containing indicated amounts of primary antibody dilutions: Anti-human CEACAM5 diluted to 10 µg/mL. Mouse IgG2a isotype control diluted to 10 µg/mL. Note: Allocate one extra well of epithelial cells as an unstained control. Follow steps 2f–2m of section EE of Part I: Alveolar Lung-Chip protocol to complete QIFIKIT® sample workflow. Follow step 3 of section EE of Part I: Alveolar Lung-Chip protocol to complete QIFIKIT® analysis workflow. Endpoint fixation with PFA Note: Prepare the workspace of the chemical fume hood prior to beginning your work, ensuring that the space within the hood is organized, free from clutter, and the path of airflow is not blocked. Follow procedure outlined in section GG of Part I: Alveolar Lung-Chip protocol (Endpoint fixation with PFA). Permeabilization and blocking Follow procedure outlined in section HH of Part I: Alveolar Lung-Chip protocol (Permeabilization and blocking). Immunofluorescent staining on fixed samples Prepare primary antibody staining solution(s) in BD perm/wash buffer: For untreated samples without TCB: Recombinant rabbit anti-CEA diluted 1:100 (v/v). Monoclonal rat anti-CD45 diluted 1:100 (v/v). For samples treated with TCB: Monoclonal rat anti-CD45 diluted 1:100 (v/v) After preparing the primary antibody solution(s), add 100 μL to the top and bottom channels, leaving pipette tips inserted in the inlet ports. Incubate chips overnight at 4 °C. After incubation, remove all pipette tips and wash each channel with 200 μL of DPBS three times. Prepare secondary antibody solution(s) in CytoPerm/wash buffer. For untreated samples without TCB: DRAQ5TM diluted 1:250 (v/v). DyLightTM 405 AffiniPure donkey anti-rat IgG (H+L) diluted 1:500 (v/v). Donkey anti-rabbit Alexa FluorTM 555 diluted 1:500 (v/v). For samples treated with TCB: DRAQ5TM diluted 1:250 (v/v). DyLightTM 405 AffiniPure donkey anti-rat IgG (H+L) diluted 1:500 (v/v). Goat anti-human Alexa FluorTM 555 diluted 1:500 (v/v). This secondary is used since the CEA target sites were bound with anti-human TCB after administration. Add 100 μL of the secondary antibody solution to the top and bottom channels, leaving pipette tips inserted in the ports as described in steps FF3 and FF4. If you are staining just one channel, add DPBS to the opposite channel. Incubate chips for 2 h at room temperature taking care to protect them from light. After incubation, remove all pipette tips and wash each channel with 200 μL of DPBS three times. Data analysis Colon-Chip Quantifying PBMC attachment to epithelium Note: Images acquired on Zeiss products are in Carl Zeiss Image Data (CZI) format; convert to Tag Image File Format (TIFF) to analyze. If tile images were acquired, stitch the tile images together using Zen Blue: Navigate to Processing tab. Under Method, choose “Stitching.” Under Parameters, select “Fuse Tiles.” Select “Apply” to export stitched tile images. Convert CZI to TIFF image type using FIJI. Download FIJI software and install Bio-Formats plugin (version 6.9.0 or higher) using the instructions provided here: https://docs.openmicroscopy.org/bio- formats/5.8.2/users/imagej/installing.html. Open FIJI software. Navigate to Process > Batch > Convert… Choose Input and Output directory. Select “Read Images Using BioFormats” box. Select Convert. Open TIFF images in FIJI to determine optimal threshold values for detecting PBMC (CD45) signal. Note: For best results, choose images in focus with the highest PBMC (CD45) signal. For Brightness & Contrast adjusting: Open the Brightness & Contrast window (Image > Adjust > Brightness/Contrast). Select “Auto” on an in-focus image. Note: Use the minimum and maximum values (circled in blue) for a few images to get an average (Figure 24). Figure 24. FIJI Brightness & Contrast menu with minimum and maximum histogram values circled blue. The Auto button (bottom left) sets these automatically for best signal. For PBMC intensity threshold optimizing: Subtract Background of the image: Process > Subtract Background… (radius = 50 pixels). Filter out the noise with Median Filter: Process > Filters > Median (radius= 2 pixels). Threshold the image (Image > Adjust > Threshold) and choose the method that best captures the particles of interest (Figure 25). Notes: 1) For a useful explanation comparing the Thresholding types, visit https://imagej.net/Auto_Threshold. 2) In the work published in Kerns et al. (2021), the Otsu threshold method was used. Figure 25. Determining the most accurate Auto Thresholding method (drop-down list circled in blue). Original image on left and the binary (black and white) Auto Threshold image on right. For PBMC size threshold optimizing: Using the Auto-Threshold binary image, use the “Oval” elliptical selection tool to encircle a single particle or cluster of PBMC. Use Analyze > Measure to get particle area in microns2 . Repeat for multiple particles until you have a range of PBMC cluster sizes. Note: In the work published in Kerns et al. (2021), the PBMC cluster size was found to be 15.9–106 mm2 with images taken at 40× magnification . After intensity and size threshold have been set, quantify number of clusters of PBMC using the pre-determined values (Figure 26): Note: Use a macro in Fiji to batch process multiple images. Open image in FIJI. Image > Adjust > Brightness/Contrast > Set. Set Minimum and Maximum displayed value to values determined in step 3. Process > Subtract Background… (radius = 50 pixels). Process > Filters > Median (radius= 2 pixels). Image > Adjust > Threshold (use previously determined method). Analyze > Analyze Particles… Size micron2 = previously determined range in step 5. Select “Display Results” option. Plot total count of PBMC clusters per sample. Figure 26. Sample quantification of PBMC clusters on Colon Intestine-Chip. (A) Excel output file from FIJI analysis with quantified number of PBMC clusters per image. (B) Typical plotted data using GraphPad Prism to compare treatment groups. Flow cytometric analysis of PBMC using FlowJo List of flow cytometry panel for PBMC: CD3: Anti-human CD3 HIT3a APC-Cy7 CD4: Anti-human CD4 OKT4 BV785 CD8: Anti-human CD8 SK1 PE/Dazzle-594CD69: Anti-human CD3 HIT3a APC-Cy7 CD25: Anti-human CD25 BC96 PerCP-Cy5.5 Live/dead yellow: BV605 Open all samples in FlowJo software and copy single stain control samples into compensation group. If compensation of signals has not already been performed, use FlowJo Compensation window to calculate a new compensation matrix and apply to all samples. Apply gating strategy to samples, using the unstained and compensation control samples for guidance: Open the unstained control sample and view in SSC-FSC dot plot. Place a polygon gate to encircle the lymphocyte population [you can also use CD3+ (APC-Cy7+) signal to determine this gate]. Within lymphocyte population, determine the live/dead negative (BV605-A-) population for live cells. In the lymphocyte+/live/dead- population, create a gate for CD8+ (PE/Dazzle-594+) signal to isolate the cytotoxic T cell population. Within the CD8+ gate, create a gate for CD69+ (APC+) to assess the activation of CD8+ T cells. Report statistics of frequency of parent to Excel for plotting: % lymphocyte+ % lymphocyte+/live/dead- % lymphocyte+/live/dead-/CD8+ % lymphocyte+/live/dead-/CD8+/CD69+ Note: It is recommended to only include data with at least 500 counts (#Cells) for analysis. Cytokine analysis Collect at least 50 µL of pod inlet and outlet media at 0, 24, 48, and 72 h post PBMC-TCB administration. Immediately freeze samples at -80 °C until measurement. Using a customized Invitrogen ProcartaPlex multiplex immunoassay, follow manufacturer instructions to assess levels of IFNγ, TNFα, Granzyme-B, IL-2, IL-4, and IL-8. Follow procedure outlined in Invitrogen documentation publication number MAN0017081 (Revision B.0 (33)). Acquire samples on Luminex BioPlex-200 system (Bio-Rad) using BioPlex Manager software. Plot the expected concentration of the standards against the mean fluorescent intensity generated by each standard and best curve fit and calculate the concentrations of the unknown samples (in pg/mL). Export the data in Excel and plot in GraphPad Prism. Notes This protocol has been validated specifically using the Emulate Organ-Chips with Zoë® Culture Module and Orb Hub Module. This cohesive system supports continuous fluid flow to two microfluidic channels while providing physiological stretch, which are crucial to the development of the Alveolus Lung-Chip and Colon Intestine-Chip. Using an alternative technology is not recommended as this would result in a different and unproven model. The recommended reagents in the above procedure specify the carcinoembryonic antigen (CEA) and folate receptor 1 (FLOR1) antigen targets for the colon and alveolar lung, respectively. The specification of these targets is based on the work described in the associated publication (Kerns et al., 2021); however, other relevant cellular targets and reagents can be considered and deployed using the approaches described in this protocol. PBMCs isolated from donor whole blood are the cells’ source used in the protocol above to assess T cell activation and killing of targeted epithelial cells. PBMCs are comprised of a variety of specialized immune cells; the largest fraction of which are lymphocytes that include T cells, B cells, and Natural Killer Cells. While other immune cell types do express the T-cell receptor (TCR), expression of the TCR is the defining characteristic of T cells and they are therefore the cell type most significantly impacted by the TCR targeting function of TCB antibodies. This is a logical conclusion that is supported by the T cell–specific responses that are observed upon the co-administration of TCBs and PBMCs. An acceptable variation to the protocol described here would be to perform steps to further purify the PBMC population and select for T cells specifically for administration in the experiment. The choice made in the current protocol version reflects the desire to capture more of the physiological context and variation that may be present in a practical clinical administration of TCBs to patients. The Zen software package was used extensively for the image analyses used in the TCB-mediated T cell activation and killing assessment described here. However, other software packages with similar functionality can be used to process and analyze images for an accurate assessment of T cell activation and killing. The Zoë Culture Module sustains cells within Emulate Organ-Chips by controlling the flow rate in each channel, air–liquid interfaces, and the frequency and strain of cyclic stretch, depending on experimental needs. It is recommended that filtered pipette tips are used throughout all steps of the protocol to help maintaining a sterile culture environment except where not appropriate (e.g., aspiration steps). The Alveolar and Colon Intestine-Chip protocols have different PBMC dosing media formulations despite having identical nomenclature. The PBMC dosing media formulation to be used is implied by the Organ-Model being utilized, either Alveolar-Lung or Colon Intestine-Chip, respectively. Recipes Part I: Alveolar-Chip PBMC culture media Prepare an appropriate volume of RPMI-1640 media with 10% v/v FBS and 1% v/v Pen-Strep. ER-1 solution Note: ER-1 is light-sensitive. Prepare ER-1 solution immediately before use and discard any remaining ER-1 solution 1 h after reconstitution. Failure to protect from light or use of ER-1 solution that has not been freshly prepared will lead to failure of chips. ER-1 is an eye irritant and must be handled in the BSC with proper gloves and eye protection. Turn off the light in BSC and allow the ER-1 and ER-2 reagents to equilibrate to room temperature before use (approximately 10–15 min). Use a 15 mL amber conical tube or wrap an empty sterile 15 mL conical tube with aluminum foil to protect it from light. Remove the small vial of ER-1 powder from the packet and briefly tap the vial to concentrate the powder at the bottom. Add 8 mL of ER-2 buffer to a covered 15 mL conical tube. Add 1 mL of ER-2 buffer to the ER-1 vial and transfer contents directly to the 15 mL conical tube. Note: The color of the solution transferred to the conical tube will be deep red. Add an additional 1 mL of ER-2 buffer to the ER-1 vial, cap the bottle, and invert to collect any remaining ER-1 powder from the lid. Transfer the collected solution to the conical tube, bringing the total volume in the tube to 10 mL ER-1 solution. The final working concentration of ER-1 should be 0.5 mg/mL. Pipette gently to mix without creating bubbles. ER-1 should be fully dissolved within the ER-2 solution prior to use. Alveolus ECM stock solutions Note: Aliquot ECM reagents prior to use to avoid multiple freeze-thaw cycles of the stock solutions. Dilute fibronectin in cell culture grade water to a final concentration of 1 mg/mL. Dilute collagen IV in cell culture grade water to a final concentration of 1 mg/mL. Dilute laminin in cell culture grade water to a final concentration of 1 mg/mL. Aliquot to each ECM to single-use volumes and store at -20 °C (collagen IV and fibronectin) and -80 °C (laminin). Alveolus ECM working solutions Note: The ECM solution is prepared fresh each time by combining the individual ECM components with cold DPBS to the final working concentrations. The ECM solution will be used to coat both the top and bottom channels. Calculate total volume of ECM solution needed to coat all chips, approximately 100 μL/chip. Thaw an appropriate quantity of fibronectin (1 mg/mL), collagen IV (1 mg/mL), and laminin (1 mg/mL) aliquots on ice. Always maintain all ECM components and mixture on ice. For the top channel of the Alveolus Lung-Chip, the final ECM working concentrations are: 200 μg/mL collagen IV, 30 μg/mL fibronectin, and 5 μg/mL laminin in DPBS. For the bottom channel of the Alveolus Lung-Chip, the final ECM working concentrations are: 200 μg/mL collagen IV and 30 μg/mL fibronectin in DPBS. Keep the ECM solution on ice until ready to use. HPAEC culture medium or complete SAGM culture medium Prepare 500 mL SAGM complete medium with SABM basal medium and SAGM SingleQuots supplement pack. Filter using 0.22 μm filters. Store at 4 °C. Use within 30 days of preparation. HPAEC maintenance media Prepare 500 mL SAGM complete medium with SABM basal medium and SAGM SingleQuots supplement pack. Add sterile, heat-inactivated FBS to a final concentration of 2%. Add dexamethasone to a final concentration of 100 nM. Add KGF to a final concentration of 5 ng/mL. Add 8-br-cAMP to a final concentration of 50 µM. Add IBMX to a final concentration of 25 µM. Filter using 0.22 μm filters. Store at 4 °C. Use within 15 days of preparation. HMVEC-L culture medium or complete EGM-2MV culture medium Prepare 500 mL of EGM2-MV complete medium with EBM-2 basal medium and EGM-2MV SingleQuots supplement pack. Filter using 0.22 μm filters. Store at 4 °C. Use within 30 days of preparation. ALI culture medium Prepare 500 mL of medium 199. Add EGF to a final concentration of 10 ng/mL. Add FGF to a final concentration of 3 ng/mL. Add VEGF to a final concentration of 125 pg/mL. Add hydrocortisone to a final concentration of 1 µg/mL. Note: Hydrocortisone is omitted from the ALI culture media in the presence of PBMCs. Add heparin to a final concentration of 10 µg/mL. Add di-butyryl to a final concentration of 80 µM. Add L-Glutamax to a final concentration of 1 mM. Add dexamethasone to a final concentration of 20 nM. Add Pen-Strep to a final concentration of 1%. Add FBS to a final concentration of 2%. Store at 4 °C. Use within 15 days of preparation. CMFDA cell tracker green Add 10.75 μL of DMSO to one 50 μg vial. PBMC dosing media Prepare 100 mL of medium 199 with a final concentration of 2% FBS. FACs buffer Prepare appropriate volume of DPBS with a final concentration of 2% FBS. 1% PFA solution To prepare 20 mL of 1% PFA solution, add 5 mL of 4% PFA solution to 15 mL of DPBS. Part II: Colon-Chip Y-27632 (ROCK inhibitor) Resuspend 5 mg of Y-27632 compound in 1.56 mL of 0.1% BSA in DPBS according to the manufacturer’s instructions, yielding a stock concentration of 10 mM. The final concentration of Y-27632 used in IntestiCult expansion media will be 10 μM. Aliquot reconstituted Y27632 to single-use volumes and store at -20 °C. CHIR99021 (GSK-3 inhibitor) Resuspend 10 mg of CHIR99021 compound in 4.29 mL of DMSO according to manufacturer’s instructions, yielding a stock concentration of 5 mM. The final concentration of CHIR99021 used in IntestiCult expansion media will be 5 μM. Aliquot to single-use volumes and store at -20 °C. Matrigel-growth factor reduced The stock bottle of matrigel must be thawed overnight on ice at 4 °C. After thawing, aliquot matrigel to suitable single-use volumes based on the specific stock concentration and amount needed in experiment. Always keep all materials on ice. Use pre-chilled pipettes, tips, and tubes at -20 °C prior to aliquoting. Freeze aliquots immediately at -20 °C. Thaw aliquots on ice just prior to use. Once aliquots are thawed, do not re-freeze. Note: Prepare aliquots of 1.4 mL for organoids expansion and 100 μL for chip ECM coating. Collagen IV Resuspend 5 mg of collagen IV in 5 mL of sterile cell culture grade water for a final concentration of 1 mg/mL and incubate at 4 °C until dissolved. Aliquot the next day 300 μL aliquots and store at -20 °C. Fibronectin Resuspend 5 mg of fibronectin in 5 mL of sterile cell culture grade water and leave the mix at room temperature for 30 min to dissolve (avoid harsh agitation or vortexing) for a final concentration of 1 mg/mL. Swirl gently before aliquoting. Store aliquots at -20 °C. 3KDa Dextran Cascade Blue Resuspend 10 mg of Dextran Cascade Blue 3000 MW in 1 mL of sterile water cell culture grade to obtain 3 KDa Dextran Cascade Blue working solution at 10 mg/mL concentration. The final concentration in medium is 10 μg/mL or 1:100 dilution. One vial of 10 mg of 3 KDa Dextran Cascade Blue is sufficient for 100 mL. Any remaining working solution can be frozen at -20 °C. PBMC culture media Prepare appropriate volume of RPMI-1640 media with 10% v/v FBS and 1% v/v Pen-Strep. ER-1 solution Note: ER-1 is light-sensitive. Prepare ER-1 solution immediately before use and discard any remaining ER-1 solution 1 h after reconstitution. Failure to protect from light or use of ER-1 solution that has not been freshly prepared will lead to failure of chips. ER-1 is an eye irritant and must be handled in the BSC with proper gloves and eye protection. Turn off the light in BSC and allow the ER-1 and ER-2 reagents to equilibrate to room temperature before use (approximately 10–15 min). Use a 15 mL amber conical tube or wrap an empty sterile 15 mL conical tube with aluminum foil to protect it from light. Remove the small vial of ER-1 powder from the packet and briefly tap the vial to concentrate the powder at the bottom. Add 8 mL of ER-2 buffer to a covered 15 mL conical tube. Add 1 mL of ER-2 buffer to the ER-1 vial and transfer contents directly to the 15 mL conical tube. Note: The color of the solution transferred to the conical tube will be deep red. Add an additional 1 mL of ER-2 buffer to the ER-1 vial, cap the bottle, and invert to collect any remaining ER-1 powder from the lid. Transfer the collected solution to the conical tube, bringing the total volume in the tube to 10 mL ER-1 solution. The final working concentration of ER-1 should be 0.5 mg/mL. Pipette gently to mix without creating bubbles. ER-1 should be fully dissolved within the ER-2 solution prior to use. Colon ECM stock solutions Note: Aliquot ECM reagents prior to use to avoid multiple freeze-thaw cycles of the stock solutions. Dilute fibronectin in cell culture grade water to a final concentration of 1 mg/mL. Dilute collagen IV in cell culture grade water to a final concentration of 1 mg/mL. Aliquot to each ECM to single-use volumes and store at -20 °C. Colon ECM working solutions Note: The ECM solution is prepared fresh each time by combining the individual ECM components with cold DPBS to the final working concentrations. The ECM solution will be used to coat both the top and bottom channels. Calculate the total volume of ECM solution needed to coat all chips, approximately 100 μL/chip. Thaw appropriate quantity of fibronectin (1 mg/mL), collagen IV (1 mg/mL), and matrigel aliquots on ice. Always maintain all ECM components and mixture on ice. For the top channel of the Colon-Chip, the final ECM working concentrations are: 200 μg/mL collagen IV and 100 μg/mL matrigel in DPBS. For the bottom channel of the Colon-Chip, the final ECM working concentrations are: 200 μg/mL collagen IV and 30 μg/mL fibronectin in DPBS. Keep the ECM solution on ice until ready to use. IntestiCult expansion media Combine IntestiCultTM OGM human component A with IntestiCultTM OGM human component B. Supplement with: Y-27632 at final concentration of 10 µM CHIR99021 at a final concentration of 5 µM Primocin at a final concentration of 100 µg/mL Store at 4 °C. Use within seven days of preparation. IntestiCult maintenance media Combine IntestiCultTM OGM human component A with IntestiCultTM OGM human component B. Supplement with: Primocin at a final concentration of 100 µg/mL Store at 4 °C. Use within seven days of preparation. Cell counting solution 20 µL of trypan blue 20 µL of cells suspension Maintain counting solution at room temperature. Prepare in Eppendorf tube fresh for each use. HIMEC culture medium In the EGM-2MV SingleQuots supplement pack, replace: The supplied FCS with FBS The supplied antibiotic with primocin at a final concentration of 50 µg/mL Prepare 500 mL of EGM2-MV complete medium with EBM-2 basal medium and EGM-2MV SingleQuots supplement pack with the replacements above. Note from Lonza: Once thawed, SingleQuotsTM kit should be stored at 2–8 °C and added to basal medium within 72 h. Filter using 0.4 μm Steriflip filters. Store at 4 °C. Use within 30 days of preparation. CMFDA cell tracker green Add 10.75 μL of DMSO to one 50 μg vial. PBMC dosing media Prepare 100 mL of medium 199 with a final concentration of 2% FBS. FACs buffer Prepare appropriate volume of DPBS with a final concentration of 2% FBS. Colonoid Thawing Media Prepare 500 mL Advanced DMEM supplemented with 1% v/v Pen-Strep. Colonoid Dissociation Solution Prepare 2 mL of Dissociation Solution for each 24-well plate of Colonoids being passaged by mixing a 1:1 v/v solution of TrypLE with DPBS supplemented with Y-27632 at a final concentration of 10 µM. Live/dead fixable yellow dead stain Add 50 µL DMSO to vial to prepare working solution. Dilute working solution 1:1,000 in DPBS to produce 2 mM live/dead solution. Acknowledgments We thank Donald Ingber for useful scientific discussions; Antonio Varone, Ionnis Moriannis, David Conegliano, and Lian Leng for their contributions to developing the Alveolus Lung-Chip model; Magdalena Kasendra, Raymond Luc, and Athanasia Apostolou for their contributions to developing the Colon Intestine-Chip model; Robin Friedman, Alicia J Stark, Abhishek Shukla, and José Fernandez-Alcon for their contributions to image analysis; and Gurpreet Brar for her contributions to flow cytometry analysis. This protocol is derived from the original research paper (Kerns et al., 2021). Competing interests The authors declare the following competing interests: S. Jordan Kerns is a former employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Is a named inventor on patent application number: 20210003559, published Jan 7, 2021, covering the methods for assessing a compound interacting with a target on epithelial cells. Chaitra Belgur is a former employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Is a named inventor on patent application number: 20210003559, published Jan 7, 2021, covering the methods for assessing a compound interacting with a target on epithelial cells. Debora B. Petropolis is a former employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Is a named inventor on patent application number: 20210003559, published Jan 7, 2021, covering the methods for assessing a compound interacting with a target on epithelial cells. Riccardo Barrile is a former employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Is a named inventor on patent application number: 20210003559, published Jan 7, 2021, covering the methods for assessing a compound interacting with a target on epithelial cells. Marianne Kanellias is a current employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Dimitris V. Manatakis is a current employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Will-Tien Street is a is a former employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Lorna Ewart is a current employee of Emulate, Inc and holds equity interests or options to obtain equity interests in Emulate Inc. Nikolche Gjorevski is a named inventor on patent application number: 20210003559, published Jan 7, 2021, covering the methods for assessing a compound interacting with a target on epithelial cells. Lauriane Cabon is a named inventor on patent application number: 20210003559, published Jan 7, 2021, covering the methods for assessing a compound interacting with a target on epithelial cells. Ethics We the authors certify the quality and integrity of your research; will respect the confidentiality and anonymity of your research respondents and can show that your research is independent and impartial. References Bacac, M., Fauti, T., Sam, J., Colombetti, S., Weinzierl, T., Ouaret, D., Bodmer, W., Lehmann, S., Hofer, T., Hosse, R. J., et al. (2016). A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors. Clin Cancer Res 22(13): 3286-3297. Bjornson-Hooper, Z. B., Fragiadakis, G. K., Spitzer, M. H., Madhireddy, D., McIlwain, D., and Nolan, G. P. (2019). A comprehensive atlas of immunological differences between humans, mice and non-human primates. BioRxiv, 574160. Brischwein, K., Schlereth, B., Guller, B., Steiger, C., Wolf, A., Lutterbuese, R., Offner, S., Locher, M., Urbig, T., Raum, T., et al. (2006). MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Mol Immunol 43(8): 1129-1143. Champiat, S., Dercle, L., Ammari, S., Massard, C., Hollebecque, A., Postel-Vinay, S., Chaput, N., Eggermont, A., Marabelle, A., Soria, J. C., et al. (2017). Hyperprogressive Disease Is a New Pattern of Progression in Cancer Patients Treated by Anti-PD-1/PD-L1. Clin Cancer Res 23(8): 1920-1928. Clynes, R. A. and Desjarlais, J. R. (2019). Redirected T Cell Cytotoxicity in Cancer Therapy. Annu Rev Med 70: 437-450. Gayer, C. P. and Basson, M. D. (2009). The effects of mechanical forces on intestinal physiology and pathology. Cell Signal 21(8): 1237-1244. Goebeler, M. E. and Bargou, R. C. (2020). T cell-engaging therapies - BiTEs and beyond. Nat Rev Clin Oncol 17(7): 418-434. Gong, J., Chehrazi-Raffle, A., Reddi, S. and Salgia, R. (2018). Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. J Immunother Cancer 6(1): 8. Hegde, P. S. and Chen, D. S. (2020). Top 10 Challenges in Cancer Immunotherapy. Immunity 52(1): 17-35. Hodi, F. S., O'Day, S. J., McDermott, D. F., Weber, R. W., Sosman, J. A., Haanen, J. B., Gonzalez, R., Robert, C., Schadendorf, D., Hassel, J. C., et al. (2010). Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363(8): 711-723. Huh, D., Matthews, B. D., Mammoto, A., Montoya-Zavala, M., Hsin, H. Y. and Ingber, D. E. (2010). Reconstituting organ-level lung functions on a chip. Science 328(5986): 1662-1668. Ishiguro, T., Ohata, H., Sato, A., Yamawaki, K., Enomoto, T. and Okamoto, K. (2017). Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications. Cancer Sci 108(3): 283-289. Kasendra, M., Luc, R., Yin, J., Manatakis, D. V., Kulkarni, G., Lucchesi, C., Sliz, J., Apostolou, A., Sunuwar, L., Obrigewitch, J., et al. (2020). Duodenum Intestine-Chip for preclinical drug assessment in a human relevant model. Elife 9: e50135. Kasendra, M., Tovaglieri, A., Sontheimer-Phelps, A., Jalili-Firoozinezhad, S., Bein, A., Chalkiadaki, A., Scholl, W., Zhang, C., Rickner, H., Richmond, C. A., et al. (2018). Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids. Sci Rep 8(1): 2871. Kebenko, M., Goebeler, M. E., Wolf, M., Hasenburg, A., Seggewiss-Bernhardt, R., Ritter, B., Rautenberg, B., Atanackovic, D., Kratzer, A., Rottman, J. B., et al. (2018). A multicenter phase 1 study of solitomab (MT110, AMG 110), a bispecific EpCAM/CD3 T-cell engager (BiTE(R)) antibody construct, in patients with refractory solid tumors. Oncoimmunology 7(8): e1450710. Kennedy, L. B. and Salama, A. K. S. (2020). A review of cancer immunotherapy toxicity. CA Cancer J Clin 70(2): 86-104. Kerns, S. J., Belgur, C., Petropolis, D., Kanellias, M., Barrile, R., Sam, J., Weinzierl, T., Fauti, T., Freimoser-Grundschober, A., Eckmann, J., et al. (2021). Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies. Elife 10: e67106. Klinger, M., Benjamin, J., Kischel, R., Stienen, S. and Zugmaier, G. (2016). Harnessing T cells to fight cancer with BiTE(R) antibody constructs--past developments and future directions. Immunol Rev 270(1): 193-208. Labrijn, A. F., Janmaat, M. L., Reichert, J. M. and Parren, P. (2019). Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18(8): 585-608. Lutterbuese, R., Raum, T., Kischel, R., Hoffmann, P., Mangold, S., Rattel, B., Friedrich, M., Thomas, O., Lorenczewski, G., Rau, D., et al. (2010). T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer cells. Proc Natl Acad Sci U S A 107(28): 12605-12610. Milling, L., Zhang, Y. and Irvine, D. J. (2017). Delivering safer immunotherapies for cancer. Adv Drug Deliv Rev 114: 79-101. Naidoo, J., Page, D. B., Li, B. T., Connell, L. C., Schindler, K., Lacouture, M. E., Postow, M. A. and Wolchok, J. D. (2015). Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies. Ann Oncol 26(12): 2375-2391. Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., Van Es, J. H., Van den Brink, S., Van Houdt, W. J., Pronk, A., Van Gorp, J., Siersema, P. D., et al. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141(5): 1762-1772. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld. S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-82. Schadendorf, D., Hodi, F. S., Robert, C., Weber, J. S., Margolin, K., Hamid, O., Patt, D., Chen, T. T., Berman, D. M. and Wolchok, J. D. (2015). Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma. J Clin Oncol 33(17): 1889-1894. Sontheimer-Phelps, A., Chou, D. B., Tovaglieri, A., Ferrante, T. C., Duckworth, T., Fadel, C., Frismantas, V., Sutherland, A. D., Jalili-Firoozinezhad, S., Kasendra, M., et al. (2020). Human Colon-on-a-Chip Enables Continuous In Vitro Analysis of Colon Mucus Layer Accumulation and Physiology. Cell Mol Gastroenterol Hepatol 9(3): 507-526. Trabolsi, A., Arumov, A. and Schatz, J. H. (2019). T Cell-Activating Bispecific Antibodies in Cancer Therapy. J Immunol 203(3): 585-592. Waldman, A. D., Fritz, J. M. and Lenardo, M. J. (2020). A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol 20(11): 651-668. Wolchok, J. D., Chiarion-Sileni, V., Gonzalez, R., Rutkowski, P., Grob, J. J., Cowey, C. L., Lao, C. D., Wagstaff, J., Schadendorf, D., Ferrucci, P. F., et al. (2017). Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med 377(14): 1345-1356. Yang, Y. (2015). Cancer immunotherapy: harnessing the immune system to battle cancer. J Clin Invest 125(9): 3335-3337. Article Information Copyright Kerns et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Immunology > Immune mechanisms > Ex vivo model Biological Engineering > Biomedical engineering Cell Biology > Cell-based analysis > Extracellular microenvironment Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood AKM Ashiqul Haque [...] Justin S Antony Dec 20, 2020 3755 Views Electrophysiological Measurements of Isolated Blood Vessels Samuel A Molina [...] Michael Koval Mar 20, 2022 1318 Views In Vitro Assay to Examine Osteoclast Resorptive Activity Under Estrogen Withdrawal Cara Fiorino [...] Rene E. Harrison Jan 5, 2025 218 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,580	https://bio-protocol.org/en/bpdetail?id=4580&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Quantitative Analysis of Gene Expression in RNAscope-processed Brain Tissue MS Maria E. Secci TR Tanner Reed VQ Virginia Quinlan NG Nicholas W. Gilpin EA Elizabeth M. Avegno Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4580 Views: 1425 Reviewed by: Arnau Busquets-GarciaFanny Ehret Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Addiction Biology Jul 2021 Abstract Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope. Graphical abstract Keywords: RNAscope Image analysis Brain Analysis threshold Automatic quantification QuPath Slide scanning microscope Background RNAscope is a powerful technology that allows for the identification of thousands of RNA targets visualized as puncta, each representing a single RNA transcript, in multiple types of tissues. Use of this assay has steadily increased since its introduction, allowing researchers across biological disciplines to perform rapid and precise quantification of RNA transcripts using a highly sensitive assay. In the neuroscience field, RNAscope is becoming an essential tool. Despite several publications in the field utilizing this technique, there is no standard methodology for processing brain tissue and quantifying RNA transcripts and transcript-positive cells using RNAscope. Many publications provide limited information regarding the calculation of quantification thresholds, preventing replication of prior analyses. Here, we outline a standardized protocol that can be used to establish criteria for quantifying cells that are positive for targets of interest using RNAscope, hopefully allowing for consensus across studies and improving reproducibility across the field. There are many ways in which RNAscope assays and analyses can be conducted; the purpose of this protocol is not to define a correct way, but rather to provide one potential methodology that can be used going forward and, perhaps more importantly, to start a conversation regarding unification and standardization of this approach across neuroscience labs. Manual quantification of labeled cells is a tedious and time-consuming process. Many available tools used for image analysis (e.g., ImageJ) are often not suitable for analysis of large image files produced in RNAscope studies, and some open-source tools (e.g., dotdotdot or Cell Profiler) require advanced computational skills to run the workflow, thereby limiting their use. Here, we explain how to analyze RNAscope signal in rat neurons using the open-source bioimage quantification software QuPath (Bankhead et al., 2017). This software includes several features such as 1) annotation and visualization tools using a JavaFX interface, 2) built-in algorithms for common tasks, such as cell detection, and 3) interactive machine learning. Our method can be utilized for whole brain quantitative analysis or for analysis in selected brain regions as we performed in Avegno et al. (2021). Although we describe all the steps to generate RNAscope tissue from fresh frozen brains, the same method can be applied to fixed brain preparations with some modifications. Here, we present the optimization of just one specific parameter important for automated cell detection, the fluorescence intensity threshold. However, we provide the framework for adding optimization steps for additional cell detection parameters, if desired. Collectively, the method presented here offers a user-friendly and cost-effective framework for automated quantification of transcript-positive cells in whole tissue sections, without the need for manual counting. Materials and Reagents Tissue Preparation Rodent brains (we used n = 6 adult brains from female Wistar rats purchased from Charles River, catalog number: 003) Isoflurane, USP, or any other anesthetic (Piramal Critical Care, catalog number: 6679401710; or any other brand) 250 mL glass beaker (Fisherbrand, catalog number: FB101250; or any brand) Aluminum foil (Reynold Wraps, catalog number: 458742928317; or any brand) Ultralow temperature digital thermometer with stainless-steel probe (Fisherbrand, catalog number: 15-077-32) 2-methylbutane (isopentane) ReagentPlus® ≥99% (Sigma-Aldrich, catalog number: M32631; or any brand) Dry ice pellets Cryostat (Thermo Scientific, USA, Cryostar NX50; or any brand capable of producing 10 µm slices) Fisherbrand Superfrost Plus microscope slides (Fisher Scientific, catalog number: 12-550-15) Tissue-Tek O.C.T. compound (Sakura, catalog number: 4583) Tissue-Tek manual slide staining set (Sakura, catalog number: 4451) Paraformaldehyde 32% aqueous solution (Electron Microscopy Sciences, catalog number: 15714S) Distilled water Fisherbrand Superslip coverslips (Fisher Scientific, catalog number: 12-545-89P) Fluoro-Gel II with DAPI (Electron Microscopy Sciences, catalog number: 17985-50) RNAscope Preparation RNAscope® Fluorescent Multiplex reagent kit v1 (Advanced Cell Diagnostics, catalog number: 320850) for fresh frozen applications. Note that for fixed brain preparations, additional products are needed: RNAscope Target Retrieval and RNAscope Protease III (available in the RNAscope Universal Pretreatment kit, Advanced Cell Diagnostics, catalog number: 322380) RNAscope® RTU Protease IV reagent (Advanced Cell Diagnostics, catalog number: 322340) RNAscope target probes (catalog number varies depending on the need; we used Rn-Hcrtr1-C1, Rn-Th-C2, and Rn-Fos-C3). Note that in order to independently detect mRNA transcripts in a multiplex assay, each target probe must be in a different channel (C1, C2, or C3) and one of the target probes must be in the C1 channel. Channel C1 target probes are ready-to-use (RTU), while channel C2 and C3 probes are shipped as a 50× concentrated stock. The 50× probes for C2 or C3 must be mixed with a C1 RTU probe. If no specific C1 probe is used, then a blank probe diluent (assay dependent) is used to dilute the probes. Assignation of channels can be changed upon researcher request. RTU probe diluent (optional, see above; Advanced Cell Diagnostics, USA, catalog number: 300041) RNAscope 3-plex negative control probes (Advanced Cell Diagnostics, catalog number: 320871) Immedge® hydrophobic barrier pen (Vector Labs, catalog number: 310018) RNase AWAY (Thermo Scientific, catalog number: 14-375-35) or any other decontaminant (e.g., bleach) Notes: The items listed below are intended for fresh frozen tissue collection only. If researchers plan to use formalin-fixed tissue, there will be additional steps to be followed (refer to manufacturer manuals for more details). RNAscope kits for fresh frozen and fixed tissues are slightly different, as the fixed tissue follows a different preparation and pretreatment. Target probes listed here are merely indicative. Any type of marker can be utilized for this purpose. Customized probes (from available genome databases) can be made upon request at additional costs. Equipment Brain extraction tools: Straight scissors (Roboz, catalog number: RS-6806), rongeurs (Roboz, catalog number: RS-8300), rounded spatula (Millipore Sigma, catalog number: HS15906), small straight or curved forceps (Roboz, catalog number: RS-5136), and long forceps (Fisherbrand, catalog number: 13-820-077; see Figure 1) Slide scanner (Carl Zeiss, AxioScan Z.1, Germany; or any other microscope capable of scanning and digitizing fluorescent slides) Computer: 64-bit Windows or Linux with minimum 16 GB RAM and a fast multicore processor (e.g., Intel Core i7 or i9) HybEZ II system hybridization oven [Advanced Cell Diagnostics, USA; see Figure 2. The system comprises: HybEZ oven (PN 321710/321720), a humidity control tray (PN 310012), and HybEZ humidifying paper (2 sheets, PN 310025), EZ-Batch wash tray (PN 321717), and EZ-Batch slide holder (PN 321716)] Note: This protocol will prospectively work with a less powerful computer or with lower RAM, but analysis will be slow and may encounter memory errors. Figure 1. Brain extraction tools. Straight scissors, rongeurs, rounded spatula, and curved and long forceps indicated. Figure 2. HybEZ II system. Hybridization oven, humidity control tray, humidifying paper, slide staining/washing set (EZ-Batch wash tray and EZ-Batch slide holder), and hydrophobic pen indicated. Software Zeiss ZEN blue v2.6 or higher for images acquisition and export (https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html#downloads) QuPath 0.3.2 open-source software (https://qupath.github.io/) Microsoft Excel or similar GraphPad Prism 9.4.1 (https://www.graphpad.com/) or similar Procedure Brain collection (fresh frozen) and storage In RNase-free conditions (achieved by cleaning the area with RNase AWAY or any other decontaminant such as low concentration bleach), prepare a beaker containing 300 mL of 2-methylbutane, which will sit on dry ice pellets in a stereo foam container under a chemical hood. The temperature of the 2-methylbutane is monitored with an ultralow temperature thermometer. Allow 30–45 min for the 2-methylbutane to reach the temperature of -30 °C, which is optimal to protect the brains from cracking during cryostat slicing and the mRNA from degradation (see Note 1). Deeply anesthetize the rat with isoflurane (or any other anesthetic) and perform sacrifice by decapitation. Quickly remove the brain from the skull, removing bone and membrane residues that can potentially damage the tissue. Immediately drop the brain in chilled 2-methylbutane (-30 °C) and snap-freeze for 25 s (see Note 2). Grab snap-frozen brain with long tweezers and immediately wrap it in aluminum foil. Avoid any thawing of the tissue and immediately store brains on either dry ice or at -80 °C (see Note 3). Store brains at -80 °C for up to 12 months. It is not recommended to use brains stored for longer periods, as the mRNA degrades progressively. Notes: If researchers intend to fix the brains using formalin, a different protocol including transcardial perfusion must be followed. Brains perfused in formalin should be post-fixed with formalin overnight, transferred to 30% sucrose to cryoprotect the tissue, and snap-frozen in 2-methylbutane for long-term storage. Cracks in the tissue will make slicing the samples unmanageable for subsequent assay. Freezing the brains in liquid nitrogen (ultralow temperature) will also create cracks, making the tissue unusable. This time is critical to prevent splitting of the brain’s hemispheres and mRNA degradation. This procedure allows an even and quick freezing of brain tissue and preserves morphology and 100% of the targeted mRNA, especially if the target is low-to-moderately expressed. Avoid any contact with tools that have not been previously decontaminated. Preparation of fresh-frozen brain sections Move brains from -80 °C to -20 °C prior to slicing. Depending on the laboratory conditions, warming the tissue will take a shorter or longer period of time (30 min to a few hours). Transfer brains from -20 °C to the cryostat’s chamber, whose temperature was previously optimized (-13 °C). Leave brains inside the cryostat for 2–3 h before starting the slicing. The optimal cryostat temperature for brain slicing depends on the device’s model (user manuals typically provide this information). The above equilibration time is sufficient to allow slicing without cracks. If brain is not ready, increase equilibration time. It usually takes longer when the brain is snap-frozen at temperatures lower than -40 or -50 °C. Decontaminate the external cryostat area in which SuperFrost® Plus slides will be positioned before mounting. It is recommended to leave slides outside the cryostat to avoid them being too cold and prevent tissue adhesion after slicing (this step very much depends on the environment and cryostat). Add 2–3 drops of O.C.T. compound in the specimen stage and immediately place the frozen brain at the right orientation and angle to provide an accurate slicing. Make sure the tissue is perpendicular to the stage to avoid uneven sections. Cut 10 µm coronal slices from the desired region of interest [here, we collected sections containing the substantia nigra (SN)] and immediately mount them by adhesion onto the SuperFrost® Plus slides. Each slide can contain up to three (depending on the orientation) rat brain sections. Overcrowding the glass slide with tissue sections will obstruct the area necessary to draw the hydrophobic barrier for the subsequent RNAscope assay. Keep the sections inside the cryostat to dry and store them in an air-tight slide box wrapped with aluminum foil at -80 °C. Use sectioned tissue within three months. Sectioned tissue is more prone to faster mRNA degradation. RNAscope assay Multiplex assays are performed according to the manufacturer protocols (we outline specific details below). All incubations are performed at 40 °C using a humidity control chamber (HybEZ oven, Advanced Cell Diagnostics). The steps listed below are similar for mRNA target probes and negative controls, unless noted otherwise. It is recommended to run negative controls (one or two sections are sufficient) within each batch of tissue. Target probes were used as follows: RNAscope 3-plex negative control probe set (320871): bacterial DapB (in C1, C2, and C3 probe sets) RNAscope probe set: Rn-Hcrtr1-C1, Rn-Th-C2, and Rn-Fos-C3. Note that probe channels and fluorophores should be selected considering the expression levels of desired targets. For example, a higher expression gene may be chosen for a wavelength with high autofluorescence in your sample (e.g., 488/green). Below, we describe the most representative steps of this procedure. Slides are immersed in slide staining/washing set for each step, unless noted otherwise. Fix the sections: remove sections that were previously stored at -80 °C and immediately immerse them in pre-chilled fixative [4% paraformaldehyde (prepared by diluting a 32% stock solution of paraformaldehyde into 1× PBS) at 4 °C] for 40 min. This step must be performed in a fume hood given the toxicity of paraformaldehyde. Dehydrate the sections: place sections in 50% (1×), 70% (1×), and 100% (2×) ethanol for 5 min each at room temperature. Create a hydrophobic barrier around each brain section and let it dry for 1 min at room temperature. Prepare materials necessary to run the assay (wash buffer, target probes, reagents, and hybridization oven). Pretreat samples by applying protease IV (approximately five drops directly from the bottle; no dilution needed) for 30 min at room temperature inside the humidifying tray and close lid. Add desired probe to each section and incubate for 2 h at 40 °C inside the humidifying tray (this temperature is the same for all type of probes, including the negative controls). Importantly, apply the negative control probe to one section every time you run an assay. Hybridize probe(s) using Amp FL-1, -2, -3, and -4 (we used Amp FL-4-Alt A) for 15 or 30 min (see manufacturer’s instructions) at 40 °C. Each amplification step occurs inside the humidifying tray. After each amplification step, the Amp reagents are removed by washing the sections with wash buffer through slide solution wells. Counterstain using Fluoro-Gel II with DAPI and cover the sections with coverslips. Make sure the sections are almost completely dry before applying the mounting media to avoid creation of bubbles that can prevent target identification and thus quantification. Store slides in the dark at 4 °C until image evaluation (optimal time is up to one week). Note: It is possible to combine the RNAscope with IHC techniques, allowing the researcher to further clarify cell identity. In this particular combination, only formalin-fixed brain tissue can be used, and IHC will be done as a second step. Advanced Cell Diagnostics provides technical notes for this purpose that can be found here: https://acdbio.com/system/files_force/gated/TN_323100_TechNote_Dual_ISH_IHC_manual_Multiplex_Fluorescent_V2_06222017.pdf.pdf?download=1. For pure RNAscope applications that do not require further cellular characterization, it is recommended to use fresh frozen tissue isolations, as explained in this protocol. Advantages include: 1) less supplies needed, which is a cost-saving factor; 2) fewer steps needed to achieve the same result; 3) better tissue quality that potentially translates to high-quality mRNA targeting, which will simplify the quantification process. In formalin-fixed tissue, it is necessary to add a target retrieval step (performed at high temperatures), which can cause tissue damage and prevent a successful probe targeting. Disadvantages of using fresh frozen tissue isolations include an inability to combine IHC assays that require formalin-fixed tissues (see beginning of this note for more details). Imaging Turn on scanner and computer. Start ZEN v2.6 software. Load slides in tray and insert inside the scanner. Inspect the slide magazine for accuracy. Click on wizard to start scan setup. Select region of interest (ROI)—in this protocol, we analyzed the SN. When selecting ROI, do not include solely the desired section, but expand to include part of the adjacent areas as well. In brightfield view, inspect contiguous brain areas to make sure you are in the correct ROI. In the fluorescent view, select desired channels based on the RNAscope assay. We have DAPI (blue), GFP (green), Cy3 (red), and Cy5 (far red). For particle counts, exposure times were calculated to allow clear detection of small foci in all channels. These are optimal settings for our scanner (see Note 1): DAPI: 6–8 ms EGFP: 1,400–1,800 ms Cy3: 600–800 ms Cy5: 1,000–1,400 ms Focus each channel and set parameters for z-stacking. Z-stacking: RNAscope sections were originally at 10 µm thickness; however, sections lose a few micrometers from the original thickness post processing. We do not need to set up the z-stacking depth to 10 µm, as that depth is not accurate anymore. In order to achieve a valuable number of slices that contain high-resolution transcripts, we set the z-stacking as follows: Depth: 5.20 µm Interval between slices: 0.85 µm Number of slices: 7 Start scan—a few to several hours will be necessary to scan all the sections, depending on the selected ROI. Save files for subsequent analysis in QuPath (see Note 2). Notes: These are the steps we followed to image our sections using a Zeiss Axioscan Z.1 within the ZEN software environment. Any high-resolution fluorescent or confocal microscope capable of producing z-stack slices can be utilized to achieve similar results. For all analyses, images were captured via 20× objectives that readily enabled identification of discrete foci corresponding to individual transcripts, while retaining sufficient axial resolution to allow all elements of the tissue section to remain well focused. Although we used these specific parameters for capturing a small brain region such as the SN, the same settings can be used for larger regions, obtaining similar results. Limitations of imaging larger regions are related to longer scanning sessions and increased number of focal points to be selected. It is recommended to not vary settings in a single batch of slides. This will assist in maintaining consistency through the automatic quantification, minimizing variability in the subcellular thresholds, and identifying potential loss of mRNA signal in sections that are older. The ZEN software automatically saves the scanned images in the .czi format, which includes several types of information, thus creating large files that require specific data management. Most importantly for the next step, CZI files can be opened in QuPath without the need to merge or process the images with other software (especially if ROIs are selected). QuPath will create as many files as the number of scenes scanned in ZEN. If larger ROI is selected and if a unique focal point is missing, it might be necessary to merge the z-stack slices (with maximum projection) to reach the desired focus in each image tile. RNAscope subcellular quantification using QuPath (user manual: https://qupath.readthedocs.io/en/stable/) Open QuPath-0.3.2. Under the Project tab on the left of the screen, click Create project or Open project to retrieve previous projects (Figure 3). Notes: While searching for files, you may have to change file type from “QuPath Files” to “All Files” to access. If creating a new project, name and save your file, then import images by choosing File -> Project -> Add images -> Choose files -> Import (you may have to expand the window to full screen to see all options). Figure 3. Screen capture of QuPath. Path to create or open project indicated. Double-click the image you want to analyze from the image list. Under the View tab select Brightness/Contrast . Check the DAPI, EGFP, Cy3, and Cy5 channels (see Figure 4). Zoom in and out using the scroller on your mouse. Zoom into an area showing a decent amount of activity from all DAPI, Cy3, and Cy5 channels. Use the adjustable scrolling tool at the top left of the image (z-slice selector) to change the layer of the region you are analyzing. It is imperative to get the clearest image possible to give the most accurate count when analyzing cells and subcellular components. Figure 4. Screen capture of QuPath. Path to adjust brightness and contrast indicated. Once best clarity is achieved, go back to View and Brightness/Contrast and use only the EGFP or Cy5 channels to best view anatomical structures on the slide. Select one of the shapes on the top ribbon and drag to select your ROI (see Figure 5). Double-click outside of your region to have the outline turn white and the region locked. You can also use the minimum and maximum display scales to achieve best brightness of your data. It is best to have similar settings across all channels when analyzing images. Figure 5. Screen capture of QuPath. Shapes on the top ribbon are used to outline the ROI. Click the Analyze tab, then Cell Detection and Cell Detection again (see Figure 6). Change parameters to match the list below and then click Run . Background radius: 10 µm (if background subtraction is considered, 0 means no background subtraction). Median filter radius: 2.0 µm (reduce image texture/smooth intensity vibrations). Sigma: 2.0 µm [control/reduce the noise effect; with a smaller number you will get more nucleus (more fragments)]. Maximum area: 300 µm2 (define the range of nucleus size). Threshold: 30 (intensity parameter for nucleus objects). Figure 6. Screen capture of QuPath. Parameters used for cellular detection are indicated. After analyzing for Cell Detection , you will need to zoom in and inspect the quality of the segmentation. Some cells may have been grouped together and only counted as one, and other cells may have been segmented to count as multiple cells (see Figure 7). You can alter the median filter radius, sigma, and threshold and re-run the cell detection until best segmentation is achieved (see Figure 8). Poor segmentation can indicate that you need to alter the parameters of your run, or that you need to achieve better clarity of your ROI. Figure 7. Representative QuPath image with poor (left) and good (right) nuclear segmentation Figure 8. Representative probe expression and nuclear/subcellular segmentation in QuPath. Top left panel shows representative expression of DAPI (blue), Hcrtr1 (green), Th (red), and Fos (white) in the substantia nigra. Individual probe expression shown alone (left) or with DAPI (middle). Right panel shows the cellular and sub-cellular identification through QuPath for each channel. Scale bar = 20 µm. Click Analyze , then Cell Detection , and then Subcellular Detection (see Figure 9). Parameters should be set as below; then, click Run . Note: Threshold for DAPI should be set to 0; other thresholds shown here depend on the dot’s intensity for each channel. Channel 2 threshold: 600 Channel 3 threshold: 600 Channel 4 threshold: 600 Check all three boxes that say “smooth before detection,” “split by intensity,” and “split by shape.” Expected spot size: 0.5 µm2 Min spot size: 0.5 µm2 Max spot size: 15 µm2 Figure 9. Screen capture of QuPath. Parameters used for subcellular detection are indicated. After analyzing for subcellular detection, inspect again for quality and accuracy of data collection. Alter parameters of maximum spot size and threshold to increase quality (see Figure 9). You can return to View and Brightness/Contrast and double-click on a channel to change its color, as sometimes the subcellular components and the circle identifying them after analysis are the same color. Once best quality of data is collected, go to Measure and Show Detection Measurements (see Figure 10). Choose Copy to Clipboard and open Excel to paste all the data. The only other piece of data to collect from inside QuPath is the number of cells. You can access this by clicking Annotations on the left side of the screen; at the top-left you will see Annotation (Ellipse) (e.g., 1006/3650 objects) . The first of the two numbers is the number of cells identified in QuPath. You can double-check this by viewing how many nuclei area you have in the Excel data Figure 10. Screen capture of QuPath. Pathway to detection measurements indicated. In Excel (Figure 11), the only columns you need to keep for data collection are: Nucleus: Area Subcellular channel 2: Number of single spots Subcellular channel 3: Number of single spots Subcellular channel 4: Number of single spots Find average nucleus area by selecting the letter of the column in which the nuclei area is listed; under Sort and Filter select Sort Smallest to Largest . Insert the average function in an empty cell, select all the cells containing nucleus areas, and then click enter. Again, sort smallest to largest on the subcellular channel 2 column and count the number of single spots above your set threshold, including the threshold (i.e., if my threshold is 5, count all the subcellular single spots showing 5 or more spots). Repeat for subcellular channels 3 and 4. Figure 11. Screen capture of Excel. Subcellular detections are sorted for channel 2, and those cells above the threshold are highlighted in yellow. Once all the above parameters have been optimized, it is possible to expedite the analysis workflow using customized automatisms (see Figure 12). We can create scripts deriving from the steps shown above or create customized scripts, if additional parameters or extensions will be used (e.g., Stardist—deep learning models). For simplicity, we describe the steps to generate an automatism from an existing workflow. Go to Automate -> Show Workflow Command History -> Select desired steps -> Create script -> Save script. Open generated script and inspect all the lines for accuracy. Modify channel and analysis parameters according to optimized workflow. Save script and run it for any other selected ROI. Automated analysis saves minutes of work allowing to complete tasks in fractions of time. Figure 12. Example of script used in QuPath to run automated analysis Data analysis Each time you run an RNAscope assay it is critical to determine an analysis threshold, which will serve as the criteria for inclusion or exclusion of data in processed images. Punctate expression should be quantified in each channel of negative control probe-processed tissue, as described above (see Figure 13 for representative images). Calculate the average and standard deviation of puncta in each channel. The sum of these values is your threshold for all processed images. Note: Some cells will have zero puncta in a given channel; when calculating the average punctate expression in negative control probe-processed images, be sure to only include values >0; inclusion of 0 values will artificially lower your threshold and influence your data (see example in Table 1 and Figure 14). The threshold will be unique for each channel (e.g., Table 1). Figure 13. Representative negative control probe-processed tissue images. Non-specific signal in green–POLR2A (A), red–PPIB (B), white–UBC (C), and merged (D) channels shown. Images shown with increased brightness due to low fluorescent signal. Scale bar = 20 µm. Table 1. Example threshold calculations for each channel from negative control probe-processed tissue images. Threshold is determined by adding the average and standard deviation of the number of puncta per channel. Including zero values artificially lowers this threshold. Channel 2 (green) Channel 3 (red) Channel 4 (far red) Average number of puncta (including zero values) 0.76 1.90 0.43 Standard deviation 1.48 2.92 1.16 Threshold (including zero values) 2.24 4.82 1.59 Average number of puncta (excluding zero values) 2.17 3.43 2.10 Standard deviation 1.48 2.91 1.16 Threshold (excluding zero values) 3.65 6.34 3.26 Figure 14. Influence of threshold on cell counts using RNAscope-processed tissue. Figure shows the total number of neurons determined to be positive for expression of Hcrtr1 (green bars), Th (red bars), or Fos (purple bars) in regions analyzed within the substantia nigra from six rats. Using a lower threshold calculated by inclusion of zero values in the negative control probe-processed images (see Data Analysis section) results in a higher number of positive cell counts (lighter bars), compared to the use of a more stringent threshold value calculated by averaging punctate expression only in cells with ≥ 1 puncta (darker bars). Acknowledgments This protocol was adapted from Avegno et al. Addiction Biology (2021, DOI: 10.1111/adb.12990). The work described here was supported by the National Institute of Health (U01AA028709, R01AA023305 to NWG and K01AA028541 to EMA) and United States Department of Veterans Affairs, Biomedical Laboratory Research and Development Service (Merit Award I01 BX003451 to NWG). Competing interests Nicholas W. Gilpin owns shares in Glauser Life Sciences, a company with interest in developing therapeutics for mental health disorders. There is no direct link between those interests and the work contained herein. All other authors report no conflicts of interest. Ethics All procedures were approved by the Institutional Animal Care and Use Committee of the Louisiana State University Health Sciences Center and were in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). References Avegno, E. M., Kasten, C. R., Snyder, W. B., 3rd, Kelley, L. K., Lobell, T. D., Templeton, T. J., Constans, M., Wills, T. A., Middleton, J. W. and Gilpin, N. W. (2021). Alcohol dependence activates ventral tegmental area projections to central amygdala in male mice and rats. Addict Biol 26(4): e12990. Bankhead, P., Loughrey, M. B., Fernandez, J. A., Dombrowski, Y., McArt, D. G., Dunne, P. D., McQuaid, S., Gray, R. T., Murray, L. J., Coleman, H. G., et al. (2017). QuPath: Open source software for digital pathology image analysis. Sci Rep 7(1): 16878. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Neuroanatomy and circuitry > Fluorescence imaging Neuroscience > Basic technology Cell Biology > Cell-based analysis Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Simple Rescue of Opaque Tissue Previously Cleared by iDISCO Haylee Mesa [...] Qi Zhang Mar 5, 2024 825 Views Visualization and Analysis of Neuromuscular Junctions Using Immunofluorescence You-Tian Hsieh and Show-Li Chen Oct 5, 2024 610 Views Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca2+ Imaging Sergei G. Gaidin [...] Sultan T. Tuleukhanov Feb 5, 2025 46 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,581	https://bio-protocol.org/en/bpdetail?id=4581&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Sclerotinia sclerotiorum Protoplast Preparation and Transformation CL Chi Lan * LQ Lulu Qiao * DN Dongdong Niu (*contributed equally to this work) Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4581 Views: 922 Reviewed by: Zhibing LaiShweta PanchalRitu Gupta Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plant Biotechnology Journal Sep 2021 Abstract Sclerotinia sclerotiorum causes white mold, leading to substantial losses on a wide variety of hosts around the world. Many genes encoding effector proteins play important roles in the pathogenesis of S. sclerotiorum. Therefore, establishment of a transformation system for the exploration of gene function is necessarily significant. Here, we introduce a modified protocol to acquire protoplasts for transformation and generate knockout strains, which completements the transformation system of S. sclerotiorum. Keywords: Sclerotinia sclerotiorum Protoplast Preparation Transformation Gene function Mutant Background Sclerotinia sclerotiorum (Helotiales: Sclerotiniaceae), a phytopathogenic pathogen, infects more than 400 plants, including vegetables and ornamental plants (Kabbage et al., 2015). As the complete genome assembly of S. sclerotiorum is available, many genes encoding effector proteins have been preliminarily validated (Derbyshire et al., 2017). Preparation and transformation of protoplast are important measures to obtain mutants and research gene function. We introduce a protocol based on a standard protocol with minor modifications (Rollins, 2003). This method redounds to production of complete protoplasts for observation and transformation. Using this method, we successfully acquired SsVPS51, SsDCTN1, and SsSAC1 knockout mutants, respectively (Qiao et al., 2021). Therefore, polyethylene glycol–mediated transformation is suitable for protoplast transformation of S. sclerotiorum, providing a convenient method for studying its genetics. Materials and Reagents 1.5 mL microfuge tube (Axygen) 2.0 mL microfuge tube (Axygen) Petri dish (9 cm diameter) 40 µm cell strainer (Xiyan Co., Ltd., catalog number: 15-1040) Syringe filter (Pall Corporation, catalog number: 4612-0.2 µm) Sterile blade (ShangHaiLianHui Medical Instrument co., Ltd.) 50 mL centrifuge tube 50 mL conical flask 100 mL conical flask 250 mL conical flask KCl (BIODEE, catalog number: DE-0395A-250 g) CaCl2 (BIODEE, catalog number: DE-0556A-500 g) Agar A (BBI Life Science, catalog number: A600010-500 g) Potato dextrose broth (Solarbio Life Sciences, catalog number: P9240) D-sorbitol (Sigma-Aldrich, catalog number: S3889-500 g) Tris-HCl (Sigma-Aldrich, catalog number: T6666-500 g) Sucrose (BBI Life Science, catalog number: A100335-250 g) Yeast extract (BBI Life Science, catalog number: A515245-500 g) Hygromycin B (Thermo Fisher Scientific, GibcoTM , catalog number: 10687010-20 mL) Glucanex (Sigma-Aldrich, catalog number: L1412-5 g) Polyethylene glycol 3500 (Sigma-Aldrich, catalog number: 25322-68-3-250 g) Spermidine (Sigma-Aldrich, catalog number: 05292-1 mL) Heparin (Sigma-Aldrich, catalog number: H3149-500 KU) Casamino acid (Sangon Biotech, catalog number: A603060-100 g) KCl buffer (pH = 6–6.5) (see Recipes) 0.5% Glucanex (see Recipes) Phanta Super-Fidelity DNA Polymerase (Vazyme, catalog number: P501-d2) STC solution (see Recipes) SPTC solution (see Recipes) PDA medium (see Recipes) PDB medium (see Recipes) RM medium (see Recipes) RMA medium (see Recipes) Equipment 1,000 µL micropipette (Eppendorf) 200 µL micropipette (Eppendorf) 10 µL micropipette (Eppendorf) Centrifuge (Eppendorf, model: 5424R) Centrifuge (Eppendorf, model: 5810R) Hemocytometer (Devan Scientific Co., Ltd., catalog number: AP-0650010) Bacterial shaker incubator (Crystal Technology & Industries, model: IS-AX-190L) Biochemical incubator (Shanghai Yuejin Medical Device Co., Ltd, model: HPX-B400) Zeiss Axio Observer (Zeiss) Procedure Culturing and enrichment of S. sclerotiorum mycelium Cut the PDA medium (see Recipes) where S. sclerotiorum grew on for three days into mycelium blocks (about 1 mm length × 1 mm width) (Figure 1). Note: The ideal cutting area is preferably at the edge of the fungal colony, where S. sclerotiorum hyphae actively grow. Figure 1. The ideal cutting area Collect the mycelium blocks into a 250 mL conical flask containing 100 mL of PDB medium, and shake the flask to mix them together. Note: It’s better to add approximately 200 mycelium blocks to 100 mL of PDB medium. This proportion guarantees that S. sclerotiorum uptakes adequate nutrition and produces enough fresh mycelium. Fasten the conical flask on a shaking table (180 × g, 25 °C) and culture for 14–20 h, until fresh mycelium grows from the mycelium blocks (Figure 2). Figure 2. Fresh mycelium after culturing for 14–20 h. Scale bar = 1 cm. Using a tweezer, separate fresh mycelium from mycelium blocks, and collect them in a 50 mL centrifuge tube. Resuspend the mycelium with 20 mL of KCl buffer, centrifuge at 1,500 × g and 25 °C for 5 min, and then discard the supernatant. Repeat step A5 once again. Protoplast preparation Add 20 mL of 0.5% Glucanex [lysing enzyme from Trichoderma harzianum (Sigma)] to a 50 mL centrifuge tube containing 1 g of mycelium material. Incubate with gentle shaking (120 × g on the rotator) in the dark at room temperature for 2–3 h. After the enzymatic digestion, filter the digestion mixture using a cell strainer, and collect the filtrate into a 50 mL centrifuge tube (Figure 3A). Note: Place the filtrate on ice and proceed cautiously and gently with the next steps, as protoplasts are fragile and may easily rupture (Figure 3B). Figure 3. Filter the protoplasts and place the filtrate on ice Centrifuge the filtrate at 1,200 × g and 4 °C for 10 min, and carefully discard the supernatant. Wash the protoplasts gently with 5 mL of precooled KCl buffer, centrifuge at 5,000 × g and 4 °C for 2 min, and then discard the supernatant carefully. Repeat step B4 once again. Note: Steps B4 and B5 effectively remove the residual enzyme mixture. Resuspend the sediment with 1 mL of precooled STC, centrifuge at 5,000 × g and 4 °C for 2 min, and discard the supernatant by slowly decanting the tube. Resuspend the sediment gently with 800 µL of precooled STC buffer and add 200 µL of precooled SPTC buffer drop by drop with a pipette. Place the protoplast solution on ice and check the status of the protoplasts with a Zeiss Axio Observer (Figure 4). Figure 4. Protoplasts of S. sclerotiorum. Scale bar = 10 µm. Protoplast transformation Resuspend the protoplasts to a concentration of 1 × 108 protoplast per milliliter, in four parts of STC and one part of SPTC. Note: Calculate protoplast concentration with a hemocytometer, according to the manufacturer’s instructions. Add 5 µg of transforming DNA, 5 µL of spermidine (50 mM stock), and 5 µL of heparin (5 mg/mL) to 100 µL of protoplast suspension in a 1.5 mL microfuge tube, and then mix gently. Note: Mix them together by flicking the bottom of the microfuge tube instead of using a pipette. Incubate the mixture on ice for 30 min, then gently add 1 mL of SPTC solution, and incubate at room temperature for 20 min. Transfer the transformation mixture into a 50 mL centrifuge tube, and add 10–20 mL of Regeneration Medium (RM). Culture the mixture with 100 × g agitation, at 25 °C overnight. Gently mix the protoplasts with 200 mL of RMA medium at 43 °C, pour this into Petri dishes (10 mL per dish), and incubate at 25 °C for 24 h. Note: Heat up RMA medium until it melts completely, and cool it down in a water bath set to 43 °C. After, cover the plates with 10 mL of RMA medium containing 100 µg/mL hygromycin B. Culture at 25 °C for 7–10 days, and transfer the transformants to fresh PDA medium containing 100 µg/mL hygromycin B. Transfer the hyphal tip of hygromycin-resistant transformants to PDA medium containing 100 µg/mL hygromycin B. Repeat the selection at least three times (Figure 5A). Note: Pick the edge hyphae of recovered hygromycin-resistant transformants using a sterile blade for the first selection, and cut the mycelium block for the next two selections. Figure 5. Colony morphology of S. sclerotiorum grown on selective RMA medium. A. Colony morphology denoting successful transformation. The hyphal tips labeled by black arrows can be transferred to PDA medium for selection. B. Negative control plate made by adding no DNA in step C2. Extract total RNA of any colony and reverse transcript into cDNA. Perform PCR to validate positive colony by Phanta Super-Fidelity DNA Polymerase with the following cycles: Pre-denature the cDNA at 95 °C for 30 s Denature the cDNA at 95 °C for 30 s 50 °C for 15 s 72 °C for 90 s Repeat steps b-d for 25–35 cycles 72 °C for 10 min Note: Primers designed to check mutants are as follows: SsVPS51-GT-5’-F: CTTAGAGACTTGAGGAAGTC SsVPS51-GT-3’-R: CTACTTCTATTGCTATTC Recipes KCl buffer (pH =6–6.5, sterilize by syringe filter, and store at 25 °C) Reagent Final concentration Amount KCl 0.6 M 4.47 g CaCl2 50 mM 0.56 g ddH2O n/a up to 100 mL Total n/a 100 mL 0.5% Glucanex (store at 4 °C) Reagent Final concentration Amount Glucanex n/a 0.5 g KCl buffer n/a up to 100 mL Total n/a 100 mL STC solution (autoclave at 121 °C for 20 min) Reagent Final concentration Amount Sorbitol 0.8 M 145.74 g Tris-HCl (pH = 8.0) 50 mM 7.88 g CaCl2 50 mM 5.54 g ddH2O n/a up to 1,000 mL Total n/a 1,000 mL SPTC solution (autoclave at 121 °C for 20 min) Reagent Final concentration Amount Sorbitol 0.8 M 145.74 g Tris-HCl (pH = 8.0) 50 mM 7.88 g CaCl2 50 mM 5.54 g Polyethylene glycol 3500 0.11 M 400 g ddH2O n/a up to 1,000 mL Total n/a 1,000 mL PDA medium (autoclave at 121 °C for 20 min) Reagent Final concentration Amount Potato dextrose broth n/a 24 g Agar A n/a 15 g ddH2O n/a up to 1,000 mL Total n/a 1,000 mL PDB medium (autoclave at 121 °C for 20 min) Reagent Final concentration Amount Potato dextrose broth n/a 24 g ddH2O n/a up to 1,000 mL Total n/a 1,000 mL RM medium (autoclave at 121 °C for 20 min) Reagent Final concentration Amount Sucrose n/a 274 g Yeast extract n/a 1 g Casamino acid n/a 1 g ddH2O n/a up to 1,000 mL Total n/a 1,000 mL RMA medium (autoclave at 121 °C for 20 min) Reagent Final concentration Amount Sucrose n/a 274 g Yeast extract n/a 1 g Casamino acid n/a 1 g Agar A n/a 15 g ddH2O n/a up to 1,000 mL Total n/a 1,000 mL Acknowledgments This work was supported by the National Natural Science Foundation of China (32072404) and the Natural Science Foundation of Jiangsu Province (BK20211524). We thank Jeffrey A. Rollins for his work in establishing this protocol. Competing interests No financial, personal, or professional interests have influenced the work. References Derbyshire, M., Denton-Giles, M., Hegedus, D., Seifbarghy, S., Rollins, J., van Kan, J., Seidl, M. F., Faino, L., Mbengue, M., Navaud, O., et al. (2017). The complete genome sequence of the phytopathogenic fungus Sclerotinia sclerotiorum reveals insights into the genome architecture of broad host range pathogens. Genome Biol Evol 9(3): 593-618. Kabbage, M., Yarden, O. and Dickman, M. B. (2015). Pathogenic attributes of Sclerotinia sclerotiorum: Switching from a biotrophic to necrotrophic lifestyle.Plant Sci 233: 53-60. Qiao, L., Lan, C., Capriotti, L., Ah-Fong, A., Nino Sanchez, J., Hamby, R., Heller, J., Zhao, H., Glass, N. L., Judelson, H. S., et al. (2021). Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake. Plant Biotechnol Journal 19(9): 1756-1768. Rollins, J. A. (2003). The Sclerotinia sclerotiorum pac1 gene is required for sclerotial development and virulence. Molecular Plant-Microbe Interactions 16: 785-795. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > Microbial cell biology Molecular Biology Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 1 Q&A Why use Glucanex(Sigma，L1412).? 1 Answer 171 Views Jan 9, 2023 News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,582	https://bio-protocol.org/en/bpdetail?id=4582&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Parasitoid Wasp Culturing and Assay to Study Parasitoid-induced Reproductive Modifications in Drosophila MS Madhumala K. Sadanandappa SS Shivaprasad H. Sathyanarayana GB Giovanni Bosco Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4582 Views: 615 Reviewed by: Geoffrey C. Y. LauDURAI SELLEGOUNDERAnand Ramesh Patwardhan Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in PLOS Genetics Mar 2021 Abstract In nature, parasitoid wasp infections are a major cause of insect mortality. Parasitoid wasps attack a vast range of insect species to lay their eggs. As a defense, insects evolved survival strategies to protect themselves from parasitoid infection. While a growing number of studies reported both host defensive tactics and parasitoid counter-offensives, we emphasize that this parasite–host relationship presents a unique ecological and evolutionary relevant model that is often challenging to replicate in a laboratory. Although maintaining parasitoid wasp cultures in the laboratory requires meticulous planning and can be labor intensive, a diverse set of wasp species that target many different insect types can be maintained in similar culture conditions. Here, we describe the protocol for culturing parasitoid wasp species on Drosophila larvae and pupae in laboratory conditions. We also detail an egg-laying assay to assess the reproductive modification of Drosophila females in response to parasitoid wasps. This behavioral study is relatively simple and easily adaptable to study environmental or genetic influences on egg-laying, a readout for female germline development. Neither the parasitoid culture conditions or the behavioral assay require special supplies or equipment, making them a powerful and versatile approach in research or teaching laboratory settings. Graphical abstract Keywords: Drosophila Leptopilina Trichopria Innate behaviors Oviposition Stress response Parasitoid wasps Background Parasitoid wasps attack the Drosophila genus with an infection rate of 90% in the natural population (Fleury et al., 2004). Depending on the species, wasps lay their eggs in the developing stages of fruit fly larvae or pupae. If the parasitized host fails to encapsulate the newly injected parasitoid egg, these developing eggs hatch into larval wasps that devour the fly progeny innards before emerging from a Drosophila pupal case. After mating, the adult female parasitoid searches for a new host and thus begins the parasitoid life cycle (Carton and Nappi, 1997). Drosophila have evolved various defensive behavioral and cellular strategies to reduce the risk of parasitoid infection. For instance, while fly larvae display a rolling response (Hwang et al., 2007; Robertson et al., 2013) and parasitoid avoidance behavior to escape the wasp attack (Ebrahim et al., 2015), Drosophila adults show accelerated mating behavior (Ebrahim et al., 2021), avoidance response (Ebrahim et al., 2015), oviposition depression (Lynch et al., 2016; Sadanandappa et al., 2021), and ethanol-seeking behavior (Kacsoh et al., 2013; Kacsoh et al., 2015) in the presence of parasitoids. These host defensive strategies lead to parasitoid’s counter-offensive tactics, resulting in more substantial selection pressure on the host. In the wild, both the host and the parasitoid seem to be locked in a perpetual arms race to best one another's newly evolved adaptations (Wertheim, 2022). Beyond the ecological and evolutionary perspective, studying host–parasitoid biology in the laboratory is significant in economic value. Since parasitoids are used in the biocontrol of fruit flies and other insects, understanding the strategies employed by parasitoid wasps helps to reduce agricultural loss by pests. Compared to Drosophila , parasitoid wasps culturing in the laboratory can be labor intensive and demands careful planning and gentle handling of the wasps (Small et al., 2012). Here, we provide the step-by-step procedure for culturing parasitoid wasp species—Leptopilina and Trichopria—on Drosophila larvae and pupae, respectively, in laboratory conditions that already exist in a typical fruit fly research or teaching laboratory. Once established, the maintenance and scaling up to larger productions of the parasitoid cultures do not present significant challenges; optimized culture conditions are generally transferable to many parasitoid species. We also summarize the egg-laying assay used by Sadanandappa et al. (2021) to study the parasitoid-induced reproductive modifications in female fruit flies and discuss the adaptability of this behavioral prototype to examine the influence of environmental and genetic factors in germline development. Materials and Reagents Narrow Drosophila vials (Genesee Scientific, catalog number: 32-116SB) Flugs® narrow plastic vials (Genesee Scientific, catalog number: 49-102) Square bottom Drosophila bottle (Genesee Scientific, catalog number: 32-130) Flugs® plastic fly bottles (Genesee Scientific, catalog number: 49-100) Round synthetic paintbrush (Lowell hillcrest, size 4/0 or 3/0) KimtechTM science kimwipes (Kimberly-Clark Professional, catalog number: 34155, 4.4” × 8.4”) Cheesecloth (Genesee Scientific, catalog number: 53-100) Host cultures: larvae and pupae of Drosophila melanogaster (strain Canton S, Bloomington Stock Center) Larval parasitoid wasps: Leptopilina boulardi (strain Lb17) or L. heterotoma (strain Lh14) (obtained from Todd Schlenke’s laboratory at the University of Arizona) Pupal parasitoid wasp: Trichopria drosophilae (strain Trical) (from Todd Schlenke’s laboratory) Raw and unfiltered honey (Nature Nate’s Honey Co.) Industrial grade carbon dioxide (CO2) (Airgas, an Air Liquide company) Fly morgue (glass beaker containing Wescodyne® Plus diluted in water and plastic funnel) Ethanol, 70%, laboratory grade Drosophila medium (cornmeal molasses yeast medium) (see Recipes) Gelidium agar (MoorAgar, catalog number: 41076) Yeast (Phileo by Lesaffre, catalog number: 73050 SafPro Relax+YF) Corn (MP Biomedicals, catalog number: 0290141180) Molasses (Reinhart Foodservice, catalog number: DW816) Propionic acid (Fisher Scientific, catalog number: A258-500) Methyl 4-hydrobenzoate (NIPAGIN) (Sigma-Aldrich, catalog number: H5501) Equipment Stereomicroscope (Zeiss, model: Stemi 2000) Fiber optic light source (Fisher Scientific, model: LaxcoTM PIFOS150IB) Incubator with controlled temperature (25 °C), humidity (60%), and 12:12 h light/dark cycles (Percival Scientific, model: DR41VL) Fly room with controlled temperature (25 °C), humidity (60%), and 12:12 h light/dark cycles Fly pad, CO2 anesthetizing apparatus (Genesee Scientific, catalog number: 59-119) Software Prism 9 (GraphPad Software, LLC, San Diego, CA, https://www.graphpad.com) OriginPro (OriginLab Corporation, Northampton, MA, https://www.originlab.com) BioRender (BioRender, https://biorender.com) Procedure Part I: Culturing parasitoid wasps (Figure 1) Figure 1. Flow chart summarizing the culturing of parasitoid wasps in laboratory conditions Preparation of the host vials In the laboratory, culture wildtype Drosophila (strain Canton S) in plastic fly bottles containing standard cornmeal molasses yeast medium (see Recipes) and maintain in 12:12 h light/dark cycle–controlled incubators at 25 °C and 60% relative humidity, unless otherwise specified. To avoid overcrowding and stock maintenance, transfer the parental flies (P0, approximately 25 females and 10 males) into fresh fly bottles after four days of egg laying. At 25 °C, adult Drosophila progeny (F1) begins to eclose from pupal cases in the culture bottles in 9–10 days. Anesthetize a bottle of F1 adult fruit flies (preferably 3–10-days-old) with CO2 and place the flies on a fly pad. Using a microscope and a paintbrush, transfer anesthetized F1 adults—15 females and 8 males—into a vial containing fresh fly food (referred to as the host vial ) and allow the flies to lay eggs for three days at 25 °C (Figure 2C–left). After three days, either discard F1 adults from the host vials into a fly morgue or transfer F1 adults to a fresh food vial for preparing a new host vial. Culturing larval parasitoid wasp Following three days of fruit fly egg laying and removal of F1 adults from the host vials, place 3–4 drops of honey diluted 1:1 in distilled water on the inner side of the host vial plugs, to serve as a food source for the adult wasps. Using CO2, anesthetize 5–10-days-old adult larval parasitoid wasps—Leptopilina boulardi (Lb17) or Leptopilina heterotoma (Lh14)—and sort male and female parasitoids (Figure 2A and 2B). Identifying male and female Leptopilina is relatively easy: male parasitoids have longer antennas and smaller bodies than females. Additionally, on the posterior ventral side of the abdomen, female wasps have a specialized needle-like structure, an ovipositor, that they insert into the host to deposit the eggs. Gently transfer the adult parasitoids (12 females and 8 males) into the host vial containing the developing Drosophila larvae. Place the honey-supplemented plugs and leave the host vials (referred to as the infected vial ) on their side until the parasitoids recover from CO2 exposure (Figure 2C–right). Anesthetized wasps could otherwise fall into wet/damp food in an upright vial and, subsequently, be unable to extricate themselves upon waking. Maintain the wasp-infected vials in a fly room or an incubator with controlled conditions until the emergence of the adult parasitoids (Figure 2D). Figure 2. Larval parasitoid–infected host vial. Image of female (left) and male (right) larval parasitoid wasps: (A) L. boulardi (Lb17) and (B) L. heterotoma (Lh14). (C) Image of a freshly prepared host vial for an egg laying (left) and L. boulardi –infected host vial containing the developing larval stages (right). (D) Image of L. boulardi –infected vial with early (left) and late (right) stages of wasp development within a Drosophila pupal case. Note the adult fruit flies that escaped the parasitoid attack or suppressed the infection (left) and newly emerged wasps in the infected vial (right). Culturing pupal parasitoid wasps After three days of egg laying, either discard or transfer the adult F1 flies from the host vial to a new food vial. Allow the larval stages to develop in the host vial at 25 °C for approximately 3–4 days. When the third instar larvae initiate pupation (pre-pupae) in the host vials, remove the plugs and add 3–4 drops of honey diluted in distilled water. Anesthetize 5–10-days-old adult pupal parasitoids [ Trichopria drosophilae (Trical)] using CO2 and sort male and female parasitoids on a fly pad. Like Leptopilina , Trical males also have longer antennas and smaller bodies than females (Figure 3A). Similar to larval parasitoid infection, carefully transfer the Trical wasps (12 females and 8 males) into the host vial containing Drosophila pre-pupae and developing larval stages. Cover the freshly infected host vial with honey-supplemented plugs and leave the vials on their side. After parasitoids wake from CO2 exposure, keep the vials vertically and maintain the wasp-infected vials in a fly room or an incubator with controlled conditions (Figure 3B). Figure 3. Pupal parasitoid–infected host vial. Image of (A) T. drosophilae female (left) and male (right) pupal parasitoid wasp and (B) the parasitoid-infected vial with early (left) and late (right) stages of development within the fly pupal case. Note the wasps used for infecting the fly pupae (left) and newly emerged ones (right) in the infected vials. Collecting and maintaining parasitoid wasps Leptopilina females infect the developing Drosophila larval stages, whereas Trichopria females attack fruit fly pupae to lay their eggs (Small et al., 2012). Successful parasitoid infections suppress the host’s immune response and delay fruit fly development. Consequently, the parasitoid egg hatches, develops, and molts, and the adult wasp emerges from the Drosophila pupal case. Depending on parasitoid species and environmental conditions—temperature and humidity—the adult wasp ecloses 25–30 days post-infection. Alternatively, if the host’s immune response encapsulates the parasitoid egg, it blocks the wasp egg development, and Drosophila adults emerge normally (Carton and Nappi, 1997). While parasitoid eggs are developing in the host vial, the fruit flies’ progeny that successfully defended against the parasitoid infection or escaped the wasp attack ecloses before the wasp’s emergence (see Figure 2D). Discard these adult fruit flies into a fly morgue. To collect and maintain adult parasitoid wasps, prepare a culture vial by inserting a folded Kimwipe sheet inside fresh fly food vials to absorb excess moisture and supplement the plugs with diluted honey. Anesthetize the wasps that emerged in the infected vial on a fly pad and gently place them (approximately 50 wasps/vial) in the culture vials (Figure 4). Repeat the above step once every 4–5 days to collect the newly emerged wasps from the infected vials and maintain the culture vials at room temperature. Then, collected wasps are used for experiments (see below) and to start new wasp cultures by infecting the host vial (Figure 2C and 3B). To avoid adult parasitoid wasp death due to deteriorating fly food, gradual dryness of the plugs, or accumulation of moisture in the vials, transfer wasps to fresh culture vials as needed. Figure 4. Parasitoid culture vials. Image of newly emerged male and female L. boulardi (left) and T. drosophilae (right) parasitoids collected from the respectively infected host vials. Notes Since parasitoid wasps are sensitive to CO2 anesthetization, we recommend using lower CO2 pressure (approximately 10 psi, depending on the fly pad and setup of CO2 station) than for fruit flies and avoiding prolonged CO2 exposure. Also, handle the wasps gently. Before its use for parasitizing the host, female wasp mating is crucial. Therefore, allowing newly collected male and female wasps to cohabitate in culture vials prior to infection is important for producing fertilized wasp eggs (Figure 4). Parasitoid wasps follow the haplodiploidy sex determination: males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. Allowing time for wasp mating and egg fertilization before infection ensures an appropriate female proportion in the next generation of wasps. This way, the ratio of female to male parasitoids in the host vials during infection is less important. As mentioned above, the presence of parasitoid wasps triggers various behavioral and physiological modifications in Drosophila (Kacsoh et al., 2013; Robertson et al., 2013; Ebrahim et al., 2015; Kacsoh et al., 2015; Ebrahim et al., 2021; Sadanandappa et al., 2021). Therefore, we recommend limiting exposure by maintaining the wasp and Drosophila cultures in different locations and using a dedicated CO2 station and materials for wasp handling. Otherwise, thoroughly clean the fly station with 70% ethanol after wasp handling. Part II: Egg-laying assay in Drosophila Culturing experimental stocks As stated, culture all experimental fly lines in 12:12 h light/dark cycle–controlled incubators at 25 °C and 60% relative humidity. To achieve age synchronization of flies for the behavioral analysis, discard all the adult flies into a fly morgue and collect newly eclosed flies (both male and female) within 12 h of clearance into fresh fly bottles (approximately 50 flies/bottle). Avoid experimental fly collection from old, overcrowded, or fungal-infected bottles. Maintain these bottles, containing 0–12-h-old fruit flies, in an incubator with controlled conditions (25 °C, 60% humidity, and 12:12 h light/dark cycles), followed by five days of aging before subjecting the flies to the reproductive assay. Performing egg-laying assay Using a microwave (approximately 30–45 s), liquefy the food in a fresh fly bottle and immediately pour approximately 1 mL of the media into empty Drosophila narrow vials (referred to as egg-laying vial ). To avoid stray flies entering the egg-laying vial and accumulating water vapor as the food solidifies, cover the vial’s mouth with a cheesecloth. Leave the tray containing the egg-laying vials at room temperature for 30–60 min. When the fly media solidifies, remove the cheesecloth and place a fresh plug in the vial. Using CO2, anesthetize adult parasitoid wasps (recommended age: 4–7-days-old) from the culture vials. Using a paintbrush, gently place three female Leptopilina or Trical parasitoids into the egg-laying vials and leave them on their side. Anesthetize all age-(5–6 days) and genotype-matched Drosophila (Canton S) and transfer five female and two male flies to the egg-laying vials that contain parasitoids (wasp-exposed vials). To prepare the mock vials (unexposed controls), follow the steps above, except for introducing the parasitoids. When all flies recover from anesthetization, place the wasp-exposed and mock vials in different fly racks and leave them undisturbed in an incubator at 25 °C, 60% humidity, and 12:12 h light/dark cycles. After 24 h, discard all the flies, including the wasps, to the fly morgue and manually document the total number of eggs laid in each vial using a stereomicroscope (Figure 5A and 5B). The Drosophila egg is approximately 0.5 mm long, ovoid in shape, with dorsal appendages on the anterior side (Figure 5C). Compared to unexposed mock controls (Figure 5A), 24 h L. boulardi –exposed (Figure 5B) female flies show reduced mean egg laying (Figure 6) (Sadanandappa et al., 2021). This behavioral assay can be easily adaptable to study the environmental—nutrition, temperature, daylight, infections, etc.—or genetic effects on germline development (Kurz et al., 2017). By transferring the experimental flies to a fresh vial, the continued egg-laying assay informs the prolonged influence of these factors on oviposition and reproductive behaviors (Pang et al., 2022). Figure 5. Egg-laying assay in Drosophila. Representative images showing 24 h of egg-laying in (A) mock and (B) L. boulardi (Lb17) parasitoid–exposed vials. Images are adapted from Sadanandappa et al. (2021, PLOS Genet) under CC BY 4.0. (C) Schematic of Drosophila egg. Notes While setting up the behavioral experiments, avoid exposing the mock flies to the wasp-exposed group and limit prolonged CO2 exposure as it influences the egg laying in Drosophila females. Exclude egg-laying documentation from the vials with dead flies/wasps. To avoid biases, we recommend double blinding the egg-laying vials before documentation and then decoding the egg count data after counting. Parasitoids used for the exposure experiments were not given any hosts for infection and were never used again after the assay. Previous studies demonstrated that Drosophila adults respond differently to female and male parasitoids. Therefore, we used only female parasitoids in this protocol. Data analysis Any subtle variation in environmental conditions—fly medium, temperature, humidity, nutrition, overcrowding—, infections, and genetic background may influence the egg-laying assay. We recommend at least 3–5 experimental sets along with unexposed genotype controls per replicate for reproducibility and accuracy. Using the Prism or the OriginPro software, the average number of eggs laid by unexposed controls and parasitoid wasp–exposed females can be plotted as a column graph with the standard error mean calculated from at least three independent experiments (Figure 6). Perform the two-tailed unpaired t-test with Welch’s correction to determine the statistical significance between the groups egg-laying means. Figure 6. Larval parasitoid–induced egg laying reduction in Drosophila. Histogram showing the 24 h percentage of egg laying in unexposed (light grey) and Lb17 parasitoid–exposed (dark grey) wildtype Canton S flies. The egg laying responses are represented as a percentage of the mock egg lay, and error bars are ± standard error mean (N = 35, three independent experiments). ** p = 0.0098, calculated using a two-tailed unpaired t-test with Welch’s correction. Recipes Cornmeal molasses yeast medium Reagent Final concentration Amount Agar 1% 10 g Cornmeal 7.6% 76 g Molasses 7.6% 76 g Yeast 5% 50 g ddH2O n/a 1 L Total n/a 1 L Cook the fly medium until it begins to boil and allow it to cool down to below 60 °C before adding NIPAGIN (0.4%) and propionic acid (0.2%). Allow food in bottles or vials to dry overnight at room temperature, covered with a sterile cheesecloth before capping with clean plugs. Fly food can be stored for up to one week in a cold room or fridge between 4 °C and 18 °C. Culture vials and bottles should be brought to room temperature and free of condensation before introducing the flies. Acknowledgments This project was supported by the Human Frontier Science Program Long-Term Fellowship to M.K.S and the National Institute of Health, Pioneer grant 1DP1MH110234 and the Defense Advanced Research Projects Agency grant HR0011-15-1-0002 to G.B. We thank Todd Schlenke (University of Arizona) for providing the wasp strains and Victoria L. Marlar, our lab technician, for all the help. The protocol is adapted from Small et al. (2012) and Sadanandappa et al. (2021). BioRender.com was used for making the graphic. Competing interests The authors of this work have no conflicts of interest to declare. References Carton, Y. and Nappi, A. J. (1997). Drosophila cellular immunity against parasitoids. Parasitol Today 13(6): 218-227. Ebrahim, S. A. M., Dweck, H. K. M., Stökl, J., Hofferberth, J. E., Trona, F., Weniger, K., Rybak, J., Seki, Y., Stensmyr, M. C., Sachse, S., et al. (2015). Drosophila avoids parasitoids by sensing their semiochemicals via a dedicated olfactory circuit. PLOS Biol 13(12): e1002318. Ebrahim, S. A. M., Talross, G. J. S. and Carlson, J. R. (2021). Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila. Nat Commun 12(1): 2453. Fleury, F., Ris, N., Allemand, R., Fouillet, P., Carton, Y. and Boulétreau, M. (2004). Ecological and genetic interactions in Drosophila–parasitoids communities: A case study with D. melanogaster, D. simulans and their common Leptopilina parasitoids in Southe-astern France. Genetica 120(1-3): 181-194. Hwang, R. Y., Zhong, L., Xu, Y., Johnson, T., Zhang, F., Deisseroth, K. and Tracey, W. D. (2007). Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr Biol 17(24): 2105-2116. Kacsoh, B. Z., Lynch, Z. R., Mortimer, N. T. and Schlenke, T. A. (2013). Fruit flies medicate offspring after seeing parasites. Science 339(6122): 947-950. Kacsoh, B. Z., Bozler, J., Ramaswami, M. and Bosco, G. (2015). Social communication of predator-induced changes in Drosophila behavior and germ line physiology. eLife 4: e07423. Kurz, C. L., Charroux, B., Chaduli, D., Viallat-Lieutaud, A. and Royet, J. (2017). Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition. eLife 6: e21937. Lynch, Z. R., Schlenke, T. A. and de Roode, J. C. (2016). Evolution of behavioural and cellular defences against parasitoid wasps in the Drosophila melanogaster subgroup. J Evol Biol 29(5): 1016-1029. Pang, L., Liu, Z., Chen, J., Dong, Z., Zhou, S., Zhang, Q., Lu, Y., Sheng, Y., Chen, X. and Huang, J. (2022). Search performance and octopamine neuronal signaling mediate parasitoid induced changes in Drosophila oviposition behavior. Nat Commun 13(1): 4476. Robertson, J. L., Tsubouchi, A. and Tracey, W. D. (2013). Larval defense against attack from parasitoid wasps requires nociceptive neurons. PLoS One 8(10): e78704. Sadanandappa, M. K., Sathyanarayana, S. H., Kondo, S. and Bosco, G. (2021). Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila. PLOS Genet 17(3): e1009456. Small, C., Paddibhatla, I., Rajwani, R. and Govind, S. (2012). An introduction to parasitic wasps of Drosophila and the antiparasite immune response. J Vis Exp (63): e3347. Wertheim, B. (2022). Adaptations and counter-adaptations in Drosophila host–parasitoid interactions: advances in the molecular mechanisms. Curr Opin Insect Sci 51: 100896. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Behavioral neuroscience Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,583	https://bio-protocol.org/en/bpdetail?id=4583&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Cartographic Tool to Predict Disease Risk-associated Pseudo-Dynamic Networks from Tissue-specific Gene Expression CC Chixiang Chen BS Biyi Shen LZ Lijun Zhang TY Tonghui Yu MW Ming Wang RW Rongling Wu Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4583 Views: 493 Reviewed by: Prashanth N SuravajhalaAmei AmeiJayaraman Valadi Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Bioinformatics May 2022 Abstract Understanding how genes are differentially expressed across tissues is key to reveal the etiology of human diseases. Genes are never expressed in isolation, but rather co-expressed in a community; thus, they co-act through intricate but well-orchestrated networks. However, existing approaches cannot coalesce the full properties of gene–gene communication and interactions into networks. In particular, the unavailability of dynamic gene expression data might impair the application of existing network models to unleash the complexity of human diseases. To address this limitation, we developed a statistical pipeline named DRDNetPro to visualize and trace how genes dynamically interact with each other across diverse tissues, to ascertain health risk from static expression data. This protocol contains detailed tutorials designed to learn a series of networks, with the illustration example from the Genotype-Tissue Expression (GTEx) project. The proposed toolbox relies on the method developed in our published paper (Chen et al., 2022), coding all genes into bidirectional, signed, weighted, and feedback looped networks, which will provide profound genomic information enabling medical doctors to design precise medicine. Graphical abstract Flowchart illustrating the use of DRDNetPro. The left panel contains the summarized pipeline of DRDNetPro and the right panel contains one pseudo-illustrative example. See the Equipment and Procedure sections for detailed explanations. Keywords: Gene regulatory network Genotype-Tissue expression Quasi-dynamic Ordinary differential equations Statistical algorithm Risk prediction Background Differential expression of genes across different tissues has been thought to play an important role in shaping human diseases (Oliva et al., 2020). An increasing body of studies has begun to characterize how genes are co-expressed in tissues, to rewire transcriptional regulatory networks as key molecular mechanisms underlying human disease (Saha et al., 2017; Malatras et al., 2020; Consortium, 2020). However, existing approaches for reconstructing gene regulatory networks are limited in capturing the full properties of gene–gene interactions, which are essential for a mechanistic understanding of disease etiology. For example, correlation-based approaches can only estimate the strength of gene–gene interactions, failing to identify the causality of the interactions, whereas Bayesian networks can identify the causality, but cannot characterize the sign of the interactions and feedback cycles. All these properties can be recovered using dynamic modeling of gene expression. However, it is impossible and ethically impermissible to collect temporal transcriptional data from human tissues. We have developed a series of quasi-dynamic models that can identify the aforementioned properties in gene–gene interactions from static data (Chen et al., 2019; Griffin et al., 2020; Wu and Jiang 2021; Chen et al., 2021). More recently, we leveraged these models to recover tissue-specific and pseudo-dynamic gene regulatory networks across the spectrum of disease risk (Chen et al., 2022). In this article, we develop a software pipeline called D isease R isk-associated pseudo-D ynamic Networks Bio-Pro tocol (DRDNetPro). This pipeline provides a detailed tutorial, enabling researchers to reconstruct pseudo-dynamic networks from static gene expression data, which helps to identify context-specific and personalized networks, and further the mechanistic understanding of diseases. The software and detailed tutorials with illustrative data examples can be found in our GitHub repository (https://chencxxy28.github.io/DRDNetPro/articles/NAME-OF-VIGNETTE.html). Equipment Computational requirements The users need to prepare a desktop/laptop computer with R version 4.1.0 or above installed. Install packages (The summary of the tutorial in the GitHub page repository) Run the function “install.packages()” to install the required packages, including “pROC”, “np”, “splines2”, “grpreg”, “Matrix”, “igraph”, “ggplot2”, and the optional packages “ranger”, “XGBoost”, and “dplyr”. Run the function “devtools::install_github()” to install the package “DRDNetPro”. Run the function “install.packages()” to install the package “devtools”, if it has not been installed yet. Software Software list DRDNetPro (Chixiang Chen, https://github.com/chencxxy28/DRDNetPro) graph (The igraph core team, https://igraph.org/r/) ggplot2 (Hadley Wickhan, https://ggplot2.tidyverse.org/) Datasets for demonstration The demo data for method illustration can be downloaded from the website https://chencxxy28.github.io/DRDNetPro/articles/web/data.html, which is from the GTEx project originally. Gene expression data were collected from blood vessels and de-identified subsamples of phenotype information from donors. Procedure To predict disease risk-associated pseudo-dynamic networks, we need the following inputs from each subject: predicted disease risk (named “agent” below), one covariate of interest (e.g., smoking), and pre-processed gene expression data. The detailed procedures of how to generate the agent, train the network model, and visualize the results are listed below. All the programs, demo data for illustration, and brief tutorials for the following procedures can be found in our GitHub repository (https://chencxxy28.github.io/DRDNetPro/articles/NAME-OF-VIGNETTE.html). Predict the agent (Tutorial 1 in the GitHub repository) Prepare the data for training prediction values of the agent Before predicting the agent, users need to prepare the data for model training. The data needs to be a matrix, including the outcome in the first column and all potential predictors in the remaining columns. Do not include the intercept as one column. The outcome in this protocol has values of 0 and 1, corresponding to healthy and diseased subjects, respectively. More complicated outcomes with multiple categories are not considered in the current bio-protocol, as we regard this as future work. One remedy for the case of multiple categories are to categorize the outcome into binaries, which is also often applied in practice. All values in the matrix should be numeric. The missing data is allowed in this training process, though it is always recommended to have complete data as an input. For demonstration, we have prepared a toy data example in this tutorial, which can be accessed from https://chencxxy28.github.io/DRDNetPro/articles/web/data.html. See details in the Data analysis section below. Select a training method There is no unique way to predict the agent. Either statistical regression models or machine learning algorithms can be applied. In our program, we allow the users to specify the following commonly used methods: conventional logistic regression, random forest, and XGBoost. Logistic regression—This is the most conventional statistical tool to predict disease risk, which will lead to a probability-scaled risk. Run the function “glm()” in R to fit the training model, and then run “fitted()” to extract the predicted agent. Random forest—This is a machine learning method with an ensemble of decision trees. It builds and combines multiple decision trees to produce more accurate predictions. It is a non-linear classification algorithm. There are several R packages available for implementing random forest. We provide demo codes based on the package “ranger”. The data required for “ranger” should be in a matrix form, where the outcome should be a factor, to enable running classification trees. After preparing the data, run the function “ranger()” to train the model, and run the function “predict()” to extract the predicted agent. XGBoost—This is another machine learning method with an efficient implementation of the gradient boosting framework. It provides built-in k-fold cross-validation (CV) Stochastic Gradient Boosting Machines, with column and row sampling (per split and per tree) for better generalization. We provide demo codes based on the package “XGBoost”. A specific type of input data is required by “XGBoost”, where the response and predictors should be separately stored. The series codes to prepare the required data are provided in this tutorial (see details in Tutorial 1). After preparing the data, run the function “xgb.cv()” to get an initial prediction model. Then, run the function “dplyr::summarise()” to extract the tuned parameter, i.e., the number of trees. Finally, run the function “xgboost()” to train the data, and “predict()” to extract the predicted agent. Predict and evaluate the agent Prepare the training and testing data—When the agent model is fitted, it is better to check its prediction performance. Note that the pseudo-dynamic network is sensitive to the values of agent, and the agent with good prediction for the disease risk is preferred. To evaluate its predictive performance, we consider the metric of the area under the receiver operating characteristic curve (AUC-ROC). To avoid the overfitting issue, we need a testing data set. For illustration purposes, we artificially created training and testing data by randomly splitting the original data, with no overlap between the two constructed data sets. The training data is used for data fitting, whereas the testing data is used for prediction evaluation. Run the function “sample()” to generate these two datasets. In real applications, another way to validate prediction performance is to use identical but independently sampled data from an external source as the testing set. Generate the AUC-ROC plot—After fitting the model based on the training data, by running the function “glm()”, extract the predicted agent based on the testing data, by running the function “predict()”. Run the function “roc” to generate the plot. A larger AUC-ROC value indicates a better prediction model. Train the network model (Tutorial 2 in the GitHub repository) Prepare the data for training the network Filtering and transformation—To construct the gene–gene network, the users need to preprocess the gene expression data, by normalizing, excluding low-expressed genes, and, if needed, transforming to relieve skewness and reduce variance. Screening—To learn the disease risk-associated network, genes showing changing expression patterns as the agent changes should be included, which is different from association-based network. Two selection procedures can be considered: To test differentially expressed genes, run the function “test_screen()” to locate genes showing the difference between disease groups, after adjusting for covariates and applying the Hochberg multiple test correction (adjusted P-value <0.05). To screen the genes, run the function “spearman_screen()” to find genes showing high associations between expression and the predicted agent. Pick the top (e.g., top 40) most correlated genes. Perform separate screening to check if more than one homogeneous group is considered. Determine the final genes by combining the two selection procedures. Optional screening can be done based on prior biological knowledge (i.e., the GO enrichment analysis). Train the varying coefficient model Refine the data—Before constructing the network, each selected gene should be fitted by the varying coefficient model. This model estimates dynamic features of coefficients in terms of a certain agent by Kernel/Spline regression, which fits the context of reconstructing pseudo-dynamic networks well. We refer readers to Chen et al. (2022) for more technical details. When two subjects have the same agent value, it means the expression data from these two are not informative. Run the R codes in this tutorial to refine the data, by deleting the data with agents too close to each other. Fit the model—After refining the data, run the function “vc.fit()” to fit the varying coefficient model with a covariate (e.g., smoking). Check the fitted curves—Run the function “plot()” and “lines()” to check how well the data is fitted. The users can also use the package “ggplot2” for visualization. Generate a base matrix for the network model The base matrix is the key component for the network to learn in a non-parametric manner. Run the function “base.construct()” to construct the base matrix for the varying intercept and base matrices for the varying coefficient of the covariate (e.g., smoking). The default knots are lower quartile and upper quartile with degree =3, which is related to the cubic spline. The output—A list of four matrices based on varying intercepts (X_big), varying covariate effects (X_big_cov), varying intercepts with a column containing ones (X_big_int), and varying covariates with a column containing ones (X_big_int_cov). Train the network After constructing the base matrices, the users should run the function “network.learn()” to train the network structure. This function requires the input of the design matrix, the outcome, and the group information, as well as the method to select the tuning parameters. The design matrix—Extract the output results from the previous step of construction of base matrices, which includes data_observe: the gene expression matrix. x_cov: the vector including the numerical values of the covariate of interest (e.g., smoking: 0 and 1). X_big_int: the matrix based on varying intercepts, with a column containing ones. X_big_int_cov: the matrix based on varying coefficients, with a column containing ones. Agent: the predicted disease risk obtained from Tutorial 1. Determine the tuning parameters—Multiple methods are available to tune the hyperparameters, including 5-fold CV (CV5), 10-fold CV (CV10), Akaike information criterion (AIC), Bayesian information criterion (BIC), and empirical BIC (EBIC). Use the argument “cv” in the function “network.learn()” to specify the preferred method. Output from the network learning—This output includes information on node size, interaction, and covariate effect. All of them will be used in the next step for visualizing the results. Node size information: self-node size for baseline, self-node size for the covariate effect, overall self-node size. Interaction information: gene interaction effects from the baseline, gene interaction effects from the covariate, overall gene interaction effects. Covariate effect information: covariate effect, trend effect for baseline, trend effect from the covariate. Visualize the results (Tutorial 3 in the GitHub repository) Extract the results from the trained network model Read the output data from the network learning in Tutorial 2, which includes network.output, data.list.t3, and gene.names. Visualize the networks with a given range of agent values Baseline networks—To visualize the pseudo-dynamic network given an agent (disease risk) value, one useful tool is the package “igraph”. It allows for a self-designed network structure. More details about “igraph” can be found in the tutorial (https://igraph.org/r/). Run the demo code to visualize the recovered baseline network (e.g., the non-smoking group), where the agent is changing from no risk to high risk. Covariate-effect networks—Run the demo code for visualizing three recovered covariate-effect (e.g., effect caused by smoking) networks, where the agent is changing from no risk to low/moderate/high risk (data-driven or subjective cut-points selected for risk levels). Visualize the interaction behaviors for a given gene over the spectrum of the agent Select one specific gene of interest, and run the demo code to plot the trend effect and interaction effects over the whole spectrum of the agent. Data analysis The goal of this section is to apply the procedure detailed above to learn and visualize the hypertension risk-associated pseudo-dynamic gene–gene networks in blood vessel tissue. The example data for illustrating the above procedure is from the GTEx project and consists of gene expression data from blood vessels and de-identified subsamples of phenotypic information from donors. Note that the raw phenotype data is confidential and protected. The illustrating data used in this protocol is de-identified and a subset of the raw data. The detailed information on data analysis is in the original paper (Chen et al., 2022), as well as in our GitHub repository. A summary is listed below. Use the phenotype data (named “pheno_used”) to predict the risk of having hypertension as the agent. The predicted disease risk can be found in Tutorial 1 in the GitHub repository. Based on the imputed disease risk of having hypertension, use the gene expression data (named “data vessel”) to fit the varying coefficient model, construct the base matrices, and then train the network learning model. Visualize the baseline network (the non-smoking group), as the risk of having hypertension is changing from no risk to high risk (figure 1 in Tutorial 3 in the GitHub repository and figure 3 in the original paper). Visualize the smoking-effect-associated networks, as the risk of having hypertension is changing from no risk to low risk, moderate risk, and high risk (figure 2 in Tutorial 3 in the GitHub repository and figure 3 in the original paper). Additionally, we pick the C3orf70 gene to visualize interaction behaviors over the whole spectrum of the risk of having hypertension for illustration. This gene is selected in a previous screening step and was shown to be associated with hypertension and neural and neurobehavioral development in literature (Samblas et al., 2019). In figures 3–6 in the GitHub repository, smoking has a negative effect on the expression of the target gene, and CTSD, GALNT4, NDUFA4L2, and RCN3 genes are detected as inhibiting the expression of the target gene. Acknowledgments Wu’s work was partially supported by Grant U01 HL119178 from the National Heart, Lung and Blood Institute (NHLBI) and 5R01HD086911-02 from the National Institute of Child Health and Human Development (NICHD), the National Institute of Health. Wang’s research was partially supported by Grants KL2 TR000126 and TR002015 from the National Center for Advancing Translational Sciences (NCATS) and start-up funding from Case Western Reserve University. Yu’s work was supported by the Fundamental Research Funds for the Central Universities of China (Grant No. JZ2022HGQA0151). The content is solely the responsibility of the authors. The original research paper from which this protocol was derived: Chen et al. (2022) published in Bioinformatics. Competing interests The authors declare that no competing interests exist. References Chen, C., Jiang, L., Fu, G., Wang, M., Wang, Y., Shen, B., Liu, Z., Wang, Z., Hou, W., Berceli, S. A. and Wu, R. (2019). An omnidirectional visualization model of personalized gene regulatory networks. NPJ Syst Biol Appl 5: 38. Chen, C., Jiang, L., Shen, B., Wang, M., Griffin, C. H., Chinchilli, V. M. and Wu, R. (2021). A computational atlas of tissue-specific regulatory networks. Front Syst Biol https://doi.org/10.3389/fsysb.2021.764161. Chen, C., Shen, B., Ma, T., Wang, M. and Wu, R. (2022). A statistical framework for recovering pseudo-dynamic networks from static data. Bioinformatics 38(9): 2481-2487. Griffin, C., Jiang, L. and Wu, R. (2020). Analysis of quasi-dynamic ordinary differential equations and the quasi-dynamic replicator. Physica A 555: 124422. Consortium, G. T. (2020). The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science 369(6509): 1318-1330. Malatras, A., Michalopoulos, I., Duguez, S., Butler-Browne, G., Spuler, S. and Duddy, W. J. (2020). MyoMiner: explore gene co-expression in normal and pathological muscle. BMC Med Genomics 13(1): 67. Oliva, M., Munoz-Aguirre, M., Kim-Hellmuth, S., Wucher, V., Gewirtz, A. D. H., Cotter, D. J., Parsana, P., Kasela, S., Balliu, B., Vinuela, A., et al. (2020). The impact of sex on gene expression across human tissues. Science 369(6509): eaba3066. Saha, A., Kim, Y., Gewirtz, A. D. H., Jo, B., Gao, C., McDowell, I. C., Consortium, G. T., Engelhardt, B. E. and Battle, A. (2017). Co-expression networks reveal the tissue-specific regulation of transcription and splicing. Genome Res 27(11): 1843-1858. Wu, R. and Jiang, L. (2021). Recovering dynamic networks in big static datasets. Phys Rep Volume 912: 1-57. Samblas, M., Milagro, F. I. and Martinez, A. (2019). DNA methylation markers in obesity, metabolic syndrome, and weight loss. Epigenetics 14(5): 421-444. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Systems Biology > Connectomics Drug Discovery Computational Biology and Bioinformatics Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,584	https://bio-protocol.org/en/bpdetail?id=4584&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Staining and Scanning Protocol for Micro-Computed Tomography to Observe the Morphology of Soft Tissues in Ambrosia Beetles ES Ellie J. Spahr SM Sarah L. McLaughlin AT Alyssa M. Tichinel MK Matt T. Kasson TK Teiya Kijimoto Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4584 Views: 329 Reviewed by: Khyati Hitesh ShahSailendra SinghJegan Sekar Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in PLOS ONE Sep 2020 Abstract Advances in imaging technology offer new opportunities in developmental biology. To observe the development of internal structures, microtome cross-sectioning followed by H&E staining on glass slides is a common procedure; however, this technique can be destructive, and artifacts can be introduced during the process. In this protocol, we describe a less invasive procedure with which we can stain insect samples and obtain reconstructed three-dimensional images using micro-computed tomography, or micro-CT (µCT). Specifically, we utilize the fungus-farming ambrosia beetle species Euwallacea validus to observe the morphology of mycangia, a critical internal organ with which beetles transport fungal symbionts. Not only this protocol is ideal to observe mycangia, our staining/scanning procedure can also be applied to observe other delicate tissues and small organs in arthropods. Graphical abstract Keywords: Micro-computed tomography Ambrosia beetle Symbiosis Euwallacea validus Mycangia Soft tissue Background The study of developmental biology often requires visualization across life stages as it concerns the process through which certain organs or tissues form. Microscopy has been the core means to observe the morphology of organs under development. To observe external features, stereo microscopy is adequate to visualize structures across developmental stages; however, internal structures are often difficult to observe and measure without destructive sampling. Employing micro-computed tomography (µCT) visualization protocols allows for the observation of structures throughout their development, in hard- and soft-bodied life stages alike (Spahr et al., 2020). The success of staining and scanning protocols can allow for detailed internal visualization and quantification of structures after genetic functional analyses, where the use of a non-destructive visualization technique is integral. Here, we describe stepwise methods for µCT scanning in the fungus-farming ambrosia Euwallacea validus . Ambrosia beetle symbiosis is an emerging, global topic in forest pathology and entomology due to many species being invasive pests that vector phytopathogenic fungi across native and introduced ranges. Notably, Raffaelea lauricola , the causal agent of laurel wilt disease, is vectored by the redbay ambrosia beetle Xyleborus glabratus (Fraedrich et al., 2008). Other phytopathogenic fungi, such as Fusarium species carried by Euwallacea ambrosia beetles, cause significant agricultural and economic damages to avocado, citrus, and tea (Freeman et al., 2013; O'Donnell et al., 2015). Understanding the developmental process of tissues in beetles or fungi may lead to the development of effective means to control these pests. The beetles rely on novel pits or pouches called mycangia, in which they house and transmit fungal propagules between tree hosts. The location of these structures varies between beetle species. External mycangia, such as the pronotal pouches of the bark beetle Dendroctonus frontalis and related species, are commonly observed by electron scanning microscopy; ease of study has allowed for detailed structural descriptions (Yuceer et al., 2011). However, the internal mycangia, such as the preoral mycangia found in Euwallacea , are more difficult to observe and describe. Published studies of internal mycangia have employed microtome cross-sectioning, laser ablation tomography (LAT) scanning, and micro-computed tomography (Li et al., 2015, 2018). The destructive nature of microtome cross-sectioning can require many samples to produce a viable result, especially when using small arthropod samples. By employing scanning techniques, the artifacts caused by slicing and staining of cross-sections can be reduced, which allows us to achieve detailed results. This technique can assist other studies in observing and describing soft tissues in arthropods and can be combined with other techniques (such as RNA interference, allometry, etc.) to strengthen resources for small, internal, or otherwise difficult to measure structures. Materials and Reagents Eppendorf 1.5 mL microcentrifuge tubes (Sigma-Aldrich, catalog number: T6649) 200 μL pipette tips (Sigma-Aldrich, Corning, catalog number: CS4860) Paint brush (any fine brush) Scissors 100% ethanol (200 proof) (Sigma-Aldrich, catalog number: E7023) Deionized/sterile water (Sigma-Aldrich, catalog number: 8483331000) 5% Lugol’s solution (Fisher Scientific, Spectrum Chemical, catalog number: 18-611-063) Dental wax ortho tray strips (Kerr, Benco, catalog number: 1021-752) Insect samples 70% ethanol (see Recipes) Equipment Bruker Skyscan 1272 micro-computed tomography scanner Computer that can run the software (see Note 1 below) Software Skyscan Micro-CT scanning software (version 1.1.10, Bruker; https://www.bruker.com/en/products-and-solutions/microscopes/3d-x-ray-microscopes/skyscan-1272.html) NRecon Reconstruction (Bruker, 3D.SUITE software includes Dataviewer, CTAn, CTVol, CTVox) (Bruker, https://www.bruker.com/en/products-and-solutions/preclinical-imaging/micro-ct/3d-suite-software.html) Procedure Sample preparation and staining Retrieve fixed insects from 70% ethanol. Rinse samples once or twice in 70% ethanol and then rehydrate insects in a graded series of ethanol (70% ethanol, to 50% ethanol, to 25% ethanol, to 100% deionized water). Remove the liquid, place insect samples in a fresh Eppendorf microcentrifuge tube, and cover with 5% Lugol’s solution (approximately 300 μL; the volume is subjective, but sample should be submerged). Place in a dark cabinet overnight, from 12 to 24 h. Rinse or flush sample with water before replacing the solution with 70% ethanol. Store in freezer at -20 °C until scanning. Micro-computed tomography: preparing your sample for scanning Trim a pipette tip to approximately 12.5 mm in height to accommodate the available space in the scanning chamber. Seal the bottom of the trimmed pipette tip with dental wax, ensuring a liquid-tight seal is created. Apply a small ball of wax to the pedestal and place the wax-sealed bottom of the trimmed pipette to the wax ball. Apply light pressure to adhere the wax from both surfaces together; additional dental wax can be added to ensure the pipette will stand upright on the pedestal. Orient the pipette tip so that it is straight and centered upright. Set the pedestal on the counter and slowly rotate to visually examine for movement of the pipette tip. Adjust the orientation of the pipette tip in the wax until rotational movement is reduced and it appears centered. Pipette 70% ethanol into the trimmed pipette (approximately 50–80 μL; can start at a lower volume and add more ethanol after placing sample). Retrieve your stained insect sample from its storage tube with a paint brush and deposit it in the ethanol of the trimmed pipette. Add more ethanol if the sample is not submerged. Gently tap the base of the pedestal on the counter (pipette staying upright) to dislodge any air bubbles within the pipette and to settle the sample in the bottom of the tube. Lightly seal the top of the trimmed tube with wax; roll a small ball of wax into a flat circle, apply to the open top of the pipette, and gently press the edges around the outside wall of the pipette tip. Check the orientation pipette tip one last time to ensure it is centered on the pedestal. Open the Bruker Skyscan Micro-CT scanning software. If the lights on the micro-CT are flashing and an X-ray source dialog box opens, the X-ray source may need time to warm up if the machine has not been used recently. Ensure the X-ray source is off and open the scanner door using the Bruker scanning software top menu bar. Place the pedestal in the scanner platform and tighten the ring. Close the scanning door using the Bruker software. Micro-computed tomography: scanning In the Skyscan Micro-CT software, turn on the Grab setting (TV icon) and visual camera (lightbulb icon) along the top bar to view the sample inside the micro-CT machine during scanning setup (Figure 1). Figure 1. Screenshot of menu bar. Active features are labeled in orange. Adjust voltage and amperage to 50 kV and 200 μA, respectively. Change the binning to 2,016 × 1,344 by clicking this setting on the bottom toolbar (found on the bottom right, dimensions next to an icon of a blue box with blue arrows, Figure 2). Figure 2. Screenshot of bottom toolbar To the right of binning setting on the bottom toolbar is the filter setting. Set the filter setting to “no filter.” Using the left mouse button and the Control key, click and drag the main image to center the sample. A line will appear to indicate the direction and degree of movement. Releasing will move the sample within the Skyscan unit. Rotate the sample 360° to ensure it is upright and within the bounds of the scanning area at all degrees of rotation. If the sample is tilted or rotates outside of the camera view, shut off the source and open the chamber to reorient the sample on the pedestal. Re-center the sample if any changes are made to the pipette orientation (Figure 3). Figure 3. Inside of the scanner with a sample Click the Elevation setting on the bottom toolbar (displays a “mm” setting found on the right side, next to two green arrows) to open the Magnification and Positions menu. Slowly decrease the pixel size in a stepwise manner until it reaches 4.26 μm, ensuring sample does not contact the internal camera. Repeat step C6 to ensure the entire sample is still within the visible bounds of the screen. Repeat steps C5 and C6 if necessary. Right click on the center of the sample to bring up the Transmission reading for your sample. Return to the Magnification and Positions menu by clicking the Elevation setting on the bottom toolbar. Record the current elevation (mm) of the sample. Remove the sample from field of view by lowering the sample height number. Navigate to Scanning Modes menu from the Options tab to update the flat field correction. Adjust exposure time to obtain approximately 60% average transmission with flat fields turned off. In previous samples, 600 ms was optimal for ambrosia beetle scans. Return the sample to the field of view by entering the recorded Elevation number in the Magnification and Position menu. Click the Scan icon on the top toolbar (blue circular arrow) to begin setting the scan parameters: rotation step = 0.200 degrees; frame averaging = ON (2); Random Movement = OFF (0). Give the scan a descriptive title and use the Browse button to designate the data directory. Create a New Folder to house the scan data (Figure 4). Figure 4. An example of scan setting Press ‘Scan’ to begin scanning. Data analysis Data processing and analysis Micro-computed tomography: image processing, cropping, and reconstruction Open NRecon. Load the scan files. On the right-hand side of the main program interface, select the Fine Tuning button from the Start tab. Work stepwise through the Fine Tuning interface to adjust Post-alignment, Beam-hardening correction, and Ring-artifacts reduction settings. Select one of these options at a time. It is recommended to leave the number of steps at 5 and parameter step at 1.0 for initial fine-tuning. Press Start within the Fine Tuning window to begin correction previews. For each setting, the program will estimate the level of correction needed; even so, individual input is recommended to use this function to move through the range of provided previews to choose the optimal correction factor. On the toolbar, click through the arrows to examine the changes to the image preview at each setting. Examine how these settings change your scan data and choose the optimized image. Note the ideal setting for each correction type and change this number for reconstruction if necessary. Repeat this process for each of the three correction types before proceeding. If necessary, adjust the parameter increments on the Fine Tuning window to examine smaller changes in correction factors. In the Output tab, move the linear bounds surrounding the histogram to optimize the contrast of the scan. Below the histogram settings in the Output tab, select the Save Destination on the computer by clicking Browse. It is recommended to save the reconstruction files with the initial scan data, but in a new subfolder with a specific designation such as Reconstruction . Navigate back to the Start tab and press Start to reconstruct the scan data (or Start batch if batch correcting). Once reconstruction has finished, close NRecon and open Dataviewer. Select and open the reconstructed files saved during the previous step. Move the selected cross-section to a point within the scan that shows the full length and width of the scanned sample within the pipette tip. Adjust the bounds of the image to crop any unnecessary blank space; try to remove as much of the pipette tip or surrounding space from the scan without cropping portions of the insect. Double-check the cropped information by selecting new points across your scan data to ensure all features are retained. Save this cropped image in another new subfolder (title folder with a descriptive designation as before such as Cropped) and close the application. Open CTVox. Select the cropped, reconstructed image files as processed in the previous step. If necessary, adjust the brightness and transparency of the scan data by modifying the Contrast histogram in the left panel. Notes Since 3D images are reconstructed from many high-resolution images, high performance computers are preferred to run the software used in this protocol. Bruker recommends an average workstation computer with dual 6-Core Intel processors, 32 GB RAM, and 6 TB of available disk space. Variability can occur between samples, which will need to be reoriented for each scan. Rotating samples 360° before scanning ensures they are not askew and allows for a pre-scan check. If too much movement occurs during rotation, the pipette tip can be reoriented to minimize potential artifacts. Altering the sample binning will alter the recommended pixel size if using the same camera position; always decrease pixel size in small increments as to not damage the internal camera. Optimization of scanning parameters should be considered based on the transmission through the sample. Ideal transmission through the sample is 20%–40% minimum intensity. Adjust by changing the filter, exposure time, and voltage. Scanning samples using the same parameters can allow for comparisons between compiled models. For an overview of SkyScan 1272 micro-CT sample loading and Dataviewer, Bruker Corporation has published short training videos introducing these software packages (Bruker Corporation, 2015 and 2018). These videos may be a good resource for basic use and familiarization with the software interface. The instructions in these videos are optimized for other sample types and, as such, should be used in conjunction with the above instruction to tailor data processing to insect samples. Movies for the procedure of Dataviewer use and Reconstruction are also available from our group (McLaughlin et al., 2022a and 2022b, respectively). Examples of reconstructed scan data generated from this protocol can be found as published in Spahr et al. (2020). Video of a 3D model from this publication can be viewed at the following link: https://doi.org/10.6084/m9.figshare.12221981.v1 Recipes 70% ethanol can be prepared from 190 or 200 proof stock as follows: 70% ethanol from 200 proof ethanol stock Reagent Final concentration Amount Ethanol (absolute) 70% 700 mL H2O 30% 300 mL Total n/a 1,000 mL Acknowledgments We would like to thank Karen Martin from WVU Animal Models Imaging Facility for training and use of the Bruker Skyscan 1272 Micro-CT and accompanying software. This research was supported by funding provided by USDA-NIFA (HATCH) WVA00712. This protocol was developed and utilized in Spahr et al. (2020). Competing interests No conflict of interest is declared regarding this article. References Fraedrich, S. W., Harrington, T. C., Rabaglia, R. J., Ulyshen, M. D., Mayfield, A. E., 3rd, Hanula, J. L., Eickwort, J. M. and Miller, D. R. (2008). A Fungal Symbiont of the Redbay Ambrosia Beetle Causes a Lethal Wilt in Redbay and Other Lauraceae in the Southeastern United States. Plant Dis 92(2): 215-224. Freeman, S., Sharon, M., Maymon, M., Mendel, Z., Protasov, A., Aoki, T., Eskalen, A. and O'Donnell, K. (2013). Fusarium euwallaceae sp. nov.--a symbiotic fungus of Euwallacea sp., an invasive ambrosia beetle in Israel and California. Mycologia 105(6): 1595-1606. Li, Y., Simmons, D. R., Bateman, C. C., Short, D. P., Kasson, M. T., Rabaglia, R. J. and Hulcr, J. (2015). New Fungus-Insect Symbiosis: Culturing, Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles. PLoS One 10(9): e0137689. Li, Y., Ruan, Y., Kasson, M. T., Stanley, E. L., Gillett, C., Johnson, A. J., Zhang, M. and Hulcr, J. (2018). Structure of the Ambrosia Beetle (Coleoptera: Curculionidae) Mycangia Revealed Through Micro-Computed Tomography. J Insect Sci 18(5). O'Donnell, K., Sink, S., Libeskind-Hadas, R., Hulcr, J., Kasson, M. T., Ploetz, R. C., Konkol, J. L., Ploetz, J. N., Carrillo, D., Campbell, A., et al. (2015). Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism. Fungal Genet Biol 82: 277-290. Spahr, E., Kasson, M. T. and Kijimoto, T. (2020). Micro-computed tomography permits enhanced visualization of mycangia across development and between sexes in Euwallacea ambrosia beetles. PLoS One 15(9): e0236653. Yuceer, C., Hsu, C.-Y., Erbilgin, N. and Klepzig, K. D. (2011). Ultrastructure of the mycangium of the southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae, Scolytinae): complex morphology for complex interactions. Acta Zoologica 92(3): 216-224. Bruker Corporation. (2015). Bruker microCT training video: Introduction to dataviewer. [Video]. Youtube, https://youtu.be/qs-ec2v3tuo Bruker Corporation. (2018) SKYSCAN 1272: Just start your analysis. [Video]. Youtube, https://youtu.be/HBZuBlg9qhg McLaughlin, S., Spahr, E., Kasson, M., and Kijimoto, T. (2022a). Cropping Data Files with Dataviewer. figshare. Media. https://doi.org/10.6084/m9.figshare.21273327.v1 McLaughlin, S., Spahr, E., Kasson, M., and Kijimoto, T. (2022b). µCT Reconstruction with NRecon. figshare. Media. https://doi.org/10.6084/m9.figshare.21273342.v1 Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > RNA > RNA interference Developmental Biology > Morphogenesis Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Variable Dose Analysis: A Novel High-throughput RNAi Screening Method for Drosophila Cells Katarzyna Sierzputowska [...] Benjamin E. Housden Dec 20, 2018 4266 Views pNP Transgenic RNAi System Manual in Drosophila Fang Wang [...] Jian-Quan Ni Feb 5, 2019 6253 Views RNA Interference Method for Gene Function Analysis in the Japanese Rhinoceros Beetle Trypoxylus dichotomus Kazuki Sakura [...] Teruyuki Niimi Apr 20, 2022 1494 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,585	https://bio-protocol.org/en/bpdetail?id=4585&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed BONCAT-based Profiling of Nascent Small and Alternative Open Reading Frame-encoded Proteins XC Xiongwen Cao * YC Yanran Chen * AK Alexandra Khitun SS Sarah A. Slavoff (*contributed equally to this work) Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4585 Views: 930 Reviewed by: ASWAD KHADILKARThomas Farid MartínezMarie A Brunet Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Chemical Biology Apr 2022 Abstract RIBO-seq and proteogenomics have revealed that mammalian genomes harbor thousands of unannotated small and alternative open reading frames (smORFs, <100 amino acids, and alt-ORFs, >100 amino acids, respectively). Several dozen mammalian smORF-encoded proteins (SEPs) and alt-ORF-encoded proteins (alt-proteins) have been shown to play important biological roles, while the overwhelming majority of smORFs and alt-ORFs remain uncharacterized, particularly at the molecular level. Functional proteomics has the potential to reveal key properties of unannotated SEPs and alt-proteins in high throughput, and an approach to identify SEPs and alt-proteins undergoing regulated synthesis should be of broad utility. Here, we introduce a chemoproteomic pipeline based on bio-orthogonal non-canonical amino acid tagging (BONCAT) (Dieterich et al., 2006) to profile nascent SEPs and alt-proteins in human cells. This approach is able to identify cellular stress-induced and cell-cycle regulated SEPs and alt-proteins in cells. Graphical abstract Schematic overview of BONCAT-based chemoproteomic profiling of nascent, unannotated small and alternative open reading frame-encoded proteins (SEPs and alt-proteins) Keywords: Small open reading frame (smORF) Microprotein Alternative protein (alt-protein) Micropeptide Chemoproteomics Proteomics Protein translation Unnatural amino acid Background Thousands of previously unannotated, expressed small open reading frames (smORFs) and alternative open reading frames (alt-ORFs) have recently been identified in mammalian genomes (Orr et al., 2020). These smORFs and alt-ORFs are found in 5' and 3' untranslated regions of mRNAs, in frame-shifted ORFs overlapping protein-coding sequences, and within long noncoding RNAs (including pseudogenes and antisense RNAs) (Brunet et al., 2018). Some smORF-encoded proteins (SEPs, also termed micropeptides or microproteins) and alt-ORF-encoded proteins (alt-proteins), which we will collectively refer to as “small proteins” in this protocol (Orr et al., 2020), have been shown to play important biological roles (Chen et al., 2020; Cao et al., 2021; Magny et al., 2021), suggesting that defining the functions of small proteins represents a major opportunity to gain insights into biology. However, due to their short lengths and limited homology to protein domains of known function, the majority of unannotated small proteins remain uncharacterized. Similar to annotated proteins, the expression of some unannotated small proteins is cell-type specific (Cao et al., 2020), and is regulated by cellular stress (Jackson et al., 2018; Zhang et al., 2022). However, many prior reports on the discovery of unannotated small proteins do not provide information about differential expression or regulation. Methods to detect small proteins that are regulated by (patho)physiological processes and cellular stress should be of broad utility to enable hypothesis generation about their functions in high throughput. Bio-orthogonal non-canonical amino acid tagging (BONCAT) incorporates the methionine analog azidohomoalanine (AHA) into all cellular proteins synthesized by the endogenous protein translation machinery during the labeling window (Dieterich et al., 2006). The first reported BONCAT workflows derivatize AHA-labeled proteins with biotin-alkyne, requiring a column-based step to remove excess biotin-alkyne, which de-enriches small proteins prior to streptavidin capture of the labeled proteome (Cao et al., 2022). Here, we describe a modified approach to profile nascent small proteins, which interfaces AHA labeling with in-solution size selection, followed by click chemistry capture directly on dibenzocyclooctyne magnetic beads, enabling sensitive detection of small proteins using mass spectrometry proteomics coupled with bioinformatic methods for unannotated protein identification. In a proof-of-principle study demonstrating this approach, we identified 22 actively translated, unannotated small proteins and N-terminal extensions of canonical proteins, in a cultured human cell line under control and stress conditions; we further confirmed that one of these unannotated small proteins is post-transcriptionally upregulated by DNA damage stress, and another one is cell-cycle-regulated (Cao et al., 2022), suggesting that this method may be broadly useful to reveal the regulated synthesis of unannotated small proteins in cultured cells. Materials and Reagents 15-cm cell culture dish (Falcon, catalog number: 353025) 6-well cell culture plate (Greiner Bio-One, catalog number: 657160) 15 mL tube (Falcon, catalog number: 352096) PolyWAX LPTM column (PolyLC, catalog number: 104WX0510) Dulbecco’s modified Eagle’s medium (DMEM) (Corning, catalog number: 10-013-CV) DMEM without methionine (DMEM-Met) (Corning, catalog number: 17-204-CI) L-Azidohomoalanine (AHA) (Click Chemistry Tools, catalog number: 1066-100) Fetal bovine serum (FBS) (Sigma, catalog number: F4135) Penicillin/streptomycin (Pen/Strep) (Gibco, catalog number: 15140-122) Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (Click Chemistry Tools, catalog number: 1061-100) Copper(II) sulfate (CuSO4) (Sigma, catalog number: C1297) Biotin-alkyne (PEG4 carboxamide-Propargyl Biotin) (Click Chemistry Tools, catalog number: 1266-5) Bond Elut C8 cartridge (Agilent, catalog number: 12105028) DBCO (dibenzocycloctyne) magnetic beads (Click Chemistry Tools, catalog number: 1037-1) 2-Iodoacetamide (TCI, catalog number: I0741, CAS 144-48-9) Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma, catalog number: C4706) Sequencing grade modified trypsin (Promega, catalog number: V5111) Ethyl acetate (Sigma, catalog number: 270989) C18 spin columns (Thermo Scientific, catalog number: 89873) Triethylamine (TEA) (Sigma, catalog number: 471283) 88%–91% formic acid (FA) (Sigma, catalog number: 399388) Acetonitrile (ACN) (Sigma, catalog number: 271004) Trifluoroacetic acid (TFA) (Sigma, catalog number: 302031) Methanol (Sigma, catalog number: 34860) Chloroform (Avantor, catalog number: JT-9180-01) Triton X-100 (Amresco, catalog number: 0694) DMSO (Dimethyl sulfoxide) (Alfa Aesar, catalog number: 36480) Streptavidin-HRP (Invitrogen, catalog number: S911) Ammonium bicarbonate (NH4HCO3) (Sigma, catalog number: A6141) Urea (CH4N2O) (Sigma, catalog number: U5128) Potassium chloride (KCl) (Avantor, catalog number: 3040-01) Sodium carbonate (Na2CO3) (Sigma, catalog number: 230952) Calcium chloride (CaCl2) (MP Biomedicals, catalog number: 153502) Lysis buffer (see Recipes) 0.25 M TEAF pH 3.0 (see Recipes) RIPA buffer (see Recipes) 4× SDS loading buffer (see Recipes) Water-saturated ethyl acetate (see Recipes) Equipment Refrigerated centrifuge (Eppendorf, model: 5424 R) Refrigerated centrifuge (Eppendorf, model: 5810 R) SpeedVac vacuum concentrator (Thermo Scientific, Savant SPD10) Vortex mixer (VWR, G-560 Vortexer 2) Tube rotator (Thermo Scientific, catalog number: 05-450-127) Heat block (Thermo Scientific) 37 °C incubator (Thermo Scientific) Q Exactive Plus Hybrid Quadrupole Orbitrap (Thermo Scientific) -80 °C freezer (Thermo Scientific) -20 °C freezer (Thermo Scientific) NEB magnetic holder (NEB, S1509S) Agilent 1100 HPLC (Agilent) Additional reagents and equipment for standard immunoblotting procedures Software Thermo Scientific Xcalibur (version 4.4) Mascot (version 2.5.1) Image Lab (version 5.2) Agilent Chemstation for LC systems (version B.04.03) Procedure AHA labelling of newly synthesized proteins Seed 2.5 × 107 HEK 293T cells in a 15-cm cell culture dish, followed by culture in complete DMEM with 10% FBS and 1% Penicillin/Streptomycin in a 5% CO2 atmosphere at 37 °C overnight, to achieve 80%–90% confluency. Aspirate medium from the cells, wash once with 10 mL of room-temperature (RT) 1× PBS, then add 10 mL of pre-warmed DMEM-Met, and incubate in a 5% CO2 atmosphere at 37 °C for 30 min. Prepare 0.4 M AHA (100× stock, which is stable at -20 °C for at least 2 months) in 1× PBS. Gently remove DMEM -Met from the cells, then gently add 10 mL of pre-warmed DMEM-Met with 4 mM AHA (1×) and 10% FBS. Incubate in a 5% CO2 atmosphere at 37 °C for 2 h, to label the newly synthesized proteins with AHA. Remove the medium, wash twice with 10 mL of 1× PBS, and harvest the cells by pipetting up and down (Note: other adherent cell types likely need to be harvested with gentle scraping). Transfer the cells into a 15 mL tube, centrifuge at 800 × g and RT for 3 min, and gently aspirate the supernatant. Note: Samples can be flash frozen and stored for 1–2 months at -80 °C at this point. Validation of AHA labelling and C8 column-based size-selection Especially if performing BONCAT labeling for the first time, it is important to confirm AHA incorporation into the cellular proteome, prior to proceeding to proteomic analysis. To detect AHA-labeled proteins, we recommend that the cells from a single well of a 6-well cell culture plate, with protein concentration-normalized lysate from unlabeled or vehicle-treated cells as a negative control, be subjected to Click chemistry with commercially available biotin-alkyne, followed by streptavidin-HRP blotting, as shown in Figure 1. This step can be performed on a separate sample that has been prepared in parallel to the cells to be used for proteomic analysis. Add 200 µL of 1% w/v SDS in 1× PBS to the cell pellet, followed with boiling at 100 °C for 10 min. In parallel for all steps, process an unlabeled (non-AHA-treated) pellet of an equal number of the same cells as a negative control, to assess labeling efficiency over background. Add 1.8 mL of 1× PBS containing 0.2% Triton X-100 to the cell lysate. For 1 mL of total lysate, prepare a mixture by adding 2 µL of a 350 mM stock of CuSO4 in water, 1 µL of 200 mM TBTA stock in DMSO, and 3.5 µL of 0.5 M TCEP stock in water. Mix and incubate at RT for 10 min, to form the click chemistry catalyst in situ. Note: The remaining 1 mL of lysate can be reserved for 1–2 days at -20 °C, in case repeat analysis is required; if the remaining lysate is not required, it can be discarded. Add 1 µL of 25 mM biotin-alkyne in DMSO to the mixture from step B1c, then add this mixture to 1 mL of the cell lysate from step B1b. Incubate with rotation at RT for 6 h, to derivatize AHA-labeled proteins with biotin via click chemistry. Transfer 100 µL of the click chemistry reaction to a new 1.5-mL tube, add 400 µL of methanol, and vortex for 5 s. Add 100 µL of chloroform and vortex for 5 s. Add 300 µL of ddH2O and vortex for 5 s, followed by centrifugation at 18,407 × g at RT for 2 min. Remove and discard the top layer, add 400 µL of methanol to the remaining bottom layer, vortex for 5 s, and centrifuge at 18,407 × g at RT for 2 min. You should observe the formation of a protein pellet. Remove the supernatant without disturbing the pellet, and dry it at RT for 5 min. To resuspend the pellet containing labeled cellular proteins, add 50 µL of 1× SDS loading buffer, then boil at 100 °C for 10 min. Load the resuspended labeled proteins onto a 12% SDS-PAGE gel, followed by transfer to nitrocellulose, and western blotting with streptavidin-HRP, as previously described (Cao et al., 2021). Perform Ponceau staining of the membrane after transfer, to confirm equal loading. Figure 1. AHA labeling validation with streptavidin-HRP blotting. (A) Experimental scheme for AHA labeling validation. (B) Streptavidin-HRP blotting reflects AHA labeling, and Ponceau staining served as a loading control. Note: If AHA labeling is inefficient, AHA treatment time and concentration can be optimized. If the AHA labeling is successful in the replicate sample, add 2 mL of lysis buffer (see Recipes) to an AHA labeled HEK 293T cell pellet from a 15-cm dish from step A4 and vortex, followed by boiling at 100 °C for 10 min. Repeat vortexing and boiling three to four times, until the cell lysate becomes clear. We typically aliquot the lysate into four 1.5-mL Eppendorf tubes for the boiling step, to fit the wells of our heating block, then recombine the lysates for step B3c below, but this can be adapted to any appropriate tube size or boiling apparatus. Size select to enrich small proteins with two C8 columns, as follows (Ma et al., 2016): Add 500 µL of methanol to each of the two C8 columns, centrifuge at 200 × g for 1 min, and discard the flow-through. Add 1 mL of 0.25 M TEAF pH 3.0 (see Recipes) to each of the two C8 columns, then centrifuge at 200 × g for 1 min, and discard the flow-through. Load 1 mL of the cell lysate from step B2 to each of the two C8 columns (e.g., combined lysates from two Eppendorf tubes for each C8 column), then centrifuge at 200 × g for 2 min, and discard the flow-through. Add 1 mL of 0.25 M TEAF pH 3.0 to each of the two C8 columns, then centrifuge at 200 × g for 2 min, and discard the flow-through. Add 0.5 mL of 3:1 ACN:0.25 M TEAF pH 3.0 to each of the two C8 columns, centrifuge at 200 × g for 2 min, and retain the eluate. Repeat this step one more time, and combine all eluates from all columns. Speedvac the solution to dryness at RT. This step may take up to 3 h. Click capture on beads Wash 50 µL of DBCO bead slurry with 1 mL of RIPA buffer (see Recipes), place the tube on a magnetic holder, remove and discard the RIPA buffer. Add 1.2 mL of RIPA buffer to the size selected cell lysate from step B3f , vortex for 10 s, then resuspend the washed DBCO beads in the cell lysate. Rotate at RT for 1 h, then place the tube on a magnetic holder, remove and discard the supernatant. Wash the DBCO beads as follows: Add 1 mL of RIPA buffer to the beads, rotate at RT for 3 min, then place the tube on a magnetic holder, and remove the supernatant. Add 1 mL of 1 M KCl in ddH2O to the beads, rotate at RT for 3 min, then place the tube on a magnetic holder, and remove the supernatant. Add 1 mL of 0.1 M Na2CO3 in ddH2O to the beads, rotate at RT for 1 min, then place the tube on a magnetic holder, and remove the supernatant. Add 1 mL of 2 M urea in ddH2O to the beads, rotate at RT for 1 min, then place the tube on a magnetic holder, and remove the supernatant. Add 1 mL of RIPA buffer to the beads, rotate at RT for 1 min, then place the tube on a magnetic holder, and remove the supernatant. Repeat this step one more time. Add 1 mL of 1× PBS to the beads, rotate at RT for 1 min, then place the tube on a magnetic holder, and remove the supernatant. Repeat this step five more times. On-bead digestion Add 400 µL of 6 M urea in 1× PBS to the DBCO beads from step C3f , then add 20 µL of 0.2 M TCEP in ddH2O. Incubate the beads at 60 °C for 10 min, then cool down to RT. Add 20 µL of 0.4 M 2-iodoacetamide in ddH2O, then incubate with shaking at 37 °C for 15 min. Dilute the solution by adding 950 µL of 1× PBS, then place the tube on a magnetic holder, and remove the supernatant. Add 300 µL of a premixed solution of 2 M urea in 1× PBS, with 3 µL of 0.1 M CaCl2 in ddH2O, and 10 µL of 0.5 µg/µL trypsin; incubate in an incubator or water bath at 37 °C for 14–16 h. Place the tube on a magnetic holder, and transfer the supernatant to a new tube. Wash the beads with 50 µL of water twice to liberate any remaining bound peptides, and combine the washes with the supernatant. Add 15 µL of 88%–91% FA to stop the digestion. Speedvac the solution to dryness at RT. This step may take up to 3 h. Note: Samples can be stored for 1–2 months at -80 °C at this point. Detergent removal with ethyl acetate Add 100 µL of 100 mM NH4HCO3 in ddH2O to the digested peptides from step D8 , and vortex for 10 s. Add 1 mL of water-saturated ethyl acetate (see Recipes) to the digested peptides solution, vortex for 10 s, followed by centrifugation at 18,407 × g at RT for 30 s. Remove and discard the upper layer. Repeat this step one more time. The remaining bottom layer contains the digested peptides. Speedvac the solution to dryness at RT. This step may take 1 h. De-salt peptides with a C18 spin column Add 100 µL of 5% ACN + 0.5% TFA in ddH2O to the digested peptides from step E3, and vortex for 10 s. Add 200 µL of 50% methanol in ddH2O to a C18 spin column, then centrifuge at 200 × g for 2 min, and discard the flow-through. Add 150 µL of 5% ACN + 0.5% TFA in ddH2O to the C18 spin column, then centrifuge at 200 × g for 2 min, and discard the flow-through. Repeat this step one more time. Add the digested peptide solution to the C18 spin column, centrifuge at 200 × g for 2 min, and discard the flow-through. Wash the C18 spin column with 150 µL 5% ACN + 0.5% TFA in ddH2O, centrifuge at 200 × g for 2 min, and discard the flow-through. Repeat this step one more time. Elute the digested peptides with 45 µL of 70% ACN in ddH2O, centrifuge at 200 × g for 2 min, and retain the eluate. Repeat this step one more time, and combine the two eluates. Speedvac the solution to dryness at RT. This step may take up to 2 h. Note: Samples can be stored for 1–2 months at -80 °C at this point. Mass spectrometry analysis of nascent small proteins Optional steps: ERLIC fractionation with a PolyWAX LP column using Agilent 1100 HPLC, as shown in Figure 2. Add 55 µL of 85% ACN/0.1% FA in ddH2O to the digested peptides from step F7, vortex for 10 s, and load 50 µL of this onto a PolyWAX LP column (150× 1.0-mm; 5 µm 300 Å). Retain 10% of the total peptide suspension for prefractionation LC-MS/MS analysis, as a quality control step. Separate the peptides with a 80-min gradient, as follows (solvent A: 80% ACN 0.1% FA in ddH2O; solvent B: 30% ACN 0.1% FA in ddH2O): set the flow rate to 300 μL/min, start with 0% B for 5 min, then 0% B to 8% B over 17 min, 8% B to 45% B over 25 min, and 45% B to 100% B over 10 min. Maintain the isocratic flow at 100% B for 5 min, then gradient from 100% B to 0% B over 10 min, and maintain the isocratic flow at 0% B for 8 min. Collect fractions 1–10 at 1-min intervals, fractions 11–13 at 10-min intervals, and fractions 14–15 at 20-min intervals. Dry the fractions and the 10% unfractionated peptides in the Speedvac, and re-suspend each sample in 10 µL of 0.1% FA in ddH2O before LC-MS/MS analysis. Figure 2. ERLIC fractionation of digested peptides If ERLIC fractionation has not been performed, add 35 µL of 0.1% FA in ddH2O to the digested peptides from step F7. Centrifuge at 18,407 × g and 4°C for 30 min, and discard the pellet. If ERLIC fractionation has been performed, inject 5 µL of each fraction, as well as 5 µL of 10% unfractionated peptides, directly on a prepacked column attached to a nanoAcquity UPLC (Waters) in-line with a Thermo Scientific Q Exactive Plus Hybrid QuadrupoleOrbitrap mass spectrometer. If ERLIC has not been performed, 5 µL of the total digested peptides is injected. Separate the peptides with a 130-min gradient as follows (solvent A: 0.1% FA in ddH2O; solvent B: ACN with 0.1% FA in ddH2O): set the flow rate to 0.1 μL/min, start with 1% B for 40 min, then gradients 1% B to 6% B over 2 min, 6% B to 24% B over 48 min, 24% B to 48% B over 5 min, and 48% B to 80% B over 5 min. Maintain the isocratic flow at 80% B for 5 min, then gradient from 80% B to 1% B over 5 min, and maintain the isocratic flow at 1% B for 10 min. Set the mass range to 300–1,700 m/z with a resolution of 70,000, and the automatic gain control (AGC) target to 3 × 106. Collect the MS/MS data using a top 10 high-collisional energy dissociation (HCD) fragmentation method in data-dependent mode, with a normalized collision energy of 27.0 eV and a 1.6 m/z isolation window with a 17,500 MS/MS resolution, and a 90 s dynamic exclusion, according to the manufacturer’s instructions. Note: Mass spectrometry parameters may require optimization for the instrument available to the experimenter, for example in consultation with a mass spectrometry core facility. Data analysis Peptide and protein identification Convert raw data files to Mascot Generic File (MGF) files, and, for combined annotated and unannotated protein identification, search the data against a database comprised of a three-frame translation of assembled transcripts from RNA-seq data, as previously described (Khitun and Slavoff, 2019), plus a contaminants database provided by Mascot, using Mascot (version 2.5.1). Search the same datasets against the UniProt human proteome database plus a contaminants database separately, for quality control: in a small protein BONCAT experiment without ERLIC fractionation, a typical run detects 1,968 annotated human proteins, and 4.88% are typically <100 amino acids. Set oxidation of methionine and N-terminal acetylation as variable modifications, and carbamidomethylation of cysteine as a fixed modification. Set enzyme specificity to semiTrypsin, and allow a maximum missed cleavage of 2. Set peptide charge to 2+, 3+ and 4+. Set the peptide mass tolerance to 20 ppm, and fragment mass tolerance to 0.02 Da, as shown in Figure 3. Click Run. The search may take up to 8 h. Note: We do not set azidohomoalanine as a variable modification during the search, because those fragments containing azidohomoalanine remain bound to the beads, and were removed during digested peptides extraction. We will identify other tryptic peptides from the same protein. Figure 3. Mascot parameter setting Once the run is finished, open the Result File URL. Identify and validate the unannotated peptides as shown in our previously published protocol paper (Khitun and Slavoff, 2019). Briefly, peptide-spectral matches to annotated proteins are removed with a custom script available via Zenodo (https://doi.org/10.5281/zenodo.5921116), via matching against a locally downloaded file containing the current human proteome annotation. Remaining peptide-spectral matches are subjected to NCBI protein BLAST against the non-redundant (NR) human protein database, using default parameters for short input sequences to confirm their uniqueness relative to annotated proteins. Any peptides found to be less than two amino acids different from the nearest annotated protein (predicted proteins with NP or XP designation are not included) match are discarded, to eliminate the possibility of false positive matches due to mutations or post-translational modifications. We note that amino acids I and L are isobaric and cannot be distinguished by mass spectrometry, so peptides differing an annotated protein by two amino acids, but one of the amino acids is I or L, should also be discarded. Because small protein identifications often rely on only a single peptide-spectral match, MS/MS spectra are manually inspected for five additional key parameters (Figure 4, right). Peptide-spectral matches that do not pass these criteria are discarded. A high-quality MS/MS spectrum from a recently identified alt-protein called MINAS-60 (Cao et al., 2022) is shown in Figure 4, as an example. Peptide-spectral matches that pass the filters are computationally mapped to their encoding transcripts and unique genomic loci. Briefly, transcript sequences corresponding to the candidate unannotated peptides identified in step 3 are extracted, which can be found in the transcript database used to generate the 3-frame proteomic search database, and then translated into amino acid sequences in three frames in a format amenable to visual analysis using the ExPASy translate tool. The smORF encoding each unannotated tryptic peptide is identified based on the presence of the tryptic peptide sequence within the translated region corresponding to the smORF. Experimental validation of smORF expression at the molecular level is ultimately required to assign a novel SEP, and exclude the remaining possibility of false positive identifications (for example, resulting from multiple isobaric substitutions within an assigned peptide-spectral match), as previously described (Khitun and Slavoff, 2019). Figure 4. High-quality MS/MS spectrum from MINAS-60. Y and b ions are product ions generated from fragmentation of the parent tryptic peptide. A y ion is an ionized peptide fragment bearing charge on the C-terminus, and a b ion is a peptide fragment ion bearing the charge on the N-terminus. Recipes Lysis buffer 50 mM HCl 0.01% β-mercaptoethanol (v/v) 0.05% Triton X-100 (v/v) 0.25 M TEAF pH3.0 To make a 50 mL solution, add 2 mL triethylamine and 2 mL formic acid to 46 mL ddH2O. RIPA buffer 10 mM Tris-HCl (pH = 7.4) 1% Triton X-100 (v/v) 0.1% sodium deoxycholate (w/v) 0.1% SDS (w/v) 140 mM NaCl 4× SDS loading buffer 240 mM Tris-HCl (pH = 6.8) 40% glycerol (v/v) 8% SDS (w/v) 5% β-mercaptoethanol (v/v) 0.04% bromophenol blue (w/v) Water-saturated ethyl acetate To make a 50 mL solution, add 5 mL ddH2O to 45 mL ethyl acetate. Acknowledgments This work was supported by a Mark Foundation for Cancer Research Emerging Leader Award (21-055-ELA), a Paul G. Allen Frontiers Group Distinguished Investigator Award, a Sloan Research Fellowship (FG-2022-18417), a Searle Scholars Program Award, an Odyssey Award from the Richard and Susan Smith Family Foundation, and start-up funds from Yale University West Campus (to S. A. S.). X.C. was supported in part by a Rudolph J. Anderson postdoctoral fellowship from Yale University. A.K. was in part supported by an NIH Predoctoral Training Grant (5T32GM06754 3-12). Competing interests The authors declare no competing interests. References Brunet, M. A., Levesque, S. A., Hunting, D. J., Cohen, A. A. and Roucou, X. (2018). Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship. Genome Res 28(5): 609-624. Cao, X., Khitun, A., Harold, C. M., Bryant, C. J., Zheng, S. J., Baserga, S. J. and Slavoff, S. A. (2022). Nascent alt-protein chemoproteomics reveals a pre-60S assembly checkpoint inhibitor.Nat Chem Biol 18(6): 643-651. Cao, X., Khitun, A., Luo, Y., Na, Z., Phoodokmai, T., Sappakhaw, K., Olatunji, E., Uttamapinant, C. and Slavoff, S. A. (2021). Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24. Nat Commun 12(1): 508. Cao, X., Khitun, A., Na, Z., Dumitrescu, D. G., Kubica, M., Olatunji, E. and Slavoff, S. A. (2020). Comparative Proteomic Profiling of Unannotated Microproteins and Alternative Proteins in Human Cell Lines. J Proteome Res 19(8): 3418-3426. Chen, J., Brunner, A. D., Cogan, J. Z., Nunez, J. K., Fields, A. P., Adamson, B., Itzhak, D. N., Li, J. Y., Mann, M., Leonetti, M. D., et al. (2020). Pervasive functional translation of noncanonical human open reading frames. Science 367(6482): 1140-1146. Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A. and Schuman, E. M. (2006). Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Proc Natl Acad Sci U S A 103(25): 9482-9487. Jackson, R., Kroehling, L., Khitun, A., Bailis, W., Jarret, A., York, A. G., Khan, O. M., Brewer, J. R., Skadow, M. H., et al. (2018). The translation of non-canonical open reading frames controls mucosal immunity.Nature 564(7736): 434-438. Khitun, A. and Slavoff, S. A. (2019). Proteomic Detection and Validation of Translated Small Open Reading Frames. Curr Protoc Chem Biol 11(4): e77. Ma, J., Diedrich, J. K., Jungreis, I., Donaldson, C., Vaughan, J., Kellis, M., Yates, J. R., 3rd and Saghatelian, A. (2016). Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides. Anal Chem 88(7): 3967-3975. Magny, E. G., Platero, A. I., Bishop, S. A., Pueyo, J. I., Aguilar-Hidalgo, D. and Couso, J. P. (2021). Pegasus, a small extracellular peptide enhancing short-range diffusion of Wingless. Nat Commun 12(1): 5660. Orr, M. W., Mao, Y., Storz, G. and Qian, S. B. (2020). Alternative ORFs and small ORFs: shedding light on the dark proteome.Nucleic Acids Res 48(3): 1029-1042. Zhang, C., Zhou, B., Gu, F., Liu, H., Wu, H., Yao, F., Zheng, H., Fu, H., Chong, W., Cai, S., et al. (2022). Micropeptide PACMP inhibition elicits synthetic lethal effects by decreasing CtIP and poly(ADP-ribosyl)ation. Mol Cell 82(7): 1297-1312 e1298. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > Protein > Detection Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols In-Cell Western Protocol for Semi-High-Throughput Screening of Single Clones Arpita S. Pal [...] Andrea L. Kasinski Aug 20, 2022 2195 Views Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging Kristina Seiler [...] Mario P. Tschan Jul 20, 2023 896 Views Protein Level Quantification Across Fluorescence-based Platforms Hector Romero [...] M. Cristina Cardoso Oct 5, 2023 854 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,586	https://bio-protocol.org/en/bpdetail?id=4586&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Preparation of Caenorhabditis elegans for Scoring of Muscle-derived Exophers KB Katarzyna Banasiak MT Michał Turek WP Wojciech Pokrzywa Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4586 Views: 714 Reviewed by: Andrea PuharRama Reddy Goluguri Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in EMBO Reports Jul 2021 Abstract Utilizingresources available from the mother's body to guarantee healthy offspring growth is the fundamental reproductive strategy. Recently, we showed that a class of the largest extracellular vesicles known as exophers, which are responsible for the removal of neurotoxic components from neurons (Melentijevic et al., 2017) and damaged mitochondria from cardiomyocytes (Nicolás-Ávila et al., 2020), are released by the Caenorhabditis elegans hermaphrodite body wall muscles (BWM), to support embryonic growth (Turek et al., 2021). Employing worms expressing fluorescent reporters in BWM cells, we found that exopher formation (exophergenesis) is sex-specific and fertility-dependent. Moreover, exophergenesis is regulated by the developing embryo in utero, and exophers serve as transporters for muscle-generated yolk proteins, which can be used to nourish the next generation. Given the specific regulation of muscular exophergenesis, and the fact that muscle-generated exophers are much larger than neuronal ones and have different targeting, their identification and quantification required a modified approach from that designed for neuronal-derived exophers (Arnold et al., 2020). Here, we present a methodology for assessing and quantifying muscle-derived exophers that can be easily extended to determine their function and regulation in various biological contexts. Graphical abstract Keywords: C. elegans Body wall muscle Extracellular vesicle Exopher Fluorescent microscopy Background Maintaining protein homeostasis (proteostasis) requires the degradation of damaged or unwanted proteins and plays a key role in the function of cells and organisms. The main proteolytic component of the cellular proteostasis network is the ubiquitin-proteasome system (UPS), by which protein substrates are labelled by attachment of a small ubiquitin protein and then targeted for degradation by the proteasome. In addition, the autophagy-lysosome pathway promotes proteostasis through the turnover of aggregated proteins and obsolete organelles (Mizushima and Komatsu, 2011; Gatica et al., 2018). Recently, a novel proteostasis mechanism was described in Caenorhabditis elegans , where protein aggregates, mitochondria, and lysosomes are removed from worm neurons to the hypodermis via large vesicles (of approximately 4 μm in diameter) called exophers (Melentijevic et al., 2017). Exophers are generated independently of the endosomal sorting complexes required for transport (ESCRT) machinery, and their surface lacks phosphatidylserine, distinguishing them from exosomes or apoptotic bodies. Conditions that interfere with proteostasis, and when protein turnover or autophagy is inhibited, increase exopher production. Exophers are sorted in the ced-1, ced-6, and ced-7 engulfment pathways (Melentijevic et al., 2017). Exophergenesis is evolutionarily conserved, as the malfunctioning mitochondria are excreted via exophers by mouse cardiomyocytes (Nicolás-Ávila et al., 2020), and exophers were identified in the human and mice neurons (Siddique et al., 2021). However, the biological function of exophers is not limited to eliminating cellular waste. We have demonstrated that the body wall muscle (BWM) of C. elegans can expel cellular content via robust exophers, which is later used to nourish the next generation. We identified these using the worms’ strains with fluorescently labeled mitochondria (GFP anchored in the mitochondrial membrane through a sequence of 50 aa of TOMM-20) and proteasome subunits, RPN-5 (19S subunit), PAS-7, or PAS-4 (20S subunits), as we initially studied exophers with regard to protein turnover. Muscle exophers, like their neuronal counterparts, are jettisoned from the cell body. Some remain connected to the extruding cell by a flexible tube that permits the transfer of cellular material to the attached vesicle (Figure 1A). Muscle exophers can also contain mitochondria, which in electron microscopy (EM) images showed increased surface area and disrupted cristae organization, as in cardiac exophers. Moreover, muscular exophergenesis is also not active during the larval stages, and its maximum level is reached around the second and third day of hermaphrodite adulthood (Turek et al., 2021). In addition, its activity is also reduced by the depletion of NADPH-cytochrome P450 reductase EMB-8 and actin-binding protein POD-1 (Melentijevic et al., 2017). However, in contrast to neurons or cardiomyocytes, we observed a low number of mitochondria-containing vesicles and a lack of significant changes in exophergenesis in response to proteotoxic stress. Our further analyses showed that BWM exophers represent a transgenerational metabolic/resource management system induced by the appearance of developing embryos in utero. In response, the newly formed exophers transport muscle-generated yolk proteins that can support the development of the offspring. Our data also suggests that BWM exophers are controlled by signals from developing embryos (Turek et al., 2021). Given the above, and that BWM exophers are generally more prominent in size than neuronal ones (up to 15 µm in diameter) and extruded into the body cavity, their identification, quantification, and analysis requires a modified methodology from that described for exophers of neuronal origin (Arnold et al., 2020). Here we provide details on the strains of C. elegans , time and conditions of growth, and a step-by-step procedure for imaging and quantifying BWM exophers. We believe this protocol will allow other laboratories to easily adapt their resources to decipher the autonomous and non-autonomous regulation of the BWM exophergenesis. Materials and Reagents Pipettes and pipette tips (Eppendorf) 1.5 mL and 50 mL polypropylene conical tube (VWR) Fluorescent reporters produced from body wall muscle promoter myo-3 that label cytoplasmic proteins, and/or organelles (mitochondria or lysosomes) that will be excreted in exophers can be used to visualise and measure the exophergenesis. We commonly use ACH93 or ACH81 worm strains, where wrmScarlet is fused to proteasome subunit (RPN-5), and GFP is tagged to the TOMM-20 mitochondrial protein of the outer membrane, allowing for exopher visualization (wrmScarlet fluorescent signal) and inspection of mitochondria presence (GFP fluorescent signal) (Figure 1B–1C). We have prepared the following protocol for the C. elegans strains we use most often (Table 1), but this procedure will also be effective for other lines, as, those based on cytoplasmic GFP produced in BWM (Pmyo-3::GFP), which is also present in released exophers. Table 1. Strains we use to study muscle-derived exophers C. elegans strain Description Source ACH93 wacIs1[myo-3p::rpn-5 CAI = 0.97::GGGGS Linker-wrmScarlet::unc-54 3′UTR, unc-119(+)], wacIs14[myo-3 promoter::tomm-20_1–50aa::attB5::mGFP::unc-54-3′UTR, unc-119(+)] Turek et al. (2021) ACH81 wacIs1[myo-3 promoter::rpn-5 CAI=0.97::Optimal Linker-wrmScarlet::unc-54 3’UTR, unc-119(+)] Turek et al. (2021) Figure 1. Fluorescently labelled (wrmScarlet) exophers extruded from the body wall muscles on day 2 of adulthood. A. Characteristics of muscle-derived exophers in C. elegans . B–C. Cross-sections of ACH93 worms (with a focus on the midbody) visualized in the RFP and GFP channels of the confocal microscope (Zeiss LSM800). The square region on panel C marked with a dashed line is magnified 3.2× on its right side. The arrows point to exophers, and the asterisks indicate the position of the vulva. Structures of an irregular, non-round shape or inside the BWM are not classified as exophers. Scale bars are 20 μm in B and 50 and 20 μm in C. D. The wrmScarlet signal may also originate from scattered and smaller vesicles (< 2 μm) present in the body cavity (circled with a dotted line). In our readings, we do not count these structures as exophers, as these are coelomocytes that degrade fluorescent proteins taken up from a pseudocoelom. Scale bar is 10 µm. E. Zoom into the vulva region in separate channels: DIC, a merge of RFP and GFP, separate RFP and GFP. The square marked with a dashed line marks the boundaries of the enlarged region in the bottom left corner. The arrows indicate exophers with mitochondria. MOM—mitochondrial outer membrane. Scale bar is 20 µm. LB broth (Sigma, catalog number: L3022-1KG) Na2HPO4 (Sigma-Aldrich, catalog number: S7907) KH2PO4 (Roth, catalog number: 3904.1) NaCl (Chempur, catalog number: 117941206) MgSO4 (Sigma-Aldrich, catalog number: M5921) Peptone (BioShop, catalog number: PEP403.1) Agar (BioShop, catalog number: AGR001.1) KPO4 (Roth, catalog number: P749.1) Streptomycin (Sigma-Aldrich, catalog number: S6501) Nystatin (Sigma-Aldrich, catalog number: N1638) Tetramisole hydrochloride (Sigma-Aldrich, catalog number: L9756) Escherichia coli OP50 strain or any other bacteria that can serve as a food source (obtained from the Caenorhabditis Genetics Center, CGC) (see Recipes) M9 buffer (see Recipes) Nematode growth medium (NGM) (see Recipes) Equipment Fluorescence stereo or confocal microscope that allows for at least 200× magnification (ZEISS Axio Zoom.V16 Motorized Fluorescence Stereo Zoom Microscope) Incubator for C. elegans (Q-Cell, PolLab) Platinum home-made wire worm pick Alcohol burner (DWK Life Sciences Wheaton) Petri dishes for C. elegans cultivation (VWR) Software GraphPad Prism 9.3.1 (GraphPad Software) Excel 2019 (Microsoft Office) ZEN (Zeiss) Procedure Maintenance of C. elegans Since exophergenesis is a process that is influenced by food availability, make sure you provide constant, optimal conditions of worms’ culture: Keep worms at 20 °C for at least 2–3 generations before running an experiment (a necessary step, especially after thawing animals from -80 °C stocks). The authors avoid bleaching of gravid C. elegans followed by a short period of starvation of L1 larvae, to avoid the potential effects of bleach and food deficiency on exophergenesis in later stages of development. Instead, transfer gravid worms (or at any other desired developmental stage) to fresh NGM plates with bacterial food using a sterile platinum worm pick, or wash them off from old plates with M9 buffer and seed them using a pipette under sterile conditions. If you decide to transfer with M9, wash animals three times in the Eppendorf tube, to avoid contamination of a new plate with bacteria from the previous plate. Centrifugation at 2,000–3,000 rpm is permitted. Make sure worms have access to food ad libitum. Starvation severely affects exophergenesis. If you starve animals, maintain them for 2–3 generations with constant access to a food source before starting a new experiment. Minimize the risk of contamination by following good laboratory practices, wearing gloves, disinfecting the work surface, working beside the burner, sterilising the worm pick, etc. If you notice contamination (e.g., spots of other bacteria, or fungi on plates), discard the plate, and thaw a fresh stock of reporter strain. Otherwise, try to transfer eggs laid as far from the contamination as possible to an empty NGM plate, wait until they hatch, and transfer single worms to a fresh plate, preferably at the edge, where there is no bacterial food. Again, such worms should not be used for experiments before ensuring contamination is eliminated. Setup of worms for scoring exophers Obtain a synchronized population of either: Eggs—transfer approximately 30–50 gravid worms with a worm pick to a fresh plate, allow them to lay eggs for 2–3 h (in the 20 °C incubator), and then remove adult worms. Alternatively, from a plate that contains mixed-stage worms, transfer eggs that contain 3-fold embryos (pretzel stage embryo) to a fresh plate. Freshly hatched L1 larvae—remove all larvae and adult worms from plate(s) with an unsynchronized population (by washing them off with M9), leaving only eggs on a plate. Place plate(s) in the incubator for 30 min–2 h (depending on how many eggs were initially on the plate, and how many are needed for your experiment; the shorter the period of incubation, the better synchronized the population will be), and allow them to hatch in the meantime. Once you obtain enough synchronized eggs (method a) or freshly hatched L1s (method b), transfer them to fresh plates. Try to maintain a similar number of worms per plate. For experiments with sufficient statistical power, seeding 50 worms per 60 mm Petri dish is optimal. If you seed more, you risk insufficient access to the food throughout the experiment. Avoid plates with fluoro-deoxyuridine (FUdR), which inhibits exopher production. Conversely, less than 20 worms per plate would require more than three biological repeats to reliably visualize the differences between the groups. Maintain worms at 20 °C unless your hypothesis requires otherwise. Synchronous growth of larvae at later stages Monitor if the worms develop synchronously again at the L4 larval stage. Take the development of the vulva and its morphological differences in shape defined in subsequent sub-stages as a benchmark (Mok et al., 2015). To provide the best possible result reproducibility, sort worms at L4.5–L4.9 (approximately 43–46 h from hatching). This timing might require adjustment due to seasonal weather changes, if you do not use air conditioning set to a constant temperature throughout the year. Remove worms in other developmental stages. To ensure you do not miss any, you can transfer single worms of interest to fresh plates using a worm pick. Scoring exophers at adulthood day 2 The exophergenesis peak coincides with the highest egg-laying activity, as shown in Turek et al. (2021). The egg-laying behaviour of the hermaphrodite occurs ~65–128 h from hatching (Altun and Hall, 2012 in WormAtlas), with the maximal egg-laying at approximately 96 h. Therefore, for the highest reproducibility of the results, we score exophers on adulthood day 2, approximately 98 h (± 2 h) from hatching into L1 larvae. When worms reach the desired developmental stage, visualize them under the fluorescence microscope. At first, monitor their general fitness using brightfield—if the majority of control or single worms from the experimental group express abnormalities, do not use them for the research. Discard plates with contamination if not done earlier. A fluorescence stereomicroscope was used to score exophers in freely moving animals directly on NGM plates. We used a ZEISS Axio Zoom.V16 Motorized Fluorescence Stereo Zoom Microscope equipped with HXP 200 C fluorescence lamp (Kübler). For ACH81 and ACH93 strains, we used the Texas Red filter set to visualize wrmScarlet (wrmScarlet is fused to proteasome subunits of the BWM), and the GFP filter set (GFP is tagged to the mitochondrial outer membrane of the BWM). The GFP channel is not required if you are interested in the number of exophers, but not on the distribution of mitochondria. To acquire images with a confocal microscope, transfer worms onto freshly prepared 3% agarose (in H2O) pads on a microscope slide. Next, immobilize them with 25 μM tetramisole/levamisole and cover them with a round or square glass coverslip. We used an inverted Zeiss LSM 800 laser-scanning confocal microscope with a 40× oil objective, equipped with 488-nm and 561-nm lasers, to excite the GFP and RFP fluorescent proteins, respectively. To investigate the presence and distribution of exophers, collect Z-stacks and process them with ZEN (Zeiss) or ImageJ (Fiji) software. Score the exophers on the ≥ 200 magnification under a fluorescence stereomicroscope. You will see the biggest vesicles even at lower magnification (16×, objective 1.0), but your quantification will not be precise (Figure 1C , left panel). Most exophers will be located near the vulva region, but can also occur anywhere in the body cavity. Exophers are round vesicles that vary in size (Figure 1A). However, sometimes they remain attached to a releasing cell (Figure 1B–1C). Reject small vesicles with weak fluorescence that do not reach ~1/10 of the width of the BWM cell in quantifying exophergenesis, as well as any other irregular shaped fluorescent structures in the body cavity (Figure 1D). Data are collected as the number of exophers visible in a single worm from one experimental/control group. (e.g., if you have 40 animals on the plate, you should obtain 40 counts). Microsoft Excel is handy for the storage of the data generated this way. Maintain separate sheets for each biological repetition, to quickly detect any disturbances. Once you collect data from all repeats, we recommend you copy them to GraphPad Prism for further processing (statistical analysis, graphical visualization, Figure 2). Data analysis Each experiment consists of three independent biological replicates. We assume non-Gaussian distribution of residuals, and therefore apply nonparametric statistical tests: Mann–Whitney (when we compare only two groups) or Kruskal–Wallis test with Dunn's multiple comparisons (when we compare more than two groups). A p-value <0.05 is considered significant. Figure 2. An example of an experiment subjected to quantitative, statistical analysis, and visualization in GraphPad Prism software. The formation of muscle-derived exophers is affected by the depletion of EMB-8 (emb-8 RNAi on strain ACH93). ACH93 worms fed on HT115 E. coli bacteria with the empty vector were used as control. **** P < 0.0001, Mann–Whitney test. Notes According to “Synchronous growth of larvae at later stages”, if worms are precisely synchronized at the beginning of the experiment, usually a maximum of 10%–20% of the worms need to be removed at the L4 stage. If more, be cautious at the initial steps of the procedure. If scoring exophers in free-moving worms on a plate is too challenging initially, you may immobilize worms (with tetramisole) before you become proficient in counting them. The authors suggest removing freely moving hermaphrodites with already counted exophers directly afterward, to avoid unintentional re-measurement. Recipes Bacterial food source Prepare a bottle of 500 mL LB (according to the manufacturer’s protocol). Inoculate the LB in a flask with a single colony of OP50/HT115. Incubate with shaking at 220 rpm at 37 °C overnight and, on the following day, seed the centrifuged 5× concentrated bacteria on NGM plates. M9 buffer Reagent Final concentration Na2HPO4 42 mM KH2PO4 22 mM NaCl 86 mM MgSO4 1 mM Nematode growth gedium (NGM) (for 750 mL) Add 1.875 g peptone, 2.25 g NaCl, 12.75 g agar, and 712.5 mL of dH2O into a 1 L bottle and place a stir bar inside the bottle. Autoclave for 20 minutes. When the medium cools down to ~59 °C, add 7.5 mL of 0.1 M CaCl2 , 7.5 mL of 0.1 M MgSO4 , and 750 μL of filtered 5 mg/mL cholesterol. Stir with a magnetic stirrer for 3–5 min. Add 18.75 mL of 1 M KPO4 , 750 μL of streptomycin, and 1.9 mL of nystatin. Mix for 1 min, then pour on plates with a peristaltic pump under sterile conditions next to the flame, or maintain at ~55 °C before pouring later that day. Plates should be left to dry overnight, before seeding the worms. Acknowledgments ZEN (Zeiss), ImageJ (Fiji), and BioRender software were used to create the figures for this manuscript. Funding: K. Banasiak and W. Pokrzywa’s work is funded by the Foundation for Polish Science and co-financed by the European Union under the European Regional Development Fund [grant POIR.04.04.00-00-5EAB/18-00]. M. Turek is supported by the National Science Centre SONATA-BIS grant [2021/42/E/NZ3/00358]. This protocol was developed for the study described in Turek et al. (2021). Competing interests The authors declare no competing interests. References Altun, Z. F., and Hall, D. H. (2012). Introduction to C. elegans anatomy. WormAtlas. Arnold, M. L., Cooper, J., Grant, B. D. and Driscoll, M. (2020). Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans. J Vis Exp (163). doi: 10.3791/61368. Gatica, D., Lahiri, V. and Klionsky, D. J. (2018). Cargo recognition and degradation by selective autophagy. Nat Cell Biol 20(3): 233-242. Melentijevic, I., Toth, M. L., Arnold, M. L., Guasp, R. J., Harinath, G., Nguyen, K. C., Taub, D., Parker, J. A., Neri, C., Gabel, C. V., et al. (2017). C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542(7641): 367-371. Mizushima, N. and Komatsu, M. (2011). Autophagy: renovation of cells and tissues. Cell 147(4): 728-741. Mok, D. Z., Sternberg, P. W. and Inoue, T. (2015). Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage. BMC Dev Biol 15: 26. Nicolás-Ávila, J. A., Lechuga-Vieco, A. V., Esteban-Martinez, L., Sanchez-Diaz, M., Diaz-Garcia, E., Santiago, D. J., Rubio-Ponce, A., Li, J. L., Balachander, A., Quintana, J. A., et al. (2020). A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart. Cell 183(1): 94-109 e123. Siddique, I., Di, J., Williams, C. K., Markovic, D., Vinters, H. V. and Bitan, G. (2021). Exophers are components of mammalian cell neurobiology in health and disease. bioRxiv: 2021.2012.2006.471479. Turek, M., Banasiak, K., Piechota, M., Shanmugam, N., Macias, M., Sliwinska, M. A., Niklewicz, M., Kowalski, K., Nowak, N., Chacinska, A., et al. (2021). Muscle-derived exophers promote reproductive fitness. EMBO Rep 22(8): e52071. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Developmental Biology > Reproduction Cell Biology > Cell imaging > Fluorescence Molecular Biology > Protein > Protein shuttling Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Fixation and Immunostaining of Endogenous Proteins or Post-translational Modifications in Caenorhabditis elegans Robert O'Hagan and Irini Topalidou Oct 5, 2021 2556 Views Labelling of Active Transcription Sites with Argonaute NRDE-3—Image Active Transcription Sites in vivo in Caenorhabditis elegans Antoine Barrière and Vincent Bertrand Jun 5, 2022 1348 Views Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans Omar Peña-Ramos and Zheng Zhou Nov 20, 2022 937 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,587	https://bio-protocol.org/en/bpdetail?id=4587&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Myonecrosis Induction by Intramuscular Injection of CTX SF Simona Feno * FM Fabio Munari * GG Gaia Gherardi DR Denis Vecellio Reane DD Donato D’Angelo AV Antonella Viola RR Rosario Rizzuto AR Anna Raffaello (*contributed equally to this work) Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4587 Views: 1458 Reviewed by: Vivien Jane Coulson-ThomasDieu-Huong HoangMarielle Saclier Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Signaling Nov 2021 Abstract Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract Keywords: Skeletal muscle Regeneration Cardiotoxin Skeletal muscle injury Myonecrosis Background The final goal of regenerative medicine is to reconstitute tissue and organ functionality after injury or disease (Mao and Mooney, 2015). Tissue regeneration is a physiological process orchestrated by different infiltrating and tissue-resident cell types. Skeletal muscle is one of the most dynamic and plastic tissues of the human body and represents approximately 40% of total body weight in humans (Frontera and Ochala, 2015). Skeletal muscle not only controls posture maintenance and locomotion but plays a fundamental role in the maintenance of metabolic homeostasis, being involved in heat production and carbohydrate and amino acid storage (Schiaffino et al., 2013). In this context, muscle degeneration due to acute and chronic conditions leads to reduced mobility and strength, and to metabolic disorders (Schiaffino et al., 2013). Regeneration of skeletal muscle relies on a tightly regulated gene expression program and on the activation of specific signaling pathways that characterize muscle embryonic development (Chargé and Rudnicki, 2004). The ability of muscle to regenerate is principally due to a specific population of normally quiescent muscle stem cells called satellite cells that are strictly associated with muscle fibers (Chargé and Rudnicki, 2004). Satellite cells are myogenic precursor cells (MPCs) that reside in a quiescent state beneath the basal lamina that surrounds muscle fibers (Chargé and Rudnicki, 2004). In detail, soon after muscle damage, satellite cells escape quiescence and start to proliferate. A subset of these activated cells proliferate and differentiate, whereas others return to quiescence to reconstitute the pool of satellite cells that will be crucial for further rounds of skeletal muscle regeneration (Chargé and Rudnicki, 2004). Although satellite cells play a key role in muscle regeneration, their presence is not sufficient for efficient skeletal muscle repair, and many additional cell types play an active role in promoting tissue repair (Joe et al., 2010; Uezumi et al., 2010). Among them, key players in the complex scenario of skeletal muscle regeneration are inflammatory cells that invade muscle soon after injury and, together with satellite cells, contribute to a complete muscle regeneration (Tidball, 2017). Among the inflammatory cells, monocytes/macrophages play a major role in the repair process (Wang and Zhou, 2022). For example, macrophages release cytokines and growth factors that contribute to satellite cell activation, proliferation, and differentiation, according to their polarization state (Saclier et al., 2013). In particular, soon after muscle damage, macrophages adopt a pro-inflammatory profile, releasing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukine-6 (IL-6) and insulin-like growth factor (IGF-1) that, in turn, promote satellite cell proliferation (Tidball, 2017). Starting from three to five days post injury, these macrophages skew from a pro- to an anti-inflammatory profile, releasing anti-inflammatory cytokines that not only dampen the inflammatory response but also play trophic function by promoting satellite cell differentiation and fusion, thus contributing to the formation of new myofibers (Tidball, 2017). To date, different mouse models of acute muscle injury are available, as they represent an attractive system for investigating interactions between the immune system and satellite cells during skeletal muscle regeneration. Indeed, the onset of tissue damage is well defined and the time course of inflammation and regeneration predictable (Tidball, 2017). The most used injury models are freeze injury (FI), barium chloride injection, notexin (NTX) injection, and cardiotoxin (CTX) injection. The last one provides a useful model for sterile inflammation and, importantly, induces homogeneous damage to the whole muscle. Furthermore, it triggers the infiltration of many monocytes and macrophages participating in muscle repair, regeneration, and growth (Rigamonti et al., 2014). Here, we describe a protocol to induce muscle sterile injury by CTX injection. This method allowed us to study the influence of mitochondrial calcium uptake on macrophage metabolic profiles and on the skeletal muscle regeneration process (Feno et al., 2021). Myonecrosis is induced by intramuscular injection of CTX in tibialis anterior (TA) muscles, as performed in many other laboratories (Mounier et al., 2013; Perdiguero et al., 2011; Saclier et al., 2013). Compared with other protocols, our method has the great advantage of avoiding muscle laceration (Feno et al., 2021). Specifically, we can directly inject CTX in TA muscles without exposing them, thus avoiding surgery and consequent damage. Muscles are collected for analysis at different time points after injury (at 3, 7, and 14 days) to precisely characterize the time course of skeletal muscle regeneration. After muscle collection, regenerating TA muscles are frozen, and muscle sections are characterized by hematoxylin and eosin (H&E) staining to evaluate the efficiency of the CTX- induced skeletal muscle regeneration process. Materials and Reagents 8 weeks C57BL6N male mice (Charles River) Isoflurane 1000 mg/g (Piramal Critical Care, catalog number: 803249) 500 μM CTX (Merk, catalog number: 217503-1MG) 9% NaCl2 (Merk, catalog number: S3014) UltraPure DNase/RNase-Free distilled water (Thermo Fisher Scientific, catalog number: 10977049) 70% v/v ethanol (Merk, catalog number: 64-17-5) Isopentane (2-Methylbutane anhydrous ≥99%-1l) (Merck, catalog number: 277258) Liquid nitrogen Hematoxylin & Eosin (H&E) kit staining (Bio-Optica, catalog number: 04-061010) Optimal cutting temperature compound (O.C.T.) (Tissue-Tek, catalog number: 4583) Equipment VetFlo veterinary anesthesia apparatus Dissection tools: Fine scissors (Fine Science Tools, catalog number: 14160-10) Fine forceps (Fine Science Tools, catalog number: 11412-11) Standard forceps (Fine Science Tools, catalog number: 11150-10) Microcentrifuge tubes 1.5 mL (Sarstedt, catalog number: 72706) Cryovials (Sarstedt, catalog number: 72.694.006) Glass slides (Vetrotecnica, catalog number: 01.4230.34) 500 μL syringe 30 G (Vetrotecnica, catalog number: 11.3525.45) Plastic scoop Dewar bottle for liquid nitrogen Cryostat (Leica, model: CM1850) Procedure Depending on the experimental design, the number of mice required for experiments varies. To obtain statistically significant and reproducible results we used at least four adult (approximately 8 weeks old) male mice for each condition (e.g., not injected, and injected for 3, 7, and 14 days). In sterile conditions, prepare CTX stock solution by dissolving 1 mg of CTX powder (MW: 6.8 g/mol) in 294 μL (3.4 mg/mL) of sterile 0.9% NaCl2 to obtain a 500 µM solution of CTX. Aliquot the CTX stock solution and store it at -20 °C. To avoid freeze-thaw cycles, prepare aliquots of 10 μL. For TA injection, dilute the stock solution in sterile 0.9% NaCl2 to obtain a final solution of 10 μM CTX in a total volume of 50 μL for each TA muscle. Mix the solution by vortexing and keep it on ice until use. Note: CTX is not classified as dangerous, so no particular care must be taken. It is important to prepare and store the solution in sterile conditions. Anesthetize the animals with isoflurane, until they fall asleep. Isoflurane is administered in 100% O2 . Use an induction concentration of isoflurane of approximately 3%–4%. Maintenance concentrations are 1.25%–1.75% to avoid the overall alteration of mouse physiology. In this condition, mice can be anesthetized for up to 20–30 min. Fill a syringe (500 μL syringe 30 G, Vetrotecnica) with 50 μL of 10 μM CTX and keep it ready for use. Restrain the mouse properly and insert its head inside the anesthetic tube (Figure 1A). Shave the hindlimbs of the mouse with an electric razor and swab the area with 70% ethanol. Hold the foot of the hindlimb firmly, insert the needle of the syringe prepared in step 3 in the TA mid-belly region parallel to the muscle, and then gently pull the syringe plunger before injecting CTX to ensure that the needle has not entered a blood vessel (Figure 1B). The optimal positioning of injection sites is shown in Figure 2 by inserting the needle of the syringe parallel to the TA muscle. If no blood enters the syringe, 50 μL of CTX is to be slowly injected into TA muscle. Special care should be taken when injecting into TA to avoid going past the muscle tissue. Let the mouse recover from anesthesia and place it back in its cage. Figure 1. Anesthesia administration and CTX injection. After the induction with sufficient anesthesia in the animal, the leg is restrained (A), and injury by CTX injection can be inflicted by injection in the TA muscle (B). Figure 2. Optimal positioning of the CTX injection site (white dot) in the TA muscle At the selected time points post CTX injection, anesthetize the recipient mouse through isoflurane inhalation and sacrifice it by cervical dislocation. Regenerating TA can be collected as follows: Cut the skin below the ankle. Cut through the skin along the inner hindlimb. Expose the muscle fascia and patella by pulling the skin up, and remove the epimysium (Figure 3A). Separate the tendon of TA muscle using forceps along the inner margin of the shank (Figure 3B). Snip the tendons with the fine scissors (Figure 3C). Gently pull the TA muscle from the bone (Figure 3C). Below the TA you can see the extensor digitorum longus (EDL) muscle (Figure 3C). Cut the upper part of the TA near the patella (Figure 3D). You can proceed with TA freezing (Figure 3E and Figure 4). Figure 3. Procedures to isolate the TA muscle. Pictures depict (A) TA muscle after the removal of epimysium, (B) isolation of TA tendon, (C) cut of the TA tendon, (D) cut of the distal part of the TA, and (E) TA muscle. a. patella; b. TA; c. tendon of TA muscle; d. EDL muscle. Fill up the Dewar with liquid nitrogen and take all the material necessary for freezing muscles (isopentane, plastic scoop, standard forceps, and cryovials). Spray dissection tools and workspace with 70% ethanol. Then fill up a becher with isopentane (Figure 4A) and place it on the surface of the Dewar full of nitrogen to cool it down (to avoid the risk of solidification, the becher is laid on the surface of the liquid nitrogen) (Figure 4B). Place the isolated TA muscles on the plastic scoop and then inside the becher containing cold isopentane until the muscle is completely frozen (it takes approximately 30 s) (Figure 4C and 4D). Take a cryovial with a standard tweezer and cool it by dipping it inside liquid nitrogen (Figure 4E). Once the cryogenic vial is cold (5 s), remove the scoop from the isopentane, immediately detach the muscle from the plastic scoop, and place it into the cryovial (Figure 4F). This operation must be performed quickly to avoid thawing the muscles. The cryovials are conserved at -80 °C until use. Note: You can consider the muscle completely frozen when it whitens homogeneously. Figure 4. Procedure for freezing TA muscles for Hematoxylin and Eosin staining. Fill a small becher with isopentane (A), place it on the surface of a Dewar containing liquid nitrogen (B), place the isolated TA muscle on the plastic scoop (C), and then inside isopentane (D), immerge the cryovial in liquid nitrogen using a tweezer (E) and detach the muscle from the plastic scoop and place it into the cryovial (F). For sectioning, cut the frozen muscles transversely and embed one half, at the tendon site, on the cryostat chuck by using two drops of OCT. Allow the tissue block to equilibrate to the cryostat temperature (-20 °C) before cutting the sections. Cut 6 μm muscle slides with the cryostat and make them adhere on positively charged microscope glass slides. Dry the glass slides at room temperature until the sections are firmly adherent to the slide. Perform H&E staining, following the manufacturer’s instructions (Bio-Optica) (Figure 5). Figure 5. Hematoxylin and Eosin (H&E) staining of WT tibialis anterior (TA) muscles (Ctrl) noninjected and 3, 7, and 14 days after CTX injection. Scale bars, 50 μm. Data analysis To compare two data sets, as in Feno et al. (2021), in which we assessed the effect of mitochondrial Ca2+ on macrophage polarization during skeletal muscle regeneration, a parametric Student’s t-test and a parametric one-way analysis of variance (ANOVA) with post hoc Tukey’s multiple comparisons test were applied. Whenever the sample size was lower than five, nonparametric Mann-Whitney or nonparametric Kruskal-Wallis tests were performed to confirm the statistical analysis. P ≤ 0.05 was considered significant. Notes Intramuscular injection: During intramuscular injection, the mouse must be properly anesthetized and restrained. If the mouse is allowed to kick or struggle, this may injure the muscle or the sciatic nerve that runs along the length of the femur. Insert the needle of the syringe parallel to the TA muscle, and once injected, check muscle status. If the CTX injection has been done correctly, you should observe muscle swelling. Anesthetic procedure: Mice that are anesthetized must be monitored during the procedure to assure that they are maintained in the proper anesthetic level. Monitor the respiration and color of the mucosae membrane and exposed tissue. An increased respiration rate is a sign that the anesthesia is too light. On the contrary, a deep or irregular respiration rate means that anesthesia is too high. Acknowledgments We thank the Italian Association for Cancer Research (AIRC) (IG18633 to R.R.), the University of Padova (STARS@UNIPD WiC grant 2017 to R.R.), the Italian Telethon Association (GGP16029 to R.R. and GGP16026 to A.R.), the Italian Ministry of Health (RF-2016-02363566 to R.R. and GR-2016-02362779 to A.R.), the Cariparo Foundation (to R.R.), and ERC (grant number 3228. v23 to A.V.) for supporting this work. This protocol is derived from Feno et al. (2021), DOI: 10.1126/scisignal.abf3838. Competing interests The authors declare that there is no conflict of financial or research interests. Ethics In vivo experiments were performed in accordance with the Italian law D. L.vo n.26/2014. References Chargé, S. B. P. and Rudnicki, M. A. (2004). Cellular and molecular regulation of muscle regeneration. Physiol Rev 84(1): 209-238. Feno, S., Munari, F., Reane, D. V., Gissi, R., Hoang, D. H., Castegna, A., Chazaud, B., Viola, A., Rizzuto, R. and Raffaello, A. (2021). The dominant-negative mitochondrial calcium uniporter subunit MCUb drives macrophage polarization during skeletal muscle regeneration. Sci Signal 14(707): eabf3838. Frontera, W. R. and Ochala, J. (2015). Skeletal muscle: a brief review of structure and function. Calcif Tissue Int 96(3): 183-195. Joe, A. W. B., Yi, L., Natarajan, A., Le Grand, F., So, L., Wang, J., Rudnicki, M. A. and Rossi, F. M. V (2010). Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol 12(2): 153-163. Mao, A. S. and Mooney, D. J. (2015). Regenerative medicine: Current therapies and future directions. Proc Natl Acad Sci U S A 112: 14452-14459. Mounier, R., Théret, M., Arnold, L., Cuvellier, S., Bultot, L., Göransson, O., Sanz, N., Ferry, A., Sakamoto, K., Foretz, M., et al. (2013). AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration. Cell Metab 18(2): 251-264. Perdiguero, E., Sousa-Victor, P., Ruiz-Bonilla, V., Jardí, M., Caelles, C., Serrano, A. L. and Muñoz-Cánoves, P. (2011). p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair. J Cell Biol 195(2): 307-322. Rigamonti, E., Zordan, P., Sciorati, C., Rovere-Querini, P. and Brunelli, S. (2014). Macrophage plasticity in skeletal muscle repair. Biomed Res Int 2014: 560629. Saclier, M., Yacoub-Youssef, H., Mackey, A. L., Arnold, L., Ardjoune, H., Magnan, M., Sailhan, F., Chelly, J., Pavlath, G. K., Mounier, R., et al. (2013). Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration. Stem Cells 31(2): 384-396. Schiaffino, S., Dyar, K. A., Ciciliot, S., Blaauw, B. and Sandri, M. (2013). Mechanisms regulating skeletal muscle growth and atrophy. FEBS J 280(17): 4294-4314. Tidball, J. G. (2017). Regulation of muscle growth and regeneration by the immune system. Nat Rev Immunol 17(3): 165-178. Uezumi, A., Fukada, S., Yamamoto, N., Takeda, S. and Tsuchida, K. (2010). Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol 12(2): 143-152. Wang, X. and Zhou, L. (2022). The Many Roles of Macrophages in Skeletal Muscle Injury and Repair. Front Cell Dev Biol 10: 952249. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cell Biology > Tissue analysis > Injury model Cell Biology > Cell imaging > Cryosection Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols The Assessment of Beta Cell Mass during Gestational Life in the Mouse Yury Kryvalap and Jan Czyzyk Mar 20, 2023 446 Views Induction of Skeletal Muscle Injury by Intramuscular Injection of Cardiotoxin in Mouse Xin Fu [...] Ping Hu May 5, 2023 1623 Views Murine Double Hit Model for Neonatal Cardiopulmonary Diseases: Bronchopulmonary Dysplasia (BPD) and Pulmonary Hypertension Associated with BPD Steven P. Garrick [...] Claudia A. Nold-Petry Nov 5, 2022 1139 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,588	https://bio-protocol.org/en/bpdetail?id=4588&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Sample Preparation and Integrative Data Analysis of a Droplet-based Single-Cell ATAC-sequencing Using Murine Thymic Epithelial Cells TI Tatsuya Ishikawa * HI Hiroto Ishii * TM Takahisa Miyao KH Kenta Horie MM Maki Miyauchi NA Nobuko Akiyama TA Taishin Akiyama (*contributed equally to this work) Published: Vol 13, Iss 1, Jan 5, 2023 DOI: 10.21769/BioProtoc.4588 Views: 677 Reviewed by: Chiara AmbrogioOlli Matilainen Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE May 2022 Abstract Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis. Keywords: Single-cell ATAC-seq Single-cell RNA sequencing Thymus Thymic epithelial cells Integrative single cell analysis Nuclei isolation Background Genomic DNA is packaged into chromatin in nucleosome units by binding with histone proteins (Kornberg and Thomas, 1974; Kaplan et al., 2009). Post-translational modifications of nucleosomes regulate the activity of chromatin by controlling its accessibility to regulatory factors, such as transcription factors and chromatin remodelers (Kornberg and Thomas, 1974; Kaplan et al., 2009). To understand the accessibility of genomic DNA regions, especially of those that act as cis-regulatory elements such as promoters, enhancers, and insulators, several techniques have been invented, including the assay for transposase-accessible chromatin using sequencing (ATAC-seq) (Buenrostro et al., 2013, 2015). Ever since its introduction, ATAC-seq has become increasingly popular due to its high efficiency, which is accomplished by using a hyperactive Tn5 transposase (Yan et al., 2020; Luo et al., 2022). Tn5 transposases approach and insert sequencing adaptors into accessible DNA regions of chromatin structure. Amplification and sequencing of genomic DNA using inserted adaptors allow quantification of accessible chromatin regions in downstream analyses. Owing to the advent of ATAC-seq, scientists can now study epigenetic profiles of different cell types or tissues from healthy and diseased samples with relative ease (Yan et al., 2020). With the arrival of single-cell ATAC-seq (scATAC-seq), one can analyze chromatin accessibility at single-cell resolution. Previous ATAC-seq analysis of bulk samples failed to tease apart epigenetic profiles of specific cell types in heterogenous populations of cells. Formerly, it was impossible to comprehend developmental changes in chromatin accessibility of certain cell types on a mini-timescale (Klemm et al., 2019). To address these needs, 10× Genomics has released droplet-based scATAC-seq that allows profiling the chromatin accessibility of hundreds to thousands of individual cells. The droplet-based method uses a microfluidic device to capture each digested nucleus and to add barcodes in a gel-based emulsion. Alternatively, a bioinformatic approach can be implemented to integrate data from scATAC-seq with that of single-cell RNA-sequencing (scRNA-seq) to relate epigenetic profiles to cell types identified based on gene expression (Stuart et al., 2019). Clearly, the advantage of such integrative analysis is to transfer cell-type labels based on scRNA-seq data to scATAC-seq data, because annotation of cell types from the latter may be challenging due to inherent sparsity of genomic data. In our recently published paper in eLife, we used such an approach to determine chromatin accessibility of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using scRNA-seq (Wells et al., 2020; Miyao et al., 2022). This protocol details procedures of droplet-based scATAC-seq using the 10× Genomics platform. Using TECs as an example, we describe how cells and nuclei need to be prepared in order to perform scATAC-seq. We conclude with an example of integrating data obtained from scATAC-seq with cell clusters annotated in scRNA-seq. Materials and Reagents Cell-cultivated dish (ϕ 60 × 13.8 mm) (VIOLAMO, catalog number: VTC-D60) 50 mL conical tubes (Harmony, catalog number: CFT5000) 15 mL conical tubes (Harmony, catalog number: CFT1500) 5 mL conical tubes (Falcon, catalog number: 352008) Mice (Clea Japan, C57BL/6) LiberaseTM (Roche Diagnostics, catalog number: 05401127001) (dissolve 50 mg in 50 mL of RPMI and store at -20 °C) 0.5 M EDTA (pH 8.0) (NIPPON GENE, catalog number: 311-90075) Digitonin (5%) (InvitrogenTM , catalog number: BN2006) DNase I (Sigma, catalog number: DN25-100MG) D-PBS(-) without Ca and Mg, liquid (Nacalai, catalog number: 14249-95) Fetal bovine serum (FBS), Netherlands Origin (SRN, catalog number: S-FBS-NL-015) RPMI-1640 with L-glutamine (Wako, catalog number: 189-02025) BSA (Wako, catalog number: 013-15104) Chromium Next GEM single cell ATAC library & gel bead kit (10× Genomics, catalog number: PN-1000176) Chromium i7 Multiplex kit N, set A (10× Genomics, catalog number: PN-1000084) Chromium Next GEM Chip H single cell kit (10× Genomics, catalog number: PN-1000162) Single Index kit N, set A (10× Genomics, catalog number: PN-1000212) Acridine orange/propidium iodide stain (LUNA-FLTM, catalog number: F23001) Actinomycin D, 7-amino- (Calbiochem, catalog number: 7240-37-1) FACS antibodies Purified anti-mouse CD16/32 (BioLegend, catalog number: 101302) APC/Cyanine7 anti-mouse TER-119/erythroid cells (BioLegend, catalog number: 116223) APC/Cyanine7 anti-mouse CD45 (BioLegend, catalog number: 103116) FITC anti-mouse CD326 Ep-CAM (BioLegend, catalog number: 118208) Sodium chloride solution, 5M (SIAL, catalog number: S6546) Magnesium chloride solution for molecular biology, 1.00 M (SIG, catalog number: M1028) Digitonin, 5% (THERMO, catalog number: BN2006) Tween-20 (Wako, catalog number: 167-11515) Nonidet P40 substitute, 10% (Wako, catalog number:145-09701) Tris-HCl, 1 M, pH 7.4 (SIGMA, catalog number: T2194) FACS buffer (see Recipes) MACS buffer (see Recipes) 5% FBS-RPMI-1640 (see Recipes) 0.04% BSA-PBS (see Recipes) 1× lysis buffer (see Recipes) Lysis dilution buffer (see Recipes) Wash buffer (see Recipes) Diluted nuclei buffer (see Recipes) Equipment LUNA-FL dual fluorescence cell counter (LUNA-FLTM, catalog number: L20001) LUNATM cell counting slides, 50 slides (LUNA-FLTM, catalog number: L12001) BD FACS Aria II cell sorter (BD Biosciences, San Jose, CA) Microvolume pipettes (Rainin, models: Lite – XLS+, P200, catalog number: 17014391) NGS MagnaStand 8Ch (YS-Model) (FastGeneTM, catalog number: FG-SSMAG2) Water bath (Thermal Robo, catalog number: TR-1α) Microscope (Olympus, IX71) Software Cell Ranger (version 3.0.0, 10X Genomics Inc.) R (R Core Team, 2017; https://www.R-project.org/) R studio (R foundation, https://www.r-project.org) Seurat (Butler et al., 2018; version 4.1.0; https://satijalab.org/seurat/) Signac (Tim et al., 2021; version 1.6.0; https://satijalab.org/signac/) Procedure Sample preparation Mince murine thymus using razor blades in a 6 cm dish and transfer to a 1.5 mL tube. Add 1 mL of ice-cold RPMI-1640 to the minced thymus and gently pipette up and down. Discard supernatant as a thymocyte fraction. Note: Thymic stroma cells including TECs are contained in the pellet at this step. If necessary, keep supernatant for analysis of thymocytes. Repeat steps A2–A3 three times. Add 700 µL of Liberase/DNase I solution to the precipitate containing thymic stroma cells. Incubate the cell suspension in a 37 °C water bath for 12 min and then collect the supernatant to a 15 mL conical tube. Repeat steps A5–A6 three times. Centrifuge the cell suspension at 440 × g and 4 °C for 5 min. Discard the supernatant and resuspend the cell pellet in 1 mL of ice-cold FACS buffer. Count cells. Incubate approximately 1.0 × 108 /mL of cells with anti-mouse CD16/32 antibody at dilution 1:200 on ice for 20 min. Centrifuge the cell suspension at 1,000 × g and 4 °C for 1 min. Re-suspend the cell pellet in FACS buffer. Stain approximately 1.0 × 108 /mL of cells with APC/Cyanine7 anti-mouse TER-119/erythroid cells, APC/Cyanine7 anti-mouse CD45, and FITC anti-mouse CD326 Ep-CAM antibodies, at dilution 1:400 each, in the dark on ice for 20 min. Resuspend the cell pellet in FACS buffer and centrifuge at 1,000 × g for 1 min. Repeat step A15. Transfer cell suspension containing actinomycin D, 7-amino- to a 5 mL conical tube for cell sorting. Cell sorting Set up BD FACS Aria II cell sorter. Sort TECs based on the gating strategy shown below (Figure 1). Figure 1. Gating strategy of thymic epithelial cells. (a) The gating strategy excludes dead cells and debris. (b) Thymic epithelial cells are defined as CD45- TER119- EpCAM+ cells and sorted using a BD FACS Aria II cell sorter. Collect the sorted cells in a 1.5 mL tube containing 750 µl of 5% FBS-RPMI-1640. Check the purity by FACS analysis using 1/100 of sorted cells. Centrifuge the sorted cells at 500 × g and 4 °C for 5 min. Resuspend the cell pellet in 100 µL of 0.04% BSA-PBS. Count cells. Note: A minimum of 1.0 × 105 cells is required at this point. Nuclei isolation Centrifuge the sorted cells at 300 × g and 4 °C for 5 min. Remove the supernatant carefully. Add 100 µL of 1× lysis buffer and mix by pipetting 10 times. Note: Change the concentration of lysis buffer by diluting with lysis dilution buffer, depending on the type of tissue analyzed to prevent cell nuclei from bursting. Incubate for 3 min on ice for nuclei isolation by cell lysis. Note: Proper incubation time for cell lysis may vary depending on cell types. Immediately add 1 mL of wash buffer and mix by pipetting five times. Centrifuge the nuclei suspension at 500 × g and 4 °C for 5 min. Remove the supernatant carefully. Resuspend the cell pellet in 50 µL of diluted nuclei buffer. Count cell nuclei. Note: Efficiency of cell lysis can be monitored by cell viability. Viability must be less than 5%. Generally, viability decreases with increasing incubation time, but excessive incubation may impair nuclear quality. To confirm the quality of nuclei, check the nuclei under the microscope (Figure 2). Note: Criteria for nuclear quality is provided in the 10× Genomics protocol (Nuclei Isolation for Single Cell ATAC Sequencing). Figure 2. A representative image of high-quality nuclei prepared from TECs. (a) Nuclei observed with brightfield microscopy. (b) Nuclei observed after acridine orange/propidium iodide staining. Permeabilized nuclei emit orange fluorescence. Proceed to the scATAC-seq library construction and sequencing by following the 10× Genomics protocol (Chromium Single Cell ATAC Solution User Guide) and using the Chromium Next GEM single cell ATAC library & gel bead kit, Chromium i7 Multiplex kit N, set A, Chromium Next GEM Chip H single cell kit, and Single Index kit N, set A. Data analysis Based on the Seurat vignette (https://satijalab.org/seurat/articles/atacseq_integration_vignette.html), here we demonstrate a bioinformatic workflow to integrate results from scATAC-seq to results of scRNA-seq, using the Signac and Seurat R packages, respectively. FASTQ data generated from our recently published paper are deposited in DNA Data Bank of Japan (DRA009125, DRA012452, and DRA013875) (Miyao et al., 2022). Note: The above protocol is used for scATAC-seq analysis. For the integrative analysis below, scRNA-seq data of TECs are also necessary. The same protocol should be used up to the point of cell sorting. Then, sorted TECs should immediately be used for scRNA-seq analysis. Prepare data Load the libraries by entering the following commands in R: library(Signac) library(Seurat) library(ggplot2) Read the RDS file into a Seurat object by entering the following command: Note: RDS files are preprocessed into SeuratObjects format based on tutorials. Please refer to the following sites: Preparing scRNA-seq data: https://satijalab.org/seurat/articles/pbmc3k_tutorial.html Preparing scATAC-seq data: https://stuartlab.org/signac/articles/mouse_brain_vignette.html tec.atac <- readRDS("tec_atac.rds") tec.rna <-readRDS("tec_rna.rds") Rename cluster identification by adding “R” on scRNA-seq dataset by entering the following commands: new.Rcluster.ids <- paste0("R", levels(tec.rna$seurat_clusters)) names(x = new.Rcluster.ids) <- levels(x = tec.rna) tec.rna <- RenameIdents(object = tec.rna, new.Rcluster.ids) tec.rna$celltype <- Idents(tec.rna) Integrative analysis of scRNA-seq and scATAC-seq (Figure 3) Identify anchors between scRNA-seq and scATAC-seq datasets by entering the following commands: DefaultAssay(tec.atac) <- 'RNA' transfer.anchors <- FindTransferAnchors( reference = tec.rna, query = tec.atac, reduction = 'cca' ) Annotate scATAC-seq cells via label transfer the cluster identification from scRNA-seq data by entering the following commands: predicted.labels <- TransferData( anchorset = transfer.anchors, refdata = tec.rna$celltype, weight.reduction = tec.atac[['lsi']], dims = 2:30) tec.atac <- AddMetaData(object = tec.atac, metadata = predicted.labels) Visualize the original annotation of scATAC-seq and the predicted label-transferred annotation from scRNA-seq by entering the following commands: tec.atac$predicted.id <- factor(tec.atac$predicted.id, levels = levels(tec.rna)) p1 <- DimPlot(object = tec.atac, label = TRUE, repel = TRUE) + ggtitle('Original annotation') p2 <- DimPlot (object = tec.atac, group.by = 'predicted.id', label = TRUE, repel = TRUE) + ggtitle('Predicted annotation') p1 \| p2 Figure 3. The integrative analysis of scATAC-seq and scRNA-seq. The gene expression profile of a single cell from scATAC-seq data is predicted using the Signac package. It is then compared with that from scRNA-seq data to label transfer the annotation from scRNA-seq data to scATAC-seq data. TECs were separated as clusters R0 to R17 in the scRNA-seq analysis and clusters 0 to 11 in the scATAC-seq analysis. Label-transferred annotations predicted from scRNA-seq are exhibited in the UMAP dimension of scATAC-seq analysis in the figure on the right. Recipes FACS buffer Reagent Final concentration Amount D-PBS(-) n/a 490 mL FBS 2% 10 mL Total n/a 500 mL MACS buffer Reagent Final concentration Amount D-PBS(-) n/a 490 mL BSA 2% 10 mL Total n/a 500 mL 5% FBS-RPMI-1640 Reagent Final concentration Amount RPMI-1640 n/a 475 mL FBS 5% 25 mL Total n/a 500 mL 0.04% BSA-PBS Reagent Final concentration Amount D-PBS(-) n/a 996 µL BSA (10%) 0.04% 4 µL Total n/a 1,000 µL 1× lysis buffer Reagent Final concentration Amount Tris-HCl (1 M, pH 7.4) 10 mM 10 µL NaCl (5 M) 10 mM 2 µL MgCl2 (1 M) 3 mM 3 µL Tween-20 (10%) 0.1% 10 µL Nonidet P40 substitute (10%) 0.1% 10 µL Digitonin (5%, incubate at 65 °C) 0.01% 2 µL BSA (10%) 1% 100 µL ddH2O n/a 863 µL Total n/a 1,000 µL Lysis dilution buffer Reagent Final concentration Amount Tris-HCl (1 M, pH 7.4) 10 mM 10 µL NaCl (5 M) 10 mM 2 µL MgCl2 (1 M) 3 mM 3 µL BSA (10%) 1% 100 µL ddH2O n/a 885 µL Total n/a 1,000 µL Wash buffer Reagent Final concentration Amount Tris-HCl (1 M, pH 7.4) 10 mM 10 µL NaCl (5 M) 10 mM 2 µL MgCl2 (1 M) 3 mM 3 µL BSA (10%) 1% 100 µL Tween-20 (10%) 0.1% 10 µL ddH2O n/a 875 µL Total n/a 1,000 µL Diluted nuclei buffer Reagent Final concentration Amount Nuclei Buffer (20×) 1× 50 µL ddH2O n/a 950 µL Total n/a 1,000 µL Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from JSPS (17H04038, 17K08622, 20K07332, 20H03441) (T.A., N.A.) and CREST from Japan Science and Technology Agency (JPMJCR2011) (T.A.). The procedure and the data analysis of this protocol are modified from the 10× Genomics protocol (Chromium Single Cell ATAC Solution User Guide) and the Seurat vignette (https://satijalab.org/seurat/articles/atacseq_integration_vignette.html), respectively. The protocol in this work is used in the following paper: Miyao, T., Miyauchi, M., Kelly, S. T., Terooatea, T. W., Ishikawa, T., Oh, E., Hirai, S., Horie, K., Takakura, Y., Ohki, H., Hayama, M., Maruyama, Y., Seki, T., Ishii, H., Yabukami, H., Yoshida, M., Inoue, A., Sakaue-Sawano, A., Miyawaki, A., Muratani, M., Minoda, A., Akiyama, N. and Akiyama, T. (2022). Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator. Elife 11: e73998. Competing interests The authors declare no competing interests. Ethics All mice were handled in accordance to the Guidelines of the Institutional Animal Care and Use Committee of RIKEN, Yokohama Branch (2018-075). References Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. and Greenleaf, W. J. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10(12): 1213-1218. Buenrostro, J. D., Wu, B., Chang, H. Y. and Greenleaf, W. J. (2015). ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109: 21.29.21-21.29.29. Kaplan, N., Moore, I. K., Fondufe-Mittendorf, Y., Gossett, A. J., Tillo, D., Field, Y., LeProust, E. M., Hughes, T. R., Lieb, J. D., Widom, J., et al. (2009). The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458(7236): 362-366. Klemm, S. L., Shipony, Z. and Greenleaf, W. J. (2019). Chromatin accessibility and the regulatory epigenome. Nat Rev Genet 20(4): 207-220. Kornberg, R. D. and Thomas, J. O. (1974). Chromatin structure; oligomers of the histones. Science 184(4139): 865-868. Luo, L., Gribskov, M. and Wang, S. (2022). Bibliometric review of ATAC-Seq and its application in gene expression. Brief Bioinform 23(3): bbac061. Miyao, T., Miyauchi, M., Kelly, S. T., Terooatea, T. W., Ishikawa, T., Oh, E., Hirai, S., Horie, K., Takakura, Y., Ohki, H., et al. (2022). Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator. Elife 11: e73998. Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W. M., 3rd, Hao, Y., Stoeckius, M., Smibert, P. and Satija, R. (2019). Comprehensive Integration of Single-Cell Data. Cell 177(7): 1888-1902.e1821. Wells, K. L., Miller, C. N., Gschwind, A. R., Wei, W., Phipps, J. D., Anderson, M. S. and Steinmetz, L. M. (2020). Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells. Elife 9: e60188. Yan, F., Powell, D. R., Curtis, D. J. and Wong, N. C. (2020). From reads to insight: a hitchhiker's guide to ATAC-seq data analysis. Genome Biol 21(1): 22. Article Information Copyright Ishikawa et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Immunology > Immune cell function > General Systems Biology > Transcriptomics > RNA-seq Computational Biology and Bioinformatics Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols circFL-seq, A Full-length circRNA Sequencing Method Zelin Liu and Ence Yang Apr 20, 2022 2494 Views T Cell Clonal Analysis Using Single-cell RNA Sequencing and Reference Maps Massimo Andreatta [...] Santiago J. Carmona Aug 20, 2023 2608 Views Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes Anna Smirnova [...] Alexandre Smirnov Sep 20, 2023 414 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,589	https://bio-protocol.org/en/bpdetail?id=4589&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed PERK Pathway Inhibitors Cure Group A Streptococcal Necrotizing Fasciitis in a Murine Model AA Aparna Anand * AS Abhinay Sharma * MR Miriam Ravins AJ Atul Kumar Johri BT Boaz Tirosh EH Emanuel Hanski (*contributed equally to this work) Published: Vol 12, Iss 24, Dec 20, 2022 DOI: 10.21769/BioProtoc.4589 Views: 796 Reviewed by: Luis Alberto Sánchez VargasKarolina Subrtova Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Translational Medicine Aug 2021 Abstract Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below. Keywords: Group A streptococcus Necrotizing fasciitis murine model Endoplasmic reticulum stress PERK pathway GSK2656157 ISRIB Background Group A Streptococcus (GAS), or Streptococcus pyogenes , is a human-specific Gram-positive bacterial pathogen. It remains among the top ten pathogens causing several diseases with mild to life-threatening clinical symptoms (Bisno and Stevens, 1996;Efstratiou and Lamagni, 2016). The clinical presentation of these diseases includes pharyngitis, impetigo, erysipelas, tonsilitis, scarlet fever, necrotizing fasciitis, rheumatic fever, glomerulonephritis, septicemia, and others. More than 220 different GAS serotypes are circulating in the human population worldwide. M1T1 is the dominant serotype spread globally and causing diseases in the population of economically well-equipped countries (Aziz and Kotb, 2008; Nelson et al., 2016; Lynskey et al., 2019). GAS classification is based on variation in the N-terminal amino acid sequence of the surface M-protein. Antibiotic regimens are the only definitive strategy to cure GAS infections, because of the unavailability of any licensed universal vaccine on the market. Recent reports on the development of antibiotic resistance in GAS are worrisome and have motivated us to develop a new alternative therapeutic approach to cure its infections. Invasive GAS infections, like sepsis, bacteremic pneumonia, and necrotizing fasciitis (NF), spread in sterile sites (Metzgar and Zampolli, 2011). NF begins as a small lesion with the appearance of mild erythema, which rapidly progresses with inflammation in the subcutaneous tissue, and destructs skin and soft tissue over large body areas (Walker et al., 2014). Antibiotic administration, surgical debridement of infected tissues, and supportive care are the main line of treatment for invasive GAS diseases (Stevens and Bryant, 2017). Unfortunately, the mortality rate from NF ranges from 23 to 35% in resource-rich settings, despite quick medical interventions (Davies et al., 1996; Carapetis et al., 2005;Allen and Moore, 2010;Olsen and Musser, 2010; Cole et al., 2011;Ralph and Carapetis, 2013). Therefore, alternative therapeutic approaches are urgently needed to combat invasive soft-tissue GAS infections. Our recent studies showed that GAS causes endoplasmic reticulum stress (ER stress) and triggers the unfolded protein response (UPR) in the host cells upon infection. This results in enhanced asparagine synthesis and release, which is utilized by GAS to increase virulence and rate of proliferation. Furthermore, the PERK-eIF2α-ATF4 branch of UPR was exclusively triggered during GAS infection (Anand et al., 2021). When we treated GAS-infected mice with PERK pathway inhibitors (GSK2656157 and ISRIB), the bacteria were cleared faster than in untreated infected mice. We discuss the method of treatment using PERK pathway inhibitors in a murine model of human soft tissue infection. Materials and reagents Preparation of bacteria for mice infection 15 mL conical tubes (Greiner Bio-One CELLSTAR, catalog number: 188621) 50 mL conical tubes (MiniPlast, catalog number: 835-050-21-111) Microcentrifuge tubes (Corning® Axygen, catalog number: MCT-175-C) Spectrophotometric cuvettes (Polystyrene cuvette, 10 × 4 × 45 mm, Sarstedt AG & Co. KG, Germany, catalog number: 67.742) GAS strains (serotype M14 strain JS14, which was isolated from a patient with pneumonia, and serotype M1T1 strain 5448, a clinical isolate from a patient with necrotizing fasciitis and toxic shock syndrome) Sterile Dulbecco's Phosphate Buffered Saline (without calcium chloride and magnesium chloride, pH 7.1–7.5, Biological Industries, catalog number: 02-023-1A) THY media (see Recipes) Todd-Hewitt broth (Becton, Dickenson, and Company, catalog number: 249240) Yeast extract (Becton, Dickenson, and Company, catalog number: 212750) Blood Agar plates (see Recipes) Defibrinated sheep blood (DSB) (hylabs®, catalog number: PD049) Mice infection 1 mL syringes without needles (BD PlastipakTM 1 mL Luer, catalog number: 303172) Needle (30G × 1/2") for syringe (BD MicrolanceTM 3, catalog number: 2025-01) Bacterial culture prepared for injection (suspended in PBS) Female BALB/c OlaHsd mice, aged 3–4 weeks, weighing 10–12 g (ENVIGO RMS, Israel Ltd.) Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301) Polyethylene glycol (PEG-400) (Sigma-Aldrich, catalog number: 202398) 0.9% sodium chloride saline (Teva Medical, catalog number: AWN1324) Ketamine-xylazine mix (mixed in 1:6 ratio, respectively, Ketamine-Vetoquinol, Xylazine-20 mg/mL, Eurovet Animal Health B.V.) Isoflurane (Piramal Critical Care Inc, USA, catalog number: 66794-013-25) GSK2656157 (Sigma-Aldrich, catalog number: 504651) (see Recipes) Integrated stress response inhibitor (ISRIB) (Sigma-Aldrich, catalog number: SML0843) (see Recipes) CFU quantification of GAS in skin biopsy samples Sterile 2 mL tubes (Abdos Labtech Private Limited, catalog number: P10203) Sterile Dulbecco's Phosphate Buffered Saline (without calcium chloride and magnesium chloride, pH 7.1–7.5, Biological Industries, catalog number: 02-023-1A) 70% Ethanol (Romaical, 19-19-009102-80) 8.0-mm Punch biopsy (Acu-Punch, Acuderm Inc., catalog number: P825) Dissection kit—surgical scissors and fine forceps Blood Agar plates (see Recipes) Defibrinated sheep blood (DSB) (hylabs®, catalog number: PD049) Equipment Spectrophotometer (Amersham Biosciences, Ultraspec 2100 pro, 80-2112-21) Centrifuge with swing rotor (Sigma, model: 3-16PK) Vortex mixer (Heidolph reax top, Z655988) -80 °C freezer (Thermo Scientific, 8926) 37 °C incubator (BINDER GmbH, BD 56) Digital vernier caliper (Bar Naor Ltd., BN30087-00) Electronic homogenizer (POLYTRON® 2100 Homogenizers, Kinematica, 05400261) Electric shaver (Phillips, MG3730/15) Desiccator jar (SP BEL-ART polycarbonate/polypropylene desiccator, F42032-0000) Software GraphPad Prism 5.03 (for statistical analysis and graphical presentation) Procedure Preparation of bacteria for mice infection From a frozen glycerol stock (bacteria grown overnight were frozen in 15% v/v final strength sterile glycerol), inoculate the GAS strain into 10 mL of THY medium in 15 mL sealed conical tubes. Culture the bacteria statically at 37 °C for 16 h. Transfer 3 mL of overnight culture into 47 mL of pre-warmed fresh THY medium in 50 mL sealed conical tubes. Grow the bacteria at 37 °C without agitation to the mid-log phase, until the optical density at 600 nm (OD600 ) reaches 0.3–0.4. Centrifuge culture tubes at 4,000 × g and 14 °C for 10 min, and discard the supernatant. Wash the bacterial pellet after suspending it in 20 mL of sterile PBS. Centrifuge at 4,000 × g and 14 °C for 10 min. Repeat the washing step twice. Discard the supernatant and resuspend the bacterial pellet in 1 mL of sterile PBS. Measure the OD600 of the bacterial suspension and adjust it with PBS to 0.8 (corresponding to 1 × 109 CFU/mL). Dilute the bacterial culture 1:20 in PBS to get 5 × 107 CFU/mL (sub-lethal dose). Place the tubes on ice until use. CFU counts are determined to calculate the number of bacteria injected into the mice. First, serially dilute the bacterial inoculum to seven dilutions, by adding 100 µL of inoculum to 900 µL of PBS. Next, plate 100 µL of the 10-5 , 10-6 , and 10-7 dilutions onto blood agar plates. Finally, incubate the plates at 37 °C overnight. To determine the CFU/mL, count the number of colonies on the blood agar plates in the appropriate dilution and multiply by the dilution factor. In vivo soft-tissue model Three to four weeks old female BALB/c OlaHsd mice (weighing 10–12 g) were left to acclimatize for three days in disposable cages supplemented with enrichment, sterile food, and water, in specific pathogen-free (SPF) rooms with controlled environmental conditions. Weigh mice and distribute them evenly in cages. Mark mice on ears and shave their dorsal flanks one day before bacterial infection. Different groups of mice receive 100 µL of subcutaneous (SC) or intraperitoneal (IP) injections of GSK2656157 (100 µg/mouse/injection) or ISRIB (35 µg/mouse/injection) or control (90 µL of saline + 10 µL of DMSO and PEG 400 at 1:1 ratio). Inject GSK2656157/ISRIB subcutaneously 4 h before bacterial infection. Subsequently, SC and IP injections of the inhibitors are alternately given at an interval of 12 h post-infection (PI) until the sixth day. In the post-treatment mice experiment, inject GSK2656157/ISRIB subcutaneously for 12 h PI, followed by IP and SC injections of GSK2656157/ISRIB, which are given alternately at an interval of 12 h until the sixth day (Figure 1). Figure 1. Timeline of the injection of inhibitors and sampling procedure. BALB/c mice were placed in specific pathogen-free (SPF) conditions with controlled environmental conditions. They were weighed, shaved on dorsal flanks, and marked for identification. Mice were infected by subcutaneous (SC) administration of 5 × 106 CFU of GAS strain, and were either untreated, treated 4 h before infection, or treated 12 h post-infection (PI) with GSK2656157/ISRIB. The inhibitors were injected every 12 h alternately with dose 1 (D1) and dose 2 (D2) through SC or intraperitoneal (IP) routes for 6 days. Sampling (S1, S2, S3, and S4) for quantification of bacteria through skin biopsy was performed at different time intervals. Inject mice with a sub-lethal dose (5 × 106 CFU) of GAS to test the efficacy of GSK2656157/ISRIB. A volume of 100 µL of the bacterial solution is injected SC into the rear flank of mice. The bacterial load is confirmed by serial dilution and CFU estimation on blood agar plates. Euthanize mice by inhalation of isoflurane followed by cervical dislocation, and collect skin tissue samples using an 8.0-mm punch biopsy tool at different time points. Transfer the tissue samples to 2 mL Eppendorf tubes containing 0.5 mL of sterile PBS. Homogenize skin tissue samples using a homogenizer at a vibration speed of 20,000 ± 100 rpm for 30 s, dilute this in PBS, and plate the samples on blood agar plates. CFUs are quantified after incubation of blood agar plates at 37 °C overnight. The amount of CFU is normalized to the weight of the soft tissue sample. Measure the dermonecrotic lesion area of the mice in different groups daily, using a digital vernier caliper. The lesion area is calculated with the formula A = π/2)(length)(width) (Pinho-Ribeiro et al., 2018). The maximum permissible dermonecrotic lesion area is 2 cm2 . All the quantitative data (CFU counts and dermonecrotic lesion area) can be tested for statistical significance using GraphPad Prism 5.03 software. Notes The mice should be checked twice daily, including weighing. To minimize the suffering of the mice, a scoring method needs to be employed based on the status of their hair (straight or ruffled coat), eyes (open or closed), the activity of the mice, weight loss (loss of 15% body weight between two consecutive measurements), and severity of the lesion. Euthanize mice by injecting an overdose of ketamine/xylazine mix (total of 60 µL in a ratio of 1:6, respectively), followed by cervical dislocation, when scoring points to the need of an early humane endpoint. Recipes THY media Todd-Hewitt broth (3%) supplemented with 0.2% yeast extract in distilled water Blood Agar plates Tryptic soy agar (TSA) supplemented with 5% (v/v) of defibrinated sheep blood (DSB) GSK2656157 Dissolve GSK2656157 in DMSO, and add an equal volume of PEG 400. The aliquots can be stored at -80 °C until use (to be used within 3 months). On the day of injection, the mixture is diluted 10 times in saline (0.9% sodium chloride) to achieve a final concentration of 1 mg/mL. ISRIB Dissolve ISRIB in DMSO, and add an equal volume of PEG 400. The aliquots can be stored at -80 °C until use (to be used within 3 months). On the day of injection, the mixture is diluted 10 times in saline (0.9% sodium chloride) to achieve a final concentration of 0.35 mg/mL. Acknowledgments This work was supported by grants from the Israeli Science Foundation administered by the Israel Academy of Sciences and Humanities (ISF), the FIRST grant from ISF, the ISF-UGC Joint Scientific Research Program, and the National Research Foundation, Prime Minister’s Office, Singapore under its Campus of Research Excellence and Technological Enterprise (CREATE) program. In addition, AA and AS are thankful to the PBC Fellowship Program by the Israeli council for partial funding. This protocol was used in Anand et al. (2021) (DOI: 10.1126/scitranslmed.abd7465). Competing interests The authors declare no conflict of interest or competing interests. Ethics All the animal procedures for humane handling, care, and treatment of research animals are performed according to the ethical guidelines approved by the ethics committee of the Hebrew University of Jerusalem (Protocol number MD-18-15522-5). References Bisno, A. L. and Stevens, D. L. (1996). Streptococcal infections of skin and soft tissues. N Engl J Med 334(4): 240-245. Efstratiou, A. and Lamagni, T. (2016). Epidemiology of Streptococcus pyogenes. Ferretti, J. J., Stevens, D. L. and Fischetti, V. A. (Eds.) Streptococcus pyogenes: Basic Biology to Clinical Manifestations. Aziz, R. K. and Kotb, M. (2008). Rise and persistence of global M1T1 clone of Streptococcus pyogenes. Emerg Infect Dis 14(10): 1511-1517. Nelson, G. E., Pondo, T., Toews, K. A., Farley, M. M., Lindegren, M. L., Lynfield, R., Aragon, D., Zansky, S. M., Watt, J. P., Cieslak, P. R., Angeles, K., Harrison, L. H., Petit, S., Beall, B. and Van Beneden, C. A. (2016). Epidemiology of Invasive Group A Streptococcal Infections in the United States, 2005-2012. Clin Infect Dis 63(4): 478-486. Lynskey, N. N., Jauneikaite, E., Li, H. K., Zhi, X., Turner, C. E., Mosavie, M., Pearson, M., Asai, M., Lobkowicz, L., Chow, J. Y., et al. (2019). Emergence of dominant toxigenic M1T1 Streptococcus pyogenes clone during increased scarlet fever activity in England: a population-based molecular epidemiological study. Lancet Infect Dis 19(11): 1209-1218. Metzgar, D. and Zampolli, A. (2011). The M protein of group A Streptococcus is a key virulence factor and a clinically relevant strain identification marker. Virulence 2(5): 402-412. Walker, M. J., Barnett, T. C., McArthur, J. D., Cole, J. N., Gillen, C. M., Henningham, A., Sriprakash, K. S., Sanderson-Smith, M. L. and Nizet, V. (2014). Disease manifestations and pathogenic mechanisms of Group A Streptococcus. Clin Microbiol Rev 27(2): 264-301. Stevens, D. L. and Bryant, A. E. (2017). Necrotizing Soft-Tissue Infections. N Engl J Med 377(23): 2253-2265. Davies, H. D., McGeer, A., Schwartz, B., Green, K., Cann, D., Simor, A. E. and Low, D. E. (1996). Invasive group A streptococcal infections in Ontario, Canada. Ontario Group A Streptococcal Study Group. N Engl J Med 335(8): 547-554. Carapetis, J. R., Steer, A. C., Mulholland, E. K. and Weber, M. (2005). The global burden of group A streptococcal diseases. Lancet Infect Dis 5(11): 685-694. Allen, U. and Moore, D. (2010). Invasive group A streptococcal disease: Management and chemoprophylaxis. Can J Infect Dis Med Microbiol 21(3): 115-118. Olsen, R. J. and Musser, J. M. (2010). Molecular pathogenesis of necrotizing fasciitis. Annu Rev Pathol 5: 1-31. Cole, J. N., Barnett, T. C., Nizet, V. and Walker, M. J. (2011). Molecular insight into invasive group A streptococcal disease. Nat Rev Microbiol 9(10): 724-736. Ralph, A. P. and Carapetis, J. R. (2013). Group a streptococcal diseases and their global burden. Curr Top Microbiol Immunol 368: 1-27. Anand, A., Sharma, A., Ravins, M., Biswas, D., Ambalavanan, P., Lim, K. X. Z., Tan, R. Y. M., Johri, A. K., Tirosh, B. and Hanski, E. (2021). Unfolded protein response inhibitors cure group A streptococcal necrotizing fasciitis by modulating host asparagine. Sci Transl Med 13(605). Pinho-Ribeiro, F. A., Baddal, B., Haarsma, R., O'Seaghdha, M., Yang, N. J., Blake, K. J., Portley, M., Verri, W. A., Dale, J. B., Wessels, M. R., et al. (2018). Blocking Neuronal Signaling to Immune Cells Treats Streptococcal Invasive Infection. Cell 173(5): 1083-1097 e1022. Article Information Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Microbiology > in vivo model > Bacterium Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Murine Acute Pneumonia Model of Pseudomonas aeruginosa Lung Infection Xiaolei Pan and Weihui Wu Nov 5, 2020 3196 Views A Retro-orbital Sinus Injection Mouse Model to Study Early Events and Reorganization of the Astrocytic Network during Pneumococcal Meningitis Chakir Bello [...] Guy Tran Van Nhieu Dec 5, 2021 1698 Views Rapid in vitro and in vivo Evaluation of Antimicrobial Formulations Using Bioluminescent Pathogenic Bacteria Artur Schmidtchen and Manoj Puthia Jan 20, 2022 2556 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,590	https://bio-protocol.org/en/bpdetail?id=4590&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach CC Clément Chambaud SC Sarah J. Cookson NO Nathalie Ollat AB Amélie Bernard LB Lysiane Brocard Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4590 Views: 1119 Reviewed by: Wenrong HeYao Xiao Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plant Physiology Jan 2022 Abstract Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana, which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues. Graphical abstract Keywords: Plant grafting Correlative microscopy Arabidopsis thaliana Electron microscopy Electron tomography Background Plant grafting is a process that has been used for centuries in agronomy to combine interesting agronomical traits together, such as resistance and fruit productivity (Mudge, 2009), or to study systemic biological processes, such as epigenetic regulations and long-distance signaling (Molnar et al., 2010; Turnbull, 2010; Turnbull et al., 2002; Wu et al., 2013; Bidadi et al., 2014; Zhang et al., 2014; Yang et al., 2015). Despite its widespread use, the cytologic events required for the establishment of the graft union remain poorly understood (Gautier and Chambaud et al., 2019). During graft union formation, tissues from both partners have to undergo a process of dedifferentiation to form callus cells and set up new pathways of communication such as phloem, xylem, and plasmodesmata. The movement of dyes or fluorescent probes between the scion and the rootstock have been used to resolve the kinetics of vascular connection at the graft interface, but no ultrastructural data are available for the establishment of cell-to-cell connection via plasmodesmata (Melnyk et al., 2015; Melnyk et al., 2018). Knowledge of the ultrastructural mechanisms underpinning graft union formation is currently very limited because locating precisely the site of contact between the two grafted partners under electron microscopy is challenging due to the difficulty to distinguish the origin/identity of each cell at the graft interface, i.e. to distinguish between cells from the scion and cells from the rootstock (Kollmann et al., 1985; Kollmann and Glockmann, 1985, 1991; Pina et al., 2017; Notaguchi et al., 2020). Attempts were successfully made by Kollmann and Glockmann (1985) and Notaguchi (2020), in which plants from different species showing noticeable ultrastructural differences in the callus cells produced at the graft interface were used. To overcome the requirement of cytological differences to accurately identify cell origin and properly localize the graft interface, we propose a protocol for correlative light and electron microscopy (CLEM) to unambiguously target the graft interface at the ultrastructural level. At present, the use of CLEM approaches is still scarce for the analyses of plant tissues (Bell et al., 2013; Marion et al., 2017). Two out of the three CLEM protocols developed for plants are unfortunately not easily compatible with electron tomography due to either the weakness of the embedded resin used (glycol methacrylate) or the absence of resin (Tokuyasu method) (Marion et al., 2017). The protocol described by Bell et al. (2013) presents the advantage of being easily accessible but, because it is based on a chemical fixation, only permits a coarse ultrastructural preservation. Here, in order to combine fluorescence and high ultrastructural resolution in the same sample, we adapted the protocol described by Kukulski et al. (2012a and 2012b), an in-resin fluorescence CLEM method for plant tissues (Kukulski et al., 2012a and 2012b). This protocol allows us to preserve fluorescence and maintain the ultrastructure; further, in combination with electron tomography, this method allows to reach a level of resolution enabling the visualization of the bilayer of the plasma membrane. Moreover, this protocol is compatible with immunogold labeling and is adapted for hypocotyls and roots of plantlets of Arabidopsis thaliana. For our study, the hypocotyl of a scion and a rootstock expressing a yellow fluorescent protein (YFP) and monomeric red fluorescent protein (mRFP), respectively, fused to the endoplasmic reticulum–retention motif HDEL, were grafted (Chambaud et al., 2022). By optimizing (1) the grafting procedure, (2) the duration of the freeze-substitution (FS) steps, (3) the thickness of the ultrathin sections, (4) the sample mounting for light microscopy (LM) observation, and (5) the LM acquisition, we were able to observe fluorescence signals and precisely target the graft interface. The compatibility of our CLEM protocol with our previously described electron tomography protocol (Nicolas et al., 2018), allowed to access the three-dimensional (3D) view of ultrastructural elements specifically at the graft interface (Chambaud et al., 2022). Our CLEM protocol has the potential to unravel multiple plant cell processes at the ultrastructural level and was in fact already applied successfully to study autophagy (Gomez et al., 2022) and phase-to-phase separation processes (Dragwidge et al., 2022). Materials and Reagents Plant culture 1.5 mL Eppendorf tubes (Sarstedt, catalog number: 72.708) 12 N or 37% hydrochloric acid (Carlo Erba, catalog number: 524526) 1.5 mL Eppendorf tube rack (Ratiolab cardboard grid, inserts for cryobox, catalog number: 5020147) Square plastic culture plates for A. thaliana seedlings, vertical culture (VWR, catalog number: 391-0444) Bleach (Orapi Europe, catalog number: OR0011) Murashige and Skoog (MS) medium + vitamins (Duchefa Biochemie, catalog number: M0222.0050) for seedlings Plant agar for seedlings culture and grafting (Duchefa Biochemie, catalog number: P1001.1000) Anapore tape (Euromedis, catalog number: 135320) to seal culture plates KOH Bleach solution for seeds sterilization (Recipe 1) Culture medium for seedling and grafting (Recipe 2) Half-strength MS medium for seedlings Water medium for grafting Transgenic lines For grafting experiments, we used 35S::HDEL_mRFP and 35S::HDEL_YFP lines, which have strong fluorescent expression (Nelson et al., 2007;Lee et al., 2013). Both lines showed a fluorescence signal that was easily visible through oculars with a zoom microscope with the medium pre-set level for the excitation power and the adapted set filter. Plant grafting Round Petri dishes, 90 × 14.2 mm for grafting (generally used for bacterial culture) (VWR, catalog number 391-0439) Neutral nylon membrane hybond-N (GE Healthcare, catalog number: RPN 203 N) Paraffin film (Bemis, Parafilm ‘M’) cut into strips of approximately 1 cm wide to seal the Petri dishes High-pressure freezing (HPF) Screw-top 2 mL tubes (Sarstedt, catalog number: 72.693) Toothpicks Bovine serum albumin (BSA) fraction V (Sigma-Aldrich, catalog number: 05482) Methyl cyclohexane (MCH) (Merck Schuchardt OHG 85662 Hohenbrunn, Germany) Distilled water to cut sample Membrane carriers (100 μm deep with 1.5 mm diameter) (Leica Microsystems, catalog numbers: 16707898) 20% BSA solution for cryoprotection during HPF (see Recipe 3) Freeze-substitution (FS) Disposable regular pipettes, 1, 2, and 3 mL (VWR, catalog numbers: 612-2850, 612-2851, and 612-1681) and FS-specific 1 mL pipettes with thin tips (Ratiolab, catalog number: 2600155) Wheaton glass sample vials with snap-cap for cryomix preparation (DWK Life Sciences, WHEATON, catalog number: 225536) Personal cartridge half mask 6100 (Honeywell International, catalog number: 1029471) Aluminum foil Uranyl acetate powder (Merck, catalog number: 8473) Pure methanol (Fisher Scientific, catalog number: M/4062/17) Ultrapure 100% ethanol and acetone (VWR, catalog numbers: 83813.440 and 20066.558, respectively) Colored nail polish (color does not matter) 12.5 mL reagent containers with screw caps (Leica Microsystems, catalog number: 16707158) HM20 resin (Electron Microscopy Sciences, catalog number 14345) Reagent bath (Leica Microsystems, catalog number: 16707154) Plastic mold flow-through rings (Leica Microsystems: 16707157) Cryosubstitution uranyl-acetate stock solution (20%) (see Recipe 4) Cryosubstitution mix (see Recipe 5) HM20 solutions (see Recipe 6) Ultramicrotomy Crystallizing dish (Fisher Scientific, catalog number: 11766582) Fast absorbent paper filters (Whatman, Filter papers 41) (GE Healthcare, catalog number: 1441-070) Glass rods (Fisher Scientific, catalog number: 12441627) Gilder standard hexagonal 200 mesh copper grids (Electron Microscopy Sciences, catalog number: G200HS-Cu) to coat with parlodion or commercial grids with formvar (Electron Microscopy Sciences, catalog number: FCFT200-CU) Razor blades for coarse block preparation (Electron Microscopy Sciences, catalog number: 71990) Pasteur pipet (VWR, catalog number: 612-1720) 0.2 μm syringe filter (VWR, catalog number: 514-0061) 10 mL syringe (Terumo, catalog number: SS+10ES1) Solid parlodion (Electron Microscopy Sciences, catalog number: 19220) Isoamyl-acetate (Sigma-Aldrich, catalog number: W205532) Toluidine blue powder (Sigma-Aldrich, catalog number: T3260) Sodium borate powder Ultrapure (Milli-Q) water 2% parlodion solution for grid filming (see Recipe 7) Toluidine blue solution (see Recipe 8) for screening block sections on tabletop microscope during resin block milling Electron microscopy Gold colloid 5 nm (BBI Solution, catalog number: EMGC5) Gold fiducials solution (see Recipe 9) Equipment Plant culture Desiccator used as hermetic chamber Fume hood Glass crystallizing dish Fridge for vernalization -80°C freezer for sterilization Plant grafting Dissecting microscope with a minimal working distance of 5 cm (Leica Microsystems, model: Leica MZFLIII) Glass beaker for sterilization of tools with ethanol Spray for ethanol Dissecting micro-knives 15° 5 mm (Fine Science Tools, catalog number: 72-1551) Precision tweezers [EMS style 5X, catalog number: 78320-5X (soft) and 78520-5X (hard)] Fluorescence screening Zoom microscope (Zeiss Axiozoom.V16) equipped with a 0.5× objective with a numerical aperture of 0.125. The fluorescent part is composed by the fluorescence source Optoprim sola SE II and filter sets (GFP filter set: excitation window between 460 and 480 nm and emission window between 500 and 548 nm; mRFP filter set: excitation window between 540 and 580 nm and emission window up to 593 nm). High-pressure freezing (HPF) Liquid nitrogen, liquid nitrogen container, and adapted personal protection gear Air compressor (JUN-AIR) Leica EM-PACT1 machine (Leica Microsystems) Leica loading system Pod holders (Leica, catalog number: 16706832) Pods (Leica, catalog number: 16706838) Regular biomolecular pipettes, ranging from 2 to 1,000 µL Binocular with transmission illumination for root dissection (Nikon, model: SMZ-10A) Heating surface for quick drying of pods and pod holders during the session Insulated tweezers for manipulation in liquid nitrogen (VOMM Germany, 22 SA ESD) Metal containers for frozen sample transfer and associated screwdriver for lifting procedures (provided with EMPACT1) Torque screwdriver (TOHNICHI, Torque driver RTD60CN) set to 30 N.cm Freeze-substitution (FS) Leica automatic freeze-substitution AFS2 (Leica Microsystems, model: Leica AFS2) Leica freeze-substitution processor FSP (Leica Microsystems, model: Leica FSP) Metal socket for mold containers Microneedles for separating the membrane carriers from frozen samples [microneedle 0.025 mm (Electron Microscopy Science, catalog number: 62091-01) and 5 µm (Ted Pella, catalog number: 13625)] Ventilated fume hood Ultramicrotomy Block holders Diamond trimming knife for first milling steps to reach the sample (Drukker 8 mm Histoknife Dry, discontinued) Diamond knives Trim20 dry (Diatome, catalog number: DTB20), Ultra wet 45° (Diatome, catalog number: DU4530), and Histo wet (Diatome, catalog number: DH4560) ELMO glow discharger (optional) (CORDOUAN Technologies) Grid carriers for grid storage Leica Ultracut UC7 (Leica Microsystems, model: Leica Ultracut UC7) Tabletop microscope (Olympus, model: CX-41) Precision weight scale for parlodion weighting (Sartorius, model: TE124S) Confocal microscopy Sensitive confocal microscope (Zeiss Microscopy, model: Zeiss LSM 880) and 63× NA 1.4 oil immersion objective Electron microscopy FEI Tecnai G2 Spirit 120 kV equipped with Eagle 4K × 4K high sensibility CCD camera Advanced tomography holder (Fischione instruments, model: 2020) Software Zeiss Zen Dark version 2.3 software (https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html) used for confocal acquisition Tecnai imaging and analysis (TIA) version 4.5 software was used in conjunction with transmission electron microscopy (TEM) User Interface software version 4.5 to control and acquire micrograph with the electron microscopy (https://www.fei.com/software/) Xplore3D version 4.5 (FEI) was used for automated tilt series acquisitions (https://www.fei.com/software/) ImageJ version 1.53n (https://imagej.nih.gov/ij/download.html) with the Bio-Format version 6.9 plugin (https://www.openmicroscopy.org/bio-formats/) to open and process the Zeiss .czi files and to open the .mrc tilt series files and tomograms. IMOD suite version 4.11.6 (http://bio3d.colorado.edu/imod/). All processing of tomograms was done using the IMOD suite (Kremer et al., 1996), from the alignment of the tilt series to tomogram reconstruction For visualization purposes only, Adobe Photoshop version 23.0.1 is used for TEM mosaic assembling using photomerge function. ICY version 2.4.2.0 and its plugin ec-CLEM version 1.0.1.5 (https://icy.bioimageanalysis.org/plugin/ec-clem/) were used for LM and TEM correlation Procedure Plant grafting The A. thaliana hypocotyl grafting procedure was adapted and optimized from the Two Segment Shoot-Root Graft part of the protocol of Melnyk (2017a) . Finding the good level of humidity is the most challenging part of Melnyk’s protocol. In our hands, replacing the water-soaked Whatman paper by a water-based agar medium was key to maintain constant humidity for several days after grafting and increased our grafting success from 50% to 80%. To target the graft interface with CLEM, we grafted transgenic lines expressing fluorescent proteins fused to the endoplasmic reticulum–retention motif HDEL (35S::HDEL-YFP and 35S::HDEL-mRFP1.2) ( Dean and Pelham, 1990 ; Knox et al., 2015; Chambaud et al., 2022). The constitutive 35S promotor was used to ensure a strong initial fluorescent signal. Figure 1. Seed sterilization procedure. (A) Appropriate number of seeds in a 1.5 mL sample tube for a proper sterilization. (B) For gas sterilization, place them open in a tube rack. (C) Glass crystallizing dish for the bleach. (D) Hermetic chamber with bleach in the glass crystallizing dish, tube rack, and open Eppendorf tube containing seeds. (E) When mixing bleach with hydrochloric acid, the solution becomes yellow (white arrow) and a faint yellow haze of chloride gas is visible. Plant culture Sterilize A. thaliana seeds with chlorine gas (adapted from Lindsey et al., 2017). Aliquot a small number of seeds (approximately 100–150) into a 1.5 mL centrifuge tube labeled with a permanent pen (covered in adhesive tape to avoid washing off of the ink), close the caps, and place them for 20 min in a -80°C freezer (Figure 1A). The sterilization will take place in a hermetic chamber, typically a glass desiccator, under a fume hood. In the desiccator, place a small glass crystallizing dish containing 50 mL of bleach solution (see Recipe 1) (Figure 1C and 1D). Uncap centrifuge tubes before placing them in a tube rack in the chamber. Add 1.5 mL of 12 N hydrochloric acid to the bleach solution that is in the crystallizing dish without mixing and quickly close the chamber. The yellow haze of chlorine gas should be visible at this point (Figure 1E). Let the seeds in the chamber from 4 h to overnight (e.g., 6 pm–9 am). Open the chamber and close the centrifuge tubes. Leave the chamber open under the fume hood for one day until the reaction between hydrochloric acid and bleach ends. The reaction produces chlorine gas, sodium chloride, and water. You can then discard the solution. Note that if the seed germination seems affected, the incubation time should be reduced to 1–3 h. Under a laminar flow hood, gently sprinkle the seeds over square Petri dishes containing half-strength MS medium with vitamins. Thanks to chloride gas sterilization, homogeneous distribution of the dry seeds is easily achieved. Seal the plate with anapore tape. For vernalization and stratification, place the plates horizontally with the seeds on the upper face at 4°C in the dark for 2–5 d maximum. Transfer the plates to a growth chamber regulated at 20–23°C with photosynthetic photon flux density (PPFD) of 100 µmol·m-2·s-1 of light. Put the plates vertically so that the hypocotyls grow as straight as possible. Note that short-day growing conditions (such as 8 h of light per day) give rise to more elongated and thinner hypocotyls, which facilitate the grafting procedure and microscopy experiments (Melnyk, 2017a, 2017b). Once the two first true leaves are just visible under the binocular microscope, grafting can be done (Figure 2). In general, seedlings grown under short or long days (i.e., 8 and 16 h of light) have to be 5 or 7 days old, respectively, for grafting. At all times, you need to monitor plant growth to graft at the more appropriate time. Figure 2. The ideal Arabidopsis plantlets for grafting. An idealA. thaliana seedling for grafting has a straight hypocotyl, very small first leaves (white arrow), and cotyledons that should not bend over the hypocotyl (white arrowheads). Scale bar: 500 µm. Grafting Figure 3. Workflow of grafting experiments The absence of sucrose in the culture and grafting media makes it possible to graft in non-sterile conditions if you do not plan to keep the grafts in vitro for more than six days. In our grafting studies, we grafted on the bench in non-sterile conditions because we worked on samples at three and six days after grafting. Nevertheless, we work on a 70% ethanol pre-cleaned bench with sterilized tools to limit contamination. The dissecting microscope used must have a working distance (distance between the lens and its focal plane) of at least 5 cm to allow enough space for the manipulations. We usually use a magnification between 6× and 20× with annular illumination or optical fiber systems. Performing grafting is tedious and requires precise and quick manipulations. Try to avoid coffee and other stimulants before attempting grafting experiments. With practice, the grafting technique allows you to reach a success rate of 80% to 90% with a rate of 70 grafts per hour. Following the workflow of grafting experiments proposed (Figure 3) can help you to organize your schedule. For the grafting day, book the epifluorescence zoom microscope and the stereomicroscope and prepare the material (Figure 4). Figure 4. Grafting tools. (A) 1. Dissecting micro-knife for grafting; 2. Glass beaker for sterilization of tools; 3. Permanent marker to label Petri dishes; 4. Round Petri dishes for grafting; 5. Spray with ethanol for tools and bench cleaning; 6. Fine tweezers; 7. Sterile nylon membrane wrapped in aluminum foil; 8. Plants to graft. (B) Parafilm strips to seal Petri dishes after grafting. (C) Positioning of nylon membrane prior to grafting. Before grafting Cut a nylon membrane in 2 × 5 cm pieces and wrap them in aluminum foil. Prepare the grafting medium with 1.6% plant agar in distilled water and buffer it at pH 5.8 with 1 N KOH. Autoclave the grafting medium and the pieces of nylon membranes wrapped in aluminum foil at 120°C for 20 min. When the grafting medium is handleable (avoid burns after autoclaving), fill the round Petri dishes with approximately 20 mL of agar under a laminar flow hood. For grafting step Using a fluorescence zoom microscope with an excitation power set at the medium pre-set level, identify plantlets showing a strong expression of fluorescence with excitation power. Gather Petri dishes, micro-knives, tweezers, nylon membranes, permanent pen, and strips of parafilm (Figure 4). Clean the dissecting microscope, micro-knives, and tweezers by spraying 70% ethanol and let them dry completely before usage. If you have to graft more than two plates, repeat the cleaning step every two plates. Label your Petri dishes with the plant lines you will use as the scion and the rootstock. Put a piece of nylon membrane on the upper part of each box (Figure 4C). Among fluorescent plantlets, select the ones with homogeneous size, hypocotyls as straight as possible, and cotyledons not bent over hypocotyls (Figure 2). Put them in a row on the membrane (Figure 5). To move them, you can use tweezers or a toothpick that does not pinch the tissue. Note that you have to avoid any damage to the hypocotyl, shoot, and root. If you want to use the two genotypes as scion or rootstock, you could put six plants of the first genotype in the same row of six plants of the second genotype (Figure 5A). If you wish to use a genotype only as scion and the other only as rootstock, you could dispose the plants in two rows of 12 plants (Figure 5B). The membrane is used as a cutting surface. Cut on it to avoid damages to the micro-knife. Notes: If the micro-knife is used properly, each can be used for approximately 400–500 grafts. It must permit a sharp sectioning in only one movement. Under a cleaned dissecting microscope, cut off and discard one cotyledon (choose the smallest, most damaged cotyledon that is bent over the hypocotyl) to allow the seedling to lie flat on the membrane (Figure 6A, B, C). Cut transversally through the hypocotyl, just under the cotyledons, without crushing the plant (Figure 6D). Cutting the hypocotyl too low could impair the grafting success due to the formation of adventitious root from the scion (Video 1). Video 1. Arabidopsis thaliana hypocotyl grafting Lift the scion by the petiole with closed tweezers and place it against the rootstock of another plant (Figure 6F and 6G). Most of the surfaces of both sectioned parts have to be in contact. You can improve the positioning by gently pushing the cut scion against the cut rootstock with the closed tweezers to bring both partners in direct contact (Figure 6G and 6H). Note: We recommend that you graft together hypocotyls with similar diameters and avoid moving the cut rootstock, which is very fragile. The moisture level is crucial during graft union formation. Grafting onto grafting medium (water with 1.6% agar) keeps the moisture level constant and allows to keep the grafts in vitro six days after grafting. Figure 5. Grafting preparation. (A) When wishing to keep both directions of grafting (genotype 1/genotype 2 and genotype 2/genotype 1), a 6/6 graft scheme can be used. Scions and rootstocks will be prepared, cut, and exchanged between the two genotypes. (B) When a single direction of grafting is required (genotype 1/genotype 2 as above), a 12/12 graft scheme can be used. Bring the scions on the top row (genotype 1) before cutting and discarding their roots. The bottom row (genotype 2) contains rootstocks. Their apical part will be cut and discarded. Scions prepared from genotype 1 will be moved and grafted to rootstocks from genotype 2. Repeat steps c., d., and e. until all the plants are grafted. To save time, you can do this in a row: (1) cut and discard one cotyledon on each seedling, (2) cut all the shoots, (3) move them next to the prepared rootstocks, (4) align and push the scions onto the rootstocks. When the grafting is done, be very cautious with the plates to avoid sudden movements that could induce the scion falling off the rootstock. Close the lid and seal the plates with a Parafilm strip. You can label the plate with the time you ended grafting so that you know the age of the graft. Carefully put the plate vertically back in the growth chamber at 20–23°C in PPFD of 100 µmol·m-2·s-1 light in either short- or long-day conditions. Note: If you grafted properly, you can begin to manipulate your graft (i.e., observing it under a microscope, fix tissues, transferring it to a new medium, etc.) 48 h after grafting. After 3 d, the grafts become slightly more robust as phloem should be connected. After 6–7 d, xylem is connected, and the grafted plant becomes quite robust. When beginning grafting experiments, you may need to assess your grafting success rate at first. You can rely on several criteria to evaluate grafting success (Melnyk, 2017b), such as the resumption of root growth, usually with emergence of lateral roots, and the absence of adventitious root formation in the scion. You can also monitor the phloem connection by grafting scions expressing pSUC2::GFP [i.e., green fluorescent protein (GFP) under the phloem-specific SUC2 promoter] and observing the movement of GFP to the rootstock, or by applying carboxyfluorescein diacetate (CFDA) to the scion or rootstock to evaluate phloem and xylem connectivity as described in Melnyk (2017b). Figure 6. The step-by-step procedure of grafting. The two plants are placed side by side on a nylon membrane and one cotyledon of each plant is cut along the black dashed lines (A and B) and then discarded (C). Hypocotyls are cut just below the petiole of cotyledons along the white dashed lines (D and E). Scions are exchanged between plants by sticking them to the tip of the tweezers (F). Scion is carefully brought close to the rootstock with the tip of the tweezers (G). When properly grafted, the graft junction (black arrowhead) is barely visible (H). CLEM Before fixation, ensure that the fluorescence signals are easily visible through the oculars for individual samples with non-destructive observations (stereomicroscopy or macroscopy). HPF Note: HPF was done using the Leica EM-PACT1 machine (Studer et al., 2001). A detailed protocol is described in Nicolas et al. (2018). This procedure involves the manipulation of liquid nitrogen; if you are not familiar with its handling, please refer to the official guidelines. Moreover, we recommend fixing approximately 10 samples by condition and to process all your samples of one condition as a batch, before fixing the next one. It will help you avoid mixing up the different grafts. Moreover, we recommend starting the HPF and FS either on Monday or Tuesday to finish handling for block preparation on the next Thursday or Friday (Table 2). Prepare the EM-PACT1 machine by (1) filling its dewar with liquid nitrogen, (2) filling the MCH chamber, and (3) evacuating the air out the circuit by two blank shoots. Charge the membrane carrier (100 µm deep carriers) on the Leica loading system. Pick a grafted plant out of the Petri dish and place it on a glass slide covered with water under a binocular zoom microscope. Note: The following steps should be carried out as fast as possible to limit biological responses from the cutting and BSA drying. Fill the carrier with 20% BSA in water with a 0.5–2 µL pipette until the liquid surface is domed in the well of the carrier. Ensure that there are no air bubbles. Under the binocular zoom microscope, dissect the graft interface in water on a glass slide with a razor blade and then carefully pick it up with a toothpick. Dispose the graft interface into the BSA in the membrane carrier before pushing the membrane carrier in the pod using the Leica loading system. Close the pod using the Torque screwdriver (set on 30 N.cm) and remove the Leica loading system. Load the sample in the EM-PACT1 machine and shoot. Check the freezing rate and pressure rise on the screen by ensuring that maximum pressure is reached approximately 20 ms before reaching the minimal temperature. Store your frozen sample under at least -90°C until the FS step. Repeat steps B1b–j until all your samples are frozen. Table 1. Plant correlative light electron microscopy (CLEM): freeze substitution (FS) and resin polymerization program compared to standard plant and yeast CLEM programs Freeze-substitution (FS) Note: Our FS and embedding protocol is based on the protocol published by Kukulski et al. (2012a and 2012b); it was initially designed for yeast and mammalian cells and we adapted it for our plant tissues (Table 1). During the FS and until the dehydration of the samples, it is crucial to maintain the sample under -80°C to avoid the formation of ice crystals. For this purpose, every container, medium, and tool has to be precooled before entering in contact with the frozen samples. This procedure involves the manipulation of liquid nitrogen, uranyl acetate, and HM20. If you are not familiar with the handling of these compounds, please refer to the official guidelines. Uranyl acetate should be preserved from light to prevent the formation of precipitate. Nevertheless, there is no need to work in the dark; avoiding direct and long light exposition of solutions with uranyl acetate as much as possible is sufficient. We can process from one to four different conditions per FS experiments with five to ten samples per condition. To ease the manipulation, the samples of the very same condition are mixed together in the same FS reagent bath (Figure 7). Between each bath, do not remove all liquid, systematically letting a background of liquid to avoid drying of samples. Figure 7. Freeze-substitution (FS). (A) 1. Screwdriver used to transfer; 2. Leica transfer system and 3. its lid (B) 4. Automatic FS 2 lid covered with aluminum foil. (C) 5. Leica reagent bath containing membrane carriers; 6. Flow-through ring molds; 7. Insulated pair of tweezers. Reagent bath and flow-through ring molds are labeled with nail polish to identify the different conditions (black arrowheads). Fill the AFS2 dewar with liquid nitrogen and set its temperature to -90°C. It can take more than half an hour if the device was at room temperature. For each condition, label one reagent bath using colored nail polish and dispose them in the AFS2 chamber (Figure 7C). Under a chemical hood and away from direct light, prepare the cryomix for FS in a glass sample vial with snap-cap (see Recipe 5). Plan to have 2.5 mL/condition. Ensure that the AFS2 light is switched off and transfer the closed vials containing the FS mix to the AFS2 chamber. By handling in the AFS2 chamber, distribute 2.5 mL of the cryomix into each reagent bath with a Pasteur pipette. Again, ensure that the AFS2 light is switched off and close its chamber by placing the lid previously covered with aluminum foil (Figure 7B). Before the following steps, ensure that the FS mix is pre-cooled by waiting at least 10 min if the AFS2 is already at -90°C. When the temperature of the AFS2 chamber reaches -90°C, quickly transfer your frozen samples from the liquid nitrogen to the AFS2 chamber by using the pre-cooled Leica support (Figure 7A). With pre-cooled tweezers, dispatch the membrane carriers containing the frozen samples into the corresponding baths (Figure 7C). Before closing the AFS2 chamber, prepare a volume of pure acetone (plan to have 2.5 mL/per condition) in a supplemental FS bath that will be used for the next steps. Note: If necessary, you can switch on the light of AFS2 during the handling in the AFS2 chamber. Do not forget to switch it off when handling is finished. Set the AFS2 as follows before starting the program: Temperature Duration -90°C 30 h +3°C/h 13 h -50°C Standby After 43 h of FS in the cryomix, when the temperature reaches -50°C, wash your samples by removing the cryomix and replacing it by 2.5 mL of pre-cooled pure acetone. Refill the supplemental FS bath without sample by a new volume of pure acetone. Wait 10 min to cool down the acetone. Repeat the step B2.f. twice. After the third wash with pure acetone, fill the supplemental FS bath with pure ethanol and wait 10 min for cooling down before using ethanol to wash the different samples. Repeat ethanol washes twice. An optional step for roots, but also highly encouraged for bigger samples like hypocotyls and graft interfaces, consists of carefully separating the samples from the membrane carriers using microtools during the last ethanol wash. This step helps to optimize the resin infiltration into the biological tissues. A step-by-step guide and illustration of this procedure is described in Nicolas et al. (2018). To free space in the AFS2 chamber, remove the supplemental reagent bath you used for rinsing. For each condition, label reagent baths plus flow-through rings using colored nail polish (Figure 7C). Place them into the AFS2. Fill them with 2.5 mL of pure ethanol and let them cool down by waiting 10 min. If you have more than two different conditions, you can sequentially add more reagent bath into the AFS2 chamber to avoid an overfilled chamber, which hampers handling. Transfer your samples from the FS bath to one position of the plastic mold flow-through rings by pipetting the sample (it has to stay close to the tip of the pipette) with a disposable pipette of 3 mL. For samples that are kept in their carriers, use pre-cooled tweezers and place the sample on the upper face in one position of flow-through rings. Using the AFS2 binocular, center each sample in its well of the flow-through ring with a microtool. Under a chemical hood, prepare the first mix for the resin embedding procedure (25% of HM20 in pure ethanol) in a Leica reagent container. Plan to use 2.5 mL for each mold. Close the container before shaking it and bringing it into the AFS2 chamber for precooling. After 10 min, replace the pure ethanol in each mold by the embedding mix by slowly pipetting. To progressively lead to a pure HM20 bath, repeat the last step for the other embedding mix (2. to 6.) following this procedure: Mix Temperature Duration 1. HM20 25% -50°C 2 h 2. HM20 50% -50°C 2 h 3. HM20 75% -50°C overnight 4. HM20 100% -50°C 2 h 5. HM20 100% -50°C 2 h 6. HM20 100% -50°C 6 h When reaching the last embedding bath (sixth bath), mount the UV lamp and set the AFS2 as follows for the last embedding step and resin polymerization under UV: Mix Temperature Duration HM20 100% + UV OFF -50°C 6 h HM20 100% +UV ON -50°C 24 h HM20 100% +UV ON +60°C/h ~1 h HM20 100% +UV ON 20°C 12 h Note: For thinner samples such as root tips, the third HM20 bath could be reduced to 2 h. For our protocol, we also reduced the polymerization time under UV to limit the UV exposition of fluorescent proteins. A polymerization time of 24 h at -50°C followed by 12 h at 20°C gets resin blocks hard enough for ultramicrotomy. Once the resin is polymerized, remove the resin blocks from the AFS2 chamber and store them in the dark in a freezer at -20°C to prevent fluorescence from photo-oxidation. Note: For our graft interface, in samples stored for one to two years the YFP signal tends to be weak or even undetectable, while the red fluorescent protein (RFP) signal seems to be better preserved. In our opinion, to ensure the success of the CLEM approach, the processing and the observation of embedded samples should be done as quickly as possible. Table 2. Timetable of the cryo-fixation and freeze-substitution (FS) for plant correlative light and electron microscopy (CLEM). Ultramicrotomy Note: During the following steps, prevent your resin block from light exposure by limiting the power of ultramicrotome light and by placing an opaque cover on your block when not sectioning. We recommend performing the sectioning step and the confocal observations on the same day to avoid a loss of the fluorescence linked to oxygen exposition of the sections. Nevertheless, if sections are prepared a few days in advance, we recommend storing them at -20°C in the dark to slow the photo-oxidation reactions. In most cases, the conventional thickness of ultrathin sections (from 60 to 90 nm) did not allow us to detect enough fluorescent signal. In order to increase the quantity of fluorescent proteins in our sections, we increased their thickness up to 150 nm, which, in our conditions, is the optimal balance between the quantity of signal under the confocal microscope and the resolution under a 120 kV TEM. Thicker sections are less electron lucent, and the low signal-noise ratio impairs the resolution. Increasing the conventional thickness causes a loss of resolution that could be compensated by the use of a higher voltage for TEM and the electron tomography process. Note that if your fluorescence signal is very intense, you can reduce the thickness of the sections and reach a better resolution. In sum, you have to find a good balance for your samples to have enough fluorescent proteins in the section to be detected without impairing to much your final resolution on TEM. As samples are embedded in HM20, sections need to be harvested on film-coated grids. A detailed protocol of grid filming and resin block preparation is described in Nicolas et al. (2018). We recommend working with 200 square mesh copper grids with thin bars that give a good compromise between the field of view and the robustness. To avoid condensation on your block during sectioning, remove it from the freezer and keep it at room temperature for a few minutes in a place protected from light (a drawer for example). Set the speed at 500 mm/s and the thickness at 500 nm on your ultramicrotome. Trim your block with a cryotrim knife to remove the excess of resin and shape your pyramid. When you start to cut your sample, replace the cryotrim by a histoknife. Set the speed at 1 mm/s and the thickness at 500 nm and set the cutting window with “start” and “end” on the touchscreen. Control that you reached the region of interest (ROI) by making sections of 500 nm before staining it with toluidine blue. When the ROI is reached, replace the histoknife by an ultra-knife to make 150 nm thick sections. Harvest sections on copper grids coated with parlodion and store in the dark until dry to ensure their perfect adhesion on the parlodion film before making microscopy observations. Proceed to the mounting of the section between the glass slide and the coverslip.This step is delicate due to the weakness of the grids and sections. To be compatible with ulterior tomography acquisitions, grids have to be kept as flat as possible. Pay attention to manipulate the grids only on their peripheries with thin forceps. Moreover, homogeneous water immersion of the section appears to be crucial to ensure good confocal acquisitions. Take special care to avoid air bubble formation between the section and the coverslip during the grid immersion prior to the fluorescence acquisitions (Figure 8). Figure 8. Air bubbles formed during the mounting step. Formation of air bubbles during the mounting can deteriorate the quality of light microscopy acquisitions due to different refraction indexes. Scale bar: 20 µm. Optional: Glow discharge If you meet difficulties in the prevention of air bubble formation, use a glow discharger to turn the parlodion and the sections hydrophilic. Glow discharge your grids one by one just before mounting, as the effect will last only approximately 30 min. Turn on the glow discharger system. Clean the glow discharger chamber by doing a blank cycle: place a coverslip inside the vacuum bell and close the chamber. Close the air valve and let the pressure go down to 2 × 10-1 mbar. Press the high-voltage button and adjust the voltage until the current reaches 15–20 mA. Maintain the high voltage during 30 s before gradually breaking the vacuum by opening slowly the air valve to avoid coverslip flying due to a high depressurization. Open the vacuum bell and place the grid on the coverslip. Close the air valve and let the pressure go down to 2 × 10-1 mbar. Press the high-voltage button and maintain the high voltage during 1 min. Gradually break the vacuum by opening the air valve very slowly and pick your grid up. Mount the grids for confocal microscopy to prevent air bubbles (Figure 9A–9F, Video 2). Video 2. Mounting the TEM grids for confocal imaging Figure 9. Mounting and unmounting of the transmission electron microscopy (TEM) grids for light microscopy (LM) observation. (A–F)Mounting of the TEM grids. (A) Prior to observation, clean the slide with ethanol to eliminate dust that can pollute the TEM grids. Then, use a syringe to put a drop of filtered water. You can wet the TEM grids on both sides so it will be easier to drop it into water later. Position the ultrathin section on the top to permit the observation of fluorescence signal even for the parts that are located on the bars of the TEM grid. (B) Approach the coverslip until it touches water. (C) Carefully place the grid between the glass slide and the coverslip with very thin tweezers. Avoid the formation of air bubbles at the surface of the grid by plunging the grid into water. (D) Release the grid from the tweezers and hold the grid in the water with the tip of the tweezers so the grid does not move when lying down the coverslip. (E) Release the grid from the tip and use the tip as a guide to slowly place the coverslip. (F) Finally, dry the excess of water with blotting paper prior to imaging. (G–L)Unmounting the TEM grids. (G and H) Add filtered water with a syringe to the side of the coverslip and wait a few minutes. (I)The excess of water helps to easily detach the coverslip from the glass slide. Slowly tilt the glass slide until a corner of the coverslip is outside the slide. (J)Remove the coverslip very carefully. At this step, there should be no resistance to avoid breaking the parlodion film. (K–L) Add more filtered water until the TEM grids unstick from the slide before taking it with the tweezers. For this step, we recommend using inverted forceps that are closed by default. Take your grid with forceps and place the forceps on the table. With a syringe and a 0.2 µm filter, put a drop of water on a microscope slide pre-cleaned with ethanol. Approach a coverslip obliquely to the glass slide until it touches the water drop. Carefully take the forceps with the grid and dip the grid into the drop of water between the slide and the coverslip. Slowly open the forceps to release the grid, which should be immersed in the water, and, with the tip of the tweezers, hold the grid in the water and lay down the coverslip on the forceps. Remove the excess of water on the glass slide with blotting paper or paper towels. You can now place the slide under the LM and do your observations. Confocal microscopy During LM analyses, as the signal in the sections can be low, you could think that the fluorescence preservation failed when this is actually not the case. To detect fluorescence signal, you need to preserve and collect the maximum of photons that will be produced by your sample for your final acquisitions. In general, only one z-stack acquisition per ROI with convenient signal-noise ratio is possible for YFP. Use weak transmitted light to position you ROI in the center of the field of view rather than the fluorescent excitation source. Choose the most sensitive detector (GaAsP instead of the conventional multi-alkali photocathode) for your acquisitions. If your confocal is equipped with an AiryScan detector, it will allow you to improve the signal and the resolution. Moreover, to have the best resolution for your acquisitions, choose the objective with the highest numerical aperture (between 1.3 and 1.4) with a good fluorescence transmission and corrected to work with a coverslip. Set the larger emission window as possible to collect a maximum of emitted photons. You should have a motorized stage and consider doing Z-stack to collect the maximum of photons emitted by the section. Also, to facilitate correlation and to ensure the observation of the same region of interest, doing a tiles scan (to have an overview with good resolution) and acquiring the transmission image will help. Start your observations through the oculars with a low magnification objective (such as 10×) with transmitted light to find and center your grid. To localize your section more easily, close the aperture diaphragm to increase the contrast and find the edges of the section. Increase the resolution by placing your best objective. Adjust the focus and the positioning of your sample on the stage and correctly set the aperture diaphragm (same numerical aperture as the one of the objective in place). Shift to the confocal mode. Switch on only the mRFP excitation line and activate the detector in the transmitted position plus the most sensitive fluorescent detector. For this last one, push the signal amplification by setting the gain up to 850. Switch on only the mRFP excitation line and/or chlorophyll to preserve the maximum YFP fluorescence. Start fast-scanning to target the graft interface using the transmitted and mRFP channels and set the upper and lower Z-window limits for the Z-stack. Avoid setting a too thick Z-stack, which would induce an unproductive over-exposition of your sample. Stop the scanning and set a sequential mode acquisition: YFP and chlorophyll on one track and mRFP on a second track. Optimize the parameters for acquisitions: pinhole opening, pixel size, z-interval, and pixel dwell time around 2 µs, and push the excitation power (up to 12% for the 488 wavelength and 3% for the 561 wavelength). When all previous parameters are well set, start your Z-stack acquisition. Despite the thickness of the section, not all fluorescent regions are on the same slice of the Z-stack. To facilitate the visualization, a Z-projection is systematically achieved after the Z-stack acquisition; either maximal or sum projection can work depending on your sample. Moreover, despite all efforts made for optimizing the acquisition, YFP visualization could require a compression of intensity levels of the histogram. Removal of the EM grid (Figure 9G–9L, Video 2) Long observations under the confocal microscope can lead to evaporation of the water between slide and coverslip, with a risk for the EM grid to stick to the glass. Taking off the coverslip when it is stuck to the glass greatly increases the chance of breaking the parlodion film. To remove the coverslip without breaking the film, put water using a syringe (and filter) around the coverslip and wait until the coverslip is floating on water. Carefully remove the coverslip with the forceps. If a grid is stuck to the coverslip or the glass slide, put a drop of water around the grid and wait until the grid detaches by itself and floats before taking it gently with the forceps tip. Dry the grids by blotting it vertically on Whatman paper and put them back in the grid box for subsequent EM observations. Be sure to remember on which side of the grid your sample is, so you can observe the same side under EM (otherwise, you will have a mirror image of your sample and the correlation and localization of your region of interest will be much harder). The fluorescence pattern observed under the confocal microscope allows the identification of cells under the EM based on their shapes. TEM and electron tomography: TEM observations impair molecule conformations. Fluorescence observations as well as immuno-labeling, if it is required, have to be performed systematically before the first TEM observation. For correlation, TEM overviews can be required to match the field of view of LM for correlation. Based on the shape of cells, the ROI observed with LM is targeted under TEM. To avoid miscalling and deformation during TEM acquisitions, get help from the staff of the microscopy facility to ensure the proper alignment of the TEM and the proper setting of the eucentric height. For studies that require a high ultrastructural resolution, electron tomography has to be done on the structures of interest. It allows to obtain a 3D view and to restore the loss of resolution related to thicker sections. Only required for electron tomography: On a parafilm strip placed on the bench, put a drop of 20 µL of the gold fiducial solution (see Recipe 9). Put the grid of interest on the drop and let it float for 1 min. Remove the grid with tweezers and remove the excess of gold fiducial solution remaining on the grid by carefully blotting it at its periphery with a Whatman paper. Place the other face of the grid on the drop and let it float for 1 min before repeating step iii. Let the grid dry before inserting it into the TEM. Insert the grid into the TEM. Based on the cell shapes and their positions in relation to the mesh of the grid, target the ROI previously localized by LM. Acquire a series of images to cover the integral meshes of interest with the magnification for which the objective diaphragm can be introduced. It allows you to get a complete mosaic of the ROI with enough contrast and resolution (Figure 10A). Increase the magnification to observe your structures of interest and acquire your data. For plasmodesmata, we increased the magnification up to 41,000× and took images for intermediate magnifications to ensure success for ulterior correlations (Figure 11). If beads were added, electron tomography acquisitions can be successfully done following the process described in the Bio-protocol paper of Nicolas et al. (2018) (Figure 11D). Figure 10. Mosaic acquisition and merging allows to have a large field of view for later correlation. (A) Sixteen images are acquired with the objective diaphragm on the transmission electron microscope to have a large field of view covering the graft interface. (B) Images are automatically arranged in order by the photomerge function of Adobe Photoshop. (C) Photomerge can homogenize contrast and exposure and correct geometrical deformation for a seamless fusion. Correlation After the acquisition of images by both LM and TEM/electron tomography, correlation of both microscopies needs to be performed to get the most out of a CLEM approach. Mosaic images (acquired at step 6c.) from TEM are fused using the photomerge function of Adobe Photoshop software (Figure 10). It permits you to have a large field of view, eventually containing the whole graft interface. At this stage, it is important to keep the raw images composing the mosaic for later analysis, as Photoshop is not able to preserve image calibration. Correlation between LM and TEM is performed through the ec-CLEM plugin (Paul-Gilloteaux et al., 2017) of the Icy software (De Chaumont et al., 2012). On overviews, cellular landmarks that are easily recognizable in LM and TEM can be used (such as plastids, the nucleus, and cell corners, but also dirt particles) (Figure 11A). A detailed procedure and video tutorials on using ec-CLEM and Icy software are available (https://icy.bioimageanalysis.org/plugin/ec-clem/). Here is a step-by-step protocol on its use on grafted hypocotyl specimens. Figure 11. From correlated overview to high-resolution correlated tomogram. (A) Large CLEM field of view of a region of interest (ROI). (B) Higher magnification of the region highlighted in (A). (C) Higher magnification of the region highlighted in (B). (D) Correlated electron tomogram of the ROI corresponding to (C). Scale bars: (A) 10 µm, (B) 5 µm, (C) 0.2 µm, and (D) 0.05 µm. Copy your LM and TEM acquisitions on a new folder dedicated to the correlation where automatic xml files generated by the Icy software for automatic saving will be stored. Open your TEM and LM images in Icy using Image > Open or by dragging and dropping your images in the software. Once your images are open, run the ec-CLEM plugin. Set the plugin as follows: start working with “2D but let me update myself” to avoid slow process due to automatic updates of the transformation. This option is especially useful when the workstation is not powerful enough. Select the images to process following ec-CLEM recommendations [i.e., fluorescent microscopy (FM) image to be transformed and TEM image not to be modified] and press play to run the plugin. Place the first points on a landmark on TEM image. Adjust the position of the same landmark on the LM image. Repeat that for at least 10 points, ideally covering every region of the image, and press Update transformation . This will transform and resize the FM image based on the points you placed. Link the two images using the lock button so their display can be synchronized to place other points more easily. Working on overviews allows you to place 10–25 markers to get a more precise correlation. When all points are placed, update transformation again and then press the Stop button in the plugin window. This will automatically generate the correlated image that you can save. At this stage, the software may display an error message to inform you that the correlation is not perfect. This is due to anisotropic deformations linked to the HM-20 shrinkage under the electron beam. To refine the correlation, before closing images, go back to the ec-CLEM plugin window and change the transformation to non-rigid (2D or 3D) , press play, and update transformation. Press Stop to generate the correlated image. The image generated through this method will be anisotropically transformed so each correlation point can match. You can save your image. With the help of this correlation from overviews, TEM images at higher magnifications or electron tomography acquisitions can be used to produce high-resolution CLEM images (Figure11). Note that correlative images are generated for visualization only and are not intended for measurement as dimension of pixel could be modified at different stages of the process (merging TEM images, correlation). If you want to measure certain characteristics of your structure of interest, such as cell wall thickness in our case, or distance between organelles for example, you have to go back to the original raw TEM images. Data analysis Our grafting protocol combined with HPF and our FS protocol adapted for CLEM allowed us to preserve the fluorescence of the samples together with good ultrastructural resolution. At the graft interface of A. thaliana, the origin of the cells can now be determined without any ambiguity using the expression of different fluorescent proteins for each partner (Figure 11). In comparison with standard FS with osmium tetroxide, lipidic components such as membranes are less contrasted, but still clearly visible. Moreover, if necessary, electron tomography can be applied to improve the resolution (Figure 12), as it restores the contrast quality leading to a very high resolution. Cytoplasm and plasmodesmata observed by conventional electron microscopy were indistinct, while with electron tomography they appeared much sharper, more highly contrasted, and with a higher resolution. Details such as membranes of plasmodesmata, clear ribosomes (either free or associated with the endoplasmic reticulum), and small vesicles were clearly visible. Electron tomography allowed us to reach a high 3D level of detail for the structures of interest, such as plasmodesmata, and to study their shapes in three dimensions, while this was not possible with conventional electron microscopy images (Figure 12). Figure 12. Electron tomography and segmentation of plasmodesmata at the graft interface. (A) Tomogram of the plasmodesmata observed at the graft interface z = 0 nm. (B) Tomogram of the PD observed at the graft interface z = + 11 nm. (C) Tomogram of the PD observed at the graft interface z = + 31 nm. (D) Segmentation of the tomograms allows the visualization of the structure in 3D. Scale bars (A–C): 100 nm. Additionally, our protocol preserves antigenicity, thus enabling immunolabeling of desired structures for further identification under electron microscopy (Figure 13). Finally, this CLEM protocol was successfully applied on other plant tissues such as roots (Dragwidge et al., 2022; Gomez et al., 2022). Figure 13. Immunolocalization of the yellow fluorescent protein (YFP). (A) Confocal observations of a graft interface of HDEL_YFP grafted onto HDEL_mRFP and (B) the corresponding correlated view. (C) Magnification of the region highlighted by the white square in (B), showing the endoplasmic reticulum of HDEL_mRFP (red dotted squared) and HDEL_YFP (green dotted squared). (D) Magnification of these regions reveal the presence of the endoplasmic reticulum immunolabeled with gold particles for HDEL_YFP cell, while there are no gold particles in the endoplasmic reticulum of HDEL_mRFP cell. Scale bars: (A and B) 3 µm, (C) 1 µm, (D) 500 nm. Notes When doing the seed sterilization, you need to work with bleach and acid separately to prevent an accidental mix of the two reagents. Always use the appropriate protective equipment as gloves and a lab coat. Recipes Bleach solution for seeds sterilization Dissolve one tablet (containing 1.5 g of active bleach) in 50 mL of distilled water (3% w/v in active bleach) Culture media for seedlings and grafting Half-strength MS medium for seedlings (1 L) MS medium + vitamins: 2.2 g Plant agar: 8 g Adjust pH with 1 N KOH solution to 5.8 Autoclave flasks at 120°C for 20 min Water medium for grafting Volume (mL) 100 200 300 40 500 1,000 Approximate number of Petri dishes 5 10 15 20 25 50 Agar (g) 1.6 3.2 4.8 6.4 8 16 Adjust pH with 1 N KOH solution to 5.8. Autoclave flasks at 120°C for 20 min Put the autoclaved medium in the Petri dishes while it is still hot and liquid. Prepare only the amount of medium you need, according to the planned number of grafts. 20% BSA solution for cryoprotection during HPF Weigh 0.4 g of BSA and put it into a 2 mL screw-top sample tube. Complete with water (or your medium culture) up to 2 mL. Altern quick centrifugations and vortex until BSA is dissolved entirely. If necessary, complete with water up to 2 mL to get a final concentration of 20%. Cryosubstitution uranyl-acetate stock solution (20%, 500 µL) Weigh 0.1 g of uranyl acetate powder (gloves and dust mask required) and put in a 2 mL screw-cap tube. Add pure methanol (under a ventilated hood) up to 500 µL. Wrap the tube in tinfoil and store in the dark at -20°C in a dedicated cryosubstitution box. Cryosubstitution mix for 3 mL Compound (initial concentration) Volume (mL) Final concentration Uranyl acetate powder (20% w/v in pure methanol) 0.015 0.1% Pure acetone 2.985 - HM20 solutions Make the solution in the FS Leica plastic solvent container and cool it for 10–15 min in AFS chamber before use. HM20 concentration HM20 volume (mL) Pure ethanol (mL) 20% 2 8 50% 5 5 75% 7.5 2.5 2% parlodion solution for grid filming Prepare only a small quantity of parlodion in a small glass flask, as it is very sensitive to humidity and will not preserve properly over a long time. Weigh a small piece of solid parlodion (approximately 0.01 g) after cutting. Add isoamyl-acetate (store in a vented solvent cupboard and keep away from humidity) up to 1 mL. Wrap the flask in tinfoil and seal the lid with Parafilm. Before first use, agitate to homogenize the solution. Always seal the lid and protect from light. Toluidine blue solution Make a stock solution with 1 g of toluidine blue powder and 1 g of sodium borate in 20 mL of distilled water. Dilute 25 times to prepare the working solution. Gold fiducial solution Make a solution of 0.5% BSA in MilliQ water. Filtrate the solution with a 0.2 µm filter connected to a syringe. Mix colloidal gold solution in 0.5% BSA (1:1 ratio). Aliquot in 0.5 mL sample tubes and store at -20°C. Acknowledgments The Ph.D. thesis of C.C. was financed by grants entitled “Etude des mécanismes de l’union greffon /porte-greffe,” 50% from the French National Institute for Agronomical Research/INRAE department “Plant biology and breeding/BAP” and 50% from the Région Nouvelle Aquitaine, France. Further financial support was provided by the Starter grant “Etude des mécanismes de l’union greffon/porte-greffe” also from BAP. The Institute of Vine and Wine Science (ISVV) supported a travel grant for C.C. to go to Charles Melnyk’s lab to learn grafting procedure. Thanks to Charles Melnyk and Remi Lemoine who invited me to learn the grafting procedure of A. thaliana . This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 852136 to AB). All sample preparation and imaging was done on the Pôle Imagerie du Végétale, from Bordeaux Imaging Centre (http://www.bic.u-bordeaux.fr/). Competing interests The authors declare to have no competing interest References Bell, K., Mitchell, S., Paultre, D., Posch, M. and Oparka, K. (2013). Correlative imaging of fluorescent proteins in resin-embedded plant material. Plant Physiol 161(4): 1595-1603. Bidadi, H., Matsuoka, K., Sage-Ono, K., Fukushima, J., Pitaksaringkarn, W., Asahina, M., Yamaguchi, S., Sawa, S., Fukuda, H., Matsubayashi, Y., et al. (2014). CLE6 expression recovers gibberellin deficiency to promote shoot growth in Arabidopsis. Plant J 78(2): 241-252. Chambaud, C., Cookson, S. J., Ollat, N., Bayer, E. and Brocard, L. (2022). A correlative light electron microscopy approach reveals plasmodesmata ultrastructure at the graft interface. Plant Physiol 188(1): 44-55. De Chaumont, F., Dallongeville, S., Chenouard, N., Herve, N., Pop, S., Provoost, T., Meas-Yedid, V., Pankajakshan, P., Lecomte, T., Le Montagner, Y., et al. (2012). Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9(7): 690-696. Dean, N. and Pelham, H. R. (1990). Recycling of proteins from the Golgi compartment to the ER in yeast. J Cell Biol 111(2): 369-377. Dragwidge, J. M., Wang, Y., Brocard, L., De Meyer, A., Hudeček, R., Eeckhout, D., Chambaud, C., Pejchar, P., Potocký, M., Vandorpe, M., et al. (2022). AtEH/Pan1 proteins drive phase separation of the TPLATE complex and clathrin polymerisation during plant endocytosis. BioRxiv, 2022.03.17.484738. Gautier, A. T., Chambaud, C., Brocard, L., Ollat, N., Gambetta, G. A., Delrot, S. and Cookson, S. J. (2019). Merging genotypes: graft union formation and scion-rootstock interactions. J Exp Bot 70(3): 747-755. Gomez, R. E., Chambaud, C., Lupette, J., Castets, J., Pascal, S., Brocard, L., Noack, L., Jaillais, Y., Joubes, J. and Bernard, A. (2022). Phosphatidylinositol-4-phosphate controls autophagosome formation in Arabidopsis thaliana. Nat Commun 13(1): 4385. Lindsey, B. E., 3rd, Rivero, L., Calhoun, C. S., Grotewold, E. and Brkljacic, J. (2017). Standardized Method for High-throughput Sterilization of Arabidopsis Seeds. J Vis Exp(128): 56587. Knox, K., Wang, P., Kriechbaumer, V., Tilsner, J., Frigerio, L., Sparkes, I., Hawes, C. and Oparka, K. (2015). Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation. Plant Physiol 168(4): 1563-1572. Kollmann, R. and Glockmann, C. (1985). Studies on graft unions. I. Plasmodesmata between cells of plants belonging to different unrelated taxa. Protoplasma 124(3): 224-235. Kollmann, R. and Glockmann, C. (1991). Studies on graft unions - III. On the mechanism of secondary formation of plasmodesmata at the graft interface. Protoplasma 165(1-3): 71-85. Kollmann, R., Yang, S. and Glockmann, C. (1985). Studies on graft unions - II. Continuous and half plasmodesmata in different regions of the graft interface. Protoplasma 126(1-2): 19-29. Kremer, J. R., Mastronarde, D. N. and McIntosh, J. R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1): 71-76. Kukulski, W., Schorb, M., Kaksonen, M. and Briggs, J. A. (2012a). Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150(3): 508-520. Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. and Briggs, J. A. (2012b). Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers. Methods Cell Biol 111: 235-257. Lee, H., Sparkes, I., Gattolin, S., Dzimitrowicz, N., Roberts, L. M., Hawes, C. and Frigerio, L. (2013). An Arabidopsis reticulon and the atlastin homologue RHD3-like2 act together in shaping the tubular endoplasmic reticulum. New Phytol 197: 481-489. Marion, J., Le Bars, R., Satiat-Jeunemaitre, B. and Boulogne, C. (2017). Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots. J Struct Biol 198(3): 196-202. Melnyk, C. W. (2017a). Grafting with Arabidopsis thaliana. Methods Mol Biol 1497: 9-18. Melnyk, C. W. (2017b). Monitoring Vascular Regeneration and Xylem Connectivity in Arabidopsis thaliana. Methods Mol Biol 1544: 91-102. Melnyk, C. W., Gabel, A., Hardcastle, T. J., Robinson, S. and Miyashima, S. (2018). Transcriptome dynamics at Arabidopsis graft junctions reveal an intertissue recognition mechanism that activates vascular regeneration.PNAS 115(10). https://doi.org/10.1073/pnas.1718263115. Melnyk, C. W., Schuster, C., Leyser, O. and Meyerowitz, E. M. (2015). A Developmental Framework for Graft Formation and Vascular Reconnection in Arabidopsis thaliana. Curr Biol 25(10): 1306-1318. Molnar, A., Melnyk, C. W., Bassett, A., Hardcastle, T. J., Dunn, R. and Baulcombe, D. C. (2010). Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328(5980): 872-875. Mudge, K. (2009). A History of Grafting. Horticultural Reviews 35: 437-494. Nelson B. and Nebenführ (2007). A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126-1136. Nicolas, W. J., Bayer, E. and Brocard, L. (2018). Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues-from High Pressure Freezing Preparation to Ultrathin Section Collection. Bio-protocol 8(1): e2681. Notaguchi, M., Kurotani, K. I., Sato, Y., Tabata, R., Kawakatsu, Y., Okayasu, K., Sawai, Y., Okada, R., Asahina, M., Ichihashi, Y., et al. (2020). Cell-cell adhesion in plant grafting is facilitated by b-1,4-glucanases.Science 369(6504): 698-702. Paul-Gilloteaux, P., Heiligenstein, X., Belle, M., Domart, M. C., Larijani, B., Collinson, L., Raposo, G. and Salamero, J. (2017). eC-CLEM: flexible multidimensional registration software for correlative microscopies. Nat Methods 14(2): 102-103. Pina, A., Cookson, S. J., Calatayud, A., Trinchera, A. and Errea, P. (2017). Physiological and molecular mechanisms underlying graft compatibility. Chapter 5. Vegetable Grafting. Principles and Practices. 132-154. Studer, D., Graber, W., Al-Amoudi, A. and Eggli, P. (2001). A new approach for cryofixation by high-pressure freezing. J Microsc 203(Pt 3): 285-294. Turnbull, C. G. N. (2010). Grafting as a Research tool. Methods Mol Biol 655: 11-26. Turnbull, C. G., Booker, J. P. and Leyser, H. M. (2002). Micrografting techniques for testing long-distance signalling in Arabidopsis. Plant J 32(2): 255-262. Wu, R., Wang, X., Lin, Y., Ma, Y., Liu, G., Yu, X., Zhong, S. and Liu, B. (2013). Inter-species grafting caused extensive and heritable alterations of DNA methylation in Solanaceae plants. PLoS One 8(4): e61995. Yang, Y., Mao, L., Jittayasothorn, Y., Kang, Y., Jiao, C., Fei, Z. and Zhong, G.-Y. (2015). Messenger RNA exchange between scions and rootstocks in grafted grapevines.BMC Plant Biology 15(1): 251. Zhang, W., Kollwig, G., Stecyk, E., Apelt, F., Dirks, R. and Kragler, F. (2014). Graft-transmissible movement of inverted-repeat-induced siRNA signals into flowers.Plant J 80(1): 106-121. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant physiology > Tissue analysis Cell Biology > Cell imaging > Electron microscopy Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa William J. Nicolas [...] Elliot M. Meyerowitz Dec 5, 2022 1052 Views Fast and High-Resolution Imaging of Pollinated Stigmatic Cells by Tabletop Scanning Electron Microscopy Lucie Riglet and Isabelle Fobis-Loisy Nov 20, 2024 409 Views Cryo-SEM Investigation of Chlorella Using Filter Paper as Substrate Peng Wan [...] Jinghan Wang Dec 20, 2024 229 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,591	https://bio-protocol.org/en/bpdetail?id=4591&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Rapid Multiplexed Flow Cytometric Validation of CRISPRi sgRNAs in Tissue Culture JC John S. Chorba VX Vivian Q. Xia GS Geoffrey A. Smith AP Arun Padmanabhan Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4591 Views: 1068 Reviewed by: ASWAD KHADILKAR Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Translational Medicine Sep 2022 Abstract Genome-wide CRISPR-based screening is a powerful tool in forward genetics, enabling biologic discovery by linking a desired phenotype to a specific genetic perturbation. However, hits from a genome-wide screen require individual validation to reproduce and accurately quantify their effects outside of a pooled experiment. Here, we describe a step-by-step protocol to rapidly assess the effects of individual sgRNAs from CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. All steps, including cloning, lentivirus generation, cell transduction, and phenotypic readout, can be performed entirely in 96-well plates. The system is highly flexible in both cell type and selection system, requiring only that the phenotype(s) of interest be read out via flow cytometry. We expect that this protocol will provide researchers with a rapid way to sift through potential screening hits, and prioritize them for deeper analysis in more complex in vitro or even in vivo systems. Graphical abstract Keywords: CRISPRi Forward genetics Hit validation Flow cytometry Background Pooled screening methods leveraging the CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats) system constitute powerful tools for unbiased profiling of gene function across the coding genome (Shalem et al., 2015; Bock et al., 2022). CRISPR interference (CRISPRi) uses a nuclease-dead Cas9 (dCas9) fused to a transcriptional repressor to produce highly potent gene silencing (Qi et al., 2013; Gilbert et al., 2014), with the advantages of avoiding toxicity from double-strand DNA breaks, increased specificity for genetic targets, potential reversibility of effects, and multiplexed gene knockdown (Horlbeck et al., 2016; Mandegar et al., 2016; Reis et al., 2019). A successful genome-wide screening experiment is expected to yield a list of potential genetic hits whose perturbations affect the phenotype of interest. Yet, these hits require subsequent validation for several reasons. First, the effects of individual gene knockdowns need to be confirmed in isolated, as opposed to pooled, conditions. Second, independent confirmation of multiple small-guide RNA (sgRNA) sequences against the same target gene increases the confidence that the desired phenotype results from on-target knockdown. Third, identifying the most potent sgRNA facilitates downstream interrogation of gene function. Fourth, sequence-specific toxicity unique to a particular sgRNA must be excluded (Cui et al., 2018). Lastly, in the pooled screen, the strength of the genetic perturbation effect is only indirectly inferred from the degree of sgRNA enrichment among cells displaying a pre-defined phenotypic strength. Therefore, a direct assessment of effect strength requires a quantitative evaluation of the individual gene knockdown on the phenotype of interest, as well as a comparison to a non-targeting control. Unbiased screens may produce a hit list of hundreds of genes, requiring cloning, lentiviral generation, transduction, and phenotypic analysis of at least twice the number of sgRNAs. Interrogating this list can be time-consuming and costly, if using traditional cloning or viral generation methods. To address this bottleneck, here we describe a step-by-step protocol to generate and phenotype individual sgRNA expressing cells entirely in 96-well plates, which we have used to identify a previously undiscovered regulator of the low density lipoprotein receptor (LDLR) (Smith et al., 2022). The protocol is applicable to either the CRISPRi or CRISPR activation (CRISPRa) systems, as both use the same sgRNA delivery vector (Horlbeck et al., 2016), and offers flexibility in phenotypic analysis. The flow cytometry readout offers functional insights at the single cell level. Comparisons can be performed either against non-transduced cells in the same microenvironment, or between separately generated cell populations harboring either a targeting or control sgRNA after puromycin selection. We limit our described protocols to the measurements of cell surface markers detectable by fluorophore antibody conjugates, and the internalization of fluorescently labeled ligands. However, in principle, the protocol could be amended to use any fluorescence-based readout compatible with flow cytometry on the cell type of interest. Furthermore, as this is fundamentally a method to deliver and phenotype many clonally derived vectors in parallel, we expect that it would be easily adaptable to other moderate-sized focused lentiviral libraries. Materials and Reagents 0.2 mL PCR tubes (USA Scientific, catalog number: 1402-2900) Sterilized, low retention, LTS pipette tips (Rainin, catalog numbers: 30389213, 30389240, 30389226) 1.5 mL Eppendorf Safe-Lock centrifuge tubes (USA Scientific, catalog number: 4036-3204) Falcon 15 mL conical centrifuge tubes (Corning catalog number: 352097) Falcon 50 mL conical centrifuge tubes (Corning, catalog number: 352098) Accutec razor blades (ThermoFisher Scientific, catalog number: S17302) 14 mL culture tubes (Avantor/VWR, catalog number: 60818-689) Parafilm (ThermoFisher Scientific, catalog number: S37440) 96-well, cell culture-treated, flat-bottom microplate (Corning, catalog number: 3903) 5 mL, 10 mL, 25 mL, and 50 mL serologic pipettes (Santa Cruz Biotechnology, catalog numbers: sc-200279, sc-200281, sc-200283, sc-213234) Countess cell counting chamber slides (ThermoFisher Scientific, catalog number: C10228) Sterile 96-well v-bottom polypropylene plates (Corning, catalog number: 3357) 50 mL, 250 mL, 500 mL, and 1000 mL Nalgene Rapid-Flow sterile single use vacuum filter units (ThermoFisher Scientific, catalog numbers: 5640020, 5680020, 5660020, 5670020) 23G sterile blunt needles (ThermoFisher Scientific, catalog number: NC9031029) NORM-JECT luer lock sterile syringes (Avantor/VWR, catalog number: 53548-023) 90 micron nylon mesh (Elko Filtering, catalog number: 03-90/49) Polypropylene microtiter (“bullet”) tubes (ThermoFisher Scientific, catalog number: 02-681-376) 5 mL round bottom polystyrene with cell strainer cap (Corning, catalog number: 352235) MultiScreenHTS HV sterile filter plate, 0.45 µm (Millipore Sigma, catalog number: MSHVS4510) Aluminum foil (Avantor/VWR, catalog number: 89125-940) Desalted, custom synthesized oligonucleotides (see step A1) pCRISPRi/a v2 [Addgene, catalog number: 84832, and described in Horlbeck et al. (2016), store at -20°C] FastDigest BstXI (ThermoFisher Scientific, catalog number: FD1024, store at -20°C) FastDigest Bpu1102I/BlpI (ThermoFisher Scientific, catalog number: FD0094, store at -20°C) Agarose LE (Goldbio, catalog number: A-201-100) SYBR Safe DNA gel stain (ThermoFisher Scientific, catalog number: S33102) E.Z.N.A. gel extraction kit (Omega Bio-Tek, catalog number: D2500-02) E.Z.N.A. plasmid DNA mini kit I (Omega Bio-Tek, catalog number: D6942-02, some components of kit require storage at 4°C) Isopropanol (Millipore Sigma, catalog number: 59304) 1 kb DNA ladder (Goldbio, catalog number: D010-500, store long-term at -20°C, short-term at R.T.) KOPTEC absolute ethanol (Avantor/VWR, catalog number: 89125-172) Sodium acetate (Millipore Sigma, catalog number: S2889) TempPlate non-skirted 96-well PCR plate, 0.2 mL (USA Scientific, catalog number: 1402-9596) Adhesive PCR plate foil seals (Avantor/VWR, catalog number: 60941-076) Potassium acetate (Millipore Sigma, catalog number: P1190) HEPES (Goldbio, catalog number: H-400-100) Tris base (Goldbio, catalog number: T-400-500) Concentrated hydrochloric acid (Avantor/VWR, catalog number: JT9535) Potassium hydroxide (Millipore Sigma, catalog number: P5958) Magnesium acetate (Millipore Sigma, catalog number: M5661) T4 DNA Ligase (New England BioLabs, catalog number: M0202L, store both enzyme and buffer at -20°C) One Shot Mach1 T1 phage-resistant chemical competent E. coli (ThermoFisher Scientific, catalog number: C862003, store at -80°C) Mix & Go! E. coli transformation kit (Zymo Research, catalog number: T3001, some components of kit require storage at 4°C) LB Broth (Millipore Sigma, catalog number: L3522) Carbenicillin (Goldbio, catalog number: C-103-5, store at -20°C) LB-agar plates carbenicillin-100 (Teknova, catalog number: L1010, store at 4°C) Zyppy-96 plasmid kit (Zymo Research, catalog number: D4042, some components of kit require storage at 4°C) Lab markers (ThermoFisher Scientific, catalog number: 13-379-4) Glycerol (Millipore Sigma, catalog number: G2025) HEK-293T cells (ATCC, catalog number: CRL-3126, store at -80 to -150°C) Gibco high-glucose DMEM with pyruvate and GlutaMax supplement (ThermoFisher Scientific, catalog number: 10569010, store at 4°C protected from light) Gibco low-glucose DMEM with pyruvate and GlutaMax supplement (ThermoFisher Scientific, catalog number: 10567014, store at 4°C protected from light) Heat-inactivated fetal bovine serum (FBS) (Axenia Biologix, catalog number: F002, aliquot and store at -20°C) Lipoprotein depleted FBS (Kalen Biomedical, catalog number: 880100, aliquot and store at -20°C) Human 3,3’-dioctadcylindocarbocyanine low density lipoprotein (DiI-LDL) (Kalen Biomedical, catalog number: 770230-9, store at 4°C protected from light) Clorox bleach (Office Depot, catalog number: 217595) Lentiviral packaging vectors: pCMV-dR8.91 and pMD2.G (Addgene, catalog number: 12259, and described in (Gilbert et al., 2014), store at -20°C) ViralBoost reagent (Alstem, catalog number: VB100, store at 4°C) Opti-MEM I reduced serum medium (ThermoFisher Scientific, catalog number: 31985062, store at 4°C protected from light) TransIT-LT1 transfection reagent (Mirus Bio, catalog number: MIR2300, store at 4°C) Puromycin (Invivogen, catalog number: ant-pr-1, store at -20°C) Bovine serum albumin (Millipore Sigma, catalog number: 12659, store at 4°C) Sterile reagent reservoirs (Corning, catalog number: 4870) Polybrene infection/transfection reagent (ThermoFisher Scientific, catalog number: TR-1003-G, store at -20°C)) Trypsin-EDTA (0.25%) with phenol red (ThermoFisher Scientific, catalog number: 25-200-114, store long-term at -20°C, short-term at 4°C) Phosphate buffered saline (PBS), pH 7.4, sterile (ThermoFisher Scientific, catalog number: 10010049) Accutase (Innovative Cell Technologies, catalog number: AT104, store long-term at -20°C, short-term at 4°C) Gibco 100× penicillin-streptomycin (ThermoFisher Scientific, catalog number: 15140122, store long-term at -20°C, short-term at 4°C) DNase I (Goldbio, catalog number: D-300-100, store at -20°C) CRISPRi-ready cells (dCas9-BFP-KRAB HepG2 cells are described in (Smith et al., 2022), store at -80 to -150°C) Ghost Dye red 780 (Tonbo Biosciences, catalog number: 13-0865-T100, store at -20°C) Human LDLR Alexa Fluor 647-conjugated antibody (R&D Systems, catalog number: FAB2148R, store at 4°C) Human TfR Alexa Fluor 488-conjugated antibody (R&D Systems, catalog number: FAB2474G, store at 4°C) Dimethyl sulfoxide (Millipore Sigma, catalog number: D2650) 16% formaldehyde solution (Avantor/VWR, catalog number: 100503-917) 2× Annealing Buffer (see Recipes) 50× TAE Buffer (see Recipes) 293T growth medium (see Recipes) Viral harvest medium (see Recipes) HepG2 growth medium (see Recipes) Sterol-depleted medium (see Recipes) FACS buffer (FB) (see Recipes) DiI-LDL labeling medium (see Recipes) Equipment 2 µL, 20 µL, 200 µL, and 1000 µL LTS Pipet-Lite XLS+ manual single channel pipettes (Rainin, catalog number: 30386597) 10 µL, 20 µL, and 200 µL Pipet-Lite XLS+ manual 12-channel pipettes (Rainin, catalog numbers: 17013807, 17013808, 17013810) Pipet-Aid XP2 (USA Scientific, catalog number: 4440-5010) Milli-Q Direct 16 water purification system (Millipore Sigma, catalog number: ZR0Q016WW) ProFlex 3 × 32 well PCR thermocycler (ThermoFisher Scientific, catalog number: 4484073) Mini-Sub Cell GT horizontal electrophoresis system and PowerPac basic power supply (Bio-Rad, catalog number: 1640300) E-Gel imager system with blue-light base (ThermoFisher Scientific, catalog number: 4466612) Dry block heater with 1.5 mL microcentrifuge tube block (Avantor/VWR, catalog numbers: 75838-318, 13259-286) QIAVac 24 Plus vacuum manifold (Qiagen, catalog number: 19413) Microcentrifuge (Eppendorf, catalog number: 5425) NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scientific, catalog number: ND-2000) Heratherm compact microbiological incubator (ThermoFisher Scientific, catalog number: 50125590) New Brunswick Innova 44R incubator shaker (Eppendorf, catalog number: M12820006) Binder CO2 incubator (Avantor/VWR, catalog number: CB170) Vortex mixer (Avantor/VWR, catalog number: 10153-838) FiveEasy pH meter (Mettler Toledo, catalog number: 30266626) -20°C laboratory freezer (ThermoFisher Scientific, catalog number: 02LFEETSA) Ice buckets (Avantor/VWR, catalog number: 10146-188) Allegra X-30R benchtop centrifuge with SX4400 and S606 rotors (Beckman Coulter, catalog number: B06320) Scotsman FLAKER ice maker (CurranTaylor, catalog number: SCF1415A) Aluminum alloy cooling block for 0.2 mL PCR tubes (ThermoFisher Scientific, catalog number: 13-131-013) Laboratory refrigerator (ThermoFisher Scientific, catalog number: TSG25RPGA) Ultra low temperature freezer (ThermoFisher Scientific, Forma catalog number: 995) Countess II FL automated cell counter (ThermoFisher Scientific, catalog number: A32136) Flow cytometer, such as: LSRFortessa with HTS sampler (BD Biosciences), LSRII (BD Biosciences), or CytoFLEX (Beckman Coulter) Autoclave machine Tissue culture hood Laboratory microscope (for tissue culture) Chemical fume hood Software ApE, A plasmid Editor [Described in Davis and Jorgensen (2022) https://jorgensen.biology.utah.edu/wayned/ape/] Flow cytometer acquisition software: FACSDiva (BD Biosciences, https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software) or CytExpert (Beckman Coulter, https://www.beckman.com/flow-cytometry/research-flow-cytometers/cytoflex/software) Prism (GraphPad, https://www.graphpad.com) FlowJo (BD Biosciences, https://www.flowjo.com) Excel (Microsoft, https://www.microsoft.com/en-us/microsoft-365/excel) Procedure Pre-requisite: Perform primary CRISPRi/a screen and identify hits for further validation. Cloning of Validation sgRNA Lentiviral Vectors Note: This cloning protocol has been modified from resources at https://weissman.wi.mit.edu/crispr/, and is reproduced here for convenience. Oligonucleotide Design Use the following template to order standard de-salted oligonucleotides from a vendor of choice, pre-suspended at 100 µM concentration, with one pair (“top” and “bottom”) for each sgRNA to test. The oligos are designed to anneal into a double digest of BstXI and BlpI (Figure 1). sgRNA Template LDLR 1 (Example) Protospacer(20 nt) N1,N2 …N20 GTTACCTGCAGTCCCCGCCG “Top” Oligo(33 nt) ttg(N1,N2 …N20 )gtttaagagc ttgGTTACCTGCAGTCCCCGCCGgtttaagagc “Bottom” Oligo(40 nt) ttagctcttaaac(R.C. of N1,N2 …N20)caacaag ttagctcttaaacCGGCGGGGACTGCAGGTAACcaacaag R.C. = reverse complement Figure 1. Vector Map of pCRISPRi/a-v2. The cleavage sites for BlpI and BstXI are highlighted. Order separate 96-well plates for the “Top” and “Bottom” oligonucleotides, arraying the pair in the matched wells of the complementary plates. Note: The orientation of sgRNAs target in this plate can then be carried forward throughout the entire protocol. Multiple control sgRNAs should be included for the final phenotypic experiment, including those expected to produce a phenotypic effect (biologic controls) and those expected to have a null effect (non-targeting controls). Including non-targeting controls in the last well of every row on a 96-well plate allows for eight biological replicates per plate (Figure 2). Figure 2. Sample Layout of 96-well Plate. Each experimental sample corresponds to a unique sgRNA, with space reserved for biological controls (sgRNAs expected to produce either a gain or loss of function) and non-targeting controls (sgRNAs expected to have a null effect). After a genome-wide screen, typically two sgRNAs are validated per gene. Vector Digestion Combine the following reagents in a PCR tube. Set up separate tubes for each 5-µg vector digest required. Reagent Amount 10× FastDigest Green Buffer 5 µL pCRISPRi/a-v2 5 µg BlpI 2.5 µL BstXI 2.5 µL Milli-Q H2O to 50 µL total Note: 5 µg of purified, digested vector is required for each 96-well plate of sgRNAs to be obtained. Incubate reaction in a PCR thermocycler at 37°C for 1 h. Heat inactivate the reaction at 80°C for 10 min. Resolve entire digest over a 0.7% agarose gel run in TAE buffer, visualize, and excise the band containing the linearized vector. Gel purify the excised band using a gel purification kit, according to the manufacturer’s instructions. Quantify the concentration of purified, digested vector using NanoDrop UV-Vis spectrophotometer. Oligonucleotide Annealing Combine the following components in a fresh 96-well PCR plate, using a multichannel pipette: Reagent Amount 2× Annealing Buffer 20 µL Top Oligo (100 µM) 0.8 µL Bottom Oligo (100 µM) 0.8 µL Milli-Q H2O 18.4 µL Total Volume 40 µL Incubate the reaction in a PCR thermocycler at 95°C for 5 min. Remove the plate from the thermocycler while still at 95°C and allow to cool to room temperature (RT) on the benchtop for 15 min. Dilute 1 µL of annealed oligonucleotide mixture in 19 µL of Milli-Q H2O, in a fresh 96-well PCR plate. Note: Annealed oligonucleotides can be stored at -20°C. Ligation Create ligation master mixes for each 96-well plate of ligations, as indicated below: Note: Creation of two master mixes allows the T4 DNA ligase to be kept separate from the digested vector until in the presence of the oligonucleotide insert, reducing self-ligation. Reagent Final Amount (per well) MM1 (per 96-well plate) MM2 (per 96-well plate) 10× T4 Ligase Buffer (fresh) 1× 60 µL 40 µL Digested pCRISPRi/a-v2 vector 50 ng 5 µg - T4 Ligase 0.5 µL - 50 µL Milli-Q H2O to 10 µL to 500 µL 310 µL Total Volume 10 µL 500 µL 400 µL To each well of a 96-well PCR plate placed on ice, aliquot 5 µL of ligation MM1 , then 1 µL of diluted annealed oligonucleotide mixture, then 4 µL of ligation MM2 . Seal the top of the plate, and incubate at 16°C overnight. Optional : After incubation, heat inactivate the T4 ligase at 65°C for 10 min. Transformation On ice, aliquot 10 µL of competent Mix & Go! Mach1 E. coli cells to each well of a 96-well plate. Note: Competent E. coli can be obtained direct from the vendor, or can be made competent using the Mix & Go! transformation kit, according to the manufacturer’s instructions. Competent cells will quickly lose competency if warmed, so pipette quickly. Pre-chilling the receiver plate and pipette tips to 4°C can also improve the yield of transformants. Add 1 µL of each ligation reaction to the competent cells. Incubate on ice for 30 min. Optional if using Mix & Go! cells : Heat shock the cells in the 96-well plate in a PCR thermocycler at 42°C for 45 sec, then immediately return to ice for 2 min. Partition LB-carbenicillin agar plates into 12 segments. Eight plates will be required for each 96-well plate of sgRNAs. Spot 5 µL of competent cells at the edge and streak towards the center of the plate with a pipette tip (Figure 3). Figure 3. Partitioned LB Agar Plate. Each LB agar plate is split into 12 segments. The transformed mixture is spotted at the edge, and then streaked within the partition, to allow for the selection of individual clones. Streaking within the partitioned area will reduce the risk of contamination between plasmids. Incubate plates at 37°C overnight, to allow for colony growth. Note: Avoid prolonged incubations, as very large colony sizes will make it difficult to select clones and increase the risk of cross-contamination. Clonal Plasmid Isolation Inoculate each well of a 96-well culture block from a Zyppy-96 Plasmid kit containing 760 µL of LB-carbenicillin (at 100 µg/mL) with a colony from each sgRNA-encoding plasmid. Store culture plates at 4°C after colony picking. Note: Selecting two clones per sgRNA for small scale culture growth, each in the corresponding well of separate 96-well-blocks, will improve the chance of obtaining the desired sgRNA sequence. Seal the plate with an air-permeable cover according to the manufacturer’s instructions, and incubate in a shaker at 220–300 rpm and 37°C overnight (12–18 h). After overnight growth, make glycerol stocks for convenient regrowth of plasmids. Transfer 10 µL of each culture into 10 µL of sterile 50% glycerol (in H2O) in a PCR plate. Cover with a plate cover, and store at -80°C. Isolate plasmids from cultures in 96-well blocks using a Zyppy-96 kit, according to the manufacturer’s instructions. Determine concentration and purity of isolated plasmids using a NanoDrop spectrophotometer. Submit samples for Sanger sequencing to a vendor of choice. The following oligonucleotide can be used as the sequencing primer to read out the final protospacer sequence for each clone: 5′-CGCCAATTCTGCAGACAAA-3′. Align sequences using sequence alignment software (ApE), and determine which colonies have the correct sgRNA sequence. For sgRNAs where neither selected clone has the correct sequence, grow four additional small-scale (i.e., 2–5 mL) cultures overnight selected from individual colonies on the stored culture plates, and then purify plasmids using a standard Mini-Prep procedure, and confirm the correct sequencing by Sanger sequencing. After all desired plasmids are obtained, create a master array of the sgRNA delivery plasmids in a fresh 96-well plate in a sterile tissue culture hood. Normalize plasmid concentrations to 25 ng/µL with either Milli-Q H2O or elution buffer. Seal the plate, and store at -20°C. Note: This array offers an opportunity to re-orient the sgRNAs in the 96-well plate, if desired. Lentiviral Production The day prior to transfection, seed HEK-293T cells in tissue culture treated 96-well plates in 100 µL of growth media (high-glucose DMEM with 10% FBS) at 2.2 × 104 cells per well. Grow in 5% CO2 at 37°C overnight. Note: Aim for cells to be 70%–80% confluent at time of transfection. To optimize viral production, use low-passage HEK-293T cells that have not previously grown to over-confluence. Confirm that cells appear morphologically normal prior to transfection. On the day of transfection, prepare the DNA and transfection reagent in sterile 96-well V-bottom plates in the tissue culture hood, as follows: Aliquot 4 µL of each normalized sgRNA vector (100 ng) to a sterile 96-well plate using a multichannel pipette. Make separate master mixes of packaging and envelope plasmids, and diluted transfection reagent, in OptiMEM, as indicated. Add the transfection reagent to OptiMEM dropwise, and mix by gently flicking the tube. Allow the diluted transfection reagent to incubate at RT for 5 min. Reagent Final Amount (per well) MM1 (for 120 wells) MM2 (for 120 wells) pCMV-dR8.91 90 ng 10.8 µg - pMD2.G 11 ng 1.32 µg - Trans-LT1 0.6 µL - 72 Opti-MEM to 14 µL to 600 µL 528 µL Total Volume 14 µL 600 µL 600 µL Briefly vortex plasmid mix, and aliquot 5 µL of this to each well of the plate using a multichannel pipette. Aliquot 5 µL of diluted transfection reagent to each well of the plate. Briefly, mix by tapping the side of the plate. Cover the top of the plate, and incubate at RT for 30 min. Using a multichannel pipette, transfer all 14 µL of transfection mix to the corresponding wells of previously seeded plate(s) of HEK-293T cells. Incubate cells at 5% CO2 and 37°C for 6–8 h. Carefully remove transfection mixture and growth media from each well. Carefully replace with 100 µL of viral harvest medium (growth medium with 11 g/L BSA) supplemented with 1× ViralBoost (0.2 µL 500× ViralBoost per 100 µL medium). Note: Tilt plate and position tip at bottom of the well to avoid aspirating cells. Slowly pipette replacement media along the side of well to avoid dislodging cells . Return plate to incubator at 5% CO2 and 37°C. Forty-eight hours after transfection, add another 100 µL of fresh viral harvest medium containing 1× ViralBoost to each well. Optional : If viral yields prove inadequate, a double harvest can be performed: the viral-containing media can be removed and replaced with 100 µL of fresh viral harvest media (with ViralBoost), with the conditioned media stored at 4°C, to be pooled with the subsequent harvest. Note: Decontaminate all tips in contact with virus with 10% bleach solution. Keeping a small container of bleach in the hood is a convenient way to dispose of tips, and collect discarded virus-containing liquids. Harvest virus-containing supernatant 68–72 h after transfection: Transfer media to sterile MultiScreenHTS filter plates, centrifuge at 500 × g for 1 min, and recover the virus-containing filtrate. Cover the plate and store at 4°C for short-term use (within 24 h); otherwise, store at -80°C. Note: Timing the transduction of the CRISPRi/a-ready cells to coincide with the day of the final harvest allows transduction with the highest possible viral titer. Formally titering the lentivirus is generally not required, and the virus-containing supernatant can be used directly for transductions. Optional : If filter plates are unavailable, the media can be transferred to a sterile 96-well V-bottom plate, and centrifuged at 1,250 × g for 5 min to pellet residual cells or debris. The resulting supernatant can be transferred to a fresh plate for storage. Note: This procedure can be easily scaled to transfect more than one 96-well plate of HEK-293T cells for each sgRNA, if greater volumes of viral supernatant are required. Transduction of CRISPRi/a-ready Cells Note: CRISPRi (dCas9-BFP-KRAB) HepG2 cells are used in this protocol, but any adherent CRISPRi/a-ready cell line should be amenable to this procedure. To reduce clumping of HepG2 cells, which occurs over time, pass the cells through a 20–23G needle three times when passaging. On the day prior to transduction, seed CRISPRi/a-ready cells in 96-well plates in 100 µL of appropriate growth media. Aim for 50% confluence on day of transduction. For dCas9-BFP-KRAB HepG2 cells, seed 5 × 104 cells per well in low-glucose DMEM with 10% FBS. Note: Seeding cell density will depend on the specific cell size and type. On day of transduction, add polybrene to a final concentration of 8 µg/mL per well, accounting for both the 100 µL of growth media and additional viral supernatants. Dilute 40 µL of the 10 mg/mL polybrene stock into 460 µL of sterile PBS, to obtain a 100× solution, and then aliquot 1.5–2 µL of this to each well. Add 50–100 µL of viral supernatants directly to each well. Optional: Spinfect the cells by centrifuging plates at 800 × g at RT for 60 min, to modestly increase transduction rates. If necessary to obtain adequate transduction, the media can be removed after spinfection, and replaced with fresh media and additional viral supernatant. Incubate cells with 5% CO2 at 37°C for 48 h. Forty-eight hours after transduction, split cells 1:2 into a fresh 96-well plate: Remove media from each well, and wash cells with 100 µL of PBS. Dissociate the monolayer by adding 50 µL of 0.25% sterile Trypsin-EDTA solution. Incubate with 5% CO2 at 37°C for 5–10 min. Quench trypsin by adding 150 µL of growth media. Fully suspend cells by pipetting up and down. Transfer 50% of each suspension (~95 µL) to a fresh tissue culture plate. Return plates to incubator with 5% CO2 at 37°C. Optional : If cells are particularly sensitive to trypsin, consider quenching trypsin with 50 µL of growth media, transferring cells to a sterile 96-well V-bottom plate, and removing trypsin-containing supernatant after pelleting cells (500 × g for 5 min). Cells can then be resuspended and transferred to fresh tissue culture plates. Twenty-four hours after split, select for successful transduction with puromycin: Carefully remove the conditioned media on top of the cells. Replace with fresh growth media containing puromycin at a final concentration of 2 µg/mL. Return plates to incubator with 5% CO2 at 37°C. Note: Puromycin selection should be omitted if the downstream phenotypic experiment will directly compare transduced vs non-transduced cells within the same well (this can be distinguished by TagBFP levels induced by the sgRNA-containing pCRISPRi/a-v2 vector). If puromycin is expected to result in an adverse phenotype, adding an excess of virus can assure a high percentage of transduced cells. In this case, to assess the transduction efficiency, a test well should be reserved for puromycin selection, either within the same plate or cultured in parallel. Forty-eight hours after puromycin selection, remove puromycin, and exchange cells into fresh growth media. Monitor cell growth and prepare cells for phenotypic experiments. As cells near confluency, split cells according to the procedure above. Note: At this stage, creating three “copies” of the plates is helpful: 1) a maintenance plate for propagation, 2) an experimental plate to perform the phenotypic experiment, and 3) a storage plate in freezing medium (growth medium with 10% DMSO) for long-term storage at -150°C. Phenotyping of Cells: Receptor Labeling Note: Surface levels of the LDL and transferrin receptors are evaluated in this protocol, but labeling of any antibody-detectable cell surface marker should be feasible with this procedure. Select orthogonal fluorophores for receptor labeling, TagBFP levels, and live/dead staining within the detection capabilities of the flow cytometer available. For each plate, include one well for each single stained control and fluorescence minus one (FMO) control, and two wells for completely unstained controls. Parental (i.e., non-sgRNA-transduced) CRISPRi-ready cells can typically be used for these controls. Two additional fully stained controls (harboring control sgRNA) should also be included, to evaluate instrumental drift at the beginning and end of the flow cytometry. Each of these additional wells can be included either within the plate or cultured in parallel. Prepare experimental plate from part C as appropriate for the phenotypic experiment, and avoid over-confluency. For HepG2-derived cells, inducing sterol starvation upregulates LDL receptor abundance (Horton et al., 2002). Note: To increase throughput, trypsinize the experimental plate, and seed three daughter plates to perform the experiment in triplicate. Remove media from cells, wash with 100 µL of PBS, and replace with 75 µL of sterol-depleted media per well (low-glucose DMEM with 5% lipoprotein-deficient serum). Return plate to incubator with 5% CO2 at 37°C, and incubate overnight. Harvest cells Remove media from cells, and wash with 100 µL of PBS. Add 40 µL of Accutase per well, and incubate the plate on an orbital shaker at RT for 15 min. Note: Accutase has no effect on the levels of either the LDL or transferrin receptors detected by antibody labeling when compared to non-enzymatic or physical dissociation methods. Confirm that this method of dissociation does not affect the surface protein/epitope of interest. Add 50 µL of FACS Buffer (PBS with 1% FBS and 10 U/mL DNase I) to each well, and pipette up and down vigorously to dissociate the cells. Filter cells through a 90-µm nylon mesh taped to the top of a V-bottom plate placed on ice (Figure 4). Figure 4. Cell Filtration. Filtration is a key step to maximize singlets and prevent clogs during flow cytometry. Holding the sides of the mesh to keep it taut while pressing gently against the mesh with the pipette tips allows the liquid to travel through the mesh and into the wells. If the liquid accumulates on top of the mesh, it can mix with and contaminate neighboring wells. Centrifuge plate at 2,000 × g and 4°C for 1–2 min. Remove the mesh and discard the supernatant. Note: Invert the plate and give a quick but forceful downward shake to remove the supernatant. The cells will remain at the bottom of the wells. Wash the cells by resuspending them in 200 µL of FACS Buffer. Centrifuge the plate again at 2,000 × g and 4°C for 1–2 min, and discard the supernatant. Label cells with live/dead stain: Resuspend cells in 50 µL of a 1:1,000 dilution of Ghost Red 780 stain in PBS. Cover from light and incubate the plate on an orbital shaker on ice for 10 min. Retain one well each for a live-dead stain-negative control, additional wells for each single stained antibody control, and two wells for completely unstained controls – these wells should be resuspended in 50 µL of PBS. Note: The choice of live/dead stain depends on the fluorophores used for labeling. Add 200 µL of FACS Buffer, centrifuge the plate at 2,000 × g and 4°C for 1–2 min, and discard the supernatant. Wash the cells by resuspending them in 200 µL of FACS Buffer. Centrifuge the plate again at 2,000 × g and 4°C for 1–2 min, and discard the supernatant. Label cell surface receptors with appropriate fluorophore-conjugated antibodies: Resuspend cells in 40 µL of FACS buffer containing a 1:50 dilution of anti-LDLR-A647 and 1:100 dilution of anti-TFR-A488. Cover from light and incubate the plate on an orbital shaker on ice for 30–45 min. Retain one well for each single stained antibody control (LDLR and TFR), one well for each antibody negative FMO control (LDLR-negative and TFR-negative), and two wells for the completely unstained controls—these wells should be resuspended in FACS buffer containing a 1:50 dilution of anti-LDLR-A647, a 1:100 dilution of anti-TFR-A488, or no antibody, as appropriate. Note: The choice of antibody and fluorophore depends on the experiment. The dilution and incubation time required may also differ depending on the affinity of the antibody for its target. Add 200 µL of FACS Buffer, centrifuge the plate at 2,000 × g and 4°C for 1–2 min, and discard the supernatant. Wash the cells by resuspending them in 200 µL of FACS Buffer. Centrifuge the plate again at 2,000 × g and 4°C for 1–2 min, and discard the supernatant. Optional (Fixation): Resuspend cells in 50 µL of PBS. In a fume hood, add formaldehyde to the cell suspension to a final concentration of 4%. Fix cells at RT for 15 min. Centrifuge cells, and discard the supernatant in an appropriate waste container. Wash cells with 200 µL of PBS, centrifuge again, and discard the supernatant in appropriate waste container. Cells can then be resuspended as below, or in PBS if being stored at 4°C. If the antibody to be used is compatible with fixation, fixation can be performed prior to antibody labeling. Resuspend the cells in 75–100 µL of FACS buffer, and transfer to the flow cytometer for analysis. Keep plate on ice and covered from light. Note: If the cells are fixed and stored after labeling, rather than analyzed immediately, repeat the filtration step immediately prior to flow cytometry. Phenotyping of Cells: Ligand Uptake Note: Uptake of DiI-labeled LDL (Loregger et al., 2017) is evaluated in the protocol, but uptake of any fluorescently labeled particle should be amenable to this procedure. See the Note under part D for important considerations on fluorophores and controls. Prepare the experimental plate from part C as appropriate for the phenotypic experiment, and avoid over-confluency. For HepG2-derived cells, inducing sterol starvation upregulates LDL receptor function in addition to its abundance (Horton et al., 2002). Remove media from cells, wash with 100 µL of PBS, and replace with 75 µL of sterol-depleted media per well (low-glucose DMEM with 5% lipoprotein-deficient serum). Return plate to incubator with 5% CO2 and 37°C, and incubate overnight. Treat cells with labeled LDL: Remove media from cells and wash twice with 100 µL of warm PBS, discarding the wash fluid. Add 50 µL of DiI-LDL (5 µg/mL) in low-glucose DMEM containing 0.5% BSA. Incubate in the absence of light with 5% CO2 at 37°C for 1 h. Retain one well for each single stain negative control, and two wells for the completely unstained controls—these wells should be treated with low-glucose DMEM containing 0.5% BSA with no DiI-LDL. After 1 h of DiI-LDL labeling, harvest cells: Remove media from cells and wash twice with PBS containing 0.5% BSA, then once with PBS. Perform remainder of harvest by following steps D.2.b through D.2.f above. Label cells with live/dead stain as per steps D.3.a through D.3.c above. Optional (Fixation): Fix cells as per step D.5 above. Resuspend the cells in 75–100 µL of FACS buffer, and transfer to the flow cytometer for analysis. Keep plate on ice and covered from light. Flow Cytometry Note: This protocol is meant to be a general guide to analysis with a flow cytometer. Follow specific guidelines and instructions for the available instrument. If possible, choose an instrument that accepts samples in 96-well plates, along with the appropriate lasers and channels to read out the fluorophores of interest. If a 96-well plate adaptor is not available on the machine, samples can be run sequentially by transferring to low-volume (1.2-mL) FACS tubes (i.e., “bullet” tubes), and placed inside a typical 5-mL FACS tube for analysis. Prepare machine: Empty waste container, if necessary, and add bleach to fill ~10% of container volume. Refill sheath fluid reservoir, if needed. Turn on flow cytometer and its associated computer, and login to flow cytometry software (i.e., FACSDiva, CytExpert, etc.). Perform the appropriate start up procedure for the instrument, which typically includes priming fluidics and performing quality control with calibration beads. Begin your flow cytometry experiment: Note: Be sure to keep the cells covered from light until ready for analysis. Live cells should also remain on ice until analysis. Open a fresh experiment in the software program. Enable the appropriate channels and filters for your fluorophores of interest. i. TagBFP: λex = 402 nm, λem = 457 nm. ii. Alexa-488: λex = 490 nm, λem = 525 nm. iii. DiI: λex = 550 nm, λem = 569 nm. iv. Alexa-647: λex = 650 nm, λem = 665 nm. v. Ghost Red 780: λex = 757 nm, λem = 779 nm. Note: Viewing the excitation and emission spectra for the fluorophores is important, to determine the appropriate lasers and filter sets. When multiple colors are used in a single experiment, overlapping fluorophore spectra may cause wavelengths outside the maximum values to lead to a more specific readout. Compensation for overlapping spectra can be applied in the data analysis step, provided single stained controls are obtained. Create the following box plots to appropriately gate the cells: i. Forward scatter area (FSC-A) vs. side scatter area (SSC-A), to gate for size. Note: FSC and SSC are generally plotted on a linear scale. ii. Forward scatter height (FSC-H) vs. FSC-A, to gate for doublets. Note: Plotting forward scatter width (FSC-W) vs. FSC-A, or side scatter height (SSC-H) vs. SSC-A can also be used to gate for doublets. iii. FSC-A vs live-dead channel (i.e., Ghost Red 780) area, to gate for live/dead cells. Note: Fluorescence channels are generally plotted on a logarithmic scale. iv. FSC-A vs. TagBFP channel, to gate for successfully transduced cells. v. Additional FSC-A vs. fluorophore channels (i.e., Alexa-488, DiI, or Alexa-647), as desired for the experiment. Note: Histograms of the fluorophore channels may also be helpful for real-time evaluation of the samples . vi. Fluorophore vs fluorophore plots, for cells with experimental stains for two or more markers. vii. Draw preliminary gates (Figure 5), to provide real-time estimates of the readout of your experiments. Figure 5. Gating Strategy for Flow Cytometry. Representative example of a population of sgRNA-expressing dCas9-BFP-KRAB HepG2s, with sequential gates for cells (SSC-A vs. FSC-A, gate in green), single cells (SSC-H vs. SSC-A, gate in purple), live cells (FSC-A vs. Ghost Red 780, gate in orange), and BFP+ cells (FSC-A vs. TagBFP, gate in blue). Adjust the gains and voltages for the experiment, to achieve proper gating i. Open the instrument settings panel to adjust voltages for the appropriate channels. Note: Voltages adjust the sensitivity of the detectors on the instrument, with higher values increasing the values of the events on the axes of the plot. Discuss with the scientist maintaining the flow cytometer for beginning voltages for the cell type of interest, as these typically depend on cell size. Generally, aim to adjust voltages so that negative events range between 101 and 102 in arbitrary units, and the positive events are clearly identifiable, yet remain within the instrument’s limits of detection. ii. Transfer one well each of unstained parental cells and fully stained control cells to the 96-well plate used by the flow cytometer. Note: Resuspend cells by pipetting up and down prior to running samples, to prevent settling of cells. If no 96-well plate module is available on the flow cytometer, transfer cells to 1.2 mL “bullet” tubes that can fit inside a typical 5-mL FACS tube (the larger 5-mL FACS tube can be reused for each sample). iii. Begin running the unstained sample at low speed, and monitor readouts in real time, but do not record the data. Note: Stop the flow of cells once the voltages are established, as otherwise the machine will continue to aspirate cells and the sample will be fully consumed before the data are recorded. iv. Adjust FSC and SSC voltages to bring most of the cells into the gated region. Adjust gates as needed in real-time. Note: Debris should end up in the lower left corner of the FSC-A vs SSC-A plot, outside of the cell gate. v. Adjust the active fluorescent channel voltages other than TagBFP, to bring the values for the unstained cells to between 101 and 102. vi. Adjust the TagBFP fluorescent channel voltage, to bring the value for the unstained parental cells to between 102 and 103. Note: The parental cells contain the dCas9-TagBFP-KRAB fusion, and will be positive for TagBFP. However, the brightness of cells harboring the free TagBFP protein from the CRISPRi/a vector is significantly higher than that of the parental cells with the TagBFP fusion. This allows differentiation of transduced from non-transduced cells . vii. Run the fully stained control sample at low speed, and monitor readouts in real time, but do not record the data. viii. Confirm that the FSC and SSC voltages are appropriate for the fully stained sample. ix. Adjust the active fluorescent channel voltages, to achieve a distribution of values that enables a clear distinction of the signal, yet remain within the dynamic range of detection for the instrument. Note: For the live-dead stain, the most positively fluorescent cells are nonviable, and will be excluded from analysis. x. Save the experimental template on the software, as well as the voltages for the instrument, so that both can be easily reloaded for future experiments. Run and collect the data on both control and experimental samples: i. On the software, create and label wells (or “tubes”) for each control and experimental sample. ii. Set the amount of volume to aspirate from each well and speed with which to run samples. Note: Expect to leave about 10 µL of sample in each well to avoid aspirating air into the fluidics lines. Samples will run faster if more concentrated and reconstituted in lower volumes. While samples will also run faster if run at higher speed, the accuracy of readouts may be reduced. This should be confirmed empirically, for the samples and the particular machine used. iii. Set the number of events to record. Typically, this is 1 × 105 total events, but this number can be reduced, if necessary (e.g., if too few cells are available to record), or if a total number of “gated” events (i.e., cells) are desired. iv. Highlight and select the wells with the unstained cells, parental cells, and fully stained controls. Run these samples using an auto-record feature, if available on the machine. Note: Comparing the results of a fully stained control at the beginning of the experiment to one at the end will assess for any drift in the fluorescence channels. v. Using a multichannel pipette, transfer one row of cells (12 wells) to the 96-well plate to be used in the flow cytometer. Note: For live cells, transferring one row at a time allows cells to remain on ice longer until ready for analysis. This is not necessary for fixed cells or if the 96-well plate module for the flow cytometer is temperature controlled. Including a non-targeting control well at the end of each row permits a direct comparison between controls and experimental sgRNAs analyzed at similar timepoints after harvest and labeling. vi. Highlight and select the wells to run. Use the auto-record feature to run and record the samples. vii. Run and record a final fully stained control well at the end of the experiment. After all samples are run, clean and decontaminate the instrument: i. Export the raw data for each well as a .fcs file for data analysis. Note: Data analysis can also be performed on the machine’s software if desired. ii. Run the cleaning protocol for the instrument: 1). For 96-well plates, this typically involves 3–5 wells of 10% bleach, followed by 5 wells of water. 2). For individual tubes, this typically involves 3–5 min with a tube of 10% bleach at high speed, followed by 5 min of water at high speed. iii. Shut the machine down, according to the shut-down protocol. Data analysis Import raw flow cytometry data into the FlowJo analysis software, creating groups for experimental samples, biological controls, and non-targeting controls. Concatenate data from all non-targeting control well files from a given plate into a separate file. Import this file into the group of non-targeting controls. Note: This step creates a control sample containing all events from the non-targeting control wells over the entire plate. This can improve sensitivity to detect a phenotypic effect of an experimental sgRNA, provided there is no significant fluorophore drift over the course of the experiment. Otherwise, the concatenated file may obscure a true effect. Optional : Derive new parameters as desired for the experiment. Examples include ratiometric levels of fluorescence (e.g. Alexa-648LDLR/Alexa-488TFR), and fluorescence levels corrected for cell size (e.g. Alexa-648LDLR/FSC-A). These derived parameters allow for ratiometric readouts at the single cell level. Open the file from the first analyzed non-targeting sgRNA control, to draw gates and perform the template analysis: Gate for cells (FSC-A vs SSC-A), singlets (FSC-H vs FSC-A), live cells (FSC-A vs Ghost Red 780), and transduction (FSC-A vs TagBFP), and adjust axes as in F.b.ii. (see Figure 5). Note: Gating for live-dead cells or transduced cells can also be performed on histograms of the Ghost Red 780 or TagBFP channels, respectively. Such gating is simpler to apply, but does not account for differences in channel output correlated with cell size. On the fully gated population, open histograms of experimental fluorophore channels and derived parameters, to visualize the spread of data. Apply the desired statistics (i.e., total count, mean, median, and standard deviation) for the desired fluorophore channels and derived parameters, for the fully gated population. Assess for experimental drift by comparing the first non-targeting sgRNA control well to the last non-targeting sgRNA control well of the experiment. Note: Differences between these samples can occur due to instrumental drift or to fluorophores fading over time. Both can be minimized by using either row-matched controls, or well-matched non-transduced controls. Apply the same gates and statistics to both wells. Overlay the data from both wells on the box plots and histograms, to visualize and qualitatively assess any differences. Quantify the difference in fluorophore channels: i. With the fully gated population (transduced, live, singlet cells) of the first control selected, open the “Compare Populations” tool in FlowJo. ii. Drag the same gated population of the last non-targeting sgRNA control into the “Compare Populations” window. iii. Select the desired parameter channels to compare, to obtain the Tχ metric. Note: The Tχ metric will provide a statistical likelihood that the populations are different, but does not imply there is a biological difference. The statistical test must be combined with the qualitative visualization of the histograms, as well as knowledge of the experimental details, to infer whether the biological effect is significant. Determine the background fluorescence for each of the fluorophore channels. Apply the gates and statistics to the FMO negative control samples for each experimental parameter (i.e., Alexa-488, DiI, or Alexa-647). The mean and median of these values indicate the background fluorescence. If FMO controls are not available, the fully unstained control can be used. However, only the gates created with FSC and SSC parameters can be used to identify the desired cells. Assess for whether additional compensation is needed due to fluorescence “spillover” between channels. Compare the experimental parameters in the FMO controls to the fully stained control. Apply the same gates and statistics to these controls, if not already done. Overlay the data from each fully gated FMO control to the fully stained control in the histograms of the experimental parameters, to qualitatively assess differences. A shift of the histogram in a particular channel in the fully stained control compared to the FMO control suggests spillover. Optional: If desired, the difference can be quantified by comparing populations, as in step 5.c. If spillover exists that affects the interpretation of the experiment, create a compensation matrix using the single-stained controls in the FlowJo software. This matrix can then be applied to the entire analysis. Compare the experimental parameters in each of the experimental samples against control populations. Using non-targeting sgRNAs as controls: i. Apply the gates and statistics to the first experimental sample. ii. With the fully gated population of the first experimental sample selected, open the “Compare Populations” tool in FlowJo. iii. Drag the same gated population of the concatenated non-targeting sgRNA control into the “Compare Populations” window. iv. Select the desired parameter channels to compare, to obtain the Tχ metrics. v. Apply the gates, statistics, and comparison from the first experimental sample to the first experimental or biological control samples from each row of the plate analyzed. vi. For each of these samples, repeat the “Compare Populations” analysis with the non-targeting sgRNA control from the same row of the plate. vii. Apply all gates, statistics, and comparisons from the first experimental sample in each row of the plate analyzed to the remainder of the wells in that row. Note: The first comparison will be to the concatenated file and applicable to all experimental or biological control samples. The second comparison will be to the row-matched non-targeting sgRNA control. Using non-transduced cells from the same well as controls: Note: This comparison offers the advantage of comparing cells grown in the same well, but cannot be used if puromycin selection was performed. i. Select the population of live cells (Ghost Red 780 negative) in the first non-targeting sgRNA control sample. ii. Apply the desired statistics for the experimental parameters on both the TagBFP+ (transduced) and TagBFP- (non-transduced) cells. iii. Perform the “Compare Populations” analysis of the TagBFP+cells to the TagBFP- cells for this sample, as in 8.a. iv. Apply the same statistics and comparison to each experimental and control sample performed in the experiment. Export the data in tabular format. Open the table window in FlowJo, and select the desired statistics and comparison metrics to export. Typically, this will include mean, median, standard deviation, total counts, and proportional counts for each population, as well as Tχ metrics for each comparison. Optional : Create a graphical comparison of histograms for any biological controls or experimental samples desired [see Figures. S1B and C, S5A–C, S7A and B, S9A&B, S10A–D, and S11A–H in Smith et al. (2022) for examples]. Open the layout window in FlowJo, and drag the desired populations into the window to create the desired histograms. Given the number of samples analyzed in the experiment, a comparison of every sample is not interpretable on a single graph. For comparison within a given experiment, evaluate the following for the individual sgRNAs: Use the total count of gated events to assess whether the data from a given well is interpretable. i. Total gated counts < 10% of the non-targeting control wells may be skewed and represent an inadequate sampling of the population, and should be interpreted with caution. ii. Low total gated counts but with normal proportion of live cells suggests a technical issue with the well. Consider censoring the data from these wells. iii. Low total gated counts with low proportion of live cells suggests toxicity of either the sgRNA or the gene knockdown. For interpretable wells, use the Tχ metric to assess the significance of the effect. The Tχ metrics of the biological controls can be used as a benchmark to correlate the statistical significance to the biological relevance of the effects. Import the statistics (median, standard deviation, total counts) from the tabular data for each experimental parameter into data analysis software (e.g., Prism). Note: The median, rather than the mean, may better represent the data, given the skewed curves that typically exist in flow cytometry histograms. Normalize the data for each sgRNA, using the background fluorescence as the minimum value and the result of the appropriate control (i.e., concatenated non-targeting sgRNA well, row-matched non-targeting sgRNA well, or well-matched non-transduced sample) as the maximum value. Repeat the normalization for each of these controls. Note: Normalization of the data allows for data to be combined and compared across experiments. Compare the data by performing multiple t-tests against the selected control, adjusting the significance for multiple comparisons testing. The strength of effect (i.e., fold-change) and the adjusted p values of the biological controls can be used as a benchmark for the experimental sgRNAs. To compare across multiple experiments, export the normalized tabular data into a spreadsheet. Note: Flow cytometry permits evaluation at the single cell level, and so each “event” can be considered a biological replicate. Nevertheless, consider performing at least three replicates of the experiment to ensure robustness of the findings. Combine normalized data across multiple experiments by using the Cochrane method ( Higgins and Green, 2011), as follows: Iteratively combine data across replicate experiments. As desired, import combined, normalized data into Prism, and repeat step 14. Data from individual or combined experiments can be log 2 transformed in Prism, and then visualized in a heatmap (Figure 6). Figure 6. Representative heatmap of sgRNA validation results. Heatmap showing receptor abundance (for LDLR, TFR, and the LDLR/TFR ratio) and fluorescent LDL uptake for dCas9-BFP-KRAB HepG2 cells transduced with sgRNAs targeting each indicated gene, as described in the above protocol. In this figure, hits are grouped first by the directional effect on LDLR abundance, and then by the effect on LDL uptake. Genes targeted as known controls are shown on the right. Readouts show the log2 fold change compared to transduction with a negative control sgRNA, and represent the weighted average of the effects from two separate sgRNAs. Viability indicates the relative number of cells surviving to flow cytometry analysis in the experiments. All genes displayed had both sgRNAs independently validate for LDLR abundance, defined as p < 0.05 in the Holm-Sidak corrected t-test. Data represent summary information from four independent experiments. Figure modified from Smith et al. (2022). Notes Several online resources are available to assess for spectral overlap of fluorophores and design flow cytometry experiments, including FPbase (https://www.fpbase.org) (Lambert, 2019), and ThermoFisher Scientific’s Fluorescence SpectraViewer (https://www.thermofisher.com/order/fluorescence-spectraviewer). Flow cytometry analysis software, such as FlowJo, can be used to automate compensation for overlapping fluorophores, if needed. Recipes 2× Annealing Buffer 200 mM potassium acetate 60 mM HEPES-KOH pH 7.4 4 mM magnesium acetate To obtain proper pH, first make a 1 M stock solution of HEPES, and adjust to pH 7.4 with KOH, followed by sterile filtration. Then, combine the three reagents with an appropriate amount of Milli-Q H2O, to make the 2× annealing buffer. Store at RT. 50× TAE Buffer 50 mM EDTA 2 M Tris 1 M acetic acid Combine solid Tris base with solid EDTA disodium, and dissolve completely in ¾ of the total volume of Milli-Q H2O. EDTA will dissolve only at a mildly alkaline pH, thus the order of addition of the reagents is important. After dissolution, add glacial acetic acid, and bring to final volume with Milli-Q H2O. The pH of the final buffer should approximate 8.6, and does not require adjustment. Store at RT. 293T growth medium Add 50 mL of FBS to 500 mL of high glucose DMEM (for 10% v/v). Store at 4°C. Viral harvest medium Add 1.1 g of BSA for every 100 mL of growth medium, followed by 1/100 volume of 100× penicillin-streptomycin. Dissolve and sterile filter. Store at 4°C. HepG2 growth medium Add 50 mL of sterile FBS to 500 mL of low glucose DMEM (for 10% v/v). Store at 4°C. Sterol-depleted medium Add 25 mL of lipoprotein-deficient serum to 500 mL of low glucose DMEM (for 5% v/v) and sterile filter. Store at 4°C. FACS buffer (FB) To sterile PBS, add 1/100 volume and 10 U/mL DNase I. Mix to dissolve, and sterile filter. Store at 4°C. DiI-LDL labeling medium Add 0.5% w/v BSA to serum-free low glucose DMEM. Mix to dissolve, and sterile filter. Store at 4°C. Acknowledgments JSC receives funding support from the NIH/NHLBI (K08 HL124068, R03 HL145259, R01 HL146404, R01 HL159457), the NIH/NIGMS (R21 141609), a Pfizer ASPIRE Cardiovascular Award, the Harris Fund, and the Research Evaluation and Allocation Committee of the UCSF School of Medicine. AP receives funding support from the Tobacco-Related Disease Research Program (578649), the AP Giannini Foundation (P0527061), the NIH/NHLBI (K08 HL157700), the Michael Antonov Charitable Foundation Inc., and the Sarnoff Cardiovascular Research Foundation. This protocol was derived and validated from original research described in Smith et al. (2022). We thank Matvei Khoroshkin for feedback on protocol implementation, and Kevan Shokat and the members of the Shokat laboratory for helpful discussion. Competing interests JSC has received consulting fees from Gilde Healthcare and Eko, unrelated to the published work. The other authors have no disclosures. Ethics No human or animal subjects were used in this protocol. References Bock, C., Datlinger, P., Chardon, F., Coelho, M. A., Dong, M. B., Lawson, K. A., Lu, T., Maroc, L., Norman, T. M., Song, B., et al. (2022). High-content CRISPR screening. Nat Rev Methods Prim 2(1): 8. Cui, L., Vigouroux, A., Rousset, F., Varet, H., Khanna, V. and Bikard, D. (2018). A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat Commun 9(1): 1912. Davis, M. and Jorgensen, E. (2022). ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program. Front Bioinform 2: 818619. Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., Guimaraes, C., Panning, B., Ploegh, H. L., Bassik, M. C., et al. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 159(3): 647-661. Higgins, J. P. T. and Green, S. (Eds.). (2011). Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0. Cochrane, 2011. Available from www.training.cochrane.org/handbook Horlbeck, M. A., Gilbert, L. A., Villalta, J. E., Adamson, B., Pak, R. A., Chen, Y., Fields, A. P., Park, C. Y., Corn, J. E., Kampmann, M., et al. (2016). Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife 5: e19760. Horton, J. D., Goldstein, J. L. and Brown, M. S. (2002). SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109(9): 1125-1131. Lambert, T. J. (2019). FPbase: a community-editable fluorescent protein database. Nat Methods 16(4): 277-278. Loregger, A., Nelson, J. K. and Zelcer, N. (2017). Assaying Low-Density-Lipoprotein (LDL) Uptake into Cells. Methods Mol Biol 1583: 53-63. Mandegar, M. A., Huebsch, N., Frolov, E. B., Shin, E., Truong, A., Olvera, M. P., Chan, A. H., Miyaoka, Y., Holmes, K., Spencer, C. I., et al. (2016). CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell 18(4): 541-553. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P. and Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5): 1173-1183. Reis, A. C., Halper, S. M., Vezeau, G. E., Cetnar, D. P., Hossain, A., Clauer, P. R. and Salis, H. M. (2019). Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays. Nat Biotechnol 37(11): 1294-1301. Shalem, O., Sanjana, N. E. and Zhang, F. (2015). High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 16(5): 299-311. Smith, G. A., Padmanabhan, A., Lau, B. H., Pampana, A., Li, L., Lee, C. Y., Pelonero, A., Nishino, T., et al. (2022). Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor. Sci Transl Med 14(662): eabj8670. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Systems Biology > Genomics > Screening Medicine > Cardiovascular system Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Bacterial Microcolonies in Gel Beads for High-throughput Screening Yolanda Schaerli Jul 5, 2018 8386 Views Probabilistic Models for Predicting Mutational Routes to New Adaptive Phenotypes Eric Libby and Peter A. Lind Oct 20, 2019 3420 Views Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of Escherichia coli Colonies Kisurb Choe and Jonathan V. Sweedler Nov 5, 2023 652 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,592	https://bio-protocol.org/en/bpdetail?id=4592&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms CT Chloe A. L. Thompson-Peach * JF Johannes Foßelteder * AR Andreas Reinisch DT Daniel Thomas (*contributed equally to this work) Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4592 Views: 779 Reviewed by: Salma Merchant Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in EMBO Reports Feb 2022 Abstract Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient–derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation Keywords: Hematopoietic stem and progenitor cells Megakaryocyte differentiation Thrombopoietin TPO-independent Myeloproliferative neoplasm Collagen Interleukin-6 Interleukin-9 Background Multipotent hematopoietic stem and progenitor cells (HSPCs) are not only responsible for life-long hematopoiesis but are also found to be the origin of many hematological malignancies (Bonnet and Dick, 1997; Jamieson et al., 2006; Woll et al., 2014; Reinisch et al., 2016). Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell–derived diseases that are characterized by an aberrant proliferation of myeloid cells through constitutive activation of cytokine-signaling pathways (Tefferi and Pardanani, 2015; Spivak, 2017). In essential thrombocythemia (ET) and myelofibrosis (PMF), two common subtypes of MPNs, megakaryocyte lineage-biased differentiation and proliferation are hallmarks of disease pathogenesis (Vannucchi et al., 2013; Tefferi and Pardanani, 2019). Historically, thrombopoietin (TPO) was found to be a crucial growth factor in nearly all stages of megakaryocyte development with a profound effect on the survival, proliferation, and differentiation of committed HSPCs to the development of mature megakaryocytes (Debili et al., 1995; Choi et al., 1995; Broudy et al., 1995; Chen et al., 1995; Kojima et al., 1995). Thus, it is not surprising that the oncogenic drivers of MPNs were found to be activating mutations occurring in genes along the TPO signaling axis (MPL and JAK2), rendering cells TPO-independent and inducing increased megakaryopoiesis (Kralovics et al., 2005; Pikman et al., 2006; Woods et al., 2019). Additionally, mutations in the endoplasmic reticulum chaperone CALR, which has not been implicated in megakaryopoiesis to date, introduced structural changes, resulting in protein multimerization and binding to the TPO receptor (MPL). This binding results in an activation of downstream signaling in absence of TPO (Klampfl et al., 2013; Chachoua et al., 2016; Marty et al., 2016; Araki et al., 2016). Despite recent advances in unveiling the oncogenic transformation of healthy HSPCs, detailed mechanisms of disease pathogenesis are still poorly understood. Current methods to investigate megakaryocyte-biased cellular transformation by MPN mutations either use TPO-dependent cell lines or costly HSPC differentiation assays that are not tailored to investigate TPO-independent cell growth (Lu et al., 2005; Araki et al., 2016; Elf et al., 2016; Pronier et al., 2018). Primary progenitor cells from patients with polycythemia vera, another MPN disease subtype, often form erythroid colonies in the absence of erythropoietin (Corre-Buscail et al., 2005), but assays to show TPO-independent growth or TPO-hypersensitive growth of ET- and PMF-derived cells are not well established. Current protocols for in vitro megakaryocyte differentiation focus on optimizing culture conditions to maximize the megakaryocyte output of healthy HSPCs, but do not consider MPN-related phenotypes. Here, we describe a reproducible and cost-effective step-by-step protocol for the isolation, culture, and TPO-independent megakaryocyte differentiation of primary MPN patient–derived CD34+ HSPCs to investigate disease-relevant characteristics. This approach uses fluorescence-activated cell sorting (FACS) to purify CD34+ HSPCs from the peripheral blood of MPN patients and an animal serum-free culture system for megakaryocyte differentiation in a clonogenic semi-solid media and collagen-based colony assay, as well as in liquid culture. This protocol facilitates future MPN research, providing an opportunity to test novel therapeutic interventions in a human pre-clinical setting (Tvorogov et al., 2022), but can be easily adapted to investigate other megakaryocyte-related diseases. Materials and Reagents 50 mL conical tubes (Corning, FalconTM, catalog number: 352098) 1.8 mL cryogenic tubes (Thermo Fisher Scientific, NuncTM, catalog number: 368632) 5 mL round bottom tubes (Corning, FalconTM, catalog number: 352054) 5 mL round bottom tubes with cell strainer cap (Corning, FalconTM, catalog number: 352235) 1.5 mL screw cap micro tube (Sarstedt, catalog number: 72692005) 1.5 mL safe-lock tubes (Eppendorf, catalog number: 0030123328) Double chamber slides (Thermo Fisher Scientific, NuncTM, Lab-TekTM, catalog number: 177429) 100 mm culture dish (Corning, catalog number: 430591) 35 mm culture dish (Greiner Bio-One, catalog number: 627161) 48-well plate (tissue-culture treated) (Corning, CostarTM, catalog number: 3548) 3.5 mL transfer pipettes (Sarstedt, catalog number: 86.1171.001) Parafilm (Bemis, catalog number: PM996) Filter cards and spacers (Stemcell technologies, catalog number: 04911) Phosphate buffered saline (PBS) (Gibco, catalog number: 10010015) Lymphoprep (Stemcell technologies, catalog number: 07801) RPMI 1640 (Sigma-Aldrich, catalog number: R8758) IMDM (Pan-Biotech, catalog number: P04-20450) Fetal bovine serum (FBS) (Pan-Biotech, catalog number: P30-19475) Penicillin/streptomycin (Pan-Biotech, catalog number: P06-07100) Dimethyl sulfoxide (DMSO) (WAK-Chemie, catalog number: WAK-DMSO-10) DNase (Worthington, catalog number: LS002007) MegacultTM-C collagen and medium with lipids, without cytokines (Stemcell technologies, catalog number: 04974) Methanol (Merck, EMSURE®, catalog number: 106009) Acetone (Sigma-Aldrich, catalog number: 320110) Hydrochloric acid solution 6 M (HCl) (Merck, catalog number: 143007) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888) Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434) Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 237205) EDTA tetrasodium (Sigma-Aldrich, catalog number: 03699) TRIS (Roth, PUFFERAN®, catalog number: 5429.1) Tween 20 (Sigma-Aldrich, catalog number: P1379) Evans Blue (Sigma-Aldrich, catalog number: E2129) Serum-free expansion media II (SFEM II) (Stemcell technologies, catalog number: 09655) or StemPro 34 SFM + nutrient supplement (Thermo Fisher Scientific, catalog number: 10639011) L-Glutamine (200 mM) (Sigma-Aldrich, catalog number: 59202C) Recombinant human stem cell factor (SCF) (PeproTech, catalog number: 300-07 Recombinant human thrombopoietin (TPO) (PeproTech, catalog number: 300-18) Recombinant human interleukin 3 (IL-3) (PeproTech, catalog number: 200-03) Recombinant human interleukin 6 (IL-6) (PeproTech, catalog number: 200-06) Recombinant human interleukin 9 (IL-9) (PeproTech, catalog number: 200-09) Lipids cholesterol rich (Sigma-Aldrich, catalog number: L4646) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906) EDTA (Thermo Fisher Scientific, catalog number: AM9260G, 0.5M solution) FcR blocking reagent human (Miltenyi, catalog number: 130-059-901) Mouse IgG1, κ isotype control (BioLegend, catalog number: 400101) Mouse anti-human CD41 (BioLegend, catalog number: 303702) ImmPRESS®-AP horse anti-mouse IgG polymer detection kit (Vector Laboratories, catalog number: MP-5402) Alkaline phosphatase (AP) substrate kit, Vector® Red (Vector Laboratories, catalog number: SK-5100) Mouse anti-human CD34 FITC (BD Biosciences, PharmingenTM, catalog number: 555821) Mouse anti-human CD45 V500-C (BD Biosciences, HorizonTM, catalog number: 655873) Mouse anti-human CD2 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555328) Mouse anti-human CD3 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555334) Mouse anti-human CD19 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555414) Mouse anti-human CD14 PerCP-Cy5.5 (BD Biosciences, PharmingenTM, catalog number: 562692) Mouse anti-human CD16 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555408) Mouse anti-human CD56 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555517) Mouse anti-human CD41 APC-Cy7 (BioLegend, catalog number: 303716) Mouse anti-human CD42b APC (BioLegend, catalog number: 303912) 7-Amino-Actinomycin D (7-AAD) (BD Biosciences, PharmingenTM, catalog number: 559925) Counting beads (Thermo Fisher Scientific, CountbrightTM, catalog number: C36950, LOT-specific concentration: 0.515 × 105 beads/50 µL) RBC lysis buffer (see Recipes) Freezing media (see Recipes) Thawing media (see Recipes) Staining buffer (SB) (see Recipes) Antibody cocktail (see Recipes) Megakaryocyte (Mk) colony media (see Recipes) Megakaryocyte differentiation (Mk-diff) media (see Recipes) Isotonic NaCl solution (0.15 M) (see Recipes) TRIS buffer (0.5 M, pH 7.6) (see Recipes) Wash buffer (see Recipes) Substrate buffer (see Recipes) Evans Blue solution (0.1%) (see Recipes) Staining tray (see Recipes) Equipment Swing-bucket centrifuge (Eppendorf, model: 5810R) Tabletop centrifuge (Eppendorf, model: 5427R) Inverted light microscope (Zeiss, model: Primovert) Upright light microscope (Olympus, model: BX51) Fluorescence-activated cell sorter (BD Biosciences, model: Aria IIIu) Flow cytometer (Beckman Coulter, model: Cytoflex S or BD Biosciences FACS CantoII) Laminar flow workbench Fume hood Forceps Scalpel Software FlowJo v10 (FlowJo LLC) Prism 9 (GraphPad) Procedure Peripheral blood mononuclear cells (PBMC) collection and storage Notes: Collection of patient material has to be approved by a local ethics committee and every patient needs to give informed consent prior to material collection. Perform all experimental steps in a sterile environment using a laminar flow workbench. Alternatively, processed samples may be obtained from a biobank and used for subsequent analysis. Dilute 15 mL of whole blood from MPN patients with 15 mL of PBS and slowly overlay on top of 15 mL Lymphoprep or similar density gradient reagents in a 50 mL conical tube. Centrifuge at 800 × g at room temperature with brakes off for 30 min (excluding acceleration and deceleration times). Carefully collect PBMCs from the whitish interphase layer between Lymphoprep (lower phase) and serum (upper phase) using a sterile 3.5 mL plastic transfer pipette. Avoid contamination of the layer with Lymphoprep. Wash PBMCs by mixing with 40 mL of PBS and centrifuge at 350 × g at room temperature for 5 min. Optional: If the cell pellet contains red blood cells (RBC), perform an RBC lysis to further purify PBMCs. Suspend pellet in 10 mL of RBC lysis buffer (see Recipe 1) and incubate for 10 min at room temperature. Wash PBMCs by mixing with 40 mL of PBS and centrifuge at 350 × g at room temperature for 5 min. Resuspend pellet in 5 mL PBS and count the cells. Keep cells on ice during this step. Take a small aliquot of cells (5 × 104) and determine the CD34+ cell fraction. Resuspend cells in 50 µL staining buffer containing anti-human CD34 FITC antibody at a 1:25 dilution and incubate for 30 min at 4°C. Wash cells by mixing with 1 mL of PBS and centrifuge at 350 × g at 4°C for 5 min. Resuspend pellet in 100 µL staining buffer (see Recipe 2). Add 2 µL of 7-AAD, incubate for 5 min, and acquire cells on flow cytometer. Meanwhile, centrifuge the PBMCs at 350 × g at 4°C for 5 min, resuspend up to 5 × 106 cells in 1 mL of freezing media (see Recipe 3), and transfer to cryo tube for storage. Freeze PBMCs in liquid nitrogen for long-term storage until required. Primary human CD34+ cell isolation Note: The CD34+ cell fraction observed in the peripheral blood of MPN patients is highly variable but generally much higher than normal healthy adults. We observed CD34+ HSPCs ranging from 0.2% to 20%. If this data is not available for your samples, we recommend thawing one vial of cells and determining the CD34+ cell frequency to estimate the number of PBMC vials needed. Thaw frozen PBMCs in a water bath pre-heated to 37°C. The number of vials needed to get enough CD34+ cells for megakaryocyte differentiation is dependent on the predetermined frequency of CD34+ cells within total PBMCs. Transfer cells from two vials of the same sample (1 × 107 cells) into one 50 mL tube and add 40 mL of thawing media (see Recipe 4) dropwise to the cells while gently swirling. Incubate cells for 10 min at room temperature. The DNase in the thawing media will digest any DNA released from dead cells that would otherwise promote clumping of cells. Centrifuge cells at 350 × g at room temperature for 5 min and remove supernatant. Resuspend cell pellet in 135 µL of staining buffer (see Recipe 2). Add 15 µL of FcR blocking reagent human, mix well, and incubate for 5 min at room temperature. Add 40 µL of antibody cocktail (see Recipe 5), mix well, and incubate for 30 min at 4°C. Notes: Antibodies with different fluorescent conjugates can be used; however, lineage antibodies (CD2, CD3, CD19, CD14, CD16, CD56) should emit in the same channel as the viability dye for efficient exclusion. At the initial setup of the experiment, unstained and single CD34 and CD45 antibodies as well as lineage antibody and 7-AAD stained cells should be prepared to correctly compensate all fluorophores and establish the population gates at the flow cytometer. Wash cells once with 5 mL of staining buffer. Centrifuge cells at 350 × g at 4°C for 5 min and remove supernatant. Resuspend cells in 500 µL of staining buffer and filter the suspension through the cell strainer cap into a 5 mL round bottom tube. Add 10 µL of 7-AAD and place tubes on ice prior to cell sorting. Sort Lin- CD34+ CD45dim (Figure 1) hematopoietic stem and progenitor cells (HSPCs) into 1.5 mL screw cap tubes for downstream assays. Ideally, sort 8,000 CD34+ CD45dim HSPCs into a tube containing 400 µL of Mk colony media (see Recipe 6) and at least 100,000 CD34+ CD45dim HSPCs into a separate tube containing 200 µL Mk-diff media (see Recipe 7). Note: It may not be possible to sort such high numbers of cells for some of the samples. These experiments can be scaled depending on final numbers of cells obtained. Liquid culture differentiation assays have been carried out with as few as 1,000 cells/replicate. Figure 1. Gating strategy for CD34+ cell purification via fluorescence-activated cell sorting (FACS). (A) First, cell debris (top row, left) and remaining granulocytes (top row, middle) were excluded in SSC-A/FSC-A plots and selected single cells were gated in an FSC-A/FSC-H plot (top row, right). Next, HSPCs were pre-enriched by gating on lineage marker negative and live (7-AAD negative) cells (bottom row, right plot). HSPCs were further purified by gating on CD34+ CD45dim cells (bottom row, left plot). (B) Unstained (left plots) and single stained (center and right plots) cells were used to set up compensation controls and to identify the fluorescent threshold for accurate setup of population gates. Proceed with megakaryocyte colony formation assay (section C) and liquid culture differentiation (section D). Differentiation and quantification of TPO-independent megakaryocyte progenitors in a collagen-based colony assay Notes: This assay can be used to analyze the ability of therapeutic drugs to interfere with the megakaryocyte differentiation of MPN patient–derived HSPCs. This assay is designed to analyze samples using four replicates (two double chamber slides). To carry out more tests, it is possible to reduce the number of cells and volumes of all reagents by half and perform only duplicates. The addition of 50 ng/mL TPO is required for optimal proliferation and megakaryocyte differentiation of healthy HSPCs. While addition of TPO is not required for CALRmut or MPLmut samples, in our experience, JAK2mut HSPCs will not grow in the complete absence of TPO. A low dose of TPO (5–10 ng/mL) may be required for JAK2V617F samples and should be compared to healthy controls to measure TPO hypersensitivity rather than factor-independence. Mix sorted HSPCs in 1.7 mL of Mk colony media by pipetting (see Recipe 6). If therapeutic compounds will be tested, they should be added to the Mk colony media prior to the cell suspension. Label the double chamber slides appropriately with a pencil, as ink from standard laboratory markers or pens will become illegible during the fixation process. Using a 2 mL serological pipette, carefully add 1.2 mL of cold collagen to the Mk colony media and mix by gently pipetting, avoiding any foaming of the media. During this process, keep collagen on ice and prepare only one tube at a time to prevent solidification of collagen before seeding. Dispense 0.75 mL of the cell suspension into each well of two double chamber slides (four replicates) and distribute the volume by circular motion. Place each chamber slide along with a 35 mm dish filled with 3 mL of sterile water into a separate sterile 10 mm culture dish (Figure 2). Figure 2. Setup of a double chamber slide for the collagen-based megakaryocyte colony assay. Double chamber slide seeded with CD34+ HSPCs along with a water-filled dish to maintain humidity during the 12-day culture. Close the 10 mm culture dish and place it into a humidified incubator at 37°C and 5% CO2 atmosphere for 12 days. Check for colony formation using an inverted light microscope and proceed to fixation and staining if colonies are present (Figure 3). Figure 3. Examples of megakaryocyte colonies prior to fixation and staining. (A) Two dense, small colonies. (B) Medium sized colony. (C) Large colony. Scale bars represent 100 µm. Prepare 100 mL fixative (1:3 mixture of methanol and acetone) and place into a rectangular plastic or glass dish (at least 12 × 9 × 2 cm in size). Cover the dish with parafilm or an appropriate lid and place into a fume hood. Remove one chamber slide at a time from the incubator and proceed immediately with disassembly as collagen becomes unstable at lower temperatures (Video 1). Video 1. Disassembly of double chamber slide prior to fixation. Removal of the chamber lid and detachment of the double chamber from the slide. Remove lid from the chamber slide. Carefully lift the chamber starting at the labeled end of the slide. Use a scalpel to cut the attachment points of the underlying rubber seal connecting it to the chamber (three in total). Proceed to the other end of the slide until the chamber is disconnected. The rubber seal should still be attached to the slide. Take caution to avoid moving or turning the two collagen pieces from each chamber. In case the collagen pieces have moved from the original position (e.g., moved out of the rubber seal boundaries), use a scalpel or forceps with smooth surface to carefully bring them back in place. Use a scalpel to cut the rubber seal into 2–4 pieces (Video 2). Video 2. Dehydration of the double chamber slide. Removal of the rubber seal from the double chamber slide and dehydration of media from the collagen-embedded colonies. Carefully pull the pieces of rubber seal off the slide using forceps and avoid grabbing the collagen pieces. Collagen pieces can be re-aligned again at this point before continuing to the fixation. Remove any remaining culture media before fixation (Video 2). Place a pre-cut spacer on the slide and make sure that collagen pieces are completely covered. Align the spacer to the slide prior to touching the collagen, as the collagen attaches to the spacer and allows no further adjustment afterwards. Cover the spacer with a filter card and wait until it is fully soaked with culture media. Only apply slight pressure to the filter card if it does not soak the media on its own. Complete removal of culture media is important for proper fixation. Remove the filter card and place the chamber slide with the spacer into the fixative. The whole slide should be submerged (Video 3). Wait until the spacer starts floating. Then, grab one corner with a forceps and carefully pull it off the slide. The collagen should stay attached to the slide. If the spacer does not float off on its own, gently rock the dish containing fixative to dislodge the spacer. Video 3. Fixation of the dehydrated slide. Fixation of collagen-embedded colonies on the dehydrated double chamber slide and removal of the spacer from the slide during the fixation process. Fix slides for 20 min; then, remove them from the fixative and allow to air dry for 15 min. Drying time can be extended to allow processing of all slides and perform subsequent staining of all slides in parallel. If necessary, fixed slides can be stored at 2–8°C and stained within three days. Proceed with immunocytochemical staining of colonies. Prepare all buffers and reagents required for staining (see Recipes). Place fixed and dried slides in horizontal position on a staining tray (either commercial or self-made (see Recipe 13, Figure 4). All subsequent steps are carried out at room temperature. Figure 4. Preparation of equipment for staining of the collagen-based megakaryocyte colony assay. (A) Example of a self-made staining tray using serological pipettes in a rectangular plastic dish. (B) Example of metal staining tray mounted over a sink. Rehydrate colonies by covering the whole slide with freshly made wash buffer (see Recipe 10) and incubate for 20 min at room temperature. Do not use a wash bottle as the pressure will cause the fixed collagen to dislodge. Remove buffer by tilting the slide into a waste container. Ensure that collagen-embedded colonies are always covered with liquid in the following steps and do not dry out. Block slides with six drops of 2.5% horse serum for 20 min at room temperature to prevent non-specific binding of antibodies. Other serums may be used as long as they match the species of the secondary antibody used. Prepare 1:100 dilutions of mouse anti-human CD41 and mouse IgG1 control antibodies in wash buffer + 1% BSA. Add 500 µL of CD41 antibody to the slides and incubate for 30 min at room temperature. At least one slide per staining batch should be incubated with mouse IgG1 control to assess potential unspecific staining of colonies. Gently rinse the slides three times by covering the slide with wash buffer for 3 min and tilting off excessive liquid. Cover slides with six drops of ImmPRESS®-AP secondary antibody and incubate for 30 min at room temperature. Gently rinse the slides three times by covering the slide with wash buffer for 3 min and tilting off excessive liquid. Prepare Vector® Red AP substrate. Add two drops of Reagent 1, Reagent 2 and Reagent 3 from the AP substrate kit to 5 mL of substrate buffer (see Recipe 11) and mix well. Cover slides with 500 µL of AP substrate and incubate for 20 min at room temperature in the dark. Gently rinse the slides three times by covering the slide with wash buffer for 3 min and tilting off excessive liquid. Add 500 µL of 0.1% Evans Blue solution (see Recipe 12) and incubate for 5 min. Carefully rinse slides with double-distilled water (ddH2O) until excessive blue dye is removed. Take caution, water stream might flush off the collagen. Let slides air dry before microscopy. Count individual colonies using an upright light microscope (Figure 5). CD41+ megakaryocyte colonies appear magenta and form colonies of variable size. Colonies can be categorized based on the number of containing cells: i) 5–20 cells; ii) 21–49 cells; and iii) > 50 cells. Large colonies arise from more primitive progenitors than small ones. Accompanying myeloid colonies appear pale blue and are usually of small size. In rare cases, mixed colonies containing cells of both types might occur. How do you know the staining has worked? For each sample used, at least one well should be stained with the IgG control antibody instead of anti human-CD41 to allow for the detection of any non-specific binding. If all other slides which have been stained with the human CD41 antibody do not show signs of pink megakaryocyte colonies, but colonies were observed on the slide prior to fixation, this would indicate that something has gone wrong in the staining process and staining has failed. Additionally, we acknowledge that some patient samples will fail to differentiate or proliferate at all in culture, due to the inherent and unpredictable heterogeneity of primary patient material. In this instance, colonies may appear sparse or very small in size when analysed under a microscope prior to fixation and thus proceeding with the staining might not be reasonable. Figure 5. Examples of megakaryocyte colonies upon fixation and staining. (A) Small colony forming unit-megakaryocyte (CFU-Mk) (3–20 cells). (B) Medium sized CFU-Mk (21–49 cells). (C) Large CFU-Mk (>50 cells). (D) Large non-Mk colony near small CFU-Mk. (E) Two CFU-Mk colonies and one mixed CFU-Mk/non-Mk. (F) Non-Mk colony. Scale bars represent 100 µm. Colored arrows indicate colony types: red: Mk; blue: non-Mk; purple: mixed. Megakaryocyte-directed differentiation of HSPCs in liquid culture Notes: This assay can be used to analyze the ability of therapeutic drugs to interfere with the megakaryocyte differentiation of MPN patient–derived HSPCs. TPO is not required for expansion and megakaryocyte differentiation of CALRmut or MPLmut MPN patient–derived HSPCs. In our experience, JAK2mut HSPCs will not grow in the complete absence of TPO. A low dose of TPO (5–10 ng/mL) may be required for JAK2V617F samples and should be compared to healthy controls to measure TPO hypersensitivity rather than factor-independence. Make sure to pipette precise volumes in every step of the staining protocol to avoid any bias for the cell count via counting beads. Centrifuge sorted CD34+ HSPCs at 350 × g for 10 min at 4°C and remove supernatant to remove all sheath fluid from the sorting process. Resuspend pellet at 0.75 × 105–1.0 × 105 cells/mL in Mk-diff media (see Recipe 7). Seed cells in triplicates into a 48-well plate (200–500 µL/well, 15–20,000 cells/well, ideally) for every experimental condition. Add your compound of interest or vehicle control (e.g., DMSO) to the cell suspension. Note: If it is not possible to obtain such high numbers of CD34+ cells from samples, it is possible to scale down the number of cells and the volume of experimental setup. This experiment has been carried out with a minimum of 1,000 cells/replicate in a 96-well plate. Fill all the perimeter wells with 1 mL of sterile water to prevent excessive evaporation of media. Culture cells in a humidified incubator at 37°C and 5% CO2 atmosphere. On days 4, 7, 10, and 12 mix the content of wells gently using a pipette and transfer 100 µL of cell suspension to a 1.5 mL microfuge tube. Add 100 µL of fresh Mk-diff media into each well to replenish the culture. Wash once with 1 mL of staining buffer (350 × g, room temperature, 5 min). Resuspend cells in 50 µL of staining buffer containing human FcR blocking reagent and CD41 APC-Cy7 and CD42b APC antibodies (1:25 dilution each), and incubate for 20 min at 4°C. Notes: CD41 and CD42b antibodies can be used with different fluorescent conjugates to match flow cytometer configurations or additional fluorescent staining used in the experiment. At the initial setup of the experiment, unstained and single antibody and 7-AAD stained cells should be prepared to correctly compensate fluorophores and set the population gates at the flow cytometer. Wash once with 1 mL of staining buffer (350 × g, 4°C, 5 min). Resuspend cell pellet in 133 µL of staining buffer, mix well, and transfer to a 5 mL round bottom FACS tube. Add 2.0 µL of a viability dye (e.g., 7-AAD or DAPI) and 15 µL of counting beads resulting in 150 µL total volume. Vortex bottle of counting beads properly before use. Incubate for 10 min at 4°C and measure on flow cytometer (Figure 6). Calculate CD41+ CD42b- megakaryocyte progenitor or CD41+ CD42b+ mature megakaryocyte cell counts (see Data analysis). Figure 6. Gating strategy for evaluation of megakaryocyte differentiation via flow cytometry. (A) Counting beads (high SSC-A, low FSC-A) and cells (excluding cell debris) were gated separately (top row, left plot). Beads were additionally gated in two independent fluorescent channels (e.g., ECD and Krome Orange) to exclude any contaminating cells in the initial beads gate (bottom row, left plot). After selecting cells based on morphology (top row, middle plot) and excluding cell doublets (top row, right plot) and dead cells (bottom row, right plot), megakaryocyte progenitors and mature megakaryocytes were evaluated by gating on CD41+ CD42b- and CD41+ CD42b+ cells, respectively (bottom row, center plot). (B) Unstained (left plots) and single stained (center and right plots) cells were used as compensation controls and for the identification of the fluorescent threshold for proper setup of population gates. Data analysis Megakaryocyte-directed differentiation in liquid culture Note: We recommend performing flow cytometric data analysis using FlowJo, but software provided by cytometer manufacturers can be used as well. Subset cell populations by applying the depicted gating strategy (Figure 6) to every sample. Report the percentage of CD41+ CD42b- megakaryocyte progenitors and CD41+ CD42b+ mature megakaryocytes within live cells as an indication for the efficiency of cell differentiation. Analyze the difference in cell differentiation between experimental conditions at each time point using two-way ANOVA (e.g., GraphPad Prism or similar software). Extract the total numbers of events in the “pure beads,” the “CD41+ CD42b- megakaryocyte progenitors,” and the “CD41+ CD42b+ megakaryocytes” gate. Calculate the number of CD41+ CD42b- megakaryocyte progenitors and CD41+ CD42b+ megakaryocytes that were present in each well of the liquid culture using the following equations: Multiply the events acquired in either the “CD41+ CD42b- megakaryocyte progenitors” or the “CD41+ CD42b+ megakaryocytes” gate by the number of beads per 15 µL and by the total volume in the cell culture well. The bead concentration varies between batches and is stated on the label of each bottle. Divide the result by the events acquired in the “pure beads” gate and by the volume that was removed from the cell culture well (100 µL). Test for differences in cell numbers between experimental conditions at each time point using a two-way ANOVA. Recipes RBC lysis buffer Prepare 10× stock solution. Dissolve 9.0 g of NH4Cl, 1.0 g of KHCO3, and 37 mg of EDTA tetrasodium in 100 mL ddH2O. Filter-sterilize buffer and store at 4°C. This buffer is stable for up to one year. Dilute 10× buffer with sterile ddH2O to get a 1× concentration prior to use. Staining buffer (SB) PBS 1% BSA 2 mM EDTA Freezing media RPMI 1640 20% FBS 1% penicillin/streptomycin 10% DMSO Thawing media RPMI 1640 or IMDM 10% FBS or 20% FBS 1% penicillin/streptomycin 20 U/mL DNase Antibody cocktail Note: This recipe is calculated to stain 1 × 107 PBMCs from one sample. Scale up for more cells per samples and for as many samples as required. Mix 5 µL of each mouse anti-human fluorescence conjugated antibody: CD34 FITC CD45 V500-C CD2 PE-Cy5 CD3 PE-Cy5 CD19 PE-Cy5 CD14 PerCP-Cy5.5 CD16 PE-Cy5 CD56 PE-Cy5 Megakaryocyte (Mk) colony media Notes: This recipe is calculated for one sample with four replicates. Scale up for as many samples or experimental conditions as required. To prevent repeated freeze-thaw cycles, aliquot the MegacultTM-C medium with lipids into 15 mL tubes (1.7 mL medium each) at the first thawing and store aliquots at -20°C. Prepare cytokines and potential therapeutic compounds at 8.25× concentration in IMDM with a final volume of 400 µL. Add IL-3 and IL-6 at a concentration of 82.5 ng/mL (final concentration in the assay will be 10 ng/mL). Finalize the colony media by adding 400 µL of cytokine mix into 1.7 mL of MegacultTM-C medium with lipids and mix well. Megakaryocyte differentiation (Mk-diff) media SFEM II or StemPro 34 + nutrient supplement and 2 mM L-Glutamine 20 ng/mL SCF 10 ng/mL IL-6 10 ng/mL IL-9 0.4% lipids cholesterol rich Isotonic NaCl solution (0.15 M) Dissolve 4.38 g of NaCl in 500 mL of ddH2O. Solution is stable for several months at room temperature. TRIS buffer (0.5 M, pH 7.6) Dissolve 3.03 g of TRIS in 50 mL of ddH2O. Adjust pH to 7.6 by adding HCl 6 M dropwise under constant swirling. Buffer is stable for one month at room temperature. Wash buffer Mix nine parts of isotonic NaCl solution with one part of TRIS buffer. Prepare wash buffer always fresh on day of use. Substrate buffer Dissolve 909 mg of TRIS in 50 mL of ddH2O. Adjust pH to 8.2–8.5 by adding HCl 6 M dropwise under constant swirling. Add 50 µL of Tween 20 and mix properly. Buffer is stable for one month at room temperature. Evans Blue solution (0.1%) Dissolve 50 mg of Evans Blue in 50 mL of ddH2O. Solution is stable for several months at room temperature. Staining tray Prepare a plastic dish of around 20 × 12 × 3 cm in size and cut two serological pipettes in length so they fit in the dish. Tape the serological pipettes 2 cm apart on the bottom of the dish using adhesive tape. Slides can rest on the pipettes for staining while excessive buffer and staining solutions are caught inside the dish. Acknowledgments This work was supported by the National Health and Medical Research Council Ideas Grant APP1182564 and APP2004288, and the Australian Cancer Research Foundation Discovery Accelerator, the Leukemia & Lymphoma Society Translational Research Program, the Hospital Research Fund, the Australian Medical Research Future Fund for Rare Diseases, Rare Cancers and Unmet Need, the Austrian Science Fund (grant numbers P32783 and I5021), the Austrian Society of Internal Medicine (Joseph Skoda Fellowship), the Austrian Society of Hematology and Oncology (Clinical Research Grant), and MEFOgraz. This protocol was adapted from previous studies (Cortin et al., 2009) and manufacturer recommendations (Stemcell technologies, MegacultTM-C Assays). Several figures were generated with BioRender with permissions to be published. We thank Suraiya Onnesha for assistance with video recording. Competing interests The authors have no competing interests to declare. Ethics Collection of peripheral blood from MPN patients at the Division of Hematology, Medical University of Graz was approved by the institutional review board (IRB approval: 30-464 ex 17/18) or requested from the South Australian Cancer Research Biobank (SACRB, CALHN approval: 12986). Blood was only collected from patients with informed consent. References Araki, M., Yang, Y., Masubuchi, N., Hironaka, Y., Takei, H., Morishita, S., Mizukami, Y., Kan, S., Shirane, S., Edahiro, Y., et al. (2016). Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. Blood 127(10): 1307-1316. Bonnet, D. and Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3(7): 730-737. Broudy, V. C., Lin, N. L. and Kaushansky, K. (1995). Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood 85(7): 1719-1726. Chachoua, I., Pecquet, C., El-Khoury, M., Nivarthi, H., Albu, R. I., Marty, C., Gryshkova, V., Defour, J. P., Vertenoeil, G., Ngo, A., et al. (2016). Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood 127(10): 1325-1335. Chen, J., Herceg-Harjacek, L., Groopman, J. E. and Grabarek, J. (1995). Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin. Blood 86(11): 4054-4062. Choi, E. S., Nichol, J. L., Hokom, M. M., Hornkohl, A. C. and Hunt, P. (1995). Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood 85(2): 402-413. Corre-Buscail, I., Pineau, D., Boissinot, M. and Hermouet, S. (2005). Erythropoietin-independent erythroid colony formation by bone marrow progenitors exposed to interleukin-11 and interleukin-8. Exp Hematol 33(11): 1299-1308. Cortin, V., Pineault, N. and Garnier, A. (2009). Ex vivo megakaryocyte expansion and platelet production from human cord blood stem cells. Methods Mol Biol 482: 109-126. Debili, N., Wendling, F., Katz, A., Guichard, J., Breton-Gorius, J., Hunt, P. and Vainchenker, W. (1995). The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 86(7): 2516-2525. Elf, S., Abdelfattah, N. S., Chen, E., Perales-Paton, J., Rosen, E. A., Ko, A., Peisker, F., Florescu, N., Giannini, S., Wolach, O., et al. (2016). Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. Cancer Discov 6(4): 368-381. Jamieson, C. H., Gotlib, J., Durocher, J. A., Chao, M. P., Mariappan, M. R., Lay, M., Jones, C., Zehnder, J. L., Lilleberg, S. L. and Weissman, I. L. (2006). The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation. Proc Natl Acad Sci U S A 103(16): 6224-6229. Klampfl, T., Gisslinger, H., Harutyunyan, A. S., Nivarthi, H., Rumi, E., Milosevic, J. D., Them, N. C., Berg, T., Gisslinger, B., Pietra, D., et al. (2013). Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 369(25): 2379-2390. Kojima, H., Hamazaki, Y., Nagata, Y., Todokoro, K., Nagasawa, T. and Abe, T. (1995). Modulation of platelet activation in vitro by thrombopoietin. Thromb Haemost 74(6): 1541-1545. Kralovics, R., Passamonti, F., Buser, A. S., Teo, S. S., Tiedt, R., Passweg, J. R., Tichelli, A., Cazzola, M. and Skoda, R. C. (2005). A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 352(17): 1779-1790. Lu, X., Levine, R., Tong, W., Wernig, G., Pikman, Y., Zarnegar, S., Gilliland, D. G. and Lodish, H. (2005). Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. Proceedings of the National Academy of Sciences 102(52): 18962-18967. Marty, C., Pecquet, C., Nivarthi, H., El-Khoury, M., Chachoua, I., Tulliez, M., Villeval, J. L., Raslova, H., Kralovics, R., Constantinescu, S. N., et al. (2016). Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. Blood 127(10): 1317-1324. Pikman, Y., Lee, B. H., Mercher, T., McDowell, E., Ebert, B. L., Gozo, M., Cuker, A., Wernig, G., Moore, S., Galinsky, I., et al. (2006). MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med 3(7): e270. Pronier, E., Cifani, P., Merlinsky, T. R., Berman, K. B., Somasundara, A. V. H., Rampal, R. K., LaCava, J., Wei, K. E., Pastore, F., Maag, J. L., et al. (2018). Targeting the CALR interactome in myeloproliferative neoplasms. JCI Insight 3(22). Reinisch, A., Thomas, D., Corces, M. R., Zhang, X., Gratzinger, D., Hong, W. J., Schallmoser, K., Strunk, D. and Majeti, R. (2016). A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nat Med 22(7): 812-821. Spivak, J. L. (2017). Myeloproliferative Neoplasms. N Engl J Med 376(22): 2168-2181. Tefferi, A. and Pardanani, A. (2015). Myeloproliferative Neoplasms: A Contemporary Review. JAMA Oncol 1(1): 97-105. Tefferi, A. and Pardanani, A. (2019). Essential Thrombocythemia. N Engl J Med 381(22): 2135-2144. Tvorogov, D., Thompson-Peach, C. A. L., Foßelteder, J., Dottore, M., Stomski, F., Onnesha, S. A., Lim, K., Moretti, P. A. B., Pitson, S. M., Ross, D. M., et al. (2022). Targeting human CALR-mutated MPN progenitors with a neoepitope-directed monoclonal antibody. EMBO Reports 23(4): e52904. Vannucchi, A. M., Lasho, T., Guglielmelli, P., Biamonte, F., Pardanani, A., Pereira, A., Finke, C., Score, J., Gangat, N. and Mannarelli, C. (2013). Mutations and prognosis in primary myelofibrosis. Leukemia 27(9): 1861-1869. Woll, P. S., Kjällquist, U., Chowdhury, O., Doolittle, H., Wedge, D. C., Thongjuea, S., Erlandsson, R., Ngara, M., Anderson, K., Deng, Q., et al. (2014). Myelodysplastic Syndromes Are Propagated by Rare and Distinct Human Cancer Stem Cells In Vivo. Cancer cell 25(6): 794-808. Woods, B., Chen, W., Chiu, S., Marinaccio, C., Fu, C., Gu, L., Bulic, M., Yang, Q., Zouak, A., Jia, S., et al. (2019). Activation of JAK/STAT signaling in megakaryocytes sustains myeloproliferation in vivo. Clin Cancer Res. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Stem Cell > Adult stem cell > Hematopoietic stem cell Cancer Biology > Cancer stem cell > Cell biology assays Cell Biology > Cell isolation and culture > Cell isolation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Low-viscosity Matrix Suspension Culture for Human Colorectal Epithelial Organoids and Tumoroids Tao Tan [...] Oliver M. Sieber Apr 20, 2022 2617 Views Isolation and ex vivo Expansion of Limbal Mesenchymal Stromal Cells Naresh Polisetti [...] Günther Schlunck Jul 20, 2022 1447 Views Isolation, Purification, and Culture of Embryonic Melanoblasts from Green Fluorescent Protein–expressing Reporter Mice Melissa Crawford [...] Lina Dagnino Sep 5, 2023 547 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,593	https://bio-protocol.org/en/bpdetail?id=4593&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Evaluating Plant Drought Resistance with a Raspberry Pi and Time-lapse Photography Daniel N. Ginzburg SR Seung Y. Rhee Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4593 Views: 1543 Reviewed by: Ansul LokdarshiRicardo Urquidi CamachoWan-Jun Zhang Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plant Physiology Dec 2022 Abstract Identifying genetic variations or treatments that confer greater resistance to drought is paramount to ensuring sustainable crop productivity. Accurate and reproducible measurement of drought stress symptoms can be achieved via automated, image-based phenotyping. Many phenotyping platforms are either cost-prohibitive, require specific technical expertise, or are simply more complex than necessary to effectively evaluate drought resistance. Certain mutations, allelic variations, or treatments result in plants that constitutively use less water. To accurately identify genetic differences or treatments that confer a drought phenotype, plants from all experimental groups must be subjected to equal levels of drought stress. This can be easily achieved by growing and imaging plants that are grown in the same pot. Here, we provide a detailed protocol to configure a Raspberry Pi computer and camera module to image seedlings of multiple genotypes growing in shared pots and to transfer images and metadata via the cloud for downstream analyses. Also detailed is a method to calculate percent soil water content of pots while being imaged to allow for comparison of stress symptoms with water availability. This protocol was recently used to uncouple differential water usage from drought resistance in a dwarf Arabidopsis thaliana mutant chiquita1-1/cost1compared to the wild-type control. It is cost effective, suitable for any plant species, customizable to various biological questions, and requires no prior experience with electronics or basic software programming. Keywords: Drought resistance Phenotyping Soil water content Raspberry Pi Time-lapse photography Background Drought stress symptoms can be qualitatively and quantitatively measured via image-based phenotyping. Imaging plant growth and the visual onset of stress symptoms results in greater sensitivity and data availability than what can be achieved from visual observations or endpoint analysis alone (Mutka and Bart, 2014; Tovar et al., 2018). Many protocols to measure plant stress via image analysis involve either a high up-front cost, a large experimental workspace, or integration of and proficiency with complex analytical software (Shakoor et al., 2017; Tovar et al., 2018). In contrast to these approaches, we have developed a simple, modular, low-cost protocol using a Raspberry Pi–controlled camera (which together require approximately $40 USD) to correctly evaluate drought resistance. We define drought resistance as any combination of plant strategies to cope with insufficient water availability required for maximum plant growth and reproduction, namely drought tolerance, drought avoidance, and drought escape (Blum, 2011; Deikman et al., 2012; Lawlor, 2013). Common plant responses to low water availability include increased root growth or maintenance of cell turgor via increased osmolyte production. Imaging plants as described in this protocol cannot specifically differentiate between drought avoidance and tolerance strategies. When paired with additional assays, however, both drought tolerance and avoidance can be quantified, as we recently demonstrated (Ginzburg et al., 2022). Drought escape (via earlier flowering time in response to reduced soil water content) can readily be quantified via time course imaging. This protocol requires no previous knowledge of software programming and requires only a small amount of space (approximately 1.5 m3 needed for 16 pots with Arabidopsis seedlings; Figure 4). While this protocol is suitable for any plant species, larger species or sample sizes may require additional space. Some reports of image-based drought phenotyping platforms do not involve growing different genotypes together in the same pot or correcting for differences in % soil water content (SWC) between genotypes that are grown in separate pots (Kim et al., 2020). Experiments conducted without these crucial controls can yield potentially misleading results due to genotype-specific differences in water-use requirements (Lawlor, 2013). To account for these differences when evaluating drought resistance, soil water content for each genotype needs to be kept uniform throughout the drought treatment for all genotypes. This can be easily achieved, for example, by growing plants together in the same pot (Ginzburg et al., 2022), or, if in separate pots, by using soil moisture probes (Miralles-Crespo and van Iersel, 2011) or gravimetrically (Granier et al., 2006; de Ollas et al., 2019). The persistence of reports that equate delayed onset of stress symptoms with drought resistance goes against established knowledge and slows progress in identifying true drought phenotypes (Lawlor, 2013). The protocol described here was recently used to differentiate between drought resistance and prolonged survival due to lower water usage in a dwarf Arabidopsis thaliana mutant chiquita1-1/cost1 (Ginzburg et al., 2022). Because plant responses to drought can vary depending on plant species, developmental stage, and degree of stress, there is no single set of measurements that can capture a drought phenotype in its entirety. Therefore, this protocol does not prescribe or outline in detail any specific measurements of plant stress. Rather, it provides readers with a foundation to properly set up a comparative drought experiment and capture images throughout the experiment to use for whichever downstream analyses are most appropriate to the experimental design and biological question(s). The straightforward methodology outlined here will be a valuable resource to the plant science community at large, not only for studying plant responses to drought but for any research requiring accurate visual comparisons of plant growth and development. Equipment Raspberry Pi–related equipment Raspberry Pi (model 3 or newer) Micro SD card (should have 6 GB or more of storage) Micro USB power cable Camera module: Minimum specifications (i.e., lens resolution, pixels, frame rate, etc.) will vary based on the distance between the lens and samples, sample size, and external light source provided. An Arducam M12 lens (model B0031; 5 MP 2,592 × 1,944 resolution, 4 mm focal length) was used in this protocol to image Arabidopsis seedlings from approximately 75 cm above the pots. Camera cable Note: If using a Raspberry Pi Zero W, you will need a smaller cable connection to insert into the Raspberry Pi. Monitor with HDMI port HDMI cable Note: If using a Raspberry Pi Zero W, you will need a mini-HDMI to HDMI cable. Keyboard and mouse Mounting-related equipment Adhesive or Bungee cables or Mounting stand (see Tovar et al., 2018) Optional Case for Raspberry Pi and camera module Plant growth–related equipment Pots: Appropriate size depends on species and number of plants grown in each pot. For growing two Arabidopsis seedlings together in a single pot, it is recommended to use square pots of at least 40 cm 2. Soil: Any horticultural or potting soil/mix is suitable. PRO-MIX HP Mycorrhizae potting soil (Premier Tech Horticulture, Quakertown, PA) is suitable for a wide range of species. Additional amendments may be added based on species-specific requirements. Scale: For small plants grown in small pots, such as Arabidopsis , use a scale sensitive to 0.1 g. A scale sensitive to 1.0 g is suitable for larger plants and bigger pots. Plastic cover Oven: It should be capable of reaching 45°C. Adhesive: It should be strong enough to support the weight of the Raspberry Pi, lens, and power cable (approximately 20 g). Procedure Initial Raspberry Pi configuration Note: This protocol requires the Raspberry Pi to be connected to the internet. All third-generation models or newer with built-in Wi-Fi capabilities (which excludes the Raspberry Pi Zero and Pico) are recommended. Whichever model of Raspberry Pi used will need a Micro SD card with Raspberry Pi OS (Raspbian) installed. Micro SD cards can be purchased either with Raspbian preinstalled or the latest version of Raspbian can be installed from https://www.raspberrypi.org/downloads/raspbian/. Ensure you use a Micro SD card with at least 6 GB of storage to allow for temporary image storage on the Micro SD before being transferred to cloud storage. Insert the Micro SD card with Raspbian installed into the Raspberry Pi. Connect the monitor, keyboard, and mouse into the Raspberry Pi and then insert the power cable. Note that once the Micro SD card is inserted into the Pi, all scripts outlined in this protocol will be written to and saved onto the Micro SD card itself. No additional steps other than those outlined below will be required to perform the protocol. If starting your Pi for the first time, the Welcome to Raspberry Pi application will pop up and guide you through initial configuration, including setting a username and password, time zone, and connecting to Wi-Fi. Step-by-step instructions can also be found at https://projects.raspberrypi.org/en/projects/raspberry-pi-getting-started/1. Once initial setup is complete, click on the raspberry icon on the top left of the desktop and navigate to Preferences > Raspberry Pi Configuration. This can also be achieved by typing “sudo raspi-config” in the terminal window, which can be opened by clicking on the black monitor icon on the toolbar, located at the top of the desktop. In the interfaces tab, set camera and secure shell (SSH) to “enabled.” These configurations will allow you to take images and communicate with the Pi without connecting it to a monitor. In order to communicate with the Pi remotely via SSH, you will need to first get your Pi’s IP address. In the terminal window, type “hostname -I.” Also write down the MAC address in case your IP address is set to change and you want to confirm a given device is yours using a network scanner [i.e., Fing (https://www.fing.com/), available for Android and iOS]. Type “ifconfig” in the terminal window. In the outputted text, the MAC address is the 12-digit hexadecimal number immediately after “ether.” You can connect to your Pi via SSH directly from the terminal window of your computer (running Linux, Mac OS, or Windows 10 or later). Type “ssh pi@<IP>” replacing “IP” with your Pi’s IP address. Alternatively, you can use a free SSH client such as Termius (https://www.termius.com/), which is compatible with Linux, Mac OS, and Windows operating systems. Configuring remote data transfer Note: Before configuring the camera, it will be helpful to first configure how to transfer images from the Raspberry Pi to a cloud-based server or a local computer where images can be more easily analyzed than on the Pi itself. While there are many methods for remote data transfer, this protocol describes the configuration of rclone (https://rclone.org/), an open-source software designed for managing content on common cloud-based platforms. Open a terminal window either by clicking on the terminal icon at the top of the screen (if using an external monitor) or by typing Ctrl + Alt + T. Run the following command in the opened terminal window to download and install the latest, stable release of rclone: wget https://downloads.rclone.org/rclone-current-linux-arm.zip After successful installation, run the following configuration command: rclone config Create a new remote connection by typing “n” and then pressing Enter. Enter a name for your new remote connection, e.g., “RPi_Drive.” You will now see a list of various backend storage systems to connect to. Subsequent steps after choosing a specific storage system, e.g., Google Drive, will vary widely depending on the storage system chosen. Refer to https://rclone.org/overview/ for the various storage systems supported and corresponding documentation for how to configure each one. Mounting the Raspberry Pi and camera Note: Given the specific lighting and spacing requirements in the location where the experiment will be performed, it is recommended to first find a suitable location for the drought experiment and only then to configure the camera in that space. The available space above or adjacent to your plants where a camera can be mounted may vary across growth chambers or plant species. It is therefore recommended to use a Raspberry Pi–compatible lens with modular focusing capabilities. There are many Raspberry Pi–compatible options developed by Arducam (https://www.arducam.com/), which yield publication-quality images. Also, be sure to pick a location with uninterrupted access to a power outlet, as the Raspberry Pi will need to be powered continuously for the duration of the experiment. If planning to conduct experiments at high relative humidity (>70% relative humidity; Comizzoli et al., 1986), we recommend using a fan, protective case, or desiccant to avoid damaging the Raspberry Pi and camera components. Relative humidity values below 70% (40% was used in this protocol) should not cause any technical issues with the Raspberry Pi and camera components. When a suitable location has been identified, ensure your Raspberry Pi is powered off before connecting the camera module to the Raspberry Pi. Notes: Be very careful when inserting the camera cable into Raspberry Pi as the plastic clamp necessary for establishing and maintaining a connection is fragile and can easily bend or crack. Raspberry Pi Zero W has a smaller camera module port and thus needs a correspondingly smaller ribbon cable for connecting the camera to the Pi. The Raspberry Pi and camera module can be physically mounted or suspended above or adjacent to the experimental growth space in several manners. Regardless of the approach used, the camera should be free of physical disturbance (including vibrations) and far enough away from the pots to allow the user to comfortably pick up and weigh pots throughout the experiment. See Tovar et al. (2018) for additional information. Figure 1. Methods of mounting Raspberry Pi and camera module inside the growth chamber. (A) Pi and camera are affixed to the roof of the growth cabinet with adhesive. (B) Pi and camera are suspended from bungee cords to minimize vibrations within the growth cabinet. In both images, the camera module and Pi are encased in protective cases. Configuring image capture While connected to your Pi via SSH, make a folder for your images on the Pi’s desktop. From the terminal window, type “mkdir /home/pi/Desktop/Images”. The camera module can be controlled either from the command line (in a terminal window) or via Python scripts. This protocol recommends using the pre-installed Python picamera library due to its rich functionality and ease of configuration. From the terminal window, enter “nano [filename].py” replacing [filename] with a file name of your choice. The following scripts, which are written in the opened [filename].py file, include commands for capturing and saving images with the current time to a destination of your choice. Adding a timestamp to your images will provide valuable metadata to correlate images with time of day and duration of drought. A predictable file-naming scheme will also allow you to check if all images were captured and transferred as desired. from picamera import PiCamera from time import sleep camera = PiCamera() import datetime time = datetime.datetime.now().strftime("%Y-%m-%d-%H:%M") #adjust image resolution camera.resolution = (1950, 1900) camera.start_preview() sleep(5) camera.capture(r'/home/pi/Desktop/Images/image-' + time + '.jpg') camera.stop_preview() To save and exit, press Ctrl + X. Then, when prompted if you want to save your changes, press “Y”. Alternatively, you can save your changes by first pressing Ctrl + O and then clicking Enter, then pressing Ctrl + X. Place some pots, which will be used for the growth experiment, into the area where plants will be grown, and capture an image by typing “python [filename].py”. Images can be viewed by sending them remotely to your designated and previously configured storage system. While they can also be viewed by connecting your Pi to a monitor, a remote transfer system enables faster positioning of your camera. Return to the Desktop by typing “cd /home/pi/Desktop”. Create a new file on your Pi’s desktop, which will trigger remote image transfer. nano sync.sh Inside the newly created “sync.sh” file, invoke the rclone sync function to send images from the images folder to a folder in your online storage solution. The following code uses a Google Drive folder named “Raspberry_Pi_images” as an example: rclone sync -v /home/pi/Desktop/Images gdrive:Rapsberry_Pi_images Save your changes by pressing Ctrl + O and then clicking Enter, then pressing Ctrl + X. From the Desktop, run the script to transfer images ./sync.sh After viewing your captured image(s), you can manually adjust the focus of the camera lens and add modifications to your image-capturing script (resolution, angle, frame rate, color correction, etc.) based on the needs of your experiment and physical constraints of your working environment. Note: 300 pixels per inch (ppi) is frequently suggested as the minimum resolution for high-quality color images in scientific publications. PPI is based on both image resolution per dimension (pixels by width and height) and by image size, whereby larger images require a higher resolution to maintain a given PPI. The Arducam M12 lens (model B0031), for example, has a maximum resolution of 2,592 × 1,944 pixels. Using this resolution, any image with a diagonal length less than 27.4 cm would have a PPI greater than or equal to 300 ppi. The width of a single-column in most academic journals is 8.6 cm, meaning that even much smaller resolutions than are possible with the Arducam M12 (model B0031) would be sufficient to ensure publication-quality images. Modifications to how your image looks can be added to the Python script by once again entering “nano [filename].py” in the terminal window and entering additional lines of code as desired. Some useful image modifications can be found at https://projects.raspberrypi.org/en/projects/getting-started-with-picamera/. Figure 2. Setting up camera location within the growth cabinet and placing a representative pot below for image optimization Note that image focusing and optimization should be done with a soil-filled pot placed within an empty pot to ensure the correct distance between lens and soil for optimizing image focus. As will be described in the next section, the two-pot system allows rotating pots for reducing the effect of micro environmental variations while ensuring that the pot positions stay the same for imaging. When satisfied with how your images look, you can now schedule automated image capture and upload to the cloud. Using the job scheduler crontab (https://crontab.guru/), first open the editing table of jobs: crontab -e Write scripts (“cron jobs”) for both automated image capturing and uploading images to the cloud. You can test out how to code your desired schedule at https://crontab.guru/. Examples for both jobs are provided below. #run the python image-capture script every 15 minutes 15 * * * * python /home/pi/Desktop/Take_photo_with_timestamp.py #run the sync.sh program every 2 hours at minute 30 minutes past the hour 30 0-23/2 * * * /home/pi/Desktop/sync.sh Save your changes by pressing Ctrl + O and then clicking Enter, then pressing Ctrl + X. These settings can be viewed by entering “crontab -I” and can be modified again by entering “crontab -e”. Determining of soil characteristics Weigh 8–10 empty pots to determine the average weight of an empty pot. Fill each pot with a fixed amount of fresh soil by weight. To determine the average amount of water held within fresh soil, by weight, dry 8–10 soil samples in an oven set to at least 45°C. Measure pot and soil weights daily until there is no change in sample weight (approximately 2–3 days). Record final pot and dried soil weights. Dry soil weight (DSW) = [(Pot + dried soil) – empty pot] DSW % = DSW/[(Pot + fresh soil) – empty pot]. Slowly add water to the above-mentioned pots with dried soil. Stop adding water once water starts dripping from the bottom. Put a cover over the pots to reduce evaporation. When dripping from the bottom has ceased, pots have reached pot capacity (PC; 100% SWC). Weigh each pot to determine weight at PC. Water weight at PC = [(Pot + soil at PC) – empty pot – DSW] Soil pot capacity (SPC; g water/g dry soil) = (Total weight at PC – DSW – pot)/(DSW) Setting up pots and conducting a drought experiment While comparisons of drought resistance are only made between seedlings grown in the same pot, all seedlings should be maintained uniformly to reduce variation across replicates. With tape or some other adhesive, affix an equal number of empty pots as experimental pots to the shelf or table where plants will be grown and images captured. Arrange these empty pots in an area that can be fully captured by your camera. Some modifications to camera and pot placement may be required to optimize the number of samples that can be captured by the camera. Figure 3. Arranging empty pots into imaging space. (A) Affixed pots are evenly spaced out and fill up the entirety of the viewable space of the camera. (B) Representative image of a soil-filled experimental pot sitting within an affixed empty pot. Fill new pots with equal amounts of fresh soil by weight. Plant seeds at designated locations in each pot (i.e., top/bottom, right/left) to eliminate any potential differences in micro-environment during image capture period. Seeds should be planted such that each pot contains one wild-type (control) seedling and one seedling from at least one mutant. Figure 4. Planting evenly spaced seeds such that each pot contains one wild-type and at least one mutant seedling. Pots also contain numbers and arrows to indicate sample number and the mutant seedling, respectively. Image was taken approximately 75 cm above pots (40 cm2) using a 5 megapixel Arducam M12 lens (model B0031) at resolution of 1,950 × 1,900 (W × H). Approximate dimensions of growth cabinet space used: 63.5 cm × 63.5 cm × 75 cm (L × W × H). Place seeded pots into the pre-arranged empty pots. Weigh and add water to pots daily to maintain SWC at a designated well-watered state (i.e., 70% SWC). Well-watered weight (70% SWC) = [(Fresh soil weight × DSW% × SPC × 0.7) + (Fresh soil weight × DSW%) + empty pot] Note: Weigh pots when you know images are not being captured to ensure an uninterrupted progression of images throughout the experiment. When seedlings have reached a predetermined age, size, or developmental stage, water one last time up to the well-watered state, and then stop watering, as an example of a drought experiment. Note: This is only one type of drought experiment. Another type of drought experiment is to maintain soil water content at specific levels. Also, this protocol can be used for experiments other than drought treatment, such as exposure to high or low temperatures, salinity stress, etc. If images were not yet being captured, start automated image capture as described above. On a regular basis (i.e., daily), gently lift up and remove experimental pots from empty pots and weigh them to determine SWC. Return each experimental pot to the same empty pot. If interested in evaluating recovery from drought, saturate pots at a predetermined time, age, or degree of stress and then return them to the same empty pot for continued imaging. Combining images into a timelapse video Combining images into a video file format is not necessary to evaluate changes in plant growth throughout the course of an experiment. Depending on the desired analyses, inspection of individual images may be more helpful than a video of multiple images. However, viewing the progression of the entire experiment in a video format may aid users in more easily identifying overall trends in plant growth. A sample protocol for creating a timelapse video from individual images is therefore provided below. While many software programs can be used for this purpose, the following example makes use of the free video editing software Davinci Resolve (https://www.blackmagicdesign.com/products/davinciresolve/). Import images by clicking on File > Import > Media and select the folder containing your images. Make sure they are arranged by date and time to ensure a chronological order. With all images selected, right-click and select “Create New Timeline Using Selected Clips…” Images will now appear arranged from left to right on the bottom of the window. Without any additional modifications, a video progression can now be played using the Play button in the middle of the window. To modify the length of the video, select all images from the timeline at the bottom of the window. Right-click and select “Change Clip Duration”. The length of the video, or even parts of it based on the selected frames, can be modified by either changing the value in the “Time” or “Frames” tab. When satisfied with the duration of the video, click on the “Deliver” tab at the very bottom of the window. Here, users can select the file type, resolution, frame rate, and quality of the video to be generated, in addition to more advanced features. Once all settings have been selected and a name and destination for the file have been set, click “Add to Render Queue”. A new job will appear in the top right side of the window. Click “Render All”. Additional video editing features can be found at https://www.blackmagicdesign.com/products/davinciresolve/training. Data analysis Captured images provide the basis for all downstream analyses. Images or timelapse videos alone can be used to qualitatively evaluate relative drought resistance across genotypes, as was done previously (Ginzburg et al., 2022). Specific metrics to quantify plant growth and stress will vary by plant species, developmental stage, degree of stress, and by the biological question(s) of interest. No additional software is required for performing basic analysis of plant growth and stress symptoms, as images alone can be used to visually score stress symptoms (Diaz et al., 2006;Sarkar et al., 2021). Images can also be uploaded to image analysis software, such as ImageJ (Schneider et al., 2012) or PlantCV (Fahlgren et al., 2015), for additional analyses, such as quantifying vegetative area, relative area of stressed/diseased tissue to green tissue, etc. Utilizing the timestamps of captured images also allows for easy comparison of key developmental stage transitions, such as flowering time. This protocol also allows users to perform additional, non-destructive measurements on plants throughout the experiment to complement image capture. For example, chlorophyll fluorescence was measured on droughted seedlings grown and imaged using this protocol (Ginzburg et al., 2022) to compare drought tolerance between mutant and wild-type seedlings. Daily SWC levels can be correlated with chosen metrics of plant growth and stress onset to understand the relationship between soil water availability and stress symptoms. If evaluating the stress resistance of multiple mutant lines, SWC levels can also be used to determine relative water requirements of each genotype, as demonstrated in Ginzburg et al. (2022). Acknowledgments We thank A. Malkovskiy for helpful advice on imaging, G. Materassi-Shultz for plant growth facility support, and the Rhee lab for helpful discussions. This work was done on the ancestral land of the Muwekma Ohlone Tribe, which was and continues to be of great importance to the Ohlone people. Funding: This work was supported in part by Carnegie Institution for Science Endowment and grants from the National Science Foundation (IOS-1546838, IOS-1026003) and the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program grant nos. DE-SC0018277, DE-SC0008769, DE-SC0020366, and DE-SC0021286. Competing interests Authors declare no conflict of interest. References Blum, A. (2011). Drought Resistance and Its Improvement. Blum, A. (Ed.) Plant Breeding for Water-Limited Environments. Springer New York, 53-152. Comizzoli, R. B., Frankenthal, R. P., Milner, P. C., and Sinclair, J. D. (1986). Corrosion of electronic materials and devices. Science 234(4774): 340-345. de Ollas, C., Segarra-Medina, C., Gonzalez-Guzman, M., Puertolas, J. and Gomez-Cadenas, A. (2019). A customizable method to characterize Arabidopsis thaliana transpiration under drought conditions. Plant Methods 15: 89. Deikman, J., Petracek, M. and Heard, J. E. (2012). Drought tolerance through biotechnology: improving translation from the laboratory to farmers' fields. Curr Opin Biotechnol 23(2): 243-250. Diaz, C., Saliba-Colombani, V., Loudet, O., Belluomo, P., Moreau, L., Daniel-Vedele, F., Morot-Gaudry, J. F. and Masclaux-Daubresse, C. (2006). Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana. Plant Cell Physiol 47(1): 74-83. Fahlgren, N., Feldman, M., Gehan, M. A., Wilson, M. S., Shyu, C., Bryant, D. W., Hill, S. T., McEntee, C. J., Warnasooriya, S. N., Kumar, I., et al. (2015). A Versatile Phenotyping System and Analytics Platform Reveals Diverse Temporal Responses to Water Availability in Setaria. Mol Plant 8(10): 1520-1535. Diaz, C., Saliba-Colombani, V., Loudet, O., Belluomo, P., Moreau, L., Daniel-Vedele, F., Morot-Gaudry, J. F. and Masclaux-Daubresse, C. (2006). Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana. Plant Cell Physiol 47(1): 74-83. Ginzburg, D.N., Bossi, F., and Rhee, S.Y. (2022). Uncoupling differential water usage from drought resistance in a dwarf Arabidopsis mutant. Plant Physiol 190(4): 2115-2121. Granier, C., Aguirrezabal, L., Chenu, K., Cookson, S. J., Dauzat, M., Hamard, P., Thioux, J. J., Rolland, G., Bouchier-Combaud, S., Lebaudy, A., et al. (2006). PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit. New Phytol 169(3): 623-635. Kim, S. L., Kim, N., Lee, H., Lee, E., Cheon, K.-S., Kim, M., Baek, J., Choi, I., Ji, H., Yoon, I. S., et al. (2020). High-throughput phenotyping platform for analyzing drought tolerance in rice. Planta 252(3): 38. Lawlor, D. W. (2013). Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. J Exp Bot 64(1): 83-108. Miralles-Crespo, J., and van Iersel, M.W. (2011). A Calibrated Time Domain Transmissometry Soil Moisture Sensor Can Be Used for Precise Automated Irrigation of Container-grown Plants.HortScience 46: 889-894. Mutka, A. M. and Bart, R. S. (2014). Image-based phenotyping of plant disease symptoms. Front Plant Sci 5: 734. Sarkar, S., Ramsey, A. F., Cazenave, A. B. and Balota, M. (2021). Peanut Leaf Wilting Estimation From RGB Color Indices and Logistic Models. Front Plant Sci 12: 658621. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675. Shakoor, N., Lee, S. and Mockler, T. C. (2017). High throughput phenotyping to accelerate crop breeding and monitoring of diseases in the field. Curr Opin Plant Biol 38: 184-192. Tovar, J. C., Hoyer, J. S., Lin, A., Tielking, A., Callen, S. T., Elizabeth Castillo, S., Miller, M., Tessman, M., Fahlgren, N., Carrington, J. C., et al. (2018). Raspberry Pi-powered imaging for plant phenotyping. Appl Plant Sci 6(3): e1031. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant physiology > Abiotic stress Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique Federico Berdun [...] Eduardo Zabaleta Mar 5, 2024 658 Views Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione Lou Lambert and Antoine Danon Aug 5, 2024 412 Views A Step-by-step Protocol for Crossing and Marker-Assisted Breeding of Asian and African Rice Varieties Yugander Arra [...] Wolf B. Frommer Sep 20, 2024 428 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,594	https://bio-protocol.org/en/bpdetail?id=4594&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Methods to Detect AUTOphagy-Targeting Chimera (AUTOTAC)-mediated Targeted Protein Degradation in Tauopathies ML Min Ju Lee * SK Su Bin Kim * HK Hee Yeon Kim * SL Su Jin Lee * JL Ji Su Lee YK Yong Tae Kwon CJ Chang Hoon Ji (contributed equally to this work) Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4594 Views: 1303 Reviewed by: Gal HaimovichHyeong-Reh Choi KimNingfei An Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Communications Feb 2022 Abstract Targeted protein degradation (TPD) facilitates the selective elimination of unwanted and pathological cellular cargoes via the proteasome or the lysosome, ranging from proteins to organelles and pathogens, both within and outside the cell. Currently, there are several in vitro and in vivo protocols that assess the degradative potency of a given degrader towards a myriad of targets, most notably soluble, monomeric oncoproteins. However, there is a clear deficiency of methodologies to assess the degradative potency of heterobifunctional chimeric degraders, especially those in the autophagy space, against pathological, mutant tau species, such as detergent-insoluble oligomers and high-molecular aggregates. The protocol below describes both in vitro and in vivo biochemical assays to induce tau aggregation, as well as to qualitatively and quantitatively measure the degradative potency of a given degrader towards said aggregates, with specific applications of the AUTOTAC (AUTOphagy-TArgeting Chimera) platform provided as an example. A well-defined set of methodologies to assess TPD-mediated degradation of pathological tau species will help expand the scope of the TPD technology to neurodegeneration and other proteinopathies, in both the lab and the clinic. Graphical abstract Overview of assays observing elimination of tauP301L aggregates with AUTOTAC. (A) Description of the biological working mechanism of heterobifunctional chimeric AUTOTAC degraders. (B) Schematic illustration of assays described in this paper. Keywords: TPD AUTOTAC Detergent-insoluble/soluble fractionation in vivo oligomerization Lysosomal acidification hTauP301L-BiFC Background Targeted protein degradation (TPD) is a global protein editing tool with the potential to modulate the half-life of any proteinaceous cargoes (Chamberlain and Hamann, 2019). In addition to serving as a research tool for protein degradation and function, TPD offers an alternative—and thus attractive—means to eradicate disease-causing agents, especially those entrenched in the previously-considered undruggable proteome (Moon and Lee, 2018; Fisher and Phillips, 2018; Schapira et al., 2019; Ji et al., 2022). The various TPD platforms to date harness either the proteasome or the lysosome to facilitate the targeted elimination of a wide variety of cargoes, both within and outside the cell via distinct mechanisms, exemplified by PROteolysis-Targeting Chimera (PROTAC), Antibody-based proTAC (AbTAC), Lysosome-Targeting Chimera (LYTAC), Autophagy-Targeting Chimera (AUTAC), and AuTophagosome-TEthering Compound (ATTEC), to name just a few (Takalo et al., 2013; Thibaudeau et al., 2018; Takahashi et al., 2019; Li et al., 2019; Banik et al., 2020). Despite the rapid development of TPD platforms with distinct mechanisms of action, the large majority of focus has been centered around a relatively few number of targets, mostly oncoproteins. Even with the emergence of autophagy/lysosome-based degraders, efficient degradation has been limited to only a handful of alternative targets—especially those responsible for neurodegeneration and proteopathies in general. Consequently, development of novel autophagy-based degraders that can target a wide range of pathological hallmark proteins of neurodegeneration and proteopathies, most notably their high-molecular weight, oligomeric, aggregated, and amyloidogenic species, still remains a pressing issue. A notable obstacle to such development is the relative lack of in vitro and in vivo methodologies to analyze in depth the degradative potency and mechanism of action of such degraders. Such difficulty is exacerbated by the fact that, unlike oncoproteins, the downstream signaling cascades of hallmark proteins of neurodegeneration/proteopathies still remain murky, which accentuates even further the importance of well-defined, precise assays and models to assess the autophagic mechanism of action and potency of an autophagy-based degrader (Peeraer et al., 2015; Lim et al., 2018). To that end, we have recently reported the development and proof-of-concept characterization of a novel p62-targeting/activating TPD platform called AUTOphagy-TArgeting Chimera (Ji et al., 2022). AUTOTAC degraders can selectively recognize and target a broad range of cellular proteins to autophagic membranes for lysosomal degradation, through the selective interaction and activation of the archetypal autophagy cargo receptor p62/SQSTM1 via its ZZ domain. This interaction induces the conformational activation of inactive p62 into an autophagy-compatible form, which exposes PB1 and LIR domains, and facilitates p62 self-oligomerization in complex with targets and its interaction with LC3 on autophagic membranes (Cha-Molstad et al., 2015; Cha-Molstad et al., 2017; Ji et al., 2017, 2019, 2020; Zhang et al., 2018; Yoo et al., 2018; Heo et al., 2021, 2022). AUTOTAC technology was shown to efficiently degrade both oncoproteins and misfolded, aggregation-prone proteins of neurodegeneration. In this paper, adopted from our paper describing the proof-of-concept development of the AUTOTAC platform, we describe both in vitro methods and in vivo models to not only induce pathological tau aggregation but also assess the selective degradative potency of a given TPD platform towards said aggregates and fibrils (Ji et al., 2022). To this end, we employed SH-SY5Y-hTauP301L cell lines and murine model that can utilize various fluorescent reporters, including full or split halves of the GFP protein, as well as a tandem mRFP-GFP fusion (Shin et al., 2020). Using these models, we induced the formation of detergent-insoluble, high-molecular weight aggregates of the mutant recombinant tau both in vitro and in vivo. The AUTOTAC TPD platform was shown to successfully degrade these pathological tau species in a lysosome-dependent manner. Overall, the protocol provides a systematic approach to generating pathological tau for the testing of not only AUTOTACs but also other autophagy-based degraders and small-molecule compounds in general, which may be expanded to other hallmark proteins of misfolding and aggregation-prone proteopathies and neurodegeneration. Materials and Reagents Part I. In vitro degradation of tauP301L aggregates 5 mL pipette aid (SPL, catalog number: 91010) 10 mL pipette aid (SPL, catalog number: 91005) 1.5 mL e-tube (Axygen, catalog number: MCT-150-C) 100 mm dish (Nest, catalog number: 704002, SPL, catalog number: 30100) 6-well plate (Nest, catalog number: 703003, SPL, catalog number: 30006) 12-well plate (Nest, catalog number: 712003, SPL, catalog number: 30012) 24-well plate (Nest, catalog number: 702002, SPL, catalog number: 30024) Kim-wipe or paper towel (Yuhan-Kimberly, catalog number: 41112) Polyvinylidene difluoride membrane, 0.45 μm (Millipore, catalog number: IPVH00010) X-ray film (AGFA, catalog number: CP-BU) Microscope slide (Marienfield, catalog number: 1000612) Cover glass (Marienfield, catalog number: 0117520) PBS (Biosesang, catalog number: P2007-1) DMEM (Life Technologies, Gibco®, catalog number: 11995-065) RPMI (Life Technologies, Gibco®, catalog number: 22400-089) Opti-mem (Life Technologies, Gibco®, catalog number: 31984-070) 0.05% trypsin (Life Technologies, Gibco®, catalog number: 25300) Lipofectamine 2000 (Invitrogen, catalog number: 11668019) Sodium dodecyl sulfate (SDS) (Duchefa Biochemie, catalog number: S1377.1000) Triple-distilled water 1 M Tris-HCl, pH 6.8 (Biosesang, catalog number: TR2016-050-68) 1.5 M Tris-HCl, pH 8.8 (Biosesang, catalog number: TR4020-050-88) APS (Ammonium Persulfate) (Bio-Rad, catalog number: 1610700) TEMED (Bio-Rad, catalog number: 161-0801) 2-Propanol (Sigma-Aldrich, catalog number: L53191334) 5× Laemmli sample buffer (Elpis Biotech, catalog number: EBA-1052) Skim milk (Georgiachem, catalog number: SM2010) SuperSignalTM West PICO PLUS (Thermo Fisher Scientific, catalog number: 34578) PFF (Recombinant Human Tau P301L pre-formed fibrils) (NOVUS, catalog number: NBP2-76794) Hydroxychloroquine (HCQ) (Sigma, catalog number: A25547) Okadaic acid (Enzo, catalog number: ALX-350-003-C100) HEPES (Duchefa Biochemie, catalog number: H1504.011) PierceTM protein transfection reagent (PJ) (Thermo Fisher Scientific, catalog number: 89850) NaCl (Duchefa Biochemie, catalog number: S0520.5000) RIPA (Cell nest, catalog number: CNR001-0100) PierceTM BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23225) 4× LDS sample buffer non-reducing (Thermo Fisher Scientific, 84788) PLL solution (Sigma-Aldrich, catalog number: P4707) Albumin (Biosesang, catalog number: AC1025-100-00) Foil (SAM JIN FOIL, 30 cm × 30 m) Triton X-100 (Sigma-Aldrich, catalog number: T9284) Antifade-mounting media with DAPI (Vectashield, catalog number: H-1500) Fractionation buffer (see Recipes) SDS-detergent lysis buffer (see Recipes) SDS-PAGE gel (see Recipes) Part II. In vivo elimination of tauP301L neurofibrillary tangles Injection syringes (Becton Dickinson Medical, catalog number: 1258696) Dissection stainless steel tray (any brand) Pins or tape (any brand) Butterfly needles (Biosigma, catalog number: BSS250) 25- or 27-gauge infusion needle (KOREAVACCINE, catalog number: K201) 15 mL conical tube (Tarsons, catalog number: 546021) 1.5 mL e-tube (Axygen, catalog number: MCT-150-C) 24-well plate (Nest, catalog number: 702002, SPL, catalog number: 30024) Kim-wipe or paper tower (Yuhan-Kimberly, catalog number: 41112) Foil (SAM JIN FOIL, 30 cm × 30 m) Microscope slide (Marienfield, catalog number: 1000612) PBS (Biosesang, catalog number: P2007-1) Avertin (Sigma-Aldrich, catalog number: T48402) 0.9% saline (Sigma-Aldrich, catalog number: 08059) RIPA (Cell nest, catalog number: CNR001-0100) Protease inhibitor (Abbkine, catalog number: BMP1001) Phosphatase inhibitor (Sigma-Aldrich, catalog number: P0001) Sucrose (Sigma-Aldrich, catalog number: S0389) Sodium dodecyl sulfate (SDS) (Duchefa Biochemie, catalog number: S1377.1000) Triple-distilled water (DIW) 4% paraformaldehyde (Biosesang, catalog number: pc2031-100-00) Optional cutting temperature (OCT) compound (Sakura Tissue-Tek, catalog number: 4583) Sodium azide (Thermo Fisher Scientific, catalog number: J21610-22) Sudan black B (Sigma-Aldrich, catalog number: 199664) Hoechst (Abcam, ab228550) EtOH (DUKSAN, catalog number: 64-17-5) 0.01% PBST (see Recipes) Blocking solution (see Recipes) Injection drug (see Recipes) Primary antibodies Rabbit polyclonal anti-LC3 (Sigma-Aldrich, catalog number: L7543, 1:10,000 diluted in PBST solution) Rabbit polyclonal anti-GAPDH (Bio World, catalog number: AP0063, 1:10,000 diluted in PBST solution) Mouse monoclonal anti-Tau5 (Invitrogen, catalog number: AHB0042, 1:5,000 diluted in PBST solution) Rabbit polyclonal anti-p-Tau (Invitrogen, catalog number: 44–752G, 1:5,000 diluted in PBST solution) AT8 antibody (Abcam, catalog number: ab210703, 1:200 diluted in the blocking solution) Secondary antibodies Anti-rabbit IgG-HRP (Cell Signaling, catalog number: 7074, 1:10,000 diluted in PBST solution) Anti-mouse IgG-HRP (Cell Signaling, catalog number: 7076, 1:10,000 diluted in PBST solution) Cell HeLa (ATCC, catalog number: CCL-2): the first immortal human cells to be grown in culture and the basis for countless scientific discoveries SH-SY5Y-tauP301L-GFP (Innoprot): SH-SY5Y stable cells tagged with green fluorescence and expressing tau mutant [0N4R(P301L mutant)] Mouse TauP301L-BiFC transgenic mouse (Shin et al., 2020): the mouse model that has two non-fluorescent compartments (hTauP301L-VN173 and hTauP301L-VC155) turning on Venus fluorescence protein upon tau assembly when they are fused with TauP301L. Plasmid mRFP-GFP-hTauP301L plasmid [mRFP-GFP-LC3 plasmid (Cha-Molstad et al., 2016; Dalby et al., 2004) is modified with hTauP301L] Equipment Part I. In vitro degradation of tauP301L aggregates Tabletop centrifuge (Eppendorf, model/catalog number: 5424R) Sonicator (SONICS vibra cel) Centrifuge (Hanil fieta 4) 37°C incubator (Hera cell vios 250i CO2 incubator) Laser scanning confocal microscope (Zeiss 510 Meta) Rocker, CR300 (Finepcr) Part II. In vivo elimination of tauP301L neurofibrillary tangles Dissecting forceps (JEUNG DO BIO&PLANT CO., catalog number: SD-S-04PK) Tissue scissors (JEUNG DO BIO&PLANT CO., catalog number: S-49-16-SPK, S-54-10-SPK) Vascular clamp (Surtex Instruments) Micro spatula (Merck, catalog number: Z243213) Peristaltic pump (Ismatec., catalog number: ISM834) 7 mL Dounce tissue grinder (Wheaton, catalog number: 35742) Tissue grinder motor (Bel-Art, KA., catalog number: UB32-50) Polypropylene pestle (Bel-Art, BA., catalog number: 19923-0001) Rotor (FINEPCR, AG) Tabletop centrifuge (Eppendorf, catalog number: 5424R) -80°C deep freezer (Thermo Fisher Scientific, catalog number: TDE) Cryostat (Leica, catalog number: CM3050S) Cryomolds (Sakura Tissue-Tek, catalog number: 25608-922) Paintbrush (AnB, catalog number: P000EACE) ZEISS Axio Scan.Z1 (Carl Zeiss, Germany) Software LMS image software (Zeiss LSM Image Browser (ver. 4.2.0.121)) ImageJ (NIH, Bethesda) Prism 6 software (GraphPad) Procedure Part I. In vitro degradation of tauP301L aggregates Triton X-100-based insoluble/soluble fractionation assay Plate SH-SY5Y-TauP301L-GFP cells at 1.2 × 106 cells/mL/well in a 6-well plate. After 24 h, treat cells with DMSO or a combination of hydroxychloroquine (HCQ, 10 µM), okadaic acid (15 nM), or AUTOTAC degrader (100 nM) all together, at 37°C for 24 h. Aspirate DMEM culture media, wash cells with 1 mL of PBS, and collect cells using 0.05% trypsin. Collect trypsinized cells in a 1.5 mL e-tube. Centrifuge at 16,000 × g for 2 min to pellet the cells. Re-suspend the cell pellet in 77 µL of fractionation buffer containing 0.5% Triton X-100 and incubate on ice for 30 min. Note: Vortex cells every 15 min. Centrifuge at 16,000 × g for 10 min at 4°C. Measure the concentration of proteins in the supernatant with a BCA. Transfer 70 µL of the supernatant as the soluble fraction to a new e-tube, then boil with 30 µL of 5× Laemmli sample buffer at 100°C for 10 min. Wash the pellet from Step A6 with PBS and centrifuge at 16,000 × g and 4°C. Repeat four times to obtain the insoluble fraction pellet. Lyse the pellet from Step A6 with 70 µL of SDS-detergent lysis buffer and add 30 µL of 5× Laemmli sample buffer. Boil at 100°C for 10 min and load on an SDS-PAGE gel. Transfer onto polyvinylidene difluoride membrane at 100 V and 4°C for 2 h. Block the membrane with 5% skim milk in PBS solution at room temperature (RT) for 30 min. Incubate the membrane with indicated primary antibodies (anti-p-Tau, anti-Tau, anti-LC3, and anti-GAPDH) diluted in PBST solution on a rocker at 4°C overnight. Note: Information for primary antibodies is listed in Materials and Reagents. Wash the membrane with 1× PBST for 10 min three times. Incubate the membrane with host-specific HRP-conjugated secondary antibodies diluted in PBST solution on a rocker at 4°C for 1 h. Note: Information for secondary antibodies is listed in Materials and Reagents. Repeat Step A15. Detect the membrane with ECL solution using X-ray film (Figure 1). Calculate autophagic flux (A.F.) indices based on the ratio of Tau and phospho-Tau (p-Tau) levels in the presence or absence of hydroxychloroquine (HCQ), and assess the changes in the indices upon AUTOTAC treatment. Figure 1. Triton X-100-fractionation assay. (A) SH-SY5Y-TauP301L-GFP cells were treated with the PBA-1105 AUTOTAC compound (100 nM, 24 h), okadaic acid (phosphatase inhibitor and tau hyperphosphorylation/aggregation inducer; 15 nM, 24 h), and/or HCQ (lysosome inhibitor; 10 µM, 24 h), and analyzed for immunoblotting (total tau was subjected to long or short exposure). Autophagic degradation of p-Tau/Tau is accelerated upon treatment with PBA-1105. (B) Normalized A.F. (autophagy flux) index indicates the relative ratio of the differences in the protein level between the absence and presence of HCQ. Oligomerization assay for detection and degradation of Tau aggregates Seed SH-SY5Y-tauP301L-GFP cells at 1.2 × 106 cells/mL/well in 6-well plates. Prepare PFF (Pre-formed Fibrill) solution at a concentration of 15 µL/well of 133.33 mM PFF suspended in a total of 200 µL of PFF buffer. Note: As an example, add 60 µL PFF to 140 µL of PFF buffer for a total of four wells. Tap samples before sonication to facilitate pulsing. Sonicate the samples with 10 pulses (1 pulse: 1 s on and 1 s off at 20% amplitude). Repeat three times, tapping samples after each pulse. Add the sonicated PFF solution to a protein transfection reagent (PJ) vial. Note: One PJ vial could be used for up to two 6-well plates. Incubate at RT for 5 min. Add the PJ and the PFF mixture to 2 mL of serum-free RPMI media, mix well, and incubate with the cells. After 4 h, add 200 µL of FBS to the media (final concentration: 10%) and incubate for an additional 20 h. Alternatively, tau hyperphosphorylation and aggregation can be induced by treatment with the phosphatase inhibitor okadaic acid (15 nM, 24 h). Add the AUTOTAC degrader (100 nM) to the media after 24 h of incubation of PFF or okadaic acid. Using 0.05% trypsin, harvest cells by centrifugation at 16,000 × g for 2 min. Divide the mixture at a ratio of 1:9, re-suspend the smaller fraction with 50–100 µL of RIPA buffer, and incubate on ice for 30 min. Centrifuge the smaller fraction suspension at 16,000 × g for 20 min, to obtain the supernatant. Measure the concentration of proteins in the supernatants using the BCA. Add 4× non-reducing LDS buffer to the larger fraction samples from Step B10, and heat at 95°C for 10 min. Load 15–20 µg of larger fraction samples, according to the relative protein concentrations obtained in Step B12, and resolve the samples using a 4%–20% gradient SDS-PAGE. Note: Do not remove stacking gels when samples are transferred to membranes. Immunoblot the samples using Tau5 (Total tau) or p-Tau (S396) antibodies (Figure 2). Figure 2. Detection of Tau aggregates with in vitro oligomerization assay. (A)Western blot of total tau monomer and aggregates in SH-SY5Y-tauP301L-GFP cells treated with okadaic acid (15 nM, 24 h) and the chemical chaperone-based PBA-1105 AUTOTAC compound (based on the FDA-approved 4-phenylbutyric acid; 100 nM, 24 h). (B) Western blot of pre-formed fibrils induced aggregates of p-Tau in SH-SY5Y-tauP301L-GFP cells treated with various concentrations of the oligomeric modulator-based ATC102 AUTOTAC compound (based on a protein oligomer/aggregate-specific warhead). AUTOTAC treatment via PBA-1105 or ATC-102 preferentially targets the aggregated, high-molecular weight species of mutant Tau. Lysosomal digestion assay of Tau aggregates Seed HeLa cells at 5 × 104 cells/mL on coverslips coated with 10% poly-L-lysine (PLL), each placed in a 24-well plate. Note: Insert coverslips in each well of the plate, then incubate with 10% PLL in autoclaved triple-distilled water (DIW) for 30 min. Wash coverslips three times with DIW. Transfect the mRFP-GFP-hTauP301L plasmid in HeLa cells with Lipo2000. Note: Incubate 0.5 µg/well of plasmid with 250 µL/well of opti-MEM, and 0.75 µL/well of Lipo2000 with 250 µL/well of opti-MEM for 5 min. Mix solutions and incubate for 15 min. Add the resulting mixture to cells (Fisher et al., 2018). After 24 h of transfection, incubate cells in the presence or absence of okadaic acid (15 nM) and/or the AUTOTAC degrader (100 nM) for 24 h. Remove the culture medium by aspiration and wash the cells with 1× PBS twice. A volume of 500 µL/well of 1× PBS is recommended for a 24-well plate. Note: Wrap the 24-well plate with aluminum foil and carry out the next steps in the dark whenever possible. Fix cells with 4% paraformaldehyde (PFA) at RT for 15 min, followed by washing the coverslips three times on a rocking incubator with 1× PBS for 5 min per wash. Permeabilize cells using 0.5% Triton X-100 solution at RT for 15 min, followed by washing identical to Step C5. Mount coverslips with the antifade-mounting medium on the glass slide, and store at RT for 6 h for the medium to dry. Note: Avoid trapping air bubbles in the medium between the coverslip and the glass slide; if bubbles do form, press the coverslip only very slightly to remove them. Obtain confocal images with a laser scanning confocal microscope. A magnification of 60× or greater is required to accurately observe intracellular puncta structures. Repeat Step C8 as many times as required within three independent experiments, to count a minimum of 150 cells for statistical analysis. To analyze lysosomal targeting of tau aggregates, analyze their acidification by counting the number of yellow (mRFP-EGFP) and red (mRFP-quenched EGFP) puncta structures, thus obtaining a GFP/RFP ratio (Kimura et al., 2007; Lopez et al., 2018). Note: Since GFP fluorescence is quenched in acidic pH, a decline in the GFP/RFP ratio means digestion of tau aggregates in autolysosomes (Figures 3, 4). Figure 3. Measuring GFP/RFP ratio. (A)Schematic illustration of the design of mRFP-GFP-hTauP301L plasmid. (B) Description of measuring GFP/RFP ratio based on counting the puncta that are yellow (GFP and RFP) or red (RFP only). When autophagy is activated, the number of RFP only puncta is increased by quenching of GFP, which means lysosomal digestion. Figure 4. Examples of Tau aggregates lysosomal digestion assay. (A)Confocal image of mRFP-GFP-hTauP301L in HeLa cells, which are treated with okadaic acid (15 nM, 24 h) prior to the presence or absence of PBA-1105 (100 nM, 24 h). Scale bar, 10 µm. (B) Quantification of A for punctate formation (blue bars) and GFP/RFP ratio (red bars). Part II. In vivo elimination of tauP301L neurofibrillary tangles Intraperitoneal injection of AUTOTAC into the hTauP301L-BiFC tauopathy mouse model To evaluate the effect of AUTOTAC degraders on the degradation of tau aggregates in vivo, inject PBA-1105 (20 or 50 mg/kg) dissolved in a 5% DMSO/10% solutol/85% PBS mixture, or PBS containing 30% polyethylene glycol (PEG) into the peritoneum of the hTauP301L-BiFC mouse (Shin et al., 2020), three times a week for four weeks (Figure 5). Figure 5. Injection timeline and details of PBA-1105 in hTauP301L-BiFC murine model Brain tissue preparation Anesthetize mice with an intraperitoneal injection of 2% avertin (2,2,2-Tribromoethanol), confirming full anesthesia via the paw pinch reflex. Place mice on a dissection tray and fix each paw with pins or tapes. With forceps and scissors, open the skin and chest cavity, to expose the heart. While holding the heart with forceps, insert the needle into the tip of the left ventricle. Then, cut the right atrium with scissors, and start the peristaltic pump (speed should be similar to the heart rate). Perfuse with 0.9% saline, until the circulating fluid is clear and the liver turns white. After perfusion, decapitate the mouse with scissors. Cut the skin along the midline between the eyes of the mouse, and expose the skull. Keep the end of the scissors on the foramen magnum and carefully cut off the midline of the skull towards the nose, to expose the brain. Remove the skull covering the brain with forceps. Gently transfer the brain to a 15 mL conical tube containing 1× PBS using a micro spatula. RIPA-insoluble/soluble fractionation assay Preparation of brain lysate (Figure 6) Note: Perform all steps on ice. Place a homogenizer on ice to cool. Meanwhile, prepare the RIPA buffer mixture. Place perfused mouse hemispheres in each 1.5 mL Eppendorf tube containing 500 µL of the RIPA buffer mixture. Cut the brain tissue into small pieces with scissors. Homogenize the samples with a tissue homogenizer, adding an additional 500 µL of the RIPA buffer mixture. Incubate the samples on a rotor at 4°C for 2 h. Centrifuge the samples at 14,000 × g and 4°C for 15 min. Collect the supernatant (soluble fraction) in new 1.5 mL Eppendorf tubes and store in a -80°C deep freezer until use. Wash the pellet from Step C1f with the washing solution to completely remove the soluble fractions. Centrifuge at 14,000 × g and 4°C for 5 min. Repeat Steps C1h–i three times, but centrifuge at 14,000 × g and 4°C for 20 min for the final step. Discard the supernatant. Re-suspend pellet with 1 mL of the 2% SDS in triple-distilled water, containing 1% protease inhibitor and phosphatase inhibitor. Incubate the samples at RT for 20 min. Centrifuge the samples at 14,000 × g and 4°C for 10 min. Collect the supernatant in a new 1.5 mL Eppendorf tube, to obtain the insoluble fraction. Figure 6. Workflow for RIPA-insoluble/soluble fractionation assay Measure the concentration of proteins in the supernatant of Step C1g using the BCA and dilute proteins with RIPA buffer up to 1 µg/µL. Transfer 70 µg of the soluble fraction supernatant to a new Eppendorf tube, add 30 µL of 5× Laemmli sample buffer, and boil at 100°C for 10 min. Lyse the pellet from Step C1n with 70 µL of SDS-detergent lysis buffer and add 30 µL of 5× Laemmli sample buffer. Boil at 100°C for 10 min and load on an SDS-PAGE gel. Transfer onto a polyvinylidene difluoride membrane at 100 V and 4°C for 2 h. Block the membrane with 5% skim milk in PBS solution at RT for 30 min. Incubate the membrane with indicated primary antibodies (anti-Tau5 and anti-p-Tau) diluted in PBST solution on a rocker at 4°C overnight. Note: Information for primary antibodies is listed in Materials and Reagents. Wash the membrane with 1× PBST for 10 min three times. Incubate the membrane with host-specific HRP-conjugated secondary antibodies diluted in PBST solution on a rocker at 4°C for 1 h. Note: Information for secondary antibodies is listed in Materials and Reagents. Repeat Step C9. Detect the membrane with ECL solution using X-ray film (Figure 7). Figure 7. RIPA-insoluble/soluble fractionation assay. (A), (B) Fractionation assay in brain tissue of hTauP301L-BiFC mice intraperitoneally injected with PBA-1105 (20 or 50 mg/kg). (C), (D) Normalized densitometry of (A) and (B) for hTau levels, respectively (n = 5 mice). PBA-1105 AUTOTAC treatment selectively eliminates mutant human tau (hTau) in the RIPA-insoluble fraction, as opposed to murine tau (mTau) or RIPA-soluble human tau. Immunohistochemistry of hTauP301L-BiFC murine brain tissues Mouse brain fixation Rapidly fix the mouse hemisphere perfused with PBS containing 4% paraformaldehyde (PFA) at 4°C for 48 h. Transfer the brains to 30% sucrose in PBS and confirm full immersion for cryoprotection. Embedding in OCT compound Before embedding, place cryomolds in the active cryostat with the temperature set to -20°C. After cooling, label the samples on the surface of the cryomold with a permanent marker. Remove excess liquid surrounding the brains by absorption with a Kim-wipe or paper towel, prior to freezing. Note: Excess liquid will form ice crystals on the surface of the brains and prevent the samples from properly adhering to the frozen OCT compound during embedding, interfering with sectioning. Place a few drops of OCT onto the center of the bottom of the cryomold. Be careful to select the proper size embedding mold according to the size of the brains to be embedded. Place the brain sample on several drops of the OCT compound and adjust its position. Gently push the brain with forceps to ensure that the bottom surface of the brain is placed properly, touching the face of the bottom, and the brain is located in the center of the mold. Carefully add more OCT compound drop by drop onto the sample, until completely immersed. Note: Avoid the formation of air bubbles by removing them using forceps. Place the OCT-covered sample within the cryomold in a cryostat, to harden the block. Wrap the embedded block in foil and store in an -80°C deep freezer for storage. Sectioning Before sectioning, place paintbrushes, forceps, and cryostat block in the equipment for 20 min, to equilibrate to the chamber temperature of -20°C. Prepare a 24-well plate and add 1× PBS to all wells. Attach the sample to a circular cryostat block with a few drops of the OCT compound and equilibrate for approximately 10 min. Cut sections 50–100 µm thick to trim the OCT compound surrounding the brain until the brain is visible and then set the dial to cut sections 30 µm thick. Note: Ensure sections are horizontally sliced as equally as possible. Cut serially in 30 µm thickness coronal sections. Use a paintbrush to gently collect the sections. Create series of sections representing the brain by distributing similar sections on subsequent wells (Figure 8). Note: Up to five sections can be added to each well. Note: For storage, transfer sections to 1× PBS solution containing 0.05% sodium azide at 4°C. Figure 8. The manner of collecting brain sections. Each series includes five sections. As an example, sections 1, 7, 13, 19, and 25 are in Series #1. Series #2 includes sections 2, 8, 14, 20, and 26. Sections 3, 9, 15, 21, and 27 comprise series #3. Immunostaining: Sudan black B staining & AT8 antibody staining Note: Sudan black B staining reduces autofluorescence signals and improves the resolution of in situ hybridization-specific fluorescent signals in brain sections. Stir 0.05% Sudan black B in 70% EtOH in the dark at RT for 2 h and let stand overnight (O/N). Alternatively, stir overnight and then filter with a 0.2 µm filter. This solution can be stored at 4°C for a week. Remove the 1× PBS used in Step D3b by carefully aspirating it with a pipette tip, without touching the sections. Wash with triple-distilled water for 5 min; repeat twice. Immerse in the 0.05% Sudan black B solution for 10 min. Wash with 0.1% PBST for 30 s; repeat three times. Wash with triple-distilled water for 5 min. Incubate the sections with 0.5 µg/mL Hoechst in triple-distilled water for 30 min. Wash with triple-distilled water for 5 min; repeat twice. Incubate the sections in blocking solution at 4°C O/N, by adding 1 mL to each well. The following day, remove the blocking solution carefully, add 500 µL of anti-AT8 diluted 1:200 with the blocking solution, and incubate at 4°C O/N. The next day, wash the sections with 1× PBS on a shaker at RT for 10 min each three times. Prepare 500 µL of secondary antibody working solution by diluting 1:500 with blocking solution and incubate the sections on a shaker at 4°C O/N. Wash the sections with 1× PBS, as described in Step D4k. Carefully mount the sections on slide glasses using a paintbrush and dry at 4°C with a damp tissue. On the following day, dry them at RT, and then in the oven for 30 min. Note: When the sections move by a paintbrush, you have to be careful not to touch the sections, especially the targeting area. BiFC fluorescence and AT8-stained signals of samples represent total and phosphorylated hTauP301L aggregates and neurofibrillary tangles, respectively. Image acquisition and analysis (Figure 9) Capture a region of interest using an automated fluorescence microscope system (ZEISS Axio Scan.Z1); λex = 460–490 nm and λex = 500–550 nm. Observe the total (BiFC signal) and phosphorylated (AT8 signal) tau within the hippocampus and cortex regions, specifically focusing on intracellular neurofibrillary tangles or aggregates. For the quantitative analysis, fluorescence and total area of each puncta were measured using ImageJ. The significance of the data (p-value) was determined using two-tailed, unpaired Student’s t-test (degree of freedom = n-1) with Prism 6 software. Figure 9. Immunohistochemistry of BiFC fluorescence. (A), (B) Immunohistochemistry of BiFC fluorescence for total hTau levels or AT8 fluorescence for total phosphorylated levels in hTauP301L-BiFC mice injected with PBA-1105, as outlined in Fig. 6. Scale bar, 100 μm. (C), (D) Quantification of BiFC or AT8 punctate fluorescence signals in (A) and (B), respectively (n = 7 mice). Data analysis For all data shown, stated values represent the mean ± S.D or S.E.M. of at least three independent experiments (unless otherwise stated). For each experiment, sample size (n) was determined as stated in the figure legends. For all experiments, p-values were determined using two-tailed, unpaired student’s test (degree of freedom = n-1) with Prism 6 software (GraphPad). Detailed information is provided in the original, open-access publication (https://doi.org/10.1038/s41467-022-28520-4). Recipes SDS-detergent lysis buffer 20 mM HEPES pH 7.9 0.2 M KCl 1 mM MgCl2 1 mM EGTA 1% Triton X-100 1% SDS 10% glycerol Protease inhibitors Phosphatase inhibitors Fractionation buffer 100 mM NaCl 0.5 mM EDTA 20 mM Tris HCl pH 8 0.5% Triton X-100 SDS-PAGE Gel 30% acrylamide 1.5 M Tris-HCl pH 8.8 1 M Tris-HCl pH 6.8 10% SDS 10% APS TEMED 2-Propanol PFF buffer 10 mM HEPES 100 mM NaCl (1 M stock) Distilled water Injection drug PBA-1105 (20 or 50 mg/kg) 5% DMSO/10% solutol/85% PBS mixture 30% polyethylene glycol (PEG) 1× PBS RIPA buffer mixture RIPA 1% protease inhibitor 1% phosphatase inhibitor Washing solution RIPA 1 M sucrose 1% protease inhibitor 1% phosphatase inhibitor 0.01% PBST 1× PBS 0.01% Tween Blocking solution 1× PBS 0.3% Triton X-100 5% bovine serum albumin (BSA) Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2020R1A5A1019023 and NRF-2021R1A2B5B03002614 to Y.T.K.), the Korea Health Technology R&D Project through the Health Industry Development Institute and Korea Dementia Research Center (KDRC) funded by the Ministry of Science, ICT, and Future Planning (MSIP) (HU21C0201 to C.H.J.), and the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1A6A3A1307128711 to M.J.L. and 2022R1A6A3A13069514 to S.B.K.). This protocol was adapted from our previous work (Ji et al., 2022; https://doi.org/10.1038/s41467-022-28520-4)". Competing interests Ethics Animal protocols followed the principles and practices outlined in the approved guidelines and also received ethical approval by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Science and Technology. References Banik, S. M., Pedram, K., Wisnovsky, S., Ahn, G., Riley, N. M. and Bertozzi, C. R. (2020). Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584(7820): 291-297. Chamberlain, P. P. and Hamann, L. G. (2019). Development of targeted protein degradation therapeutics. Nat Chem Biol 15(10): 937-944. Cha-Molstad, H., Sung, K. S., Hwang, J., Kim, K. A., Yu, J. E., Yoo, Y. D., Jang, J. M., Han, D. H., Molstad, M., Kim, J. G., et al. (2015). Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat Cell Biol 17(7): 917-929. Cha-Molstad, H., Yu, J. E., Feng, Z., Lee, S. H., Kim, J. G., Yang, P., Han, B., Sung, K. W., Yoo, Y. D., Hwang, J., et al. (2017). p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis. Nat Commun 8(1): 102. Dalby, B., Cates, S., Harris, A., Ohki, E. C., Tilkins, M. L., Price, P. J. and Ciccarone, V. C. (2004). Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods 33(2): 95-103. Fisher, S. L. and Phillips, A. J. (2018). Targeted protein degradation and the enzymology of degraders. Curr Opin Chem Biol 44: 47-55. Heo, A. J., Kim, S. B., Ji, C. H., Han, D., Lee, S. J., Lee, S. H., Lee, M. J., Lee, J. S., Ciechanover, A., Kim, B. Y., et al. (2021). The N-terminal cysteine is a dual sensor of oxygen and oxidative stress. Proc Natl Acad Sci U S A 118(50): e2107993118. Heo, A. J., Ji, C. H. and Kwon, Y. T. (2022). The Cys/N-degron pathway in the ubiquitin-proteasome system and autophagy. Trends Cell Biol S0962-8924(22)00175-1. Ji, C. H., Kim, H. Y., Lee, M. J., Heo, A. J., Park, D. Y., Lim, S., Shin, S., Ganipisetti, S., Yang, W. S., Jung, C. A., et al. (2022). The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system. Nat Commun 13(1): 904. Ji, C. H. and Kwon, Y. T. (2017). Crosstalk and Interplay between the Ubiquitin-Proteasome System and Autophagy. Mol Cells 40(7): 441-449. Ji, C. H., Kim, H. Y., Heo, A. J., Lee, S. H., Lee, M. J., Kim, S. B., Srinivasrao, G., Mun, S. R., Cha-Molstad, H., Ciechanover, A., et al. (2019). The N-Degron Pathway Mediates ER-phagy. Mol Cell 75(5): 1058-1072 e1059. Ji, C. H., Kim, H. Y., Heo, A. J., Lee, M. J., Park, D. Y., Kim, D. H., Kim, B. Y. and Kwon, Y. T. (2020). Regulation of reticulophagy by the N-degron pathway. Autophagy 16(2): 373-375. Ji, C. H., Lee, M. J., Kim, H. Y., Heo, A. J., Park, D. Y., Kim, Y. K., Kim, B. Y. and Kwon, Y. T. (2022). Targeted protein degradation via the autophagy-lysosome system: AUTOTAC (AUTOphagy-TArgeting Chimera). Autophagy 18(9): 2259-2262. Kimura, S., Noda, T. and Yoshimori, T. (2007). Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3(5): 452-460. Li, Z., Wang, C., Wang, Z., Zhu, C., Li, J., Sha, T., Ma, L., Gao, C., Yang, Y., Sun, Y., et al. (2019). Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature 575(7781): 203-209. Lim, S., Kim, D., Ju, S., Shin, S., Cho, I. J., Park, S. H., Grailhe, R., Lee, C. and Kim, Y. K. (2018). Glioblastoma-secreted soluble CD44 activates tau pathology in the brain. Exp Mol Med 50(4): 1-11. Lopez, A., Fleming, A. and Rubinsztein, D. (2018). Seeing is believing: methods to monitor vertebrate autophagy in vivo. Open Biology 8: 180106. Moon, S. and Lee, B. H. (2018). Chemically Induced Cellular Proteolysis: An Emerging Therapeutic Strategy for Undruggable Targets. Mol Cells 41(11): 933-942. Peeraer, E., Bottelbergs, A., Van Kolen, K., Stancu, I. C., Vasconcelos, B., Mahieu, M., Duytschaever, H., Ver Donck, L., Torremans, A., Sluydts, E., et al. (2015). Intracerebral injection of preformed synthetic tau fibrils initiates widespread tauopathy and neuronal loss in the brains of tau transgenic mice. Neurobiol Dis 73: 83-95. Schapira, M., Calabrese, M. F., Bullock, A. N. and Crews, C. M. (2019). Targeted protein degradation: expanding the toolbox. Nat Rev Drug Discov 18(12): 949-963. Shin, S., Kim, D., Song, J. Y., Jeong, H., Hyeon, S. J., Kowall, N. W., Ryu, H., Pae, A. N., Lim, S. and Kim, Y. K. (2020). Visualization of soluble tau oligomers in TauP301L-BiFC transgenic mice demonstrates the progression of tauopathy. Prog Neurobiol 187: 101782. Thibaudeau, T. A., Anderson, R. T. and Smith, D. M. (2018). A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers. Nat Commun 9(1): 1097. Takalo, M., Salminen, A., Soininen, H., Hiltunen, M. and Haapasalo, A. (2013). Protein aggregation and degradation mechanisms in neurodegenerative diseases. Am J Neurodegener Dis 2(1): 1-14. Takahashi, D., Moriyama, J., Nakamura, T., Miki, E., Takahashi, E., Sato, A., Akaike, T., Itto-Nakama, K. and Arimoto, H. (2019). AUTACs: Cargo-Specific Degraders Using Selective Autophagy. Mol Cell 76(5): 797-810 e710. Yoo, Y. D., Mun, S. R., Ji, C. H., Sung, K. W., Kang, K. Y., Heo, A. J., Lee, S. H., An, J. Y., Hwang, J., Xie, X.-Q., et al. (2018). N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis. Proc Natl Acad Sci U S A 115(12): E2716-E2724. Zhang, Y., Mun, S. R., Linares, J. F., Ahn, J. W., Towers, C. G., Ji, C. H., Fitzwalter, B. E., Holden, M. R., Mi, W., Shi, X., et al. (2018). ZZ-dependent regulation of p62/SQSTM1 in autophagy. Nat Commun 9(1): 1-11. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Drug Discovery > Drug Screening Neuroscience > Nervous system disorders > Cellular mechanisms Cell Biology > Cell-based analysis > Autophagic activity Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,595	https://bio-protocol.org/en/bpdetail?id=4595&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Preparation and Characterization of DNA-assembled GRS-DNA-CuS Nanodandelions HW Haoze Wang * YC Yanna Cui * YZ Yongming Zhang ZX Zeyu Xiao (*contributed equally to this work) Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4595 Views: 540 Reviewed by: ASWAD KHADILKARValeria B Fernandez Vallone Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Biomaterials Mar 2022 Abstract In this study, we introduce a detailed protocol for the preparation of DNA-assembled GRS-DNA-copper sulfide (CuS) nanodandelion, a multifunctional theranostics nanoparticle. Using transmission electron microscope (TEM) and dynamic light scattering techniques, we characterize the physicochemical property of DNA-assembled GRS-DNA-CuS nanodandelions and their dissociation property after the first near-infrared (NIR) light irradiation. In addition, we systematically monitor the processes of tumor accumulation and uniform intratumoral distribution (UITD) of ultrasmall CuS photothermal agents (PAs), which are dissociated from GRS-DNA-CuS nanodandelions, by Raman imaging and photoacoustic imaging, respectively. The UITD of the dissociated ultrasmall CuS PAs can enhance the therapeutic efficiency of photothermal treatment under the second NIR light irradiation. Overall, this protocol provides a powerful tool to achieve UITD of PAs by explosively breaking the hydrogen bonds of DNA in GRS-DNA-CuS nanodandelions under NIR light irradiation. We expect DNA-assembled nanotheranostics to serve as a robust platform for a variety of biomedical applications related to photothermal therapy in the oncology field. This protocol can increase experimental reproducibility and contribute to efficient theranostics nanomedicine. Keywords: DNA assembly Photothermal therapy Hydrogen bond breakage Raman imaging Nanotheranostics Background Although traditional cancer treatments (e.g., chemotherapy, radiotherapy, and surgery) have been widely applied in clinical cancer therapy, they have many side effects (such as systemic toxicity or incomplete elimination), leading to poor therapeutic efficiency and easy recurrence and metastasis. In recent years, photothermal therapy (PTT) has been broadly investigated because of its advantages in deeper tissue penetration, higher spatial selectivity, and local tumor thermal ablation with less systemic toxicity and higher anti-tumor efficiency (Xiong et al., 2020; Zhou et al., 2021). However, due to the non-uniform tumor distribution of photothermal agents (PAs), PTT cannot kill all tumor cells (Yan et al., 2016). Therefore, it is urgent to develop a novel method to achieve uniform intra-tumor distribution (UITD) of PAs and high photothermal therapeutic efficiency. Several size shrinkage strategies based on cross-linking covalent bonds have been developed to improve UITD of PAs (Li et al., 2016; Zhou et al., 2019; Wang et al., 2020). However, covalent bonds with high bond energy cannot be broken quickly and the synthesis of size-shrinkable nanosystems is complex, which easily results in differences in physicochemical characteristics between different batches. Because of the low hydrogen bond energy in the DNA double helix and the simplicity of DNA self-assembling process, DNA-assembled nanotheranostics may be a promising platform to achieve UITD of PAs. In this protocol, we will introduce a DNA-assembled nanosystem, i.e., DNA-assembled GRS-DNA-CuS nanodandelion (as shown in Figure 1), which is composed of the core of a Gap-enhanced rod-shaped gold structure (termed as GRS) modified by single-stranded DNA (ssDNA), and the shell of ultrasmall copper sulfide (CuS) PAs modified by complementary single-stranded DNA (cssDNA) (Zhang et al., 2022). Based on the principle of Watson–Crick base pairing, GRS-DNA-CuS nanodandelions are prepared by cross-linking hydrogen bonds between GRS-ssDNA and CuS-cssDNA PAs. Under the first near-infrared (NIR) laser irradiation, hydrogen bonds in the GRS-DNA-CuS nanodandelions are explosively broken once above the DNA melting temperature, and the large-sized GRS-DNA-CuS nanodandelions are dissociated into GRS and ultrasmall CuS PAs within one minute (Figure 1). Both the rapid increase in local concentration of ultrasmall CuS PAs and the slight increase in intratumor temperature have effectively promoted UITD of CuS PAs. This UITD helps to eliminate the subcutaneous solid tumors under the second NIR light irradiation. In a word, we will present the detailed preparation process of DNA-assembled GRS-DNA-CuS nanodandelions and characterize their physicochemical and dissociation properties in order to provide a novel UITD-guided PTT strategy and advance their therapeutic and diagnostic applications in clinic. Figure 1. Schematic illustration of the dissociation process of DNA-assembled GRS-DNA-CuS nanodandelions under 808 nm NIR laser irradiation. Left: 3D structure of DNA-assembled GRS-DNA-CuS nanodandelions. GRS: Gap-enhanced rod-shaped gold structure; CuS: copper sulfide. Materials and Reagents 15 mL centrifuge tube (BIOFIL, catalog number: CFT013150-BD) 50 mL centrifuge tube (BIOFIL, catalog number: CFT010500) Gold (III) chloride hydrate (HAuCl4·4H2O) (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10010711) Cetyl trimethyl ammonium bromide (CTAB) (Aladdin, catalog number: H108983-500g) Sodium borohydride (NaBH4) (Aladdin, catalog number: S108355-25g) Sodium oleate (NaOL) (Aladdin, catalog number: S104196-250g) Silver nitrate (AgNO3) (Aladdin, catalog number: S116268) Hydrogen chloride (HCl) (Merck, Sigma-Aldrich, catalog number: 258148) Ascorbic acid (AA) (Aladdin, catalog number: 50-81-7) 4-Nitrobenzenethiol (4-NBT) (Aladdin, catalog number: N298977) Cetanecyl trimethyl ammonium chloride (CTAC) (Aladdin, catalog number: H105309) mPEG5k -SH (JenKem Technology, catalog number: 729108-1G) Tween 20 (Aladdin, catalog number: T104863-100ml) Sodium citrate dihydrate (Aladdin, catalog number: S434901) Cupric chloride (CuCl2) (Aladdin, catalog number: C106774-50g) Single-stranded DNA (ssDNA, sequence: 5′-TAG GTG TAG TGA GGA AAA AAA AAA A-C6-SH-3′, 200 μL, 10 μM) (Sangon Biotech Limited Co. Shanghai, China, personalized customization) Complementary single-stranded DNA (cssDNA, sequence: 5′-TCC TCA CTA CAC CTA AAA AAA AAA A-C6-SH-3′, 380 μL, 10 μM) (Sangon Biotech Limited Co. Shanghai, China, personalized customization) Vernier caliper (Shanghai Shenhan Measures, catalog number: 43528447520) Pipettes (Biosharp, catalog number: BS-XG-0.3L) Capillary cell (MALVERN, catalog number: DTS1070) PBS (Sangon Biotech, catalog number: B640435) Balb/c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China)) Equipment Centrifuge (Thermo Fisher, model: Heraeus Fresco 21) -80°C freezer (Thermo Fisher, model: ORMA 88000 SERIES) Water purification system (Millipore, model: Direct-Q5) Hot plate magnetic stirrer (IKA, model: C-MAG HS7 digital) Electron microscopy (FEI, model: FEI-TEM) Malvern laser particle size analyzer (Malvern 3000, Zetasizer Nano ZS) Inductively coupled plasma–mass spectrometry, ICP-MS (VARIAN, model: 710-ES) Renishaw inVia confocal Raman spectrometer (Renishaw, model: inVia) Ultracentrifuge (Eppendorf, model: Centrifuge 5804R) Ultrasonic cell disruptor system (SONICS, model: S-450D) Rapid mixer (Kylin-Bell, model: Vortex-5) Ultrasonic cleaner (Scientz, model: SB-5200DTS) Thermal imaging camera(FLIR, model: AX5) Ultracentrifuge (Optima, model: XPN-80) Procedure Preparation of DNA-assembled GRS-DNA-CuS nanodandelions Synthesis of gold nano-rods (GRs) Mix 5 mL of HAuCl4·4H2O (0.2 mg/mL) with 5 mL of CTAB (0.2 M). Dilute 20 μL of fresh NaBH4 (0.6 M) in 1 mL of ice-cold water and add it to the mixture under vigorous stirring at 1793 × g for 1 min. Then, the seed solution CTAB-stabilized gold core is obtained. Add 0.9 g of CTAB and 0.15 g of NaOL into 25 mL of double-distilled water (ddH2O) at 50°C under magnetic stirring for 1 h. Note: ddH2O should be warmed to 50°C in advance. Cool the solution from the previous step to 30°C and add 2.4 mL of AgNO3 (4 mM) and 25 mL of HAuCl4·4H2O (0.4 mg/mL). Let them stand at 30°C for 10 min and stir again for 15 min at 747 × g . Note: Add the ice to the water bath in order to cool down the hot water. Real-time monitor the temperature of water bath by thermometer so that the reaction temperature is kept at 30°C. Add 0.24 mL of HCl (37 weight percent) and stir for another 15 min at 747 × g . Then, add 125 μL of AA (0.064 M) and stir for 1 min to obtain the growth solution . Let the obtained growth solution stand for 12 h at 30°C for GR growth. Collect the resulting products by centrifugation at 9,710 × g for 20 min, wash with 40 mL of ddH2O, re-dissolve into 50 mL of ddH2O, and obtain gold nano-rods (GRs). Note: Rotating speed should be 9,710 × g in order to totally collect GRs. Finally, mix 5 mL of GRs solution with 5 mL of CTAC (0.2 M) and centrifuge at 11,203 × g for 10 min. Wash the precipitate with 10 mL of CTAC (0.1 M) three times and re-disperse them in 10 mL of CTAC solution (0.1 M) for completely replacing CTAB. CTAC-capped GRs are obtained. Note: Both the long sonication time and high solution temperature are helpful to completely dissolve CTAC and CTAB in the ddH2O. The better temperature for dissolving them is 45°C. Optimal sonication time is 30 min. In addition, during the displacement of CTAB with CTAC, the samples should be dispersed by sonication before proceeding to the next step, as they easily adsorb on the surface of tube. Prepare Raman molecule–modified GR Mix 10 mL of CTAC-capped GRs with 500 μL of Raman molecule 4-NBT (1 mM) under vigorous ultrasonication in an ice-water bath in an ultrasonic cleaner for 15 min. Note: Raman molecule 4-NBT should be dissolved in DMSO in advance. The concentration of 4-NBT is 1 mM. Add 10 mL of CTAC (0.1 M) to the solution and centrifuge at 8,963 × g for 10 min. Wash the precipitate with 10 mL of CTAC solution (0.05 M) twice and re-disperse the precipitate in 10 mL of CTAC solution (0.1M). The precipitate is Raman molecule-modified GR . Synthesis of GRS by modifying seed-mediated growth method Sequentially add 2.5 mL of HAuCl4·4H2O (2 mg/mL) and 2.5 mL of AA (0.04 M) to 50 mL of CTAC (0.1 M). Add the mixture to 5 mL of Raman molecule-modified GR solution drop by drop under continuous sonication. Let the mixture solution stand at room temperature for 24 h, centrifuge at 4,481 × g for 10 min, and re-disperse them in 50 mL of ddH2O. Then, GRS is obtained. Synthesis of single-stranded DNA (ssDNA)-modified GRS (GRS-ssDNA) Centrifuge the obtained GRS (20 mL) at 7469 × g for 10 min and wash twice with ddH2O. Then, add 200 μL of mPEG5k-SH (10 μM) to the GRS solution. Subsequently, add 20 mL Tween 20 (0.01 weight percent) to PEG-modified GRS solution, sonicate for 30 s, and severely mix by vortex. Repeat this step four times. Note: The processes including adding Tween 20, sonicating, and severely mixing by vortex should be repeated four times in order to completely remove the CTAC from the surface of PEG-modified GRS. Zeta potential of PEG-modified GRS should be changed from positive to negative. Add 200 μL of single-stranded DNA (ssDNA, sequence: 5′-TAG GTG TAG TGA GGA AAA AAA AAA A-C6-SH-3′, 10 μM) and 2 mL of citrate sodium dihydrate (1 M) to the obtained GRS solution, and then mix them for 30 s by vortex. Let the solution stand at room temperature for 2 h. Note: 2.894 g of citrate sodium dihydrate is dissolved in 10 mL of ddH2O to obtain citrate solution (1 M). Centrifuge the ssDNA-modified GRS, wash three times with ddH2O, and re-dissolve in 20 mL of 1× PBS. GRS-ssDNA is obtained. Synthesis of ultrasmall CuS PAs Add 13.45 mg of CuCl2 and 20 mg of citrate sodium dihydrate to 100 mL of ddH2O. Add 8.68 mg of Na2S into the mixture from the previous step under stirring. Five minutes later, heat the reaction solution to 90 and stir for 15 min. Keep the citrate-modified CuS nanoparticles in ice-cold water for 30 min and store at 4 for less than one week. Note: Ultrasmall CuS nanoparticles should be prepared prior to use. The storage time of CuS at 4 should be no more than one week because CuS is easily degraded. Synthesis of the complementary single-stranded DNA (cssDNA)-modified CuS (CuS-cssDNA) Add 380 μL of the complementary single-stranded DNA (cssDNA, 5′-TCC TCA CTA CAC CTA AAA AAA AAA A-C6-SH-3′, 10 μM) to 50 mL of CuS solution and gently shake at room temperature at the incubator shaker at 60 rpm for 12 h. Centrifuge the mixture at 232,580 × g for 30 min and re-disperse in 50 mL of 1× PBS. CuS-cssDNA is obtained. Synthesis of GRS-DNA-CuS nanodandelions Mix 20 mL of GRS-ssDNA with 10 mL of CuS-cssDNA and incubate at 37 for 12 h to generate GRS-DNA-CuS nanodandelions. Collect the GRS-DNA-CuS nanodandelions by centrifugation in 50 mL centrifuge tubes at 8,963 × g for 10 min and re-dissolve in 20 mL of 1× PBS. Add 200 μL of mPEG5k-SH (10 μM) to GRS-DNA-CuS nanodandelions for improvement of their stability. Wash the PEG-modified GRS-DNA-CuS with 20 mL of 1× PBS and re-dissolve in 20 mL of 1× PBS. The whole procedure for preparation of GRS-DNA-CuS nanodandelions is illustrated in Figure 2 . Figure 2. Flow chart of the whole procedure for preparation of GRS-DNA-CuS nanodandelions Characterization of DNA-assembled GRS-DNA-CuS nanodandelions Characterize the morphology Dribble 10 µL of the obtained GRS-DNA-CuS solution onto the formvar-coated grid. Dry the sample at room temperature for 30 min. Monitor the morphology of GRS-DNA-CuS by transmission electron microscopy (TEM). Measure particle size Dilute 5 μL of the obtained GRS-DNA-CuS into 1.5 mL of ddH2O. Add the sample to the cuvette without any bubbles. Insert the cuvette into the particle analyzer chamber. Detect the size of GRS-DNA-CuS using the Malvern laser particle size analyzer. Measure zeta potential Dilute 5 μL of GRS-DNA-CuS nanodandelions into 1.5 mL of ddH2O. Add the sample to a capillary cell. Cover the capillary cell with a plastic cap. Place the capillary cell into the chamber of the zeta potential analyzer. Measure the zeta potential value of GRS-DNA-CuS (-32.2 mV) using the Malvern laser particle size analyzer. Measure Raman signal intensity of GRS-DNA-CuS nanodandelions Dilute 5 μL of GRS-DNA-CuS nanodandelions in 150 μL of ddH2O. Drop 20 μL of the diluted GRS-DNA-CuS solution onto the foil-wrapped slide. Place slide in the instrument and measure Raman signal intensity of GRS-DNA-CuS by Renishaw inVia Raman microscope in StreamLine mode (5× objective, 62.6 mW) with 5 s of exposure time under 830 nm laser excitation, as shown in Figure 3 . Figure 3. Process for measuring Raman signal intensity of GRS-DNA-CuS nanodandelions Measure the photothermal effect of GRS-DNA-CuS nanodandelions and CuS PAs Irradiate GRS-DNA-CuS nanodandelions and CuS nanoparticles (NPs) by 808 nm laser (2.8 W/cm2) for 4 min. Use a FLIR Ax5 camera to record the temperature changes in each group at an interval of 30 s during the laser irradiation. Measure the accumulation of GRS-DNA-CuS nanodandelions at tumor tissues Intravenously inject GRS-DNA-CuS nanodandelions at the dose of 50 μmol/kg body weight into A549 tumor-bearing Balb/c male nude mice (approximately 20 g, 6 weeks old). Note: A549 cells (5 × 106 cells/mouse in 100 μL PBS) are subcutaneously injected into the back of Balb/c nude mice and allowed to grow to the tumor size of 125 mm3 for the imaging and therapeutic studies. Collect Raman signals of GRS-DNA-CuS nanodandelions at the tumor tissues at 0, 1, 2, 4, 8, 12, and 24 h post-injection, by Renishaw inVia Raman microscope in StreamLine mode (5× objective, 62.6 mW) with 5 s of exposure time under 830 nm laser excitation. Measure the intratumoral penetration of GRS-DNA-CuS nanodandelions Intravenously inject GRS-DNA-CuS nanodandelions (50 μmol/kg body weight) into A549 tumor-bearing Balb/c male nude mice (approximately 20 g, 6 weeks old). Irradiate the A549-tumors on Balb/c nude mice for 2 min at 2.8 W/cm2 under 808 nm laser at 12 h post-injection. Image the photoacoustic signals at the tumor tissues by photoacoustic imaging system at 0, 0.5, 6, and 12 h of post-laser irradiation Note: In the control group, tumors on mice are intratumorally infected with RS-DNA-CuS nanodandelions without NIR laser irradiation. Evaluate the photothermal therapeutic efficiency of GRS-DNA-CuS nanodandelions Intravenously inject GRS-DNA-CuS nanodandelions (50 μmol/kg body weight) into A549 tumor-bearing Balb/c male nude mice (approximately 20 g, 6 weeks old). Irradiate the tumors at 12 h after injection of GRS-DNA-CuS nanodandelions by the first 808 nm NIR laser. Note: This first 808 nm NIR laser irradiation is for the dissociation of GRS-DNA-CuS nanodandelions that are accumulated at the tumors. Six hours later, tumors are irradiated again by the second 808 nm NIR laser. Note: This second 808 nm NIR laser irradiation is used for the tumor photothermal treatment. Measure the tumor size by vernier caliper and calculate according to the equation: V = L × W2/2, where the L and W are the tumor size at the longest and widest point, respectively. Data analysis Characterize the physiochemical properties (such as morphology, size, zeta potential, Raman signal, and photothermal conversion properties, etc.) of DNA-assembled GRS-DNA-CuS nanodandelions. TEM images in Figure 4 show that the numerous CuS PAs are clearly coated on the surface of GRS, indicating that GRS-DNA-CuS is successfully prepared. Scanning transmission electron microscopy-energy dispersive X-ray spectrometry (STEM-EDX ) elemental images in Figure 5 also indicate the successful formation of GRS-DNA-CuS. The size of GRS-DNA-CuS is approximately 135 nm in Figure 6 . GRS-DNA-CuS exhibits the highest Raman signal intensity at 1331 cm−1, which is beneficial for detecting their intratumoral distribution by Raman imaging technique. The photothermal conversion efficiency of GRS-DNA-CuS is approximately 47.44%, indicating a better photothermal property. Meanwhile, temperature of GRS-DNA-CuS solution increases rapidly from room temperature to 46.8 within 1 min under 808 nm near-infrared irradiation with a laser power density of 2.8 W/cm2 , suggesting that the generated heat is sufficient to induce the explosive breakage of hydrogen bonds of DNA (Tm = 42.82°C) in GRS-DNA-CuS nanodandelions. Figure 4. Representative TEM image of GRS-DNA-CuS nanodandelions Figure 5. STEM-EDX elemental mapping images of GRS-DNA-CuS nanodandelions Figure 6. Size distribution of GRS-DNA-CuS nanodandelions Detect the dissociation of DNA-assembled GRS-DNA-CuS nanodandelions after NIR light irradiation. TEM images in Figure 7 show a complete dissociation of large-sized GRS-DNA-CuS into GRS-ssDNA and ultrasmall CuS-cssDNA PAs within 1 min after 808 nm NIR laser irradiation with a laser power density of 2.8 W/cm2. Figure 7. TEM images for detecting morphological changes of GRS-DNA-CuS before and after dissociation under 808 nm NIR laser irradiation Monitor the tumor accumulation process of DNA-assembled GRS-DNA-CuS nanodandelions by Raman imaging technique (as shown in Figure 4A–C in Zhang et al., 2022). GRS-DNA-CuS nanodandelions reach maximum Raman intensity in tumor tissues after 12 h and then remain unchanged until 24 h, demonstrating that NIR-triggered hydrogen-bond thermal breakage in large-sized GRS-DNA-CuS nanodandelions can be conducted between 12 and 24 h after intravenous injection. Analyze the intra-tumor diffusion process of the ultrasmall CuS PAs dissociated from GRS-DNA-CuS nanodandelions by photoacoustic (PA) imaging. At 12 h post-injection of GRS-DNA-CuS, mice in the NIR-irradiated group are irradiated by NIR laser with a power of 2.8 W/cm2 for 2 min. Accumulated GRS-DNA-CuS nanodandelions at the tumor are entirely dissociated into ultrasmall CuS PA under the first NIR laser irradiation. Higher PA red-positive signals are observed near the tumor center after 6 h of irradiation (Figure 8), indicating that NIR-triggered dissociation contributes to better tumor dispersibility of ultrasmall CuS PAs. The diffusion depth of ultrasmall CuS PAs is greater at 6 h than at 0.5 h after laser irradiation (Figure 8), demonstrating that ultrasmall CuS PAs penetrate at the tumor tissues in a time-dependent manner. Figure 8. Photoacoustic images of GRS-DNA-CuS nanodandelions at tumor tissues after 0.5 and 6 h post-irradiation. Evaluate UITD-guided PTT efficiency. In the GRS-DNA-CuS-treated group with two NIR laser irradiations, three-fifths of the tumors are completely eliminated and no longer recurred, indicating the role of UITD in PTT-treated tumors (Figure 5B and 5C in Zhang et al., 2022). Meanwhile, the above physicochemical characterizations and the obtained main results about GRS-DNA-CuS nanodandelions are also illustrated in Table 1. Table 1. Physicochemical characterizations and the obtained main results of DNA-assembled GRS-DNA-CuS nanodandelions Characterization content Equipment Obtained main results 1 Morphology of GRS-DNA-CuS Transmission electron microscopy (TEM) GRS-DNA-CuS has spherical structure 2 Size of GRS-DNA-CuS Malvern laser particle size analyzer Hydrodynamic size of GRS-DNA-CuS is 135 nm 3 Zeta potential value of GRS-DNA-CuS Malvern laser particle size analyzer Zeta potential of GRS-DNA-CuS is -32.2 mV 4 Raman signal intensity of GRS-DNA-CuS Renishaw inVia Raman microscope GRS-DNA-CuS has the highest Raman signal intensity at 1331 cm−1 5 Photothermal effect of GRS-DNA-CuS 808 nm laser and FLIR Ax5 camera GRS-DNA-CuS exhibits photothermal conversion efficiency of 47.44% and higher photothermal stability 6 Intratumoral accumulation Renishaw inVia Raman microscope The accumulation amount of GRS-DNA-CuS at tumors can reach the maximum after 12 h of intravenous injection 7 Intratumoral penetration Photoacoustic imaging system GRS-DNA-CuS distributes around the whole tumor after 6 h of irradiation 8 Photothermal therapeutic efficiency Vernier caliper In the treated group with two laser irradiations, three-fifths of the tumors are completely eliminated and no longer recurred Acknowledgments This protocol is based on our previous publication (Zhang et al., 2022). We are supported by National Key Research and Development Program of China (NO. 2020YFA0210800), National Natural Science Foundation of China (NO. 21874092, 52161160307, and 31671003), Startup Fund for Young Faculty at Shanghai Jiao Tong University (NO. 21X010501069), and Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases. We also thank the Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine for assistance with in vitro experiments. Competing interests The authors declare no conflicts of interest. Ethics All the animal procedures were approved by Shanghai Jiao Tong University School of Medicine Ethics Committee and carried out in the experimental animal Center of Shanghai Jiao Tong University School of Medicine. References Zhou, C., Zhang, L., Sun, T., Zhang, Y., Liu, Y., Gong, M., Xu, Z., Du, M., Liu, Y., Liu, G. and Zhang, D. (2021). Activatable NIR-II Plasmonic Nanotheranostics for Efficient Photoacoustic Imaging and Photothermal Cancer Therapy. Adv Mater 33(3): e2006532. Xiong, X., Xu, Z., Huang, H., Wang, Y., Zhao, J., Guo, X. and Zhou, S. (2020). A NIR light triggered disintegratable nanoplatform for enhanced penetration and chemotherapy in deep tumor tissues. Biomaterials 245: 119840. Yan, F., Duan, W., Li, Y., Wu, H., Zhou, Y., Pan, M., Liu, H., Liu, X. and Zheng, H. (2016). NIR-Laser-Controlled Drug Release from DOX/IR-780-Loaded Temperature-Sensitive-Liposomes for Chemo-Photothermal Synergistic Tumor Therapy. Theranostics 6(13): 2337-2351. Li, H. J., Du, J. Z., Liu, J., Du, X. J., Shen, S., Zhu, Y. H., Wang, X., Ye, X., Nie, S. and Wang, J. (2016). Smart Superstructures with Ultrahigh pH-Sensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration. ACS Nano 10(7): 6753-6761. Wang, G., Zhou, Z., Zhao, Z., Li, Q., Wu, Y., Yan, S., Shen, Y. and Huang, P. (2020). Enzyme-Triggered Transcytosis of Dendrimer-Drug Conjugate for Deep Penetration into Pancreatic Tumors. ACS Nano 14(4): 4890-4904. Zhou, Q., Shao, S., Wang, J., Xu, C., Xiang, J., Piao, Y., Zhou, Z., Yu, Q., Tang, J., Liu, X., et al. (2019). Enzyme-activatable polymer-drug conjugate augments tumour penetration and treatment efficacy. Nat Nanotechnol 14(8): 799-809. Zhang, Y., Cui, Y., Li, M., Cui, K., Li, R., Xie, W., Liu, L. and Xiao, Z. (2022). DNA-assembled visible nanodandelions with explosive hydrogen-bond breakage achieving uniform intra-tumor distribution (UITD)-guided photothermal therapy. Biomaterials 282: 121381. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > Nanoparticle > Plan-derived nanoparticles Biological Engineering > Biomedical engineering Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells Jingen Zhu [...] Venigalla B. Rao Apr 5, 2020 3781 Views Extracellular Vesicles Tracking and Quantification Using CT and Optical Imaging in Rats Shaowei Guo [...] Shulamit Levenberg Jun 5, 2020 4187 Views Preparation and Characterization of Ginger Lipid-derived Nanoparticles for Colon-targeted siRNA Delivery Junsik Sung [...] Didier Merlin Jul 20, 2020 3249 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,596	https://bio-protocol.org/en/bpdetail?id=4596&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Lysate-to-grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging IC Ian Cooney DM Deirdre C. Mack AF Aaron J. Ferrell MS Michael G. Stewart SW Shuxin Wang HD Helen M. Donelick DT Daniela Tamayo-Jaramillo DG Dakota L. Greer DZ Danyang Zhu WL Wenyan Li PS Peter S. Shen Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4596 Views: 1364 Reviewed by: Laxmi Narayan MishraSashikantha Reddy PulikalluNityanand Srivastava Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Aug 2019 Abstract Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow. Here, we present a protocol using budding yeast (S. cerevisiae), in which a tractable immunoprecipitation tag (3xFLAG) is appended at the endogenous locus of a gene of interest (GOI). The modified gene is expressed under its endogenous promoter, and cells are grown and harvested using standard procedures. Our protocol describes the steps in which the tagged proteins and their associated complexes are isolated within three hours of thawing cell lysates, after which the recovered proteins are used directly for cryo-EM specimen preparation. The prioritization of speed maximizes the ability to recover intact, scarce complexes. The protocol is generalizable to soluble yeast proteins that tolerate C-terminal epitope tags. Graphical abstract Overview of lysate-to-grid workflow. Yeast cells are transformed to express a tractable tag on a gene of interest. Following cell culture and lysis, particles of interest are rapidly isolated by co-immunoprecipitation and prepared for cryo-EM imaging (created with BioRender.com). Keywords: Cryo-EM Yeast transformation Co-immunoprecipitation Protein purification Protein complexes Vitrification Background Structural determination of biological macromolecules is essential for providing insights into their function. Over the past decade, revolutionary advances in hardware and software have ushered in a new era of single-particle electron cryo-microscopy (cryo-EM) (Kühlbrandt, 2014; Shen, 2017). The field is amidst a golden age, as evidenced by recent structures that have achieved atomic resolution (Nakane et al., 2020; Yip et al., 2020). Such resolution has traditionally only been accessible through other established methods, such as X-ray crystallography and nuclear magnetic resonance, which also generally require a substantial amount of material (e.g., on the order of several milligrams). In contrast, single particle cryo-EM only requires enough material to cover the support grid, and a few microliters of sample between 0.1 and 1.0 micromolar concentration is often sufficient for each specimen. Common approaches to generate sufficient material for structural studies typically depend on overexpressing proteins in heterologous systems. While overexpression systems are effective in producing large quantities of material, the purified proteins may lack relevant binding partners or post-translational modifications that do not exist in such systems. The relatively low sample volume and concentration requirements for cryo-EM make it an attractive method to determine structures of protein complexes that are isolated directly from their native sources. Indeed, cryo-EM imaging of proteins from cell extracts is increasingly becoming more feasible as a tool for discovery biology (Ho et al., 2020; Skalidis et al., 2022). Such top-down approaches enable the characterization of particles in a more native-like context that is inherently missing in bottom-up in vitro reconstitution methods. Yet, achieving sufficient material of scarce or short-lived complexes remains a major barrier to preparing high-quality cryo-EM specimens. By leveraging the advantages of working with the budding yeast model organism (S. cerevisiae), including genetic tractability and throughput of cell growth, we have optimized a generalizable procedure that enables the rapid isolation of endogenous complexes from cell lysates. The procedure balances between speed and purity, and samples are ready for cryo-EM specimen vitrification within three hours. The prioritization of speed enables the recovery of multi-component complexes that may otherwise fall apart in lengthier procedures, such as those with overnight steps. Our procedure was used to isolate scarce complexes trapped in their active, functional states, including capturing the processes of mRNA-independent peptide synthesis by the ribosome-associated quality control complex (~300 copies per cell; Shen et al., 2015) and protein unfolding by the Shp1-Cdc48 AAA+ ATPase complex (~3,000 copies per cell; Cooney et al., 2019; protein copy numbers estimated from Ghaemmaghami et al., 2003). In both of these studies, the rapid isolation of native complexes enabled the structure determination of multi-component complexes in novel and biologically important functional states. Here, we describe our detailed protocol of how native multi-component, soluble complexes are quickly isolated from yeast lysates and used for cryo-EM imaging. A C-terminal 3xFLAG tag is inserted into the endogenous locus of a gene of interest (GOI) via homologous recombination. Cells are grown and harvested and their lysates are prepared for co-immunoprecipitation (co-IP) experiments. The target protein and its associated complexes are recovered by competitive elution and used directly for cryo-EM specimen preparation. In principle, our protocol can be applied to isolate and image any soluble yeast protein and their associated complexes as long as their function is not disrupted by the C-terminal tag and the particles are of sufficient size to be visible by cryo-EM. Materials and Reagents General supplies 250 mL Erlenmeyer flasks (Corning, catalog number: 4980-250) 2.8 L Fernbach culture Pyrex flasks (Sigma-Aldrich, catalog number: CLS44202XL) 1.5 mL Eppendorf/microcentrifuge tubes (Fisher Scientific, catalog number: 14-282-302) 15 mL conical tubes (Greiner Bio-One, catalog number: 188271) 50 mL conical tubes (Greiner Bio-One, catalog number: 227270) 15 mL glass vials (Corning, catalog number: 9820-16X) Small growth platform shaker (Lab-Line Instruments, Inc., catalog number: 3590-1) Large growth platform shaker (Barnstead \| Lab-Line, A-Class., catalog number: SHKA3000) 1 L centrifuge bottle and caps (Beckman Coulter, catalog number: C31597) Styrofoam box Membrane filter, 0.22 μm pore size (Millipore, catalog number: GSWP04700) For yeast transformation (section A) pFA6a plasmid (Longtine et al., 1998). We use pTF272 (pFA6a-TEV-6xGly-3xFLAG-HphMX) (Addgene plasmid # 44083; http://n2t.net/addgene:44083; RRID: Addgene_44083) iProof HF 2X Master mix (Bio-Rad, catalog number: 1725310) GeneRuler 1 kb Plus DNA ladder (ThermoFisher Scientific, catalog number: SM1331) EZVision One Dye-as-Loading buffer, 6× (VWR, catalog number: 97064-190) Agarose (GoldBio, catalog number: A-201-500) Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: AC118432500) Acetic acid (Fisher Chemical, catalog number: BP2401C-212) S. cerevisiae yeast strain BY4741 (derived from S288C strain; genotype MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) Yeast extract (Fisher Scientific, catalog number: BP9727-2) Peptone (Fisher Scientific, catalog number: BP9725) Adenine (Sigma-Aldrich, catalog number: A8626) Glucose (Sigma-Aldrich, catalog number: G7021) Agar (Becton Dickson & Company, catalog number: 2013-03-31) Poly(ethylene glycol) (PEG) 3350 (Sigma-Aldrich, catalog number: 202444) Sheared salmon sperm DNA (Ambion, catalog number: 1009027) Lithium acetate (Sigma-Aldrich, catalog number: 517992) Hygromycin B (Sigma-Aldrich, catalog number: 10843555001) For transformation validation (section B) Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166) 4%–15% precast protein gel (Bio-Rad, catalog number: 4561086) 2× Laemmli sample buffer (Bio-Rad, catalog number: 1610737) Beta-mercaptoethanol (Bio-Rad, catalog number: 161-0710) Methanol (Fisher Scientific, catalog number: A412P-4) PVDF membrane (Bio-Rad, catalog number: 1620177) Precision plus protein dual color standards (Bio-Rad, catalog number: 161-0374) Thick blot filter paper (Bio-Rad, catalog number: 1703969) Roller (ThermoFisher Scientific, catalog number: 0084747) Skim milk powder (MP Bio, catalog number: 902887) Tween 20 (Sigma-Aldrich, catalog number: 11332465001) Incubation box (Li-Cor, catalog number: 929-97301) Monoclonal ANTI-FLAG® M2 antibody produced in mouse (Sigma-Aldrich, catalog number: F1804) Goat anti-mouse IgG secondary antibody (Li-Cor, catalog number: 926-32210) Sodium azide (Sigma-Aldrich, Catalog number: S2002) For large scale culture, harvest, and lysis (sections C–D) Glycerol (Fisher Bioreagents, catalog number: BP229) HEPES (Fisher Bioreagents, catalog number: BP310-500) Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190) Magnesium acetate [Mg(Oac)2] (Sigma-Aldrich, catalog number:M5661) Calcium chloride (CaCl2) (Fisher Chemical, catalog number: C79-500) D-sorbitol (Sigma-Aldrich, catalog number: 1003101761) Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626) Aprotinin (GoldBio, catalog number: A-655) Pepstatin (GoldBio, catalog number: P-020) Leupeptin (GoldBio, catalog number L-010) Dithiothreitol (DTT) (GoldBio, catalog number DTT) Liquid nitrogen For co-immunoprecipitation (section E) Magnesium chloride hexahydrate (MgCl2·6H2O) (Acros Organics, catalog number: 41341-500) Igepal CA-630 (Sigma-Aldrich, catalog number I3021) Anti-FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220) 3xFLAG peptide (APExBio, catalog number: A 6001) For quality control and cryo-EM specimen preparation (sections F–G) Formvar/Carbon 200 mesh grids (Ted Pella, Inc., catalog number: 01801) 595 Vitrobot filter paper (Ted Pella, Inc., catalog number: 47000-100) Uranyl acetate salt (EMS, catalog number: 22400) Glutaraldehyde (EMS, Catalog number: 16000) Tris hydrochloride (Tris-HCl) (Sigma-Aldrich, Catalog number: 9310-OP) Quantifoil R1.2/1.3 or UltrAuFoil R1.2/1.3 300 mesh grids (SPT Labtech) Compressed nitrogen gas cylinder Compressed ethane gas cylinder Stock solutions 50× TAE (see Recipes) 5× TBS (see Recipes) 10× transfer buffer (see Recipes) 1 M HEPES-KOH, pH 7.4 (see Recipes) 1 M KOAc (see Recipes) 1 M Mg(Oac)2 (see Recipes) 1 M CaCl2 (see Recipes) 1 M sorbitol (see Recipes) 1 M MgCl2 (see Recipes) 1 M DTT (see Recipes) 2× IP buffer (see Recipes) 1 M lithium acetate (LiOAc) (see Recipes) Gels 1% agarose gel (see Recipes) Buffers 1× TAE (see Recipes) Working transfer buffer (see Recipes) Blocking solution (see Recipes) TBST solution (see Recipes) Yeast lysis buffer (see Recipes) Wash buffer 1 (see Recipes) Wash buffer 2 (see Recipes) Mixes Transformation PCR mix (according to Bio-Rad iProof HF manual) (see Recipes) Colony PCR mix (see Recipes) PEG mix (see Recipes) Reagents Lithium acetate (LiOAc) (see Recipes) 1% uranyl acetate (see Recipes) Tris base (Sigma-Aldrich, catalog number: TRIS-RO) Glycine (Sigma-Aldrich, catalog number: 50046) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888) Dextrose (Sigma-Aldrich, catalog number: G7021) Plates and Media YPAD plates (see Recipes) YPAD + hygromycin plates (see Recipes) YPD liquid media (see Recipes) Equipment General equipment and supplies Pipettes (VWR P10, P20, P200, P1000; catalog numbers: 89079-962, 89079-964, 89079-970, 89079-974) Floor centrifuge (Beckman Coulter, model: Avanti J-26 XP) Fixed angle centrifuge rotor (Beckman Coulter, model: JLA 8.1) Refrigerated microcentrifuge (Sorvall Biofuge Fresco) 42°C water bath 37°C incubator 30°C incubator Vortex mixer (Scientific Industries Inc.) Nutator (Clay Adams, catalog number: 421105) Spectrophotometer (ThermoFisher Scientific, catalog number: ND-ONE-W) Gel electrophoresis apparatus Micro spatula GelDoc-IT (VWR) Benchtop centrifuge (Beckman Coulter, model: Allegra 6R; GH-3.8 rotor) For yeast transformation Thermal cycler (Applied Biosystems, catalog number: 4484073) Cell culture rotator 3 mm solid glass beads (Sigma-Aldrich, catalog number: Z143928) Replica plate tool (Cole Parmer, catalog number: EW-14210-00) Velveteen squares (Cole Parmer, catalog number: EW-14210-50) Cryogenic vial (VWR, catalog number: 89094-800) For whole-cell immunoblotting Trans-Blot turbo transfer system (Bio-Rad, catalog number: 1704150) Orbital shaker (Vevor) Odyssey CLx imaging system (Li-Cor) For cell lysis (freezer mill) Cryo-gloves (Tempshield) Face shield/goggles Freezer/mill cryogenic grinder (Spex) Grinding vial sets (polycarbonate cylinders, impactors, and end plugs; Spex, catalog number: 6751) Freezer mill magnetic extractor (Spex, catalog number: 6791) Freezer mill vial opener (Spex, catalog number: 6754) For cryo-EM specimen preparation/vitrification Pelco easiGlow unit (Ted Pella, Inc., catalog number: 91000) DUMONT clamping ring medical tweezers (Ted Pella, catalog number: 38825) Vitrobot (ThermoFisher Scientific, catalog number: VITROBOT) Vitrobot tweezers (Ted Pella, catalog number: 47000-500) Stainless steel long tweezers (Xducom) Cryo-EM grid storage boxes (MiTeGen, catalog number: M-CEM-CGBSW1) Grid box storage dewar (Worthington 25LDB Storage Dewar, Cole-Parmer, catalog number: EW-03773-57) Procedure Yeast transformation (Figure 1) Figure 1. Workflow to generate a new tagged strain. Yeast cells are transformed with PCR products via homologous recombination. After quality control and vetting successful transformation, the cells are ready to be used for downstream purification (created with BioRender.com). A1. Primer design for tagging GOI Choose a forward adapter sequence to amplify the entire 3xFLAG tag and a linker sequence from a pFA6a plasmid (Longtine et al., 1998), e.g., pTF272 (Addgene). This adapter should anneal to the 5′ end of the 3xFLAG tag and be roughly 24 bases (8 codons) in length, ensuring that the reading frame of the tag is preserved. Example using the pTF272 plasmid: 5′ ggctctagagactacaaggaccac 3′ Choose a gene-specific sequence for your GOI to direct tagging to the C-terminal residue of the encoded protein. This sequence should be between 35 and 60 bases in length and end directly before the stop codon to preserve the reading frame of the tag. This gene-specific sequence is combined with the forward adapter sequence to form the final forward tagging primer. Example for the SHP1 gene as reported in Cooney et al. (2019): 5′ GATCTGCTGAACTCCGTTGTCGTGCAAAGATGGGCAggctctagagactacaaggaccac 3′ (upper case: GOI sequence; lower case: pTF272 sequence). Choose a reverse adapter sequence to amplify the hphMX6 selection cassette and terminator from the plasmid. This adapter should represent the reverse complement of the sequence from the 3′ end and anneal 3′ of the TEF terminator and be roughly 24 bases in length. Example from the pTF272 plasmid: 5′ cagcagtatagcgaccagcattca 3′ Choose another gene-specific sequence ending directly after the stop codon of the GOI. This sequence should be between 35 and 60 bases in length. Combine the reverse complement of the gene-specific sequence with the reverse adapter sequence to form the final reverse tagging primer. Example for the SHP1 gene: 5′ AGTCTTTTCCCGTTTCTGTTTTTGTATATTTATGCcagcagtatagcgaccagcattca 3′ (upper case: GOI sequence; lower case: pTF272 sequence). For a graphical representation of the regions of homology for each primer, see Figure 2. Figure 2. Overview of primer design for yeast transformation. Primers are designed with the forward strand being homologous to the 3’ end of the gene of interest (GOI) just before the endogenous stop codon and the reverse strand being homologous with a segment immediately following the stop codon. The primers anneal to a region of a pFA6a plasmid (e.g., pTF262; see Materials) that encode a 3xFLAG tag and selectable marker (in this case, hygromycin). Following PCR amplification and yeast transformation, the cells now express a 3xFLAG-tagged version of the GOI and a selectable marker. A2. Amplification of PCR product Prepare the PCR reaction in a PCR tube using transformation PCR mix (see Recipes). Run PCR using settings recommended by the iProof Master mix. Annealing temperatures should be determined by the calculated melting temperatures of the oligonucleotide sequences that anneal to the plasmid, which should be between 55 and 72°C. We recommend running a gradient of temperatures to determine the best settings to maximize PCR product (Figure 3). Load 5 µL of PCR reaction and 5 µL of DNA ladder (each mixed with 1 µL of EZVision One Dye-as-Loading buffer, 6×) into separate wells of a 1% agarose gel. Run the gel electrophoresis apparatus with the gel submerged in 1× TAE buffer at 120 V until the leading dye reaches the bottom of the gel (estimated 30 min; see Recipes). Image gel on GelDoc-IT. Confirm the presence of a band at the expected size of the PCR product. Store PCR product at -20°C for up to 24 h until cells are ready for transformation. Figure 3. Example of PCR product to be used for yeast transformation. A gradient of annealing temperatures is used to identify optimal settings for PCR amplification. In this example, the expected PCR product of approximately 1,700 bp is generated using annealing temperatures between 68 and 72°C. These products would then be pooled for downstream yeast transformation. A3. Transformation For general guidelines about yeast culture in liquid or solid media, consult the Sigma-Aldrich Yeast Protocols sites: https://www.sigmaaldrich.com/US/en/technical-documents/protocol/microbiological-testing/pathogen-and-spoilage-testing/yeast-growth-protocols. The transformation protocol is adapted from Gietz and Schiestl (2007). Using aseptic technique with a sterilized pipette tip, scrape off a small amount of glycerol stock of BY4741 yeast cells and streak it out on a YPAD plate. Be careful to prevent the glycerol stock from thawing. Incubate plate at 30°C until single colonies appear (1–2 days). Using aseptic technique, pick one isolated colony of BY4741 from the plate by gently scraping with a sterile pipette tip. Inoculate yeast by placing the pipette tip into 3 mL of YPD media in a 15 mL culture tube. Gently pipette up and down until the colony is suspended in the media. Place the glass vial on a cell culture rotator and incubate at 30°C overnight. After approximately 16 h of cell growth, prepare OD600 measurement by diluting the overnight growth 1:20 in YPD, e.g., 50 µL of cell culture into 950 µL of YPD. Blank the spectrophotometer with YPD and measure the OD600 of the diluted cells. The target OD600 after dilution should be approximately 1.0, i.e., the actual OD600 is approximately 20. Back dilute the overnight growth by pipetting 100 µL of the overnight growth into 10 mL of YPD in a 15 mL glass vial (1:100 dilution). Grow cells for approximately 3 h on a cell culture rotator at 30°C. Remove the vial after the OD600 has reached approximately 1.0. While cells are growing, prepare PEG mix with the PCR product from section A2 step 6 (see Recipes). To dissolve PEG 3350, add double-distilled water (ddH2O), vortex the mixture thoroughly, then incubate at 37°C for 5 min. Make sure that the PEG has completely dissolved and continue vortexing if needed. Place salmon sperm DNA in a 95°C water bath for 5 min and then chill on ice prior to addition to PEG mix. Transfer yeast culture to a 15 mL conical tube and centrifuge at 2,000 ×g for 5 min at room temperature. Decant and discard the media supernatant. Resuspend the cell pellet in 5 mL of ddH2O. Mix by pipetting. Centrifuge at 2,000 × g for 5 min at room temperature. Decant and discard the water supernatant. Resuspend the cell pellet in 1 mL of 100 mM LiOAc by pipetting and then transfer to a 1.5 mL Eppendorf tube. Pellet the cells at 4,500 × g for 1 min at room temperature and then discard the supernatant. Cut 2 cm off the end of a 1,000 µL pipette tip using a razor blade (Figure 4). CAUTION: Use care when handling sharps. Use this tip to pipette the viscous PEG mixture over the cell pellet. Vortex vigorously to resuspend the cell pellet into the PEG mixture. Figure 4. Preparing pipette tips for viscous solutions. Comparison of an uncut 1,000 µL pipette tip (top) and a tip with approximately 2 cm cut off with a razorblade (bottom). Incubate the resuspended cells in a 42°C water bath for 40 min. Centrifuge at 4,500 ×g for 30 s at room temperature and then discard the supernatant. Resuspend the cell pellet in 600 µL of ddH2O. Pipette 300 µL of resuspended cells onto two separate YPAD agar plates each (without antibiotic). Note: This step is important to allow cells to recover after transformation. Use autoclaved glass beads to spread the cells evenly across the plates. Label the plates and incubate at 30°C for 24 h. A4. Replica plating A lawn of cells should be visible after 24 h of growth. The cells are now ready to transfer onto selection plates. Place the sterilized velvet on top of the replica plate tool and firmly secure the velvet using the replica plate tool ring. Transfer cells onto velvet by gently pressing the plate onto the velvet. Separate the plate from the velvet and then gently press the selection plate (YPAD + hygromycin, see Recipes) onto the cell-covered velvet. Store plates in a 30°C incubator for 2–3 days until single colonies begin to appear. After colonies appear, streak out multiple individual colonies on new YPAD + hygromycin plates. We recommend streaking eight colonies per plate. Store plates in a 30°C incubator until cell growth is visible. Cell growth indicates that cells are expressing the selection marker and suggests that transformation was successful. To rule out false positives, validate the transformation by colony PCR and/or whole-cell immunoblot (Section B, below). Transformation confirmation B1. Colony PCR. Protocol adapted from Akada et al. (2000) Design colony PCR primers that are specific to the transformed gene, such that one primer anneals to the GOI and the other primer anneals to the inserted sequence. We recommend targeting a PCR product approximately 300 bp in size. The colony PCR forward primer should be approximately 35 bases in length and within the GOI. Example from the SHP1 gene: 5′ CTATCAAACCAATAAGCAACGATGAGACAACATTG 3′ The reverse primer should be designed using the reverse complement sequence from the 3′ end corresponding to approximately 35 bases within the inserted DNA. Example from the pTF272 plasmid: 5′ GAGGTGTGGTCAATAAGAGCGACCTCATACTATAC 3′ Using a pipette tip, transfer an isolated yeast colony to 20 µL of 0.25% SDS in an Eppendorf tube. We recommend repeating this for multiple colonies. Vortex thoroughly to suspend the cells in SDS. Place the tubes containing cells in a 95°C water bath for 3 min. Add 60 µL of ddH2O to each tube. Spin the cells at 17,000 × g for 1 min at room temperature in a microcentrifuge. Add 1.5 µL of the supernatant containing genomic DNA to 18.5 µL of colony PCR Master mix (see Recipes) in a PCR tube. Repeat steps 4–9 for the desired number of colonies to screen. We recommend up to 15 colonies. Perform PCR and confirm presence of PCR product as in section A2 steps 3–5 (see Figure 3 for an example of PCR confirmation). Note that absence of PCR products may be interpreted as false negatives. We recommend running whole-cell immunoblots as an additional measure of validation. B2. Whole-cell immunoblot For each colony, add 10 mL of YPD to a 15 mL culture tube using aseptic technique. Inoculate each tube with a separate colony from the plate. Place the tubes on a cell culture rotator at 30°C and grow cells overnight. Centrifuge 1 mL of culture at 4,000 × g for 5 min at 4°C. Resuspend cell pellet in 100 µL ddH2O and 100 µL 2× working Laemmli buffer. Heat at 95°C for 2–3 min. Avoid overboiling to prevent protein precipitation. Centrifuge at 4,000 × g for 5 min at 4°C. Load 15 µL of supernatant onto a precast 4%–15% SDS-PAGE gel. Run SDS-PAGE gel at 170 V for 35 min. Prepare 100 mL of working transfer buffer (see Recipes). Prepare 50 mL of 100% methanol. Soak the SDS-PAGE gel in 20 mL of working transfer buffer. Cut a PVDF membrane to be 2 cm larger in length and width than the SDS-PAGE gel. Soak the PVDF membrane in the 100% methanol solution. Cut two pieces of thick blot filter paper to be 2 cm larger in length and width than the PVDF membrane. Soak the filter paper in the remaining transfer buffer solution. In an immunoblot transfer system cassette, assemble the transfer stack in this order from bottom to top: filter paper, PVDF membrane, SDS-PAGE gel, filter paper. Pour the remaining working transfer buffer over the transfer stack to keep it hydrated. Use a roller to remove any trapped air bubbles from the transfer stack. Place the top of the cassette on the transfer stack, lock it in place, and insert the cassette into the transfer system. Transfer the contents of the gel onto the PVDF membrane at 25 V for 30 min. Prepare 20 mL of the blocking solution (see Recipes). Transfer the PVDF membrane to an opaque incubation box. Add the blocking solution to the box and incubate at room temperature for 30 min with gentle agitation. Prepare the mouse anti-FLAG primary antibody by diluting it 1:1,000 in a 1× TBST with 5% milk. Pour off the blocking buffer, add 10 mL of the anti-FLAG primary antibody, and then incubate with gentle agitation for at least 1 h at room temperature (or overnight at 4°C). Pour the anti-FLAG antibody back into a 15 mL conical tube. The antibody solution may be stored at -20°C and re-used several times. Wash membrane by adding 15 mL of TBST solution to the membrane and incubate with gentle agitation for 5 min at room temperature. Repeat the TBST wash step three times, discarding the TBST after each wash. Prepare the goat anti-mouse secondary antibody by diluting the antibody 1:10,000 in 1× TBST with 5% milk. Add 10 mL of the secondary antibody and incubate for 1 h at room temperature. Pour the secondary antibody into an opaque 15 mL conical tube. The antibody solution may be stored in the presence of 0.01% sodium azide at -20°C and re-used several times. Repeat the washing steps in steps 28 and 29. Image the membrane using the Odyssey CLx imaging system. B3. Prepare glycerol stock Using aseptic technique, pick one confirmed isolated colony by gently scraping with a sterile pipette tip. Add to 10 mL of YPD and incubate overnight on a cell culture rotator at 30°C. Add 500 µL of sterilized 30% glycerol (in water, v/v) to a cryogenic vial. Add 500 µL of culture to the cryogenic vial. Discard excess culture. Close vial and invert to mix. Label vial and store at -80°C. The stock may be stored indefinitely and used when a fresh plate needs to be prepared. Large-scale yeast cell culture and harvest (Figure 5) Figure 5. Workflow to grow and prepare cells for cell lysis (created with BioRender.com) Add 100 mL of YPD to two 250 mL flasks each using aseptic technique. Inoculate each flask with a colony from a plate. Place flasks on a platform shaker at 30°C. Grow cells for approximately 16 h or until saturation. Using aseptic technique, add 30 mL of saturated culture to 970 mL of fresh YPD in 2.8 L flasks. Repeat this for six flasks. The target starting OD600 should be approximately 0.3. Grow cells on a platform shaker at 30°C for approximately 4 h until they reach a mid-log phase OD600 of approximately 1.5. Measure the OD600 periodically by retrieving 500 µL of culture to estimate when cells will reach the proper OD600. After OD600 of approximately 1.5 has been reached, harvest cells by transferring the yeast culture from flasks into 1 L bottles. Pellet the cells by centrifugation at 4,500 × g for 6 min at 4°C. After the cells are pelleted, pour off the media supernatant. Resuspend all cells in a total of 30–45 mL of chilled ddH2O using an automatic pipette and then transfer the resuspended cells to a 50 mL conical vial. Pellet the cells by centrifuging the conical vial at 4,500 × g for 5 min at 4°C. Pour off and discard the water supernatant. Record the weight of the cell pellet by subtracting the weight of an empty tube from the weight of tubes with the cell pellet. Typically, six liters of cell culture harvested at OD600of approximately 1.5 will weigh 10–20 g. Add 1 mL of chilled lysis buffer and protease inhibitors (see Recipes) per 6 g of cell pellet. Mix by agitating with a pipette tip and keep on ice. Fill a small Styrofoam box with liquid nitrogen. CAUTION: Protective equipment such as eye protection and cryogenic gloves should be used when handling liquid nitrogen. Using a cut pipette tip (Figure 4), slowly drip the resuspended cell pellet into the liquid nitrogen (Figure 6, left). Maintain separation from the liquid nitrogen to avoid freezing the pipette tip. The yeast should form small pellets as they contact the liquid nitrogen. For best results, scatter the dripping throughout the box to prevent drops from clumping together. Figure 6. Preparing yeast pellet for freezer mill lysis. (Left) Photo showing how the resuspended cell pellet is dripped into liquid nitrogen to create small frozen cell pellets. (Right) Photo of resulting small frozen cell pellets. Fill another larger Styrofoam container with liquid nitrogen and chill a 50 mL conical tube with the lid removed, a funnel, and long tweezers in liquid nitrogen. Hold the tube with a tube clamp so that it is mostly submerged in liquid nitrogen. Pour the yeast pellets into the clamped tube through the chilled funnel. It is okay if the tube overflows with liquid nitrogen. Use the chilled long tweezers to scrape off any remaining pellets that are stuck to the box. Use scissors to puncture the lid of the 50 mL conical tube (Figure 7). This is important to allow liquid nitrogen to vent from the tube after it is capped. Allow 80%–90% of the liquid nitrogen to evaporate from the 50 mL tube and then cap the tube with the punctured lid. The remaining liquid nitrogen will vent through the punctured lid. Store the capped tube at -80°C until cells are ready to be lysed (section D). Figure 7. Preparing a 50 mL conical tube for frozen yeast pellets. Photo of a 50 mL conical tube and punctured lid to store frozen yeast pellets. The punctured lid allows liquid nitrogen to vent after cell pellets have been poured into the tube. Cell lysis via freezer mill Attach a connecting hose between the liquid nitrogen tank and the freezer mill. Open the valve to the liquid nitrogen tank to pre-chill the machine. CAUTION: Eye protection and cryogenic gloves should be used when handling liquid nitrogen. Fill a Styrofoam box with liquid nitrogen. Chill freezer mill polycarbonate cylinders with one end plugged and impactors in the box containing liquid nitrogen. Be careful to not leave liquid nitrogen inside the cylinder. Add the chilled impactor and frozen yeast pellets to the cylinder. Fill each cylinder up to half capacity (Figure 8). Note that filling the tube more than halfway may reduce the lysis efficiency. Figure 8. Assembling freezer mill polycarbonate cylinders. (Left) Photo showing how a freezer mill polycarbonate cylinder is loaded with an impactor and frozen cell pellets. (Right) Photo showing the tube filled to half capacity and ready to be capped. Plug the open end of the freezer mill cylinder, ensuring that there is no remaining liquid nitrogen inside of tubes (Figure 9). CAUTION: Leaving liquid nitrogen inside the tubes can cause the plugs to pop off if there is a sudden temperature change, causing injury. Place the sealed freezer mill cylinders inside the freezer mill. Each run can accommodate four cylinders (Figure 9). Figure 9. Preparing the freezer mill cylinders for pulverization. (Left) Photo showing the process of plugging the open end of the freezer mill cylinder. (Middle) Photo showing a capped freezer mill cylinder being submerged in liquid nitrogen before placing it inside the freezer mill. (Right) Photo showing all four cylinders placed inside the freezer mill. Set the freezer mill settings to six cycles, 10 cps (impacts per second), 3 min grind, and 2 min cool. Start the run, which will take approximately 45 min to complete. When the run has completed, remove cylinders from the freezer mill and place them in a -80°C freezer for 20 min. This step is important to prevent the end plugs from popping off due to a sudden temperature change. Fill a Styrofoam box with liquid nitrogen. Pre-chill 50 mL conical tubes, a funnel, and a micro spatula in the box. Remove the caps from the freezer mill tubes and transfer the lysed yeast powder into a pre-chilled 50 mL conical tube over liquid nitrogen using a micro spatula (Figure 10). We recommend dividing the yeast powder in 2 g aliquots. This should occupy approximately 5 mL per 50 mL conical tube (Figure 10). Take care to keep the powder at -80°C or colder until they are used for co-IP. Powder should stay loose and fluffy if handled at the appropriate temperature. Each 2 g aliquot is used as a single co-IP experiment (section E). More powder may be used if higher co-IP yields are desired. Powder can be stored at -80°C for several months. Note: An overview video of freezer mill usage and principles by Spex SamplePrep is available through the following link: https://youtu.be/Q8600XCBsnQ. Figure 10. Handling yeast powder. (Left) Photo showing the loose and fluffy consistency of the lysed yeast powder when handled and stored at -80°C or colder. (Right) Example 2 g aliquot of yeast powder stored in a 50 mL conical vial. Co-immunoprecipitation and elution (Figure 11) Figure 11. Workflow to purify samples, assess their quality, and prepare cryo-EM specimens (created with BioRender.com). All steps in this section (section E) are performed at 4°C. Prepare wash buffer 1 (see Recipes) immediately before beginning this stage of the protocol. Retrieve the yeast powder aliquot from the -80°C freezer and immediately place on ice. Resuspend the powder in wash buffer 1 at a 1:1 (v/w) ratio. Mix thoroughly by stirring with a pipette tip into a homogeneous slurry. The quantity of powder needed per experiment will depend on the expression level of the tagged protein of interest. We recommend 2 g of powder as a starting point. Transfer the viscous slurry to 1.5 mL Eppendorf tubes on ice using a cut pipette tip (Figure 4). Clarify the lysate by centrifuging at 5,000 × g for 5 min in a microcentrifuge. Transfer supernatant to a new 1.5 mL Eppendorf tube. Discard the insoluble pellet. Clarify the lysate again by centrifugation at 10,000 × g for 10 min. Transfer supernatant to a new 1.5 mL Eppendorf tube. Discard the insoluble pellet. Repeat the lysate clarification by centrifugation at 15,000 × g for 10 min. Discard the insoluble pellet. While the centrifugation step is running in step 10, equilibrate 30 µL of anti-FLAG M2 affinity gel by resuspending it in 1 mL of wash buffer 1 in a 15 mL conical tube. Spin at 1,000 × g for 2 min to pellet the resin. Pipette off and discard the supernatant, making sure not to disturb or remove the resin bed. Transfer the clarified lysate supernatant to the equilibrated anti-FLAG M2 affinity gel and resuspend the affinity gel by gentle pipetting. Place the tube containing affinity gel and clarified lysate on a nutator and incubate for 1 h with gentle rocking. After incubation, centrifuge resin at 1,000 × g for 2 min. Remove and discard the unbound material by pipetting, taking care to not disturb the resin bed. Add 1 mL of wash buffer 1 to affinity gel and resuspend by gentle pipetting. Transfer the resuspended resin from the 15 mL conical tube to a fresh 1.5 mL Eppendorf tube by pipetting. Centrifuge resin at 1,000 × g for 2 min. Pipette off supernatant, being careful not to disturb the resin. Repeat steps 15, 17, and 18 five times with wash buffer 1 and five times with wash buffer 2 (i.e., five washes in each wash buffer). Note that the number of washes may require optimization to achieve the desired balance between protocol duration and sample purity. Add 30 µL of 3xFLAG peptide at a concentration of 1.5 mg/mL in wash buffer 2 and incubate for 30 min on a nutator. Pellet the resin at 1,000 × g for 2 min. Carefully transfer 30 µL of the eluate to a fresh 1.5 mL Eppendorf tube and use immediately for quality control (section F) or cryo-EM specimen preparation (section G) (step 8). See Figure 12 for an example of the quantity of eluate to recover. Figure 12. Elution from 3xFLAG resin. (Left) Photo of co-immunoprecipitation after addition of 3xFLAG peptide to the anti-FLAG affinity resin bed. The eluate (supernatant layer) should be carefully removed by pipetting. (Right) Photo of remaining resin after separating the eluate. Quality control (recommended, optional) F1. Silver stain SDS-PAGE Dilute protein ladder 1:50 in working Laemmli buffer and load 6 µL of the diluted ladder into an empty lane of a precast polyacrylamide gel. Mix 5 µL of eluate with 5 µL of 2× working Laemmli buffer. Place on a heat block for 3 min at 95°C. Load the denatured sample onto precast gel. Run gel at 170 V for 35 min or until the leading dye approaches the bottom of the gel. We recommend the SilverQuest Silver Staining kit (Thermo Fisher Scientific) to perform silver staining of co-IP elutions because the high sensitivity of this method uses relatively low quantities of material. Follow the “Fast Staining” protocol according to the manufacturer’s handbook. For examples of silver-stained SDS-PAGE, see Shen et al. (2015) or Cooney et al. (2019). Protein identities can be confirmed by mass spectrometry proteomics or immunoblot. F2. Negative stain TEM Negative stain transmission electron microscopy (TEM) provides a quick and simple way to assess particle quality before committing resources to cryo-EM. Consult your institution’s TEM facilities for access to instrumentation. For a general overview of negative stain TEM, including protocol and staining options, please see the Negative Stain module of the cryo-EM 101 website: https://cryoem101.org/chapter-1/#part5 (Shen and Iwasa, 2018). Negative stain grids are prepared at room temperature. Place continuous carbon (Formvar/carbon 200 mesh) grids with the sample application side face up on a glass slide wrapped in parafilm (Figure 13). Figure 13. Preparation of grids for glow discharging. (Left) Grids are placed on a Parafilm-wrapped glass slide. (Right) Slide is inserted in Pelco easiGlow glow discharge chamber. Place the slide on the glow discharge platform and cover with the glass chamber. Glow discharge the grids at 15 mA for 30 s. Picking up the grid with DUMONT tweezers by handling the outer rim of the grid. Clamp the tweezers with the clamping ring. Apply 3.5 µL of the sample onto the grid for one minute. Gently blot off the sample using 595 Vitrobot filter paper. Wash the grid by submerging the side the sample was applied to into a droplet of water. Remove the excess water by blotting with filter paper. Pipette 3.5 µL of 1% uranyl acetate (see Recipes) onto the grid for 20 s. Blot off the uranyl acetate solution with the filter paper. Note: All disposables in contact with uranyl acetate must be disposed according to institutional safety guidelines. Allow the grid to air-dry for several minutes before storing. Image the grid using a transmission electron microscope to confirm that particles are visible and at a sufficiently high concentration for cryo-EM (Figure 14). Figure 14. Example of negative stain TEM image of Cdc48 complexes isolated by co-immunoprecipitation of Shp1-3xFLAG (Cooney et al., 2019). Image recorded on a Tecnai T12 transmission electron microscope equipped with a Gatan Orius CCD camera. Particles meet criteria to proceed with cryo-EM specimen preparation, including monodispersity, concentration, and size. Cryo-EM specimen preparation Sample crosslinking (optional) Crosslinking may be necessary to maintain structural integrity of complexes during cryo-EM sample preparation (Monroe et al., 2017). Particle exposure to the hydrophobic air–water interface after blotting is well known to induce common problems associated with cryo-EM, including preferred orientations (Noble et al., 2018) and particle denaturation (D’Imprima et al., 2019). If performing a crosslinking reaction, prepare a 0.2% stock of glutaraldehyde. Add 1.4 μL of 0.2% glutaraldehyde to 30 μL of eluate. Final concentration of glutaraldehyde is 0.009% (v/v; approximately 0.9 mM). Mix by pipetting and incubate crosslinking reaction at room temperature for 10 min. Alternatively, the reaction may be performed on ice instead. The crosslinking conditions (including glutaraldehyde concentration, temperature, and incubation time) may require optimization to achieve the desired extent of crosslinking. Quench the reaction using 1 μL of 100 mM Tris pH 7.4 (final Tris concentration is approximately 3 mM) and place the sample back on ice. Confirm the extent of crosslinking by silver stained SDS-PAGE (section F). For an example, see Cooney et al. (2019). Cryo-EM specimen preparation A video of the vitrification procedure using the Mk. II Vitrobot is available at https://youtu.be/gbA5BytYBhc (credit, Helen M. Donelick). Other general guidelines and advice about grid vitrification are available on CryoEM 101: https://CryoEM101.org (Shen and Iwasa, 2018). Glow discharge grid (Quantifoil or UltrAufoil) by following the steps in section F2, steps 1–3. Note that the glow discharge settings may require optimization. Open the compressed nitrogen gas tank and turn on Mk. II Vitrobot. Fill the humidifier cup to the proper volume with distilled water and attach to Vitrobot. Set the Vitrobot humidity to 100% and temperature to 4°C. Place new blotting paper onto the blotting pads. Allow 30 min for the Vitrobot to equilibrate at the specified temperature and humidity. Adjust the various plunge freeze settings in the software. Set the instrument to the following settings: Wait time: 20 s Plunge time: 4 s Blot time: 4 s for UltrAuFoil (gold) grid or 7 s for Quantifoil (carbon) grid Blotting offset: -1 mm Note that these settings may require optimization to achieve the desired ice thickness. Fill a dewar with fresh liquid nitrogen. Prepare the freezing apparatus: place the brass cup and the grid box pedestal in the insulated bowl, place the metal spider on top of the brass cup, and place the float around the grid box pedestal. Place a labeled grid box into one of the locations on the grid box pedestal. Fill the insulated bowl with liquid nitrogen. This may need to be repeated a few times as the nitrogen will evaporate quickly during the initial cooling. Pre-chill the brass cup using liquid nitrogen. After the liquid nitrogen in the brass cup has evaporated, place the glass pipette tip from the ethane tube into the brass cup and open the valves to the compressed ethane tank. As the ethane starts to liquify it will make a whistling sound. Fill the brass cup with liquid ethane until the level of liquid ethane reaches the spider. Close the ethane tank and allow the liquid ethane in the brass cup to start chilling. Remove the spider immediately after ethane begins to turn to a slush. Make sure that the ethane does not completely freeze solid; if this happens, place a separate room temperature spider upside down on the brass cup to melt the ethane into a slush. Load a glow-discharged grid using the Vitrobot loading tweezers onto the Vitrobot. Be sure to pick up the grid by its outer rim to prevent damage to the sample application area. Click “Place New Sample” to lower the Vitrobot arm into position. Place the tweezers on the Vitrobot arm and click “Continue.” Make sure the sample application side of the grid is facing the side opening on the Vitrobot. Place the freezing apparatus on the Vitrobot and click “Continue.” Click “Start Process” and apply 3.5 μL of sample onto the grid through the side opening on the Vitrobot. Click “Continue” to proceed with grid blotting and plunge-freezing into the liquid ethane. Transfer the grid in a swift motion from ethane to a labeled storage container submerged in liquid nitrogen, taking care to not bend the grid. Repeat steps 19–25 for the desired number of grids. Each grid storage container can accommodate four cryo-EM specimens. Store the grid storage containers in a storage dewar until they are ready to be loaded into a cryo-TEM. Shut down Vitrobot according to the manufacturer's instructions. Data analysis Data processing for cryo-EM will be dependent on instrumentation, particle size, and available computational hardware and software. An example of a cryo-EM data processing workflow can be found in the supplementary materials of Cooney et al. (2019). Available for free on PubMed: PMCID: PMC7362759. Limitations This protocol is designed for the isolation of endogenous soluble proteins and their associated complexes. We have not tested the suitability of our protocol for membrane proteins, which would require additional optimization steps with detergents. Troubleshooting The procedures described in this protocol are recommended as starting points. Many variables may need to be explored to optimize yield and purity for each sample. Such variables may include buffer pH, salt concentration, quantity of starting lysate and immobilizing resin, incubation times, the number of washes, and elution conditions, each of which may need to be explored to generate suitable samples for cryo-EM. Recipes Stock solutions 50× TAE 242 g of Tris base 57.1 mL of glacial acetic acid 100 mL of 0.5 M EDTA solution, pH 8.0 Fill with ddH2O to a final volume of 1 L. This buffer can be stored at room temperature for months. 5× TBS 12 g of Tris base 44 g of NaCl Fill with ddH2O to a final volume of 1 L. This buffer can be stored at room temperature for months. 10× transfer buffer 30.2 g of Tris base 144 g of glycine Fill with ddH2O to a final volume of 1 L. This buffer can be stored at 4°C for months. 1 M HEPES-KOH, pH 7.4 238.30 g of HEPES Fill with ddH2O to 800 mL. Adjust pH to 7.4. Fill with ddH2O to 1,000 mL. Store at 4°C. 1 M KOAc 98.2 g of KOAc Fill with ddH2O to a final volume of 1 L. Store at 4°C. 1 M Mg(OAc)2 214.46 g of Mg(OAc)2 Fill with ddH2O to a final volume of 1 L. Store at 4°C. 1 M CaCl2 147.02 g of CaCl2 Fill with ddH2O to a final volume of 1 L. Store at 4°C. 1 M D-sorbitol 182 g D-sorbitol Fill with ddH2O to a final volume of 1 L. Store at 4°C. 1 M MgCl2 203.3 g of MgCl2·6H2O Fill with ddH2O to a final volume of 1 L. Store at 4°C. 1 M DTT 3.86 g of DTT Fill with ddH2O to a final volume of 25 mL. Prepare 100 μL aliquots and store at -20°C. 2× IP buffer 50 mM of HEPES-KOH, pH 7.4 200 mM of KOAc 20 mM of MgCl2 Store at 4°C. 1 M lithium acetate (LiOAc) 10.2 g of LiOAc Fill with ddH2O to a final volume of 100 mL. Store at 4°C. Gels 1% agarose gel 100 mL of 2× TAE (50× TAE diluted with ddH2O) 2 g of agarose Fill with ddH2O to a final volume of 200 mL. Dissolve by boiling before pouring. Buffers 1× TAE Diluted with ddH2O from 50× TAE stock. Final volume depends on the size of the electrophoresis tank. Prepare sufficient volume to submerge agarose gel. Working transfer buffer 5% methanol 1× transfer buffer (diluted with ddH2O from 10× transfer buffer stock) Blocking solution 5% milk powder (w/v) 1× TBS (diluted from 5× TBS stock) 0.05% Tween 20 Fill with ddH2O to a final volume of 25 mL. TBST solution 1× TBS (diluted with ddH2O from 5× TBS stock) 0.1% Tween 20 Fill with ddH2O to a final volume of 1 L. Yeast lysis buffer 50 mM of HEPES-KOH pH 6.8 150 mM of KOAc 2 mM of Mg(OAc)2 1 mM of CaCl2 200 mM of sorbitol Fill with ddH2O to a final volume of 1 L. Store at 4°C. Wash buffer 1 1× IP buffer (diluted with ddH2O from 2× IP buffer stock) 5% glycerol 0.1% Igepal (CA-630) 1 mM of DTT Protease inhibitor cocktail: 0.5 μg/mL leupeptin, 0.5 μg/mL aprotinin, 0.7 μg/mL pepstatin, and 16.67 μg/mL PMSF Fill with ddH2O to a final volume of 20 mL (per co-IP). Note on preparation: Add DTT and PMSF last. Wash buffer 2 1× IP buffer (diluted with ddH2O from 2× IP buffer stock) 1 mM DTT Fill with ddH2O to a final volume of 10 mL (per co-IP). 2× working Laemmli sample buffer 2× Laemmli sample buffer Add 50 μL of beta-mercaptoethanol per 950 μl of 2× Laemmli sample buffer. Mixes Transformation PCR Mix (according to Bio-Rad iProof HF manual) For each PCR reaction: 25 µL of 2× iProof Master mix 2.5 µL of forward primer (from 10 µM stock) 2.5 µL of reverse primer (from 10 µM stock) 10 ng of pTF272 plasmid Add ddH2O to a final volume of 50 µL. Colony PCR Mix For each PCR reaction: 10 µL of 2× iProof HF Master mix 1 µL of forward primer (from 10 µM stock; final concentration 0.5 µM) 1 µL of reverse primer (from 10 µM stock; final concentration 0.5 µM) 1.5 µL of SDS-released genomic mix Add ddH2O to a final volume of 20 µL. PEG mix 240 μL of 50% PEG 3350 36 μL of 1 M LiOAc 10 μL of ssDNA (10 mg/mL) 25 μL of PCR product (use half of a 50 μL PCR reaction product) Add ddH2O to a final volume of 350 µL. Reagents Lithium acetate (LiOAc) For each yeast transformation reaction: 100 mM of LiOAc (diluted with ddH2O from 1 M LiOAc stock) Final volume of 1 mL. 1% uranyl acetate 10 mg of uranyl acetate salt Add ddH2O to a final volume of 1 mL. Notes on preparation: Mix by vortexing and incubate solution at 37°C for 2 h until dissolved. Filter using 0.22 µm pore size membrane filter. Aliquot final solution into 250 µL aliquots. Store in an opaque tube at 4°C. *HAZARDOUS MATERIAL: Uranyl acetate is a radiological hazard and requires safety precautions. Consult your institutional guidelines when handling this material. Plates and media YPAD plates 5.5 g of yeast extract 11 g of peptone 28 mg of adenine (Note: Adenine is not required to support growth of the BY4741 yeast strain. We use it to support other yeast strains.) 11 g of agar 50 mL of sterile 20% glucose Add ddH2O to a final volume of 500 mL. Sterilize by autoclave, allow the media to cool to 55–60°C, and then pour into sterile Petri dishes. Allow agar to solidify at room temperature and then store the dishes at 4°C. YPAD + hygromycin plates Follow YPAD plate recipe. After autoclaving, allow the media to cool to 55–60°C, and then add hygromycin B to a final concentration of 0.3 mg/mL just before pouring dishes. CAUTION: Hygromycin B is toxic, handle in hood. YPD liquid media (per liter) 20 g of peptone 20 g of dextrose (glucose) 10 g of yeast Add ddH2O to a final volume of 1 L, followed by autoclave sterilization. Acknowledgments This work was supported by grants to PSS (NIH R35 GM133772, R25 EY029124), IC (NIH F31 CA254427), DM (NIH Ruth L. Kirschstein Institutional National Research Service Award T32GM122740), and HMD (NIH Ruth L. Kirschstein Institutional National Research Service Award T32GM122740). This protocol was derived from original research reported in Shen et al. (2015) and Cooney et al. (2019). Competing interests The authors declare no competing interests. References Akada, R., Murakane, T. and Nishizawa, Y. (2000). DNA extraction method for screening yeast clones by PCR. Biotechniques 28(4): 668-670, 672, 674. Cooney, I., Han, H., Stewart, M. G., Carson, R. H., Hansen, D. T., Iwasa, J. H., Price, J. C., Hill, C. P. and Shen, P. S. (2019). Structure of the Cdc48 segregase in the act of unfolding an authentic substrate. Science 365(6452): 502-505. D'Imprima, E., Floris, D., Joppe, M., Sanchez, R., Grininger, M. and Kuhlbrandt, W. (2019). Protein denaturation at the air-water interface and how to prevent it. Elife 8: e42747. Ghaemmaghami, S., Huh, W. K., Bower, K., Howson, R. W., Belle, A., Dephoure, N., O'Shea, E. K. and Weissman, J. S. (2003). Global analysis of protein expression in yeast. Nature 425(6959): 737-741. Gietz, R. D. and Schiestl, R. H. (2007). Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 35-37. Ho, C. M., Li, X., Lai, M., Terwilliger, T. C., Beck, J. R., Wohlschlegel, J., Goldberg, D. E., Fitzpatrick, A. W. P. and Zhou, Z. H. (2020). Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu. Nat Methods 17(1): 79-85. Kühlbrandt, W. (2014). Biochemistry. The resolution revolution. Science 343(6178): 1443-1444. Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14(10): 953-961. Monroe, N., Han, H., Shen, P. S., Sundquist, W. I. and Hill, C. P. (2017). Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase. Elife 6: e24487. Nakane, T., Kotecha, A., Sente, A., McMullan, G., Masiulis, S., Brown, P., Grigoras, I. T., Malinauskaite, L., Malinauskas, T., Miehling, J., et al. (2020). Single-particle cryo-EM at atomic resolution. Nature 587(7832): 152-156. Noble, A. J., Wei, H., Dandey, V. P., Zhang, Z., Tan, Y. Z., Potter, C. S. and Carragher, B. (2018). Reducing effects of particle adsorption to the air-water interface in cryo-EM. Nat Methods 15(10): 793-795. Shen, P. S. (2018). The 2017 Nobel Prize in Chemistry: cryo-EM comes of age. Anal Bioanal Chem 410(8): 2053-2057. Shen, P. S., Park, J., Qin, Y., Li, X., Parsawar, K., Larson, M. H., Cox, J., Cheng, Y., Lambowitz, A. M., Weissman, J. S., et al. (2015). Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347(6217): 75-78. Shen, P. S., Iwasa, J. H. (2018). CryoEM 101: https://cryoem101.org/. Skalidis, I., Kyrilis, F. L., Tuting, C., Hamdi, F., Chojnowski, G. and Kastritis, P. L. (2022). Cryo-EM and artificial intelligence visualize endogenous protein community members. Structure 30(4): 575-589 e576. Yip, K. M., Fischer, N., Paknia, E., Chari, A. and Stark, H. (2020). Atomic-resolution protein structure determination by cryo-EM. Nature 587(7832): 157-161. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biophysics > Microscopy > Cryogenic microscopy Biochemistry > Protein > Isolation and purification Biochemistry > Protein > Imaging Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Isolation of Mitochondria from Ustilago maydis Protoplasts Juan Pablo Pardo [...] Lucero Romero-Aguilar Jan 5, 2022 1625 Views Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris Adam Turner [...] Angie Gelli Mar 5, 2023 1395 Views Purification of Human Cytoplasmic Actins From Saccharomyces cerevisiae Brian K. Haarer [...] Jessica L. Henty-Ridilla Dec 5, 2023 521 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,597	https://bio-protocol.org/en/bpdetail?id=4597&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Establishment of Restraint Stress–induced Anorexia and Social Isolation–induced Anorexia Mouse Models IP Ingrid Camila Possa-Paranhos KC Kerem Catalbas JB Jared Butts KO Kyle O’Berry PS Patrick Sweeney Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4597 Views: 794 Reviewed by: Edgar Soria-GomezAbraam YakoubMiao He Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Translational Medicine Apr 2021 Abstract Anorexia nervosa (AN) is a devastating neuropsychiatric disease with a prevalence rate of approximately 0.3%–1% among women and morbidity and mortality rates among the highest of all neuropsychiatric disorders. The disease etiology is complex but primarily characterized by reduced food intake and body weight, and intense anxiety and fear associated with gaining weight. Existing rodent models of AN are useful and capture features of the disease, but either require specialized genetic mouse models or are difficult to implement in mice. Here, we describe two simple mouse models of stress-induced anorexia that are easy to implement in basic research labs, and capture core features associated with AN, such as reduced food intake in the context of social/physical stress and increased anxiety-related behavior. These protocols provide reproducible and robust assays for stress-induced anorexia and may be implemented with additional assays to probe the neural circuitry mediating the effects of psychological stress on feeding in mice. Graphical abstract Keywords: Anorexia nervosa Eating disorders Mouse models Feeding Restraint Stress–induced anorexia Neural circuits Background Feeding behavior is controlled by multiplex neural circuitry throughout the brain, including the hypothalamus, hindbrain, and mesolimbic dopamine circuitry (Sohn et al., 2013). While dedicated feeding circuits exist in the brain, substantial experimental evidence indicates that these are bidirectionally connected to neural circuitry controlling stress and emotion (Sweeney and Yang, 2017). These interactions are especially important when considering the biological mechanisms governing neuropsychiatric eating disorders such as anorexia nervosa (AN), since eating disorders are characterized by alterations in both feeding and emotional state (Keski-Rahkonen, 2007). As such, the underlying biological causes of eating disorders may be mediated by neural circuit alterations in brain circuits at the intersection of feeding and emotion. Thus, mouse models of stress-induced anorexia have value in dissecting the neural circuit and molecular mechanisms for normal and pathological feeding responses to stress. Anorexia nervosa is a serious neuropsychiatric disorder characterized by reduced feeding and body weight and co-morbid neuropsychiatric traits, such as increased anxiety and depressive-related behaviors (Bulik et al., 2006). The disease occurs in approximately 0.3%–1% of women in the United States and is associated with a high rate of morbidity and mortality (Bulik et al., 2006). Since the disease is characterized by reduced feeding in the context of psychological and psychosocial stress, substantial efforts have been made to model aspects of AN in rodents (Scharner and Stengel, 2020; Zhang and Dulawa, 2021). Although AN is a complex disease with human-specific etiology, important components of the disease can be effectively modeled in rodents, such as reduced feeding in the face of stress and anxiety, compulsive exercise, and elevated anxiety and depressive behavior. These models provide an important entry point towards understanding the biological mechanisms mediating dysregulated eating and may provide a pathway towards novel therapeutic options for eating disorders. One commonly used rodent model of AN is the activity-based anorexia (ABA) mouse model (Carrera et al., 2014). The ABA model subjects rodents (mice or rats) to restricted food access in the presence of a running wheel. In this model, a subset of mice will stop eating and exercise excessively, leading to starvation and death. This model is useful as it incorporates multiple characteristics of AN, such as excessive exercise and voluntary anorexia, and is more effective in female rodents (approximately 90% of AN cases occur in females). However, the model relays on the presence of a running wheel and restricting food access to a user-defined time window (usually between 1 and 4 h/day). Rodents in this model develop gradual hypothermia and may exercise excessively to generate heat. As such, the ABA model does not work when supplemental heat is provided to the rodents (Gutierrez et al., 2008). Thus, although hypothermia is often observed in AN, the ABA model may more closely model physiological forms of anorexia resulting secondarily to hypothermia. Further, the model can be difficult to implement in mice (as opposed to rats), and only a subset of mice are sensitive to the ABA model. Given the limitations of the ABA model, additional attempts have been made to develop mouse models that capture the AN phenotype more accurately. One model that shows promise is the “GED” (gene, environment, deprivation) model developed in the lab of Lori Zeltser (Madra and Zeltser, 2016; Madra et al., 2015). The GED model uses a transgenic mouse line containing a BDNF-Val66Met mouse mutation that has been associated with neuropsychiatric disorders. In the GED model, the BDNF-Val66Met mice are subjected to social isolation during adolescence (environmental manipulation) and given 20%–30% calorically restricted food access during a user-defined time window of 3 h twice a day. Thus, this model incorporates a genetic component (BDNF-Val66Met mutation), an environmental stressor (social isolation), and food deprivation by both caloric restriction and limited food access to produce anorexia-related behaviors. A subset of mice exposed to the GED model voluntarily stop eating and develop severe anorexia. Like in the ABA model, in the GED model the voluntary starvation phenotype observed in AN is captured and the starvation phenotype is more common in female mice. However, this model relays on a transgenic mouse line, which limits the use of some modern behavioral neuroscience approaches, such as optogenetics and chemogenetics, which often require additional transgenic mouse lines and/or multiple breeding steps. Therefore, there is a need for simplified stress-induced anorexia models which can be implemented in all basic biology labs and transgenic mouse lines (Table 1). Here, we describe two simple models of stress-induced anorexia that are easy to implement in mice: restraint stress–induced anorexia and social isolation–induced anorexia. These models do not require specialized equipment or transgenic mouse strains and can be implemented in most biology and neuroscience laboratories. Both of these assays may serve as useful models for disorders characterized by reduced feeding in the face of physical or psychosocial stress, such as the reduced appetite associated with stress-related mood disorders or the impaired food-seeking and consumption observed in AN. Table 1. Strengths and weaknesses of common models of stress-induced anorexia Method Advantages Disadvantages Activity-based anorexia -Easy to implement -Self-induced anorexia and starvation -Excessive exercise -More severe in female rodents -Model may rely on physiological anorexia secondary to hypothermia, as opposed to psychologically induced anorexia -Difficult to implement in mice (relative to rats) G.E.D. model -More severe in females -Self-induced anorexia and starvation in a subset of mice -Anorexia due to a combination of genetic and environmental factors -Requires a specialized transgenic mouse line Restraint induced–anorexia -Easy to implement -Reliably induced anorexia and weight loss in mice -Easily combined with advanced behavioral neuroscience approaches (i.e., optogenetics, chemogenetics) -Not sexually dimorphic -Usually does not induce self-starvation -May not capture psychologically induced anorexia Social isolation–induced anorexia -Easy to implement -Anorexia resulting from chronic psychological stress -Anorexia phenotype is usually mild -Better suited for determining factors that enhance psychological-induced anorexia -Unknown sexual dimorphism Subjects and housing: Adult male and female C57/BL6J littermate mice may be used for both social isolation and restraint stress–induced anorexia studies. Given that neuropsychiatric eating disorders are highly sexually dimorphic, it is important to include both sexes in the experimental design and to determine if behavioral phenotypes are differentially expressed in males and females. For restraint stress–induced anorexia assays, littermate mice should be group-housed with 3–4 mice per cage. Lights in the housing facility should be on a 12:12 h light/dark cycle. Since mice are nocturnal and consume most of their food during the dark period, it is recommended to perform feeding assays at the start of the dark cycle, although light cycle measurements may also be performed. Materials and Reagents Group-housed mice 50 mL conical tubes with holes cut into the sides (for restraint stress–induced anorexia; Figure 1) Figure 1. Setup of restraint stress–induced anorexia apparatus. Separate cages are used for male and female mice, respectively Clean mouse cages Standard chow mouse diet (Formulab Diet 5008 or similar approved mouse diet) Standard home cage water bottles Standard food hoper Cage enrichment (such as nestlets) Scale for measuring food intake and body weight (Satorius Entris II Essential Precision Balance or similar) Lab notebook for recording food intake and body weight Instructions to make restraint stress apparatus A simple restraint stress–anorexia apparatus can be constructed from 50 mL conical tubes (Figure 1). Make 6–12 holes, depending on how many you can fit, on the sides of the conical tubes to enable mice to breathe. Care should be taken to make the holes big enough for air flow, but not big enough for the mice to fit their nose into, so they do not hurt themselves in the borders. Holes can be made by using a tool such as a heat pen for melting plastic. The mouse identifying information (i.e., ear tag number and sex) can be written on the side of each tube. During restraint sessions, the mice can be placed into the restrainers within their resident cage (Figure 1). Following each restraint session, the restraint tubes should be cleaned with soap and water and allowed to dry before re-using on each mouse. Procedure Restraint stress–induced anorexia Group house mice, 3–4 per cage and separated by sex; acclimate the mice to housing conditions for one week prior to beginning food intake measurements. During this period, mice should be handled daily by the investigator to acclimate the mice to handling-associated stress. To acclimate the mice to handling, pick up each mouse and scruff gently. Remove the water bottle and food for one hour prior to baseline feeding measurements (to control for the one-hour restraint period in which mice will not have access to food and water). Place mice in a clean cage immediately following the removal of the food and water so that mice do not have access to any food crumbs that may be on the bottom of the cage. Note: All feeding measurements should be performed at the same time of day in the same experimental location. After one hour of food and water removal, add a water bottle to the clean cage with fresh bedding and mouse enrichment (i.e., nestlet or wooden block) and between 20 and 30 g of food to the food hopper (mice eat approximately 3–5 g/day/mouse). Measure the body weight of the mice and amount of food immediately prior to adding the food and water to the cages. Measure the amount of food left in the food hopper 1, 2, 4, and 24 h after adding the food. Food intake per mouse is calculated at each time point by dividing the amount of food consumed by the number of mice in each cage. Measure the body weight of the mice 24 h after adding the food. Repeat steps A2–A4 for 3 to 5 days to establish baseline levels of food intake for each mouse prior to restraint stress. On restraint stress day (following baseline food intake measurements), grab the mouse by the tail and place each mouse in a 50 mL conical tube (see Figure 1) for sixty min, inserting the head of the animal first followed by the rest of its body, including the tail. For the control animals, only remove the food and water to give them a similar environment of food and water restriction as the restraint mice for sixty minutes. Notes: Institutional animal care and use approval (IACUC) must be obtained to perform experimental work on mice. It is essential to monitor each mouse during the restraint period to ensure that mice are breathing adequately. To obtain the food intake of individual mice with and without restraint stress, the above procedure (steps 1–7) may also be performed on singly housed mice. However, mice should be single-caged for a minimum of one week prior to starting restraint stress–anorexia experiments in order to acclimate mice to single housing. Following sixty minutes of restraint, return the mice to the home cages with their littermates and add food and water as described in step A3. Measure food intake as described in step A4. Repeat steps A7–A9 for an additional 7–10 days to examine the effects of chronic restraint–stress on food intake and body weight. Social isolation–induced anorexia Group house mice with 3–4 per cage and acclimate the mice to housing conditions for one week prior to beginning food intake measurements. During this period, mice should be handled daily by the investigator to acclimate the mice to handling-associated stress. Change mice to a fresh cage, with fresh bedding and mouse enrichment. Measure the body weight of each mouse. Add 20–30 g of food to the cages and measure the amount of food added to each cage. Return to measure the amount of remaining food 1, 2, 4, and 24 h later. Calculate the amount of food consumed by each mouse by dividing the total amount of food consumed by the number of mice in the cage. Note: All feeding experiments should be performed at the same time of day in the same experimental location. Since mice are nocturnal and consume most of their food during the dark cycle, optimal results will be obtained by performing feeding experiments at the onset of the rodent’s dark cycle. Repeat step B2 for 3–5 consecutive days to establish a baseline measurement of food intake and body weight prior to social isolation. At the same time of day as steps B2-B3, single-cage each mouse, measure each mouse’s body weight, and add 10–15 g of food to each cage. Return to measure the amount of food in each cage 1, 2, 4, and 24 h later. For the control group, continue to calculate the amount of food consumed by each mouse by dividing the total amount of food consumed by the number of mice in the cage. Note: Mice consume food based on a circadian structure. Care must be made to perform all feeding measurements at the same time each day. Repeat step B4 for an additional 3–14 days. Data analysis Exclusion criteria: During stress-induced anorexia experiments, researchers must abide by IACUC approved exclusion criteria for removing animals from experimental conditions. These exclusion criteria will vary at each research institute and individual investigators should discuss exclusion criteria with veterinary staff prior to initiating experiments. Generally, mice are removed from experimental sessions when signs of pain or distress, such as reduced activity or piloerection, are observed or if any of the animal’s body weight drops by more than 20 percent. Following the completion of restraint stress induced–anorexia or social isolation–anorexia experiments, food intake and body weight is compared for each mouse following no-restraint vs. restraint-stress (paired, within samples comparisons). It is expected that restraint stress will reduce food intake and body weight in mice. For restraint stress–induced and social isolation–induced anorexia, we typically observe 10%–20% changes in food intake in the hours immediately following restraint stress, although results will vary depending on mouse strain, sex, and age of mice. The effects of pharmacological or other manipulations to reduce or enhance stress-induced anorexia can also be evaluated by determining the effect of the manipulation on food intake and body weight. The specific experimental design will vary slightly depending on the specific research question. Conclusion In this article, we describe two simple mouse models for inducing stress-induced anorexia. These models provide a valuable experimental tool for researchers interested in determining the neural mechanisms connecting stress with feeding behavior. Acknowledgments This work was funded in part by awards R00 DK127065 (P.S.), Brain and Behavior Research Foundation Young Investigator Award (P.S.), and the Foundation for Prader Willi Research (P.S.). The original methods in this paper are based on our prior publication (Sweeney et al., 2021; DOI: 10.1126/scitranslmed.abd6434). Competing interests There are no conflicts of interest or competing interests. Ethics All experiments were approved by the University of Illinois Institutional animal care and use committee (IACUC). References Bulik, C. M., Sullivan, P. F., Tozzi, F., Furberg, H., Lichtenstein, P. and Pedersen, N. L. (2006). Prevalence, heritability, and prospective risk factors for anorexia nervosa. Arch Gen Psychiatry 63(3): 305-312. Carrera, O., Fraga, A., Pellon, R. and Gutierrez, E. (2014). Rodent model of activity-based anorexia. Curr Protoc Neurosci 67: 9 47 41-11. Gutierrez, E., Cerrato, M., Carrera, O. and Vazquez, R. (2008). Heat reversal of activity-based anorexia: implications for the treatment of anorexia nervosa. Int J Eat Disord 41(7): 594-601. Keski-Rahkonen, A., Hoek, H. W., Susser, E. S., Linna, M. S., Sihvola, E., Raevuori, A., Bulik, C. M., Kaprio, J. and Rissanen, A. (2007). Epidemiology and course of anorexia nervosa in the community. Am J Psychiatry 164(8): 1259-1265. Madra, M. and Zeltser, L. M. (2016). BDNF-Val66Met variant and adolescent stress interact to promote susceptibility to anorexic behavior in mice. Transl Psychiatry 6: e776. Scharner, S. and Stengel, A. (2020). Animal Models for Anorexia Nervosa-A Systematic Review. Front Hum Neurosci 14: 596381. Sohn, J. W., Elmquist, J. K. and Williams, K. W. (2013). Neuronal circuits that regulate feeding behavior and metabolism. Trends Neurosci 36(9): 504-512. Sweeney, P., Bedenbaugh, M. N., Maldonado, J., Pan, P., Fowler, K., Williams, S. Y., Gimenez, L. E., Ghamari-Langroudi, M., Downing, G., Gui, Y., et al. (2021). The melanocortin-3 receptor is a pharmacological target for the regulation of anorexia. Sci Transl Med 13(590): eabd6434. Sweeney, P. and Yang, Y. (2017). Neural Circuit Mechanisms Underlying Emotional Regulation of Homeostatic Feeding. Trends Endocrinol Metab 28(6): 437-448. Zhang, J. and Dulawa, S. C. (2021). The Utility of Animal Models for Studying the Metabo-Psychiatric Origins of Anorexia Nervosa. Front Psychiatry 12: 711181. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Neuroscience > Behavioral neuroscience Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,598	https://bio-protocol.org/en/bpdetail?id=4598&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Reliable and Consistent Method to Quantify Percent Lethality and Life Span in Drosophila melanogaster PK Priyanka Kumari * UA Ushashi Ain * HF Hena Firdaus (contributed equally to this work) Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4598 Views: 929 Reviewed by: Nafisa M. JadavjiChetan PaliwalDjamel Eddine Chafai Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Chemosphere Sep 2019 Abstract Drosophila melanogaster is a classic model organism to study gene function as well as toxicological effects. To study gene function, the expression of a particular gene of interest is disrupted by using the widely explorable Drosophila genetic toolkit, whereas to study toxicological effects the flies are exposed to a particular toxicant through diet. These experiments often require the quantification of lethality from embryonic to adult stages, as well as the assessment of the life span in order to check the role of the gene/toxicant of interest in Drosophila. Here, we propose an experimental protocol that enables a consistent and rigorous assessment of lethality and life span of cadmium chloride (CdCl2)–exposed or genetically perturbed flies [downregulation and overexpression of the cytosolic Cu, Zn superoxide dismutase (SOD1) gene], consecutively. The protocol insists upon the requirement of one single experimental setup that is unique, distinctive, and cost-effective as it engages minimal laboratory equipment and resources. The described methods lead to the smooth observation of the embryos, their successive stagewise transition, and life span of the adult flies post eclosion. Additionally, these methods also facilitate the assessment of crawling and climbing behavioral parameters of the larvae and adults, respectively, and allow the calculation of lethal concentration (LC50) for the mentioned toxicant as well as median survival of the flies, which can be a determining factor in proceeding with further stages of experiments. Graphical abstract Keywords: Drosophila Toxicant exposure Mutant developmental analysis Lethality Life span Background Drosophila melanogaster, a holometabolous, invertebrate model organism, has contributed extensively towards analyzing the roles of various phylogenetically conserved genes (Green et al., 2018; Dubey et al., 2020). Toxicological studies utilizing flies have also aided in the assessment of the effects of heavy metals that interfere with important biological processes (Nanda and Firdaus, 2021; Nanda and Firdaus, 2022), including immune system (Nanda et al., 2019). Drosophila research has also paved a way for the exploration of innumerable scientific questions in various branches of biological sciences. Notably, Drosophila melanogaster genome is 60% homologous to humans (Adams et al., 2000). Analysis of Drosophila health and activity is a fundamental aspect to quantify any adversities either due to genetic manipulation or dietary exposure to toxicant. Among the various life traits, quantifying stagewise lethality, checking life span (Carey et al., 2006), and examining locomotor and reproductive capabilities (Rogina et al., 2007) are the biomarkers most often used (Landis et al., 2020; Olcott et al., 2010). Several behavioral assays also facilitate the analysis of a typical mutant from embryonic to adult stage (Ain and Firdaus, 2021). The Cu, Zn superoxide dismutase (SOD1) is an antioxidant enzyme that provides protection against excessive reactive oxygen species (ROS) in Drosophila melanogaster. The three isoforms of SOD in Drosophila, namely SOD1, mitochondrial Mn superoxide dismutase SOD 2 (SOD2), and extracellular Cu Zn superoxide dismutase (SOD 3), are encoded by genes located on separate chromosomal loci (Duttaroy et al., 1994; Blackney et al., 2014). In this protocol, we used Drosophila wherein the cytosolic SOD1 gene expression was either overexpressed or downregulated in a ubiquitous manner through genetic crosses between the driver Act-5c-GAL4 and the responder RNAi/overexpression fly lines. This enabled the study of the role of SOD1 gene in life span, lethality, and motility behaviour of Drosophila. We describe a simple continuous method that requires a single experimental setup to perform both the lethality and life span assays of the genetically altered or toxicant-exposed Drosophila. The protocol is unique and significant, due to the fact that it highlights the usage of minimal laboratory equipment and uses one single experimental setup for continuous monitoring of lethality followed by survival analysis and crawling and climbing assays in larvae and adults, respectively. Materials and Reagents Autoclavable glass vials (Borosil, custom made, RIVIERA brand) Black craft paper (120 GSM) Brush (soft and synthetic bristles) Cylinder with spout, CL B (Borosil, catalog number: 3022006) Conical flask (Borosil) Graph paper (0.2 mm2) Measuring cylinder (Borosil) Non-absorbent cotton Petri plate (Borosil, catalog number: 3165077 Pipette and microtips (100–1,000 μL) (Thermo Scientific, catalog number: NH27702) Stirrer (Borosil) Tapping pad (self-made with soft, cushion material) Tissue roll, 20 m (Indiamart, catalog number: 128587378) Watch glass, 75 mm (MRSC Borosilicate) Agar agar, type I (HiMedia, catalog number: 0000227116) Agarose low EEO, regular grade (SRL, catalog number: 9303545) Cadmium chloride (CdCl2) (Rankem, catalog number: N041M14) Corn flour (single batch) (Dr. RBL’s Corn Flour Atta) Propionic acid (Qualigens, catalog number: 26955) Sucrose (TITAN, CAS-57-50-1) Yeast extract: TM media Drosophila stocks (Bloomington Drosophila Stock Center): a) BS #24750: w[1]; P{w[+mC]=UAS-Sod1.A}B37 (express SOD1 under UAS control); mentioned as the simplified name w;+;UAS-SOD1 b) BS #24493: w[];P{w[+mC]=UAS-Sod1.IR}F103/SM5 (express dsRNA of SOD1 under UAS control); mentioned as w;UAS-SOD1-RNAi;+ c) w;Act-5c-GAL4/CyO-GFP;+ (ubiquitous GAL4) d) Oregon R (wild-type strain of Drosophila) Drosophila food media (100 mL) (see Recipes) Egg-laying media (20 mL) (see Recipes) Petri plate for crawling assay (2% agarose) (see Recipes) 10 mM CdCl2 stock solution (see Recipes) 30 mM CdCl2 stock solution (see Recipes) 100 mM CdCl2 stock solution (see Recipes) Equipment Autoclave (Equitron, catalog number: 7431STWL.AFH.430) BOD incubator (Thermotech, catalog number: TH-7004) Fluorescence microscope (RADICAL Scientific, catalog number: RXLr-5-50) Lab electric heater: eiSCO, 220 V, up to 450°C Stereomicroscope (Magnus Analytics, MSZ-Bi, catalog number: 16G1070) Water distillation unit (BOROSIL-HD-XL, Yuwinsil) Weighing balance (Mettler Toledo, catalog number: ME204/A04) Thermometer (LABWORLD, mercury thermometer) Software GraphPad Prism 9 Procedure Egg-laying media preparation Measure the required quantities of components for the egg-laying media (see Recipes). Boil the media in a conical flask at 100°C. Let the media cool down and then add propionic acid. Pour 2 mL of media in the required number of autoclaved vials and seal the vials with autoclaved cotton plugs. Add yeast paste (yeast extract dissolved in water) at the corner of each vial in order to enhance egg-laying capacity. Setting up crosses for egg collection Collect 10 virgin females from each of the following fly lines: w;+;UAS-SOD1, w;Act-5c-GAL4/CyO-GFP;+ (ubiquitous driver), and w;UAS-SOD1-RNAi;+. Collect the virgins with the help of a stereomicroscope, either in the pupal stage by identifying the male testes as two black dots or in adult stage by detecting meconium and gonads in the posterior. Set three vials containing egg-laying media as follows: (a) SOD1 overexpression: 2 days old, 10 virgin females of w;+;UAS-SOD1 and 5 males of w; Act-5c-GAL4/CyO-GFP;+, (b) 10 virgin females of w;+;UAS-SOD1 and 5 males of Oregon R, and (c) 10 virgin females of w;Act-5c-GAL4/CyO-GFP;+ and 5 males of Oregon R. The first filial generation (F1) of the above-mentioned crosses (a), (b), and (c) will be referred to as M1, C1, and C2, respectively. Write the date of crossing and genotype in each vial using a marker. Similarly, set up egg collection for SOD1 downregulated progenies: (a) SOD1 downregulation: 10 virgin females of w;UAS-SOD1-RNAi;+ and 5 males of w;Act-5c-GAL4/CyO-GFP;+ (F1 named as M2), and (b) 10 virgin females of w;UAS-SOD1-RNAi;+ and 5 males of Oregon R (C3 progenies). In order to assess the effect of various concentrations of CdCl2 on Drosophila, set up egg-laying media vials containing Oregon R males and females in a 1:2 ratio. Drosophila food media preparation Measure the required quantities (see Recipes) and mix in a flask with a glass stirrer. Boil the media for 15 min on a heater (up to 100°C). Autoclave the media along with the required number of washed glass vials and non-absorbent cotton. Allow the media to cool for a few minutes; then, add the required amount of propionic acid with continuous stirring. Pour 3 mL of media to each vial and seal with cotton plugs. In case of metal media preparation, follow steps C1–C3 without pouring the media in the vials. Prepare CdCl2 stock solutions and pour the required volumes in separate media flasks to obtain working concentrations of 0.05 and 0.1 mM (from a 10 mM stock solution), 0.3, 0.6, and 0.75 mM (from a 30 mM stock), and 1 and 1.5 mM (from a 100 mM stock solution) of CdCl2 (see Recipes). Pour 3 mL of media of each concentration to separate vials and seal with cotton plugs. Egg collection and rearing Collect the eggs from the vial by gently stroking with a fine brush soaked in water (Figure 1a). Do not rupture the eggs (check whether they are healthy by microscopic observation of intact micropyle in the egg structure) (Figure 1b). Do not keep any damaged eggs as it may lead to erroneous results in lethality quantification. Place the collected eggs from the experimental setup B2 and B3 in a small square black paper, in cohorts of 40 eggs per vial. Mark the vials with the number of eggs collected, date of collection, and genotype using a marker. Maintain the egg-containing vials at a constant temperature in the incubator. Confirm temperatures using a laboratory thermometer. Notably, GAL4 activity gets enhanced and best expressed at 29°C while the life cycle of Drosophila is fast-forwarded to 10 days. Hence, for maximal SOD upregulation and downregulation, raise flies at 29°C. Figure 1. Egg collection setup (Figure 1a) and eggs after collection (Figure 1b) Screening Genetic crosses often require segregation of markers as per mating schemes given in step B2. Here, green fluorescent protein (GFP)–tagged embryos were separated from non-GFP embryos in experimental setups B2 before performing step D2. Place the healthy eggs monitored in step D1 in watch glasses. Mark the watch glasses with the genotype, so as not to mix up eggs. Use the fluorescence microscope to segregate GFP-positive from non-GFP eggs. Remove the GFP-positive eggs gently with a brush and discard them. Place a black paper containing non-GFP eggs in the respective media vials and follow step D3. Observation and counting of stagewise lethality Check the egg-containing vials each day. Observe under the stereomicroscope for lethality in embryonic (yellowish to blackish appearance) or larval stage (no movement of mouth hook). Transfer 3rd-instar larvae (n = 20) of control and metal-treated (0.3 and 0.6 mM CdCl2) from extra vials kept for performing behavioral assays at larval stage. Observe and count the number of healthy pupae as well as degenerated pupae. Note down the stage of pupal degeneration: early, mid, or late stage. Encircle pupae with a marker to count their number. Make a note of number and dates of fly eclosion. Also, mark any lethality in adults during their eclosion. Maintenance of adult flies for survival assay Collect flies immediately after their eclosion. Transfer them to fresh media vials. Write the date of eclosion of each genotype on the vial with a marker. Transfer CdCl2-raised flies to normal media post eclosion for survival analysis. Record the death events every day until all flies are dead in one of the groups. Keep aside 20 flies from CdCl2 and normal media vials for behavioral analysis. Crawling assay Dissolve 2% agarose in distilled water and boil it. Pour the dissolved solution quickly in a glass Petri plate and allow to solidify. Place the Petri plate over a 0.2 mm2 graph paper. Wash 3rd-instar wandering larvae (n = 20), as collected in step F2, one at a time with distilled water; remove the excess water with tissue paper. Transfer one larva at a time to the center of the Petri plate (Figure 2) and allow it to acclimatize for 1 min. Then, start a one-minute timer and note down the number of grids crossed by the larva in the stipulated time. Return the larva to the center and, after a 1 min interval, note down the second reading for the same larva in terms of number of grids crossed in one minute. Repeat the above-mentioned process with each of the 20 larvae collected from control, 0.3 mM, and 0.6 mM CdCl2. Figure 2. The figure shows a third-instar larva (a) as well as its enlarged view (b), transferred to the center of a Petri plate, coated with 2% agarose and placed over a 0.2 mm2 graph paper Climbing assay Collect the adults, as mentioned in step G2, two days post eclosion. Introduce one fly at a time in a 10 mL measuring cylinder, seal it with a cotton plug (Figure 3), and allow the fly to acclimatize for 1 min. Tap the cylinder thrice before recording the time taken by the fly to climb a distance of 10 cm. Repeat after a period of 1 min to get a second reading of the same fly. Figure 3. Image of the adult fly climbing assay, wherein a distance of 10 cm has been marked in the cylinder (black line). Time taken for a fly to climb the marked distance was recorded. Data analysis Developmental lethality, delayed progression to next molt, shortening of life span, and disrupted locomotor activity are some of the most important indicators of adverse effects of any mutation or toxicant exposure in Drosophila. These can be experimentally analyzed using the protocols described in the above sections. Quantification of lethality Open the software GraphPad Prism to plot “Grouped” graph and perform a 2-way ANOVA analysis in order to verify significant lethality of SOD1 mutant flies as well as cadmium-treated flies compared to their respective controls. Lethality plot allows to comprehend the decrease in number of live individuals during egg to pupae transition and then further death events during pupal development, leading to declined emergence of adult fly when exposed to varying concentrations of CdCl2 (Figure 4). In each triplicate, the number of pupae as well as the eclosed fly was counted to quantify the stagewise lethality due to either toxicant exposure or SOD1 level manipulation. LC50 can be calculated for the egg-to-pupae and pupae-to-adult fly transition (Nanda and Firdaus, 2022). The percentage of embryos that displayed lethality during their metamorphosis to pupal stage were 8.3%, 25%, 43.3%, 66.67%, 80%, 88.3%, and 100% for concentrations of 0.05, 0.1, 0.3, 0.6, 0.75, 1, and 1.5 mM of CdCl2, respectively. During pupal metamorphosis, 11%, 35%, 55%, 81.6%, 83.33%, and 95% pupae degenerated from the CdCl2 concentrations of 0.05, 0.1, 0.3, 0.6, 0.75, and 1 mM, respectively. The embryos raised in 1.5 mM CdCl2 media displayed 100% lethality. SOD1-overexpressed flies showed severe lethality during early as well as pupal development; approximately 68% embryos were able to form pupae, of which only 39.6% successfully metamorphosed to adult flies (Figure 5). SOD1 downregulation led to pupal stage lethality with no effect on early development (Figure 4), with only 45.2% embryos successfully surviving until the adult stage. Figure 4. Percent survival of Drosophila in varying CdCl2 concentrations. The graph shows survival percentages of Drosophila at two different developmental stages when eggs were transferred to metal media (taken from Nanda and Firdaus, 2022). Figure 5. Percent survival of SOD1-overexpressed (M1, w;Act-5c-Gal4/+;UAS-SOD1/+) and downregulated (M2, w;Act-5c-Gal4/UAS-SOD1-RNAi;+) Drosophila in comparison to their respective controls C1 (w/+; +; UAS-SOD1/+), C2 (w/+; Act-5c-GAL4/+;+), and C3 (w/+;UAS-SOD1-RNAi/+;+) Life span assay To analyze life span of SOD1 mutant flies as well as cadmium-treated flies, plot “Survival” graph and perform “Log-rank (Mantel-Cox test)” by using GraphPad Prism. The life span of the flies post eclosion was observed to be shortened as reflected by the decrease in their median survival with an increase in CdCl2 concentration (Figure 6). Figure 6. Life span of CdCl2-raised flies. Cd animals succumb earlier than their control counterparts and median survival decreases with increasing metal concentration (Figure taken from Nanda and Firdaus, 2022). SOD1-overexpressed flies (M1) showed median survival of 39 days and its respective controls C1 and C2 had median survival of 51 and 64.5 days, respectively. Although the survival curve of M1 was significantly different from C2, it was comparable to C1. Likewise, SOD1 downregulation (M2) led to a median survival of 56.5 days, whereas its respective controls C2 and C3 median survival was calculated as 64.5 and 66 days, respectively. Herein, M2 showed no significant differences with C2 (Figure 7). Figure 7. Life span of SOD1-overexpressed (M1) and downregulated (M2) flies with respective controls Locomotor assays Larval crawling assay To analyze locomotor activities of crawling and climbing with CdCl2-fed larvae and adults, respectively, “Column” graphs were plotted in GraphPad Prism and mobility analysis was done using non-parametric t-test. p-values were checked for calculating the significance between the two groups considered. Control larvae crossed 8.3 grid lines in 1 min on average, whereas 0.3 mM CdCl2-exposed larvae crossed 6.7 grids per minute. Significant decrease in larval mobility was observed in 0.6 mM CdCl2-treated animals, as they were able to cross only 3.55 squares per minute (Figure 8). Figure 8. Crawling assay of CdCl2-fed larvae Adult climbing assay Average time taken by the control flies to climb a distance of 10 cm was 8.45 s. However, 0.3 and 0.6 mM CdCl2-raised flies’ climbing time was 12.97 and 19.20 s, respectively (Figure 9). Using the above protocol, a significant decline in the climbing activity was observed in the CdCl2 flies. Figure 9. Climbing assay of two-days-old CdCl2 flies This protocol enabled Drosophila embryos to be monitored in various concentrations of cadmium salts mixed with media. Successive steps of the protocol led to the conclusive fact that cadmium exposure to the flies caused lethal effects, dispersed throughout their developmental stages (previously published data in Nanda and Firdaus, 2022). The protocol further facilitates the analysis of life span of the surviving animals post eclosion. Life span was observed to be shortened to a greater extent, as reflected by calculation of median survival with an increase in CdCl2 concentration (Nanda and Firdaus, 2022). Thus, cadmium exposure assessment in Drosophila under laboratory conditions indicated its biological toxicity and raises awareness towards its environmental pollution. The protocol also enabled smooth quantification of stagewise lethality in Drosophila where cytosolic Cu, Zn SOD gene was perturbed using pan-tissue specific actin GAL4 driver. SOD1-overexpressed flies showed lethality dispersed at different developmental stages, while the SOD1-downregulated flies displayed only pupal to adult stage lethality. The experiment exhibited the essentiality of SOD expression towards proper development of flies, a result that has not been published before. Previously published literature showed an increase in the life span of SOD-overexpressed flies (Orr et al., 2003) and a decrease in the life span of SOD-downregulated flies (Oka et al., 2015; Martin et al., 2009). However, in the present study, SOD1 manipulation did not lead to any significant life span differences. Since 0.3 and 0.6 mM Cd concentrations fall on either side of LC50 (Nanda and Firdaus, 2022), they were selected to further analyze the effect of cadmium on locomotor abilities of Drosophila. Our protocol enabled the collection of larvae and adults from these concentrations to perform crawling and climbing assays, respectively. Crawling ability was significantly decreased upon cadmium treatment and a similar trend was observed in adult flies’ climbing abilities. This incites future research on cadmium-led deregulation of neuromuscular functioning. Notes Virgin female isolation should be cautiously done under the stereomicroscope, either in the pupal or adult stage (Ashburner et al., 2005). Prepare media with appropriately measured quantities and concentrations as mentioned in the recipes. Take a maximum volume of 500 µL from the CdCl2 stock solution for 10 mL of media. Rigorous stirring after adding CdCl2 solution in the media is extremely essential. Collect eggs and transfer them to media vials without damaging them by improper handling. Carefully observe and note each stage of fly development until eclosion. Identify and select third-instar larvae for motility assay. Record the exact time taken by a fly to climb a distance of 10 cm, without stopping at any point or jumping over a short distance. For 100 mL media preparation, use a 250 mL conical flask. This prevents the media from sticking to the cotton plug due to frothing during autoclave. Write down each day’s data in a notebook; mention stagewise observations with the date, especially the death events post eclosion (Step F4) so as to plot the survival curve. Change the food vials every fourth day for survival assay to avoid death due to media sogginess. Recipes Drosophila food media (100 mL) Agar agar (0.8 g) Yeast extract (1.5 g) Sucrose (5 g) Corn flour (8 g) Propionic acid (400 μL) Egg-laying media (20 mL) Agar agar (0.4 g) Sucrose (0.2 g) Propionic acid (0.15 μL) Petri plate for crawling assay (2% agarose) Agarose (0.25 g) Distilled water (15 mL) 10 mM CdCl2 Stock solution CdCl2 (0.0228 g) Distilled water (10 mL) 30 mM CdCl2 Stock solution CdCl2 (0.0685 g) Distilled water (10 mL) 100 mM CdCl2 Stock solution CdCl2 (0.2284 g) Distilled water (10 mL) Acknowledgments We acknowledge Nanda and Firdaus (2022) for optimizing these protocols. Figures 4 and 6 have been reprinted from the aforementioned citation with due permission from the publisher. Competing interests The authors declare no competing interest. References Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., et al. (2000). The genome sequence of Drosophila melanogaster. Science 287(5461): 2185-2195. Ain, U. and Firdaus, H. (2021). Behavioural Assays to Analyse the Muscle Mutants of Drosophila melanogaster. 49-63. Ashburner, M., Golic, K. G., Hawley, R. S. (2005). Drosophila: A Laboratory Handbook. Second edition. Cold Spring Harbor Laboratory Press. ISBN: 0-87969-706-7 Blackney, M.J., Cox, R., Shepherd, D., Parker, J.D. (2014). Cloning and expression analysis of Drosophila extracellular Cu Zn superoxide dismutase.Biosci Rep 34(6): e00164. Carey, J. R., Papadopoulos, N., Kouloussis, N., Katsoyannos, B., Muller, H. G., Wang, J. L. and Tseng, Y. K. (2006). Age-specific and lifetime behavior patterns in Drosophila melanogaster and the Mediterranean fruit fly, Ceratitis capitata. Exp Gerontol 41(1): 93-97. Dubey, M., Ain, U. and Firdaus, H. (2020). An insight on Drosophila myogenesis and its assessment techniques. Mol Biol Rep 47(12): 9849-9863. Duttaroy, A., Meidinger, R., Kirby, K., Carmichael, S., Hilliker, A. and Phillips, J. (1994). A manganese superoxide dismutase-encoding cDNA from Drosophila melanogaster. Gene 143 (2): 223-225 Green, H. J., Griffiths, A. G., Ylanne, J. and Brown, N. H. (2018). Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila. Elife 7: e35783. Landis, G. N., Doherty, D. and Tower, J. (2020). Analysis of Drosophila melanogaster Lifespan. Methods Mol Biol 2144: 47-56. Martin, I., Jones, M. A. and Grotewiel, M. (2009). Manipulation of Sod1 expression ubiquitously, but not in the nervous system or muscle, impacts age-related parameters in Drosophila. FEBS Lett 583(13): 2308-2314. Nanda, K. P. and Firdaus, H. (2022). Dietary cadmium induced declined locomotory and reproductive fitness with altered homeostasis of essential elements in Drosophila melanogaster. Comp Biochem Physiol C Toxicol Pharmacol 255: 109289. Nanda, K. P. and Firdaus, H. (2021). Dietary cadmium (Cd) reduces hemocyte level by induction of apoptosis in Drosophila melanogaster. Comp Biochem Physiol C Toxicol Pharmacol 250: 109188. Nanda, K. P., Kumari, C., Dubey, M. and Firdaus, H. (2019). Chronic lead (Pb) exposure results in diminished hemocyte count and increased susceptibility to bacterial infection in Drosophila melanogaster. Chemosphere 236: 124349. Oka, S., Hirai, J., Yasukawa, T., Nakahara, Y. and Inoue, Y. H. (2015). A correlation of reactive oxygen species accumulation by depletion of superoxide dismutases with age-dependent impairment in the nervous system and muscles of Drosophila adults. Biogerontology 16(4): 485-501. Olcott, M. H., Henkels, M. D., Rosen, K. L., Walker, F. L., Sneh, B., Loper, J. E. and Taylor, B. J. (2010). Lethality and developmental delay in Drosophila melanogaster larvae after ingestion of selected Pseudomonas fluorescens strains. PLoS One 5(9): e12504. Orr, W. C., Mockett, R. J., Benes, J. J. and Sohal, R. S. (2003). Effects of overexpression of copper-zinc and manganese superoxide dismutases, catalase, and thioredoxin reductase genes on longevity in Drosophila melanogaster. J Biol Chem 278(29): 26418-26422. Rogina, B., Wolverton, T., Bross, T. G., Chen, K., Muller, H. G. and Carey, J. R. (2007). Distinct biological epochs in the reproductive life of female Drosophila melanogaster. Mech Ageing Dev 128(9): 477-485. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Developmental Biology > Cell growth and fate > Ageing Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Validating Candidate Congenital Heart Disease Genes in Drosophila Jun-yi Zhu [...] Zhe Han Jun 20, 2017 8298 Views How to Catch a Smurf? – Ageing and Beyond… In vivo Assessment of Intestinal Permeability in Multiple Model Organisms Raquel R. Martins [...] Michael Rera Feb 5, 2018 12011 Views Development of a Mouse Model of Hematopoietic Loss of Y Chromosome Soichi Sano and Kenneth Walsh Aug 5, 2023 618 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,599	https://bio-protocol.org/en/bpdetail?id=4599&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed In vivo Assessment of Lysosomal Stress in the Drosophila Brain Using Confocal Fluorescence Microscopy FM Felipe Martelli Published: Vol 13, Iss 2, Jan 20, 2023 DOI: 10.21769/BioProtoc.4599 Views: 672 Reviewed by: Nafisa M. JadavjiSrajan Kapoor Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Feb 2022 Abstract Lysosomes play a central role in signaling, nutrient sensing, response to stress, and the degradation and recycling of cellular content. Defects in lysosomal digestive enzymes or structural components can impair lysosomal function with dire consequences to the cell, such as neurodegeneration. A number of methods exist to assess lysosomal stress in the model Drosophila, such as specific driver and reporter strains, transmission electron microscopy, and the investigation of gene expression. These methods, however, can be time consuming and, in some cases, costly. The procedure described here provides a quick, reliable, and low-cost approach to measure lysosomal stress in the Drosophila brain. Using fluorescence confocal microscopy and the LysoTracker staining, this protocol allows for the direct measurement of lysosome size and number. This method can be used to assess lysosomal stress under a number of different genetic and environmental scenarios in the Drosophila brain. Keywords: Lysosome Lysosomal stress LysoTracker Fruit fly Brain Fluorescence microscopy Insecticide Background In the eukaryotic cell, lysosomes are a signaling hub central to the response to stress (Ballabio and Bonifacino, 2020; Bouhamdani et al., 2021; Saftig and Puertollano, 2021). Their acidic environment and repertoire of over 60 types of hydrolytic enzymes make them the main cellular degradative organelle, capable of digesting and recycling proteins, glycans, lipids, and nucleic acids (Bouhamdani et al., 2021). Through endocytosis and autophagy, the external and internal materials delivered to lysosomes allow them to collect information about a range of cellular processes. Given that, lysosomes are key components of the response to stress, sensing the nutritional environment and the presence of pathogens, and even regulating membrane receptors (Ballabio and Bonifacino, 2020; Saftig and Puertollano, 2021). As a recipient of different environmental cues, lysosomes become a platform that generates signals to help maintain cellular homeostasis (Ballabio and Bonifacino, 2020; Saftig and Puertollano, 2021). For instance, they contain hundreds of membrane proteins and signaling complexes on the cytosolic surface, which can transmit information to the nucleus. One example is the target of rapamycin complex 1 (TORC1). In a rich nutritional environment, TORC1 becomes active and localizes to the cytosolic side of the lysosomal membrane, promoting cell anabolism. Under starvation, lysosomes radically decrease in number and increase in size and TORC1 is no longer active or present in the lysosomal membrane (Demetriades et al., 2014; Ballabio and Bonifacino, 2020; Saftig and Puertollano, 2021). These organelles are, thus, highly heterogeneous. They change in size, shape, acidity, and location depending on the cellular environment and cell type (Bouhamdani et al., 2021). Genetics and environmental factors that impair lysosomal membrane integrity, enzyme activity, acidity, and size, affect protein aggregation, or increase reactive oxygen species (ROS) levels cause lysosomal stress and may lead to cell damage or death (Lin et al., 2016; Lakpa et al., 2021). Lysosomal storage disorders (LSD) are a group of over 50 disorders that arise from mutations affecting lysosomal function and structure, resulting in their inability to digest content, and triggering their enlargement (Darios and Stevanin, 2020; Barral et al., 2022). With a high metabolic activity and lack of cellular division, neurons are especially susceptible to LSD. The buildup of non-digested lysosomal material may severely affect the neuronal activity leading to neurodegeneration (Darios and Stevanin, 2020; Barral et al., 2022). The fly genetic toolkit offers a range of lysosomal proteins tagged with fluorescent markers that can be used to investigate lysosomal function, such as tagged Lamp (lysosomal-associated membrane protein) or Atg (autophagy-related gene) proteins (Rigon et al., 2021). Using these strains, however, may require crossing schemes in case recessive alleles are under investigation, which may become time consuming. Another approach is transmission electron microscopy, a reliable method that may be able to identify the cell types where lysosomal stress is taking place (Lorincz et al., 2017). Nonetheless, this method is not always available, may be costly relatively to other approaches, and requires a more in-depth understanding of cell morphology. Interrogating the expression of genes involved in lysosome function is also an alternative, such as RT-qPCR or next-generation sequencing (Rigon et al., 2021), but they provide indirect measures of lysosomal stress. The method described here involves the use of LysoTracker staining and fluorescence confocal microscopy, and it allows for the in vivo measurement of lysosomes’ size and number without the need of fly husbandry. LysoTracker is a dye for cellular acidic compartments, including lysosomes and autolysosomes (Chazotte, 2011). This method was recently implemented (Martelli et al., 2022) to measure the area occupied by lysosomes in the Drosophila larval brain in response to exposure to the insecticide spinosad. The procedure used here cannot pinpoint the cell type where lysosomal stress takes place, but it can be adapted with the use of other markers to achieve that. This protocol can also be modified to assess impacts on lysosomes under the effect of other drugs, rearing conditions, or mutations. Materials and Reagents 1.7 mL microtubes (Axygen, catalog number: MCT-175-C) 48-well Nunc cell plate (Thermo Scientific, catalog number: 150687) Micropipette 200 µL tips (Axygen, catalog number: T-200-Y) Double-sided tape (Scotch 665 12.7 mm × 22.8 m) Aluminum foil Microscopy slides (Westlab, catalog number: 663-249) Cover slips (Trajan, catalog number: 471112440M) Fly stocks of interest LysoTrackerTM Red DND-99 (Thermo Fisher Scientific, catalog number: L7528) LysoTrackerTM Blue DND-22 (Thermo Fisher Scientific, catalog number: L7525). LysoTrackerTM Green DND-26 (Thermo Fisher Scientific, catalog number: L7526). Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: P5493) Schneider's Drosophila medium 1× (Thermo Fisher Scientific, catalog number: 21720024) Spinosad (Sigma-Aldrich, catalog number: 33706) Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855-100ML) Vectashield 10 mL mounting medium (Vector, catalog number: VEH1000) Analytical standard sucrose (Merk, product number: 47289) 5% sucrose solution (200 mL) (see Recipes) 1,000 ppm spinosad solution in DMSO (see Recipes) 5× spinosad stock solution (see Recipes) 5× DMSO stock solution (see Recipes) LysoTracker 1:100 working stock solution (see Recipes) Distilled water Equipment Dissecting forceps (Fine Science Tools, Dumont #5 Forceps, catalog number:1195-10) Stereo microscope (Zeiss, model: Stemi 2000-C) Confocal fluorescence microscope (Leica TCS SP8, DM600 CS) Micropipette P200 500 mL laboratory bottles Nutating mixer (Corning, catalog number: 6720) Software Fluorescence microscope image acquisition software (LAS X) ImageJ/FIJI (NIH, https://fiji.sc/) GraphPad Prism (Dotmatics, http://www.graphpad.com) Procedure Larvae collection and insecticide exposure Using the micropipette, load a 48-well Nunc plate with 200 µL of 5% sucrose solution per well. Using a pair of forceps, gently transfer 25 early third-instar larvae per well (Figure 1A, B). For the insecticide exposure, add 50 µL of the 5× spinosad stock solution (see Recipes) to each well containing larvae to be exposed and give the plate a gentle swirl. For unexposed controls, add 50 µL of the 5× DMSO stock solution instead (see Recipes). The final concentration of insecticide used here is 2.5 ppm. Keep the plate at 25°C and covered with aluminum foil to protect it from light for 2 h (or desirable number of hours), until exposure is over. Once exposure time is over, using a pair of forceps gently transfer larvae to a new well containing 250 µL of 1× PBS. Tissue dissection and staining Transfer larvae from the PBS-containing well to a microscopy slide containing a drop of cold (4°C) Schneider's medium 1×. Under the stereo microscope, begin the dissection. Using two pairs of forceps, hold the larval body at the midpoint (Figure 1C) and pull the anterior and posterior regions apart (Figure 1D). Using a pair of forceps, gently transfer the anterior body section (Figure 1E) to a microtube containing 495 µL of cold (4°C) 1× PBS. Add 5 µL of LysoTracker Red working stock solution (see Recipes) to the microtube. Final LysoTracker concentration of 1:10,000 (Figure 1F). Wrap the microtube in aluminum foil to protect it from light and keep it at slow agitation on a nutator mixer for 7 min (Figure 1G). Using a micropipette, slowly and completely remove the staining solution without touching the sample (Figure 1H). Gently add 500 µL of 1× cold (4°C) PBS back into the microtube (Figure H) and place it back on the nutator mixer for 5 min (Figure I). Under the stereo microscope, add a drop of cold (4°C) Schneider's medium 1× to a new microscopy slide. Using a pair of forceps, gently transfer the stained sample from the microtube onto the slide (Figure 1J). Conclude the dissection by holding the anterior body section sideways with two pairs of forceps and gently pull them apart from each other to tear the cuticle. Once the brain is located, use a pair of forceps to clear the surrounding tissues (Figure 1K). Mounting slides for fluorescence microscopy Add a small drop of Vectashield on top of the brain, only enough to cover it. Using a pair of forceps, if necessary, adjust sample orientation. Cut two small pieces of double-sided tape and place them flanking the sample in Vectashield. Place the coverslip on the top, aligning its border with the double-sided tape (Figure 1L). Figure 1. Experimental procedure. A. 48-well Nunc plate with 200 µL of 5% sucrose solution and 25 larvae per well. B. Detail of well containing 25 early third-instar larvae. C. For partial dissection start by holding down a larva at midpoint. D. Pull anterior and posterior body regions apart. E. Larva anterior body region. F. Transfer the anterior body region to a microtube containing cold 1× PBS and add LysoTracker Red working stock solution to it. G. Wrap the microtube in aluminum foil and keep it at slow agitation on a nutator mixer for 7 min. H. Perform one washing step by replacing the solution in the microtube with cold 1× PBS. I. Return the microtube wrapped in foil to the nutator mixer at low agitation for 5 min. J. Transfer the partially dissected sample to a slide containing a drop of cold Schneider’s medium. K. Under the microscope, conclude dissection, and isolate the brain from other tissues. L. Mount the slide for microscopy using double-sided tape, Vectashield, and a cover slip and proceed to image acquisition immediately. Imaging Proceed to image acquisition with a confocal fluorescence microscope immediately. Given that no fixative solution is used here, time from partial dissection to acquisition of last image should be kept within 30 min to avoid tissue degradation. In some cases, LysoTracker staining may not penetrate the more central parts of the brain or may aggregate at the brain’s surface. For that reason, to maintain consistency across samples, it is desirable to acquire images from a zone that excludes the brain’s surface and avoids central areas (Figure 2A). To ensure consistency and accuracy in the measurements, set optimal values of laser power and gain with a control sample and maintain the same settings across all samples. Select the same zones for imaging across all samples. Here, to maintain consistency, images were only acquired from the optic lobes. Once the zone is localized, acquire red signal (excitation/emission 577/590 nm) using a 40× objective (NA 1.1). It is important to find consistency in the signal observed within control samples, as well as exposed samples. Lack of consistency may indicate sample degradation or inappropriate staining. Acquire at least 20 images across the z-stack for every sample with 1 µm distance between them and avoid imaging the sample’s top and bottom. Image at least seven brain samples per group/treatment condition. Data analysis Analyzing microscopy images on ImageJ Open the image files on ImageJ and, using the rectangle selection tool, create a region of interest (ROI) of 30 × 30 µm (Figure 2A). The ROI will be used to quantify the percentage of area occupied by LysoTracker. Right click on the rectangle and duplicate the selected area. On top bar menu, click Image > Adjust > Threshold. A new window will appear; select Dark background. If necessary, adjust the threshold bar to reduce background. Take note of the percentage of area occupied by LysoTracker (Figure 2B). Acquire three to five independent ROIs, randomly assigned across the z-stack and within the zone, to each sample. Measure the percentage of area occupied by LysoTracker for each ROI in the same way. LysoTracker is also available in the options blue and green. Statistical analysis and plotting Make an average of the independent measurements acquired per sample. Use these averaged values for plotting graphs and performing statistical analysis. Using a statistics software of your preference (such as GraphPad Prism), perform a Student’s unpaired t-test. Plot results as a bar plot including the dot plot for individual values (Figure 2C). Figure 2. Image analysis and data representation. A. Acquire images of the brain zone (i.e., excluding the brain’s surface and avoiding central areas) from where regions of interest (ROIs) will be later selected. B. Duplicate a 30 × 30 µm ROI and, using the threshold tool, measure the percentage of area occupied by LysoTracker (small red box). 400× magnification. C. LysoTracker area (%) (n = 7 brain samples/treatment; three image sections/brain), Student’s unpaired t-test, p-value < 0.0001. Notes Given the exposure method used here, early third-instar larvae are preferable, as late wandering third instars may crawl out of the wells or start pupation during insecticide exposure. Start the exposure of each well at regular intervals to accommodate time to perform dissections and microscopy. This will optimize the number of samples that can be assessed per experimental batch. Recipes 5% sucrose solution (200 mL) Distilled water 190 mL Analytical standard sucrose 10 g Into a 500 mL glass bottle, add 190 mL of water and 10 g of sucrose. Give the bottle a swirl until completely dissolved. The solution can be stored at 4°C for up to one month. Solution must be at room temperature for usage. 1,000 ppm spinosad solution in DMSO DMSO 1 mL Spinosad 1 mg Add 1 mL of DMSO and 1 mg of spinosad into a 1.7 mL microtube and vortex it vigorously for a few minutes until solubilized. Create ten 100 µL aliquots in separated microtubes and store them at -20°C. Avoiding multiple freeze-thaw cycles and protecting the solutions from light will increase the insecticide’s half-life. Let the solution thaw at room temperature before usage. 5× spinosad stock solution 1,000 ppm spinosad solution in DMSO 12.5 µL 5% sucrose solution 987.5 µL Add 987.5 µL of 5% sucrose solution and 12.5 µL of the 1,000 ppm spinosad solution in DMSO into a 1.7 mL microtube and vortex for 30 s. This will create a 12.5 ppm spinosad solution that will be used to dose the wells of the Nunc plate where larvae are to be exposed. 5× DMSO stock solution DMSO 12.5 µL 5% sucrose solution 987.5 µL Add 987.5 µL of 5% sucrose solution and 12.5 µL of DMSO into a 1.7 mL microtube and vortex it for 30 s. This will create a 12.5 ppm DMSO solution that will be used to generate the control exposure. LysoTracker 1:100 working stock solution 1× PBS 495 µL LysoTrackerTM Red DND-99 5 µL Thaw one LysoTrackerTM Red DND-99 vial at room temperature. To a 1.7 mL microtube add 495 µL of 1× PBS and 5 µL of LysoTrackerTM Red DND-99. Using a micropipette, gently mix the solution. Wrap the microtube in foil to protect it from light and keep it at room temperature. Prepare it immediately before usage. Acknowledgments The author was supported by a Victorian Latin America Doctoral Scholarship (Victorian Government and the University of Melbourne), an Alfred Nicholas Fellowship (University of Melbourne), and a University of Melbourne Faculty of Science Travelling Scholarship. Original research paper: Martelli, F., Hernandes, N. H., Zuo, Z., Wang, J., Wong, C. O., Karagas, N. E., Roessner, U., Rupasinghe, T., Robin, C., Venkatachalam, K., et al. (2022). Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.Elife 11: e73812. Competing interests The author declares no conflicts of interest. Ethics Genetically modified Drosophila strains were maintained in a physical containment insectary facility following the biosafety guidelines by the Office of Research Ethics & Integrity at the University of Melbourne. References Ballabio, A. and Bonifacino, J. S. (2020). Lysosomes as dynamic regulators of cell and organismal homeostasis. Nat Rev Mol Cell Biol 21(2): 101-118. Barral, D. C., Staiano, L., Guimas Almeida, C., Cutler, D. F., Eden, E. R., Futter, C. E., Galione, A., Marques, A. R. A., Medina, D. L., Napolitano, G., et al. (2022). Current methods to analyze lysosome morphology, positioning, motility and function. Traffic 23(5): 238-269. Bouhamdani, N., Comeau, D. and Turcotte, S. (2021). A Compendium of Information on the Lysosome. Front Cell Dev Biol 9: 798262. Chazotte, B. (2011). Labeling lysosomes in live cells with LysoTracker. Cold Spring Harb Protoc 2011(2): pdb prot5571. Darios, F. and Stevanin, G. (2020). Impairment of Lysosome Function and Autophagy in Rare Neurodegenerative Diseases. J Mol Biol 432(8): 2714-2734. Demetriades, C., Doumpas, N. and Teleman, A. A. (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 156(4): 786-799. Lakpa, K. L., Khan, N., Afghah, Z., Chen, X. and Geiger, J. D. (2021). Lysosomal Stress Response (LSR): Physiological Importance and Pathological Relevance. J Neuroimmune Pharmacol 16(2): 219-237. Lin, J., Shi, S. S., Zhang, J. Q., Zhang, Y. J., Zhang, L., Liu, Y., Jin, P. P., Wei, P. F., Shi, R. H., Zhou, W., et al. (2016). Giant Cellular Vacuoles Induced by Rare Earth Oxide Nanoparticles are Abnormally Enlarged Endo/Lysosomes and Promote mTOR-Dependent TFEB Nucleus Translocation. Small 12(41): 5759-5768. Lorincz, P., Mauvezin, C. and Juhasz, G. (2017). Exploring Autophagy in Drosophila. Cells 6(3). Martelli, F., Hernandes, N. H., Zuo, Z., Wang, J., Wong, C. O., Karagas, N. E., Roessner, U., Rupasinghe, T., Robin, C., Venkatachalam, K., et al. (2022). Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies. Elife 11: e73812. Rigon, L., De Filippis, C., Napoli, B., Tomanin, R. and Orso, G. (2021). Exploiting the Potential of Drosophila Models in Lysosomal Storage Disorders: Pathological Mechanisms and Drug Discovery. Biomedicines 9(3). Saftig, P. and Puertollano, R. (2021). How Lysosomes Sense, Integrate, and Cope with Stress. Trends Biochem Sci 46(2): 97-112. Article Information Copyright Martelli. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Neuroscience > Nervous system disorders > Cellular mechanisms Cell Biology > Cell staining > Organelle Cell Biology > Cell imaging > Confocal microscopy Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Dual-Color Live Imaging of Adult Muscle Stem Cells in the Embryonic Tissues of Drosophila melanogaster Monika Zmojdzian [...] Rajaguru Aradhya Feb 5, 2023 619 Views Live Imaging of Phagoptosis in ex vivo Drosophila Testis Diana Kanaan [...] Hila Toledano Mar 20, 2023 752 Views Protocol for Imaging the Same Class IV Neurons at Different Stages of Development Sonal Shree and Jonathon Howard Aug 20, 2024 460 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
46	https://bio-protocol.org/en/bpdetail?id=46&type=1	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource This is an In Press version of the protocol that has not yet been assigned to an issue. Peer-reviewed DNA Molecular Weight Calculation Fanglian He In Press Published: Mar 20, 2011 DOI: 10.21769/BioProtoc.46 Views: 71821 Ask a question Favorite Cited by Abstract This method is to roughly estimate DNA molecular weight. One of its applications is to calculate the ratio of vector to insert in a ligation reaction (please see Standard DNA Cloning protocol). Procedure Anhydrous molecular weight of each nucleotide is (see reference 1): A= 313.21 T= 304.2 C= 289.18 G=329.21 For rough estimation, typically average the molecular weights of all four, average weight of a DNA nucleotide (in salt solution) = 325 Daltons (see reference 2) MW of a single-strand DNA molecule= (#of bp) x (325 Daltons/per base) Moles of a ssDNA molecule = (grams of DNA)/ (MW in Daltons) For accurate estimation of MW of ssDNA, an online calculator (see Reference 2) is available. Acknowledgments This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613. References New England Biolabs catalog book. http://www.basic.northwestern.edu/biotools/oligocalc.html. Article Information Copyright © 2011 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > DNA > DNA quantification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Free Bio-protocol alerts Sign up to receive alerts for: . Monthly Electronic Table of Contents (eToC) . Protocol Collections . Bio-protocol Webinars . Events By clicking Subscribe, you agree to register as a Bio-protocol user and to our Terms of Service and Privacy Policy. Subscribe News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,600	https://bio-protocol.org/en/bpdetail?id=4600&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Measurement of Secreted Embryonic Alkaline Phosphatase MW Meiyan Wang XW Xinyi Wang HY Haifeng Ye Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4600 Views: 895 Reviewed by: Laxmi Narayan MishraNityanand SrivastavaSashikantha Reddy Pulikallu Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Dec 2021 Abstract Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling. Graphical abstract Schematic overview of the workflow described in this protocol Keywords: SEAP Secreted reporter Reporter gene assays Gene expression Gene regulation Background Reporter gene assays have been important for biomedical and pharmaceutical studies to monitor cellular activities associated with gene expression, regulation, and signal transduction. An ideal secreted reporter system would provide a more simple, rapid, and sensitive method to detect gene expression in a quantitative manner compared to classical intracellular reporters. Secreted embryonic alkaline phosphatase (SEAP), a C-terminal truncated variant of human placental alkaline phosphatase (PLAP), is one of the most widely used secreted mammalian reporter enzymes, engineered by deleting the 30 amino acid anchoring domain of PLAP (Berger et al., 1988). The SEAP reporter assay has superior properties: it is a simple and non-invasive procedure that does not require cell lysis and allows for long-term monitoring of gene expression in vitro and in vivo (Yang et al., 1997; Schlatter et al., 2002; Jiang et al., 2008). Moreover, transfected cells are not disturbed and remain intact for further studies during measurement of SEAP activity (Hu et al., 2022). Furthermore, the heat stability of SEAP and resistance to the phosphatase inhibitor L-homoarginine allow the samples to be pretreated at 65°C or with this inhibitor, to eliminate endogenous alkaline phosphatase activity (Tannous and Teng, 2011). Here, we describe the experimental procedures of in vitro measurement of SEAP adapted from our previously published work (Wang et al., 2021). This method can be useful for real-time monitoring of gene expression patterns in various cells and animals. Materials and Reagents 96-well plate, transparent (Corning, catalog number: 3365) 24-well plate, transparent (Corning, catalog number: 3537) T25 flask (Corning, catalog number: 430639) HEK-293T (ATCC, CRL-11268) Dulbecco's modified Eagle's medium (DMEM) (Gibco, catalog number: 31600-083) Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106) 0.25% trypsin-EDTA 1% (vol/vol) penicillin and streptomycin solution (Beyotime Inc., catalog number: ST488-1/ST488-2) p-nitrophenylphosphate (Sangon Biotech, CAS: 333338-18-4) L-homoarginine (Sangon Biotech, CAS: 1483-01-8) MgCl2·6H2O (Sangon Biotech, CAS: 7791-18-6) Diethanolamine (Sangon Biotech, CAS: 111-42-2) NaCl (Sangon Biotech, CAS: 7647-14-5) KCl (Sangon Biotech, CAS: 7647-40-7) Na2HPO4 (Sangon Biotech, CAS: 7558-79-4) KH2PO4 (Sangon Biotech, CAS: 7778-77-0) HCl (Sinopharm Chemical Reagent Co., Ltd., CAS: 7647-01-0） Polyethyleneimine (PEI, molecular weight 40,000, stock solution 1 mg/mL in ddH2O) (Polysciences, catalog number: 24765) 1× PBS (see Recipes) 2× SEAP assay buffer (see Recipes) Substrate solution (see Recipes) Plasmids (see Table 1) Table 1.Plasmids designed and used in this study Plasmid Description and cloning strategy Reference pXY137 Constitutive mammalian BldD and BphS expression vector (ITR-PhCMV-p65-VP64-BldD-pA-PhCMV-BphS-P2A-YhjH-pA-ITR) Shao et al. (2017) pZQ5 Far-red light-inducible expression vector (2crRNA binding site-PhCMVmin-SEAP-pA) This work pZQ28 Constitutive crRNA2SEAP expression vector (PU6-crRNASEAP-pA) This work pZQ113 Constitutive mammalian PhCMV-driven expression vector (PhCMV-scFv-45bp linker-p65-HSF1-pA) This work pZQ116 Far-red light-inducible expression vector (PFRL2-dAsCas12a-NLS-10×GCN4-pA; PFRL3, (OwhiG)2-PhCMVmin) This work Equipment Water bath CO2 incubator Constant temperature incubator (65°C) Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments, Inc.) Multipass pipette Reagent reservoirs Hemocytometer (Merck, catalog number: Z359629) Software Excel (Microsoft) Gen5 software (version: 2.04) Procedure Cell culture Warm DMEM supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin and streptomycin solution (hereafter referred to as complete DMEM) and 1× PBS in a 37°C water bath for at least 15 min. Wash ~90% confluent human embryonic kidney cells (HEK-293T) in a T25 flask with 3 mL of 1× PBS, aspirate the PBS, and add 0.5 mL of 0.25% trypsin-EDTA. Incubate the cells at 37°C in a 5% CO2 incubator for 3–5 min, add 3 mL of prewarmed complete DMEM, and transfer the cell suspensions to a 15 mL sterile conical tube. Spin down the cells at 300 × g for 3 min at room temperature, discard the supernatant, and resuspend the cell pellet in 15 mL of complete DMEM. Dispense 4 mL of the cell suspension into a T25 flask and incubate it for three days at 37°C in a humidified atmosphere containing 5% CO2 before subculturing again. Cell transfection The 293T cell suspension should be prepared (as described in the Cell culture procedure) the day before transfection (Day 1) (Figure 1). Figure 1. Schematic diagram of the schedule for cell transfection and SEAP analysis Count the cells using a hemocytometer and seed 6 × 104 HEK-293T cells per well in a 24-well cell culture plate; culture for 18 h. The next day (Day 2), prepare the transfection mix as follows: Add 500 ng of the corresponding plasmid (see Table 1) into 50 μL of FBS-free and antibiotic-free DMEM medium for each well of a 24-well plate. Add 1.5 μL of PEI (1 μg/μL, PEI and DNA at a ratio of 3:1) into the above plasmid DNA solution. To scale up, multiply the quantities by the number of replicates. Vortex vigorously for 30 s. Incubate the DNA-PEI mixture solution at room temperature for 15 min. Add 50 μL of transfection mix dropwise to each well. Rock the slide gently a few times and incubate at 37°C in a humidified atmosphere containing 5% CO2. Six hours after transfection, change the medium with fresh complete DMEM and incubate the 24-well plate at 37°C in a 5% CO2 incubator until Day 3. On Day 3, the transfected slides can be used to perform SEAP reporter assay. SEAP reporter assay Aspirate 200 μL of cell culture supernatant from each sample and transfer into a chamber of a 96-well plate. Heat-inactivate the cell culture supernatants at 65°C for 30 min. Prewarm 2× SEAP assay buffer (see Recipes) at 37°C and protect from light. Mix 100 μL of 2× SEAP buffer with 20 μL of substrate solution (see Recipes) in the dark. Add 120 μL of the above-mentioned mixed substrate solution into 80 μL of heat-inactivated cell culture supernatant. At least three replicates should be performed for each group. The media will change color from pink to orange in the presence of SEAP (Figure 2). Figure 2. Detection of SEAP production at the indicated time points after illumination. In the presence of SEAP, the media changes from pink to orange. The yellow color intensity correlates with light illumination time. Measure light absorbance at 405 nm at 37°C for 15 min using a multi-mode microplate reader. See Figure 3 for the detailed procedure. Figure 3. Schematic diagram of SEAP reporter assay Calculate the levels of SEAP activity using data points that yield a rate of change in light absorbance that is linear with respect to time. (If available, a computer-linked kinetic ELISA plate reader is most convenient, as this will precisely calculate the linear change in A405 in all 96 wells of a microplate over time.) Data analysis For each illumination time, data are expressed as mean ± standard deviation (SD); n = 3 independent experiments. The data can also be represented as fold change difference compared to non-stimulated cells and analyzed by one-way ANOVA followed by Tukey’s post-hoc tests. Perform statistical analysis using GraphPad Prism or other software. Figure 4. Quantification of far-red light-inducible SEAP expression kinetics. Transfected HEK-293T cells were illuminated with far-red light for different time periods (0–120 min). SEAP production in the culture supernatant was profiled at 24 h after illumination. P < 0.01, **P < 0.001 illumination groups vs. 0 min. Notes The mixture of 2× SEAP buffer and substrate solution should be protected from light, quickly added to cell culture supernatants, and detected immediately. If SEAP production is too high, the samples should be diluted with PBS buffer solution. Recipes 1× PBS buffer Reagent Final concentration Amount NaCl 137 mM 8.00 g KCl 2.7 mM 0.20 g Na2HPO4 10 mM 1.42 g KH2PO4 1.8 mM 0.24 g H2O n/a 800 mL Adjust pH to 7.4 with HCl, then add ddH2O to final volume (1,000 mL) Total n/a 1,000 mL 2× SEAP buffer Reagent Final concentration Amount L-homoarginine 20 mM 4.4938 g MgCl2 (3 M) 1 mM 0.334 mL Diethanolamine 21% (v/v) 210 mL Adjust pH to 9.8 with HCl, then add dH2O to final volume (1,000 mL) Total n/a 1,000 mL Store at 4°C and protect from light. Substrate solution Reagent Final concentration Amount p-nitrophenylphosphate 120 mM 2.226 g 2× SEAP buffer n/a 50 mL Total n/a 50 mL Aliquot, store at -20°C, and protect from light. Acknowledgments This protocol was adapted from previous work (Wang et al., 2021. DOI: 10.1126/sciadv.abh2358). This work was financially supported by grants from the National Key R&D Program of China (No. 2019YFA0904500, No. 2019YFA0110802), the National Natural Science Foundation of China (NSFC: No. 32171414, No. 31971346, No. 31861143016, No. 31870861), the Nature Science Foundation of Chongqing, China (No. CSTB2022NSCQ-MSX0461), the Open Project Program of State Key Laboratory of Dairy Biotechnology (No. SKLDB2021-001), and the Instruments Sharing Platform of the School of Life Sciences, East China Normal University. Competing interests All authors wish to declare no competing interests. References Berger, J., Hauber, J., Hauber, R., Geiger, R. and Cullen, B. R. (1988). Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66(1): 1-10. Hu, Z., Zhang, T., Jiang, S. and Yin, H. (2022). Protocol for evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase assay.STAR Protoc 3(1): 101061. Jiang, T., Xing, B. and Rao, J. (2008). Recent developments of biological reporter technology for detecting gene expression.Biotechnol Genet Eng Rev 25: 41-75. Schlatter, S., Rimann, M., Kelm, J. and Fussenegger, M. (2002). SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase. Gene 282(1-2): 19-31. Shao, J., Xue, S., Yu, G., Yu, Y., Yang, X., Bai, Y., Zhu, S., Yang, L., Yin, J., Wang, Y., Liao, S., Guo, S., Xie, M., Fussenegger, M. and Ye, H. (2017). Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Sci Transl Med 9(387): eaal2298. Tannous, B. A. and Teng, J. (2011). Secreted blood reporters: insights and applications.Biotechnol Adv 29(6): 997-1003. Wang, X., Dong, K., Kong, D., Zhou, Y., Yin, J., Cai, F., Wang, M. and Ye, H. (2021). A far-red light-inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation. Science advances 7(50): eabh2358. Yang, T. T., Sinai, P., Kitts, P. A. and Kain, S. R. (1997). Quantification of gene expression with a secreted alkaline phosphatase reporter system.Biotechniques 23(6): 1110-1114. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Biochemistry > Protein > Activity Cell Biology > Cell-based analysis > Enzymatic assay Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays Tobias Schachinger [...] Peter L. Turecek Sep 5, 2024 340 Views Measurement of the Activity of Wildtype and Disease-Causing ALPK1 Mutants in Transfected Cells With a 96-Well Format NF-κB/AP-1 Reporter Assay Tom Snelling Nov 20, 2024 272 Views Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays Tom Snelling Nov 20, 2024 257 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,601	https://bio-protocol.org/en/bpdetail?id=4601&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing MA Marcus Alvarez JB Jihane N. Benhammou SR Shuyun Rao LM Lopa Mishra JP Joseph R. Pisegna PP Päivi Pajukanta Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4601 Views: 1082 Reviewed by: Laxmi Narayan MishraNitin AmdareAmit Kumar Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Translational Medicine Dec 2021 Abstract Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types. Keywords: Nuclei isolation Liver Frozen tissue Single-nucleus RNA-seq Background The human liver performs critical functions ranging from lipid metabolism, amino acid synthesis, and drug processing to protection from portal venous bacteria and viruses (Trefts et al., 2017). Consistent with this diverse range of functions, its spatial organization is specifically tailored (Ben-Moshe and Itzkovitz, 2019). For example, lobule zonation between a central vein and portal triad divides hepatocyte and endothelial functions (Halpern et al., 2017). Diverse and specialized cell types populate the liver (Aizarani et al., 2019), and thus studies involving the liver would benefit greatly from single-cell resolution. The field of single-cell genomics has accelerated cellular composition studies of tissues and samples. Single-cell RNA sequencing (RNA-seq) has been used to discover subtypes within blood monocytes and dendritic cells (Villani et al., 2017), as well as to discover myeloid differentiation pathways (Drissen et al., 2016). In solid tissues, single-cell RNA-seq has provided insight into fibrosis mechanisms in lung (Xu et al., 2016) and kidney (Kuppe et al., 2021). Single-cell RNA-seq has also been used in liver to transcriptionally characterize the landscape of cell types (MacParland et al., 2018), as well as the immune landscape in hepatocellular carcinoma (HCC) (Zhang et al., 2019). One adaptation of single-cell RNA-seq is the use of nuclei instead of whole cells. Single-nucleus RNA-seq (snRNA-seq) has been shown to produce similar results as scRNA-seq (Habib et al., 2017). Furthermore, it can identify a greater expression diversity within cell types than scRNA-seq (Andrews et al., 2022). A main advantage of snRNA-seq is its applicability to frozen tissues. Archived tissues remove the need to coordinate tissue processing immediately after biopsy collection, which is challenging for human tissues. Archived tissues may also have additional phenotype and molecular data readily available, allowing for more thorough analyses and controlling of confounders. However, the isolation of nuclei from frozen tissues for snRNA-seq presents distinct challenges. Snap freezing can result in the formation of ice crystals, disrupting the tissue and possibly decreasing the yield of intact nuclei (Larson and Chin, 2021). This can also result in higher amounts of tissue debris and contaminating ambient RNA in the lysate and nucleus suspension. We present a specifically tailored protocol for snRNA-seq of human frozen liver tissue (Rao et al., 2021), balancing requirements for yield and debris removal. For an overview of the protocol, see Figure 1. The advantage of our protocol is the rapid isolation of nuclei with minimum equipment, which prevents RNA degradation and increases throughput. Our protocol avoids labor-intensive flow cytometry sorting, which may require high yields and decrease throughput. We also avoid density-based centrifugation, which increases run times and susceptibility to RNA degradation. In addition, we provide details on how to isolate nuclei suitable for droplet-based microfluidics applications, as well as data analysis guidelines. While optimized for snap-frozen liver tissue, this protocol can be adapted to other frozen tissue samples, such as adipose tissue (Alvarez et al., 2020). Figure 1. Overview of nucleus isolation from frozen liver. The flowchart outlines the steps from tissue lysis to nuclear suspension. Materials and Reagents 100 × 15 mm Petri dish (Corning®, Falcon®, catalog number: 351029) Single-use scalpels No. 10 (Fisher Scientific, FeatherTM, catalog number: 08-927-5A) MACS® SmartStrainers 30 μm (Miltenyi Biotec, catalog number: 130-110-915) FlowMiTM 40 μm pipette tip strainers (Bel-Art, catalog number: H13680-0040) 15 mL FalconTM conical centrifuge tubes (Corning®, catalog number: 352096) 50 mL FalconTM conical centrifuge tubes (Corning®, catalog number: 352070) DNA LoBind® 1.5 mL microcentrifuge tubes (Eppendorf, catalog number: 022431021) FisherbrandTM tweezers/forceps (Fisher Scientific, catalog number: 12-000-128) Countess cell counting chamber slides (Thermo Fisher Scientific, catalog number: C10228) Phosphate-buffered saline (PBS), 1× without calcium and magnesium, pH 7.4 (Corning®, catalog number: 21-040-CM) Nuclease-free water (not DEPC-treated) (Fisher Scientific, InvitrogenTM, catalog number: AM9932) IGEPAL® CA-630 (Millipore Sigma, catalog number: 18896-50ML) Sodium chloride (NaCl) (Millipore Sigma, catalog number: S5886-500G) Magnesium chloride (MgCl2) (Millipore Sigma, catalog number: M2393-100G) Bovine serum albumin (BSA) (Millipore Sigma, catalog number: A8806-5G) Hoechst 33342 10 mg/mL solution (Thermo Fisher Scientific, catalog number: H3570) Protector RNase inhibitor (Millipore Sigma, catalog number: 3335402001) Trizma® hydrochloride solution (Tris-HCl) 1 M pH 7.4 (Millipore Sigma, catalog number: T2194) 5 M NaCl stock (see Recipes) 1 M MgCl2 stock (see Recipes) 10% IGEPAL (see Recipes) 0.1% lysis buffer (see Recipes) Wash and resuspension buffer (WRB) (see Recipes) Hoechst stain buffer (see Recipes) Equipment Refrigerated benchtop centrifuge Note: Using a swinging bucket rotor for centrifugation helps to improve nuclei yields when compared with a fixed-angle rotor centrifuge. Countess II FL automated cell counter, or equivalent cell counting method Agilent Bioanalyzer Procedure Prepare materials and reagents Fill a rectangular ice pan and ice bucket with ice. Prepare the lysis buffer, wash and resuspension buffer (WRB), Hoechst stain buffer, and PBS. Place these reagents on ice. Set the benchtop centrifuge to 4°C and allow it to cool before use. Label the 15 mL Falcon tubes for samples and supernatant waste. Keep the forceps and scalpels chilled. Keep the tissue frozen on dry ice until lysis to avoid thawing, as this will lead to RNA degradation. Note: Ideally, liver biospecimens should be snap frozen and cryopreserved immediately after resection to prevent degradation. Tissue can be stored at -80°C or in liquid nitrogen until use. Lysis Perform all lysis steps in a biosafety cabinet. Add 2 mL of ice-cold lysis buffer per 100 mg of tissue onto a Petri dish over ice in an ice pan. Using a clean set of forceps, transfer the snap-frozen biopsy into the lysis buffer on the dish. Do not allow the biopsy to thaw before placing it into the lysis buffer (Figure 2A). Figure 2. Scalpel homogenization of liver tissue biopsies. (A) Incubation of liver biopsies in lysis buffer. (B) Liver biopsies after a 5 min incubation and manual dissociation with a scalpel. Allow the tissue to thaw in the lysis buffer for 5 min. Note: If significant clumping of nuclei is observed, the lysis time may be reduced to prevent damage to the nuclear membrane. Mince the tissue in the lysis buffer using a pair of forceps and a scalpel. The tissue should be minced into 2–4 mm pieces (Figure 2B). Using a 5 mL serological pipette, add 2 mL of ice-cold PBS to the tissue lysate on the dish. Mix by gently pipetting up and down 10 times. Aspirate the lysate and filter through a 30 μm smart strainer into a 15 mL conical centrifuge tube. Wash Centrifuge the tissue lysate at 500 × g for 5 min at 4°C. Remove the supernatant without disturbing the pellet. Note: The pellet may not be visible during this and subsequent wash steps (Figure 3). Care must be taken not to disturb and aspirate the pellet. A 5 mL serological pipette can be used to remove most of the supernatant, followed by a 1,000 μL pipette to carefully remove the remaining lysate. Figure 3. A small pellet of nuclei is present after lysis. The image shows a small pellet of nuclei after lysis, filtering, and centrifugation. The pellet may not be visible after centrifugation. Add 1 mL of ice-cold WRB and re-suspend the pellet by gently pipetting up and down 10–20 times. Filter the suspension through a 30 μm smart strainer into a new 15 mL conical centrifuge tube. Note: Tilt the strainer at a slight angle and pipette at the corner to allow for airflow and to reduce volume loss. If the suspension does not pass through, gently tap the strainer. Centrifuge at 500 × g for 5 min at 4°C. Using a 1,000 μL pipette, carefully remove the supernatant without disturbing the pellet. Add 70 μL of ice-cold WRB. Re-suspend the pellet by gently pipetting up and down 10–20 times. Transfer the nucleus suspension to a 1.5 mL LoBind microcentrifuge tube. Note: Compared to the 10× nuclei isolation protocol, our protocol includes only one wash step to accommodate samples with low yields of nuclei and to decrease run time. Additional wash steps can be included if the sample is contaminated and has a high enough yield (see below). Nucleus counting Mix 10 μL of the nucleus suspension with 10 μL of Hoechst stain buffer in a PCR tube. Transfer 10 μL of the stained nucleus suspension to a Countess cell counting chamber slide. Assess the concentration and quality of the suspension (Figure 4) using a Countess II FL automated cell counter. Note: Cell counting and quality assessment of nuclei can be done with an automated cell counter or manually. We recommend using a fluorescent DNA stain, such as Hoechst 33342 or DAPI, to discern nuclei from tissue debris. Alternatively, trypan blue staining and visualization under brightfield can be used. However, this makes reliable estimation of nucleus concentration challenging, especially if the nuclei are not intact. Figure 4. Hoechst staining and imaging of isolated nuclei. Microscope images of nuclei in the middle square of a hemocytometer under (A) brightfield and (B) fluorescent Hoechst staining are shown to provide a higher resolution and scale compared to a Countess. In (B), examples of singlet, doublet, and degraded nuclei are labeled. If the concentration of nuclei is too high, dilute with WRB to achieve a target concentration of 1,000 nuclei per microliter. For loading nuclei into the 10× Chromium Controller, the concentration range limit is 100–2,000 nuclei per microliter, and the recommended target concentration is 700–1,200 nuclei per microliter. Dilute with WRB to reduce the concentration if necessary. If the nucleus suspension contains high amounts of debris or doublets and aggregates, perform an additional wash step by repeating steps C2–C5. Note: Higher levels of debris and nuclei aggregates, especially those larger than 50 µm in diameter, lead to a higher chance of clogging the microfluidic channel, resulting in a wetting failure. However, note that wash steps can result in a significant loss in yield of approximately 50%. Using a 40 µm pipette tip strainer in place of a 30 µm smart strainer can help prevent volume loss, although clogging may occur. The centrifugation speed and spin time can also be reduced, for example to 400 × g at 4 min, to help prevent the formation of nuclei aggregates. Single-nucleus RNA-seq Prepare the suspension to load a predetermined number of nuclei. Suspensions can be diluted in nuclease-free water to achieve the target number of nuclei to load. For 10× solutions, consult the appropriate 10× user guide for nuclei recovery numbers given suspension concentrations. Note: Loading higher numbers of nuclei will result in a higher doublet rate. Doublet rates approximately follow a Poisson process (McGinnis et al., 2019) and the rate can be predicted from the number of nuclei loaded. For 10× solutions, perform the single-cell protocol detailed in the appropriate 10× user guide. The protocol should be adjusted accordingly if another platform, such as drop-seq (Macosko et al., 2015), is used. Briefly, these steps include 1) droplet/bead formation, 2) reverse transcription, 3) cDNA amplification, and 4) library construction. The details of each depend on the specific method and technology. Note: Begin the single-cell protocol as soon as possible. Droplet emulsification and reverse transcription should begin no more than a few hours after isolation. The number of PCR cycles to amplify cDNA is experiment- and sample-specific, but we recommend higher numbers to accommodate the lower RNA yields from nuclei compared to cells. Consult the single-cell protocol for appropriate ranges. For 10×, either 12 or 13 cycles are recommended. Assess the quality of the cDNA and sequencing libraries using an Agilent Bioanalyzer. Note: Ideally, there should be a smooth peak centered between 1,000 and 1,500 base pairs. However, RNA from human liver tissue is susceptible to degradation, thus cDNA traces can show significant peaks below 1,000 base pairs (Figure 5). Figure 5. Representative bioanalyzer trace of snRNA-seq library from frozen liver tissue. Figure shows a representative cDNA trace from an Agilent Bioanalyzer tape station. This frozen liver tissue sample shows RNA degradation with most cDNA fragments below a length of 1,000 base pairs. Sequencing Sequence the libraries using the appropriate platform. For Illumina sequencing, select the number of lanes to achieve a read depth of 20,000–50,000 reads per nucleus. Note: The target read depth per nucleus will depend on the desired coverage and the amount of RNA in a sample. Generally, higher sequencing depths will have a higher sensitivity for more lowly expressed genes. Samples with high amounts of RNA will require higher sequencing depths to profile lowly expressed genes. In contrast, samples with low amounts of RNA will reach saturation at lower sequencing depths. Data analysis For alignment and gene quantification, we recommend using STARsolo (Dobin et al., 2013; Kaminow et al., 2021). If there is significant RNA degradation, then adapters, polyA tails, and template switch oligos can be trimmed. Raw snRNA-seq data from frozen tissues typically require quality control filtering and processing. A barcode-rank plot can help assess the quality and extent of nucleus and RNA degradation. If an elbow point and count threshold are not easily discerned, droplets can be filtered based on their gene count distributions using DIEM (Alvarez et al., 2020) or EmptyDrops (Lun et al., 2019). Our publications on snRNA-seq of liver (Alvarez et al., 2022; Rao et al., 2021) provide details on filtering droplets using DIEM (Alvarez et al., 2020). After droplet filtering, nuclei can be clustered using Seurat (Stuart et al., 2019) or another approach (Yu et al., 2022). Adapting to different tissue samples The steps in this protocol were optimized for snap-frozen liver tissue samples. However, our protocol can be optimized for other sample types as well. Two key areas for optimization are homogenization and washing. Mincing with a scalpel helps to preserve the integrity of the nuclei and reduce ambient RNA contamination. However, other homogenization methods, such as a dounce grinder, can be tested to help increase yields. The second area of optimization includes the number and volume of wash steps. We include only one wash step with 1 mL of buffer to minimize a loss in concentration. If a higher number of nuclei is present (>1,000 nuclei per microliter) from a sample type, an additional wash step can be included to reduce tissue debris. Further optimizations to this approach will involve balancing yield and quality, where greater homogenization and less washing improves yields and decreases the quality. Cryopreservation and snap freezing of liver tissue Proper cryopreservation and snap freezing of liver tissue is critical to ensure high quality RNA and intact nuclei. For snRNA-seq, it is critical to avoid RNA degradation. Therefore, ischemic time must be minimized, and tissue should be snap frozen as soon as possible. It is also necessary to reduce ice crystal formation, as this can potentially damage nuclei and reduce the yield. Suspensions of intact, single nuclei will generate the highest quality snRNA-seq data. Therefore, snap-freezing methods that rapidly and evenly freeze the tissue to preserve morphology are preferred. Once frozen, tissues can be stored in -80 °C, or, for long-term storage, in the vapor phase of liquid nitrogen. The tissue should then be thawed in lysis buffer only once the protocol begins. Recipes 5 M NaCl stock Add 2.922 g of NaCl to 10 mL of water. 1 M MgCl2 stock Add 2.0331 g of MgCl2·6H2O to 10 mL of water. 10% IGEPAL Add 100 μL of IGEPAL to 900 μL of PBS. 0.1% lysis buffer To 9.77 mL of nuclease-free water, add 100 μL of 1 M Tris-HCl 20 μL of 5 M NaCl stock 10 μL of 1 M MgCl2 stock per 10 mL 100 μL of 10% IGEPAL 75 μL of 40 U/μL RNase inhibitor. Note: RNase inhibitor in the lysis buffer helps to prevent RNA degradation during the lysis step. Wash and resuspension buffer (WRB) Add 200 mg of BSA to a 15 mL conical centrifuge tube. Add up to 10 mL of 1× PBS. Add 50 μL of 40 U/μL RNase inhibitor. Hoechst stain buffer Add 10 μL of 10 mg/mL Hoechst stain to 990 μL to create stock A. Add 2 μL of stock A to 98 μL of PBS to get a working solution. Acknowledgements We thank the individuals who participated in the liver HCC cohort (Alvarez et al., 2022). The work was supported by NIH grants R01HG010505 and R01DK132775. M.A. was supported by an HHMI Gilliam Fellowship. References Aizarani, N., Saviano, A., Sagar, Mailly, L., Durand, S., Herman, J. S., Pessaux, P., Baumert, T. F. and Grun, D. (2019). A human liver cell atlas reveals heterogeneity and epithelial progenitors. Nature 572(7768): 199-204. Alvarez, M., Rahmani, E., Jew, B., Garske, K. M., Miao, Z., Benhammou, J. N., Ye, C. J., Pisegna, J. R., Pietiläinen, K. H., Halperin, E., et al. (2020). Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM. Sci Rep 10(1): 11019. Alvarez, M., Benhammou, J. N., Darci-Maher, N., French, S. W., Han, S. B., Sinsheimer, J. S., Agopian, V. G., Pisegna, J. R. and Pajukanta, P. (2022). Human liver single nucleus and single cell RNA sequencing identify a hepatocellular carcinoma-associated cell-type affecting survival. Genome Med 14(1): 50. Andrews, T. S., Atif, J., Liu, J. C., Perciani, C. T., Ma, X. Z., Thoeni, C., Slyper, M., Eraslan, G., Segerstolpe, A., Manuel, J., et al. (2022). Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatol Commun 6(4): 821-840. Ben-Moshe, S. and Itzkovitz, S. (2019). Spatial heterogeneity in the mammalian liver. Nat Rev Gastroenterol Hepatol 16(7): 395-410. Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M. and Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1): 15-21. Drissen, R., Buza-Vidas, N., Woll, P., Thongjuea, S., Gambardella, A., Giustacchini, A., Mancini, E., Zriwil, A., Lutteropp, M., Grover, A., et al. (2016). Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing. Nat Immunol 17(6): 666-676. Habib, N., Avraham-Davidi, I., Basu, A., Burks, T., Shekhar, K., Hofree, M., Choudhury, S. R., Aguet, F., Gelfand, E., Ardlie, K., et al. (2017). Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat Methods 14(10): 955-958. Halpern, K. B., Shenhav, R., Matcovitch-Natan, O., Toth, B., Lemze, D., Golan, M., Massasa, E. E., Baydatch, S., Landen, S., Moor, A. E., et al. (2017). Single-cell spatial reconstruction reveals global division of labour in the mammalian liver. Nature 542(7641): 352-356. Kaminow, B., Yunusov, D. and Dobin, A. (2021). STARsolo: accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data. bioRxiv: 2021.2005.2005.442755. Kuppe, C., Ibrahim, M. M., Kranz, J., Zhang, X., Ziegler, S., Perales-Paton, J., Jansen, J., Reimer, K. C., Smith, J. R., Dobie, R., et al. (2021). Decoding myofibroblast origins in human kidney fibrosis. Nature 589(7841): 281-286. Larson, A. and Chin, M. T. (2021). A method for cryopreservation and single nucleus RNA-sequencing of normal adult human interventricular septum heart tissue reveals cellular diversity and function. BMC Med Genomics 14(1): 161. Lun, A. T. L., Riesenfeld, S., Andrews, T., Dao, T. P., Gomes, T., participants in the 1st Human Cell Atlas, J. and Marioni, J. C. (2019). EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data. Genome Biol 20(1): 63. Macosko, E. Z., Basu, A., Satija, R., Nemesh, J., Shekhar, K., Goldman, M., Tirosh, I., Bialas, A. R., Kamitaki, N., Martersteck, E. M., et al. (2015). Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 161(5): 1202-1214. MacParland, S. A., Liu, J. C., Ma, X. Z., Innes, B. T., Bartczak, A. M., Gage, B. K., Manuel, J., Khuu, N., Echeverri, J., Linares, I., et al. (2018). Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations. Nat Commun 9(1): 4383. McGinnis, C. S., Murrow, L. M. and Gartner, Z. J. (2019). DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Syst 8(4): 329-337 e324. Rao, S., Yang, X., Ohshiro, K., Zaidi, S., Wang, Z., Shetty, K., Xiang, X., Hassan, M. I., Mohammad, T., Latham, P. S., et al. (2021). beta2-spectrin (SPTBN1) as a therapeutic target for diet-induced liver disease and preventing cancer development. Sci Transl Med 13(624): eabk2267. Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W. M., 3rd, Hao, Y., Stoeckius, M., Smibert, P. and Satija, R. (2019). Comprehensive Integration of Single-Cell Data. Cell 177(7): 1888-1902 e1821. Trefts, E., Gannon, M. and Wasserman, D. H. (2017). The liver. Curr Biol 27(21): R1147-R1151. Villani, A. C., Satija, R., Reynolds, G., Sarkizova, S., Shekhar, K., Fletcher, J., Griesbeck, M., Butler, A., Zheng, S., Lazo, S., et al. (2017). Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 356(6335). Xu, Y., Mizuno, T., Sridharan, A., Du, Y., Guo, M., Tang, J., Wikenheiser-Brokamp, K. A., Perl, A. T., Funari, V. A., Gokey, J. J., et al. (2016). Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis. JCI Insight 1(20): e90558. Yu, L., Cao, Y., Yang, J. Y. H. and Yang, P. (2022). Benchmarking clustering algorithms on estimating the number of cell types from single-cell RNA-sequencing data. Genome Biol 23(1): 49. Zhang, Q., He, Y., Luo, N., Patel, S. J., Han, Y., Gao, R., Modak, M., Carotta, S., Haslinger, C., Kind, D., et al. (2019). Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma. Cell 179(4): 829-845 e820. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > RNA > RNA sequencing Cell Biology > Organelle isolation > Nuclei Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,602	https://bio-protocol.org/en/bpdetail?id=4602&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS HS Hironori Saito TO Tatsuya Osaki YI Yoshiho Ikeuchi SI Shintaro Iwasaki Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4602 Views: 673 Reviewed by: Gal HaimovichBrian M. Zid Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in RNA Jun 2022 Abstract In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed. Keywords: Mitochondria Translation FUNCAT FACS Mitoribosome Background Mitochondria are important power plant organelles that produce ATP through oxidative phosphorylation (OXPHOS). Since the symbiosis of the bacterial ancestor, the mitochondrial genome has been minimized due to gene transfer to the nuclear genome. In humans, the organelle genome contains only 13 mRNAs, which all encode a subunit of OXPHOS complexes(Anderson et al., 1981). Despite the small number of mRNAs, their translation by mitochondrial ribosomes (mitoribosomes) is essential since defects in organello translation lead to several diseases, such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes, and myoclonus epilepsy associated with ragged-red fibers (Gorman et al., 2016). Therefore, mitochondrial translation has been researched by several methods [see Apostolopoulos and Iwasaki (2022) for a summary], such as35S-methionine-labeling of newly synthesized nascent chains (Chomyn, 1996;Sasarman and Shoubridge, 2012), the quantification of ribosome-associated mRNAs by quantitative reverse transcription-PCR (Antonicka et al., 2013; Fung et al., 2013; Zhang et al., 2014; Pearce et al., 2017), ribosome profiling (Rooijers et al., 2013; Iwasaki et al., 2016; Gao et al., 2017; Pearce et al., 2017; Morscher et al., 2018; Suzuki et al., 2020; Kashiwagi et al., 2021; Li et al., 2021; Schöller et al., 2021), and pulse stable isotope labeling by amino acids in cell culture for proteomic analysis (Imami et al., 2021). However, these approaches provide averaged data for thousands of cells, which are pooled for the experiments, and thus pose an analytic hurdle when assessing mitochondrial translation in each individual cell. However, an alternative method may overcome this barrier. In this technique, click-reaction-compatible methionine analogs, such as L-homopropargylglycine (HPG) and L-azidohomoalanine (AHA), are employed for nascent chain labeling. Subsequent fluorophore conjugation with the click reaction monitors mitochondrial protein synthesis (fluorescent noncanonical amino acid tagging, FUNCAT) (Dieterich et al., 2010). By shutting off cytosolic translation with compounds (such as anisomycin), only the proteins synthesized in the mitochondria (mito-FUNCAT) are labeled (Zhang et al., 2014; Estell et al., 2017; Yousefi et al., 2021; Zorkau et al., 2021). In addition to the in vitro click reaction and subsequent detection on a gel (on-gel mito-FUNCAT), in situ fluorophore conjugation provides visualization of mitochondrial protein synthesis in each cell under a microscope (in situ mito-FUNCAT) (Zhang et al., 2014; Estell et al., 2017;Yousefi et al., 2021;Zorkau et al., 2021). Recently, we further modified the in situ mito-FUNCAT to perform high-throughput measurements by applying fluorescence-activated cell sorting (FACS) on a massive number of cells (mito-FUNCAT FACS) (Kimura et al., 2022). In this manuscript, we describe the step-by-step protocol of this method, which is applicable to various cell lines. We added Cy3 to HPG-labeled polypeptides and simultaneously stained Tom20 with Alexa-Fluor 647 (AF 647) by immunostaining. Given that the abundance of Tom20 represents the mitochondrial mass and allows mitochondrial protein synthesis to be normalized, this method ensures that the alteration of synthesized protein originates from mitochondrial biogenesis or net translation. Moreover, with mito-FUNCAT FACS, the unexpected cellular heterogeneity of mitochondrial translation in cell populations can be determined. The application of this method will provide pivotal insights into the regulatory mechanisms of mitochondrial protein synthesis across cell types, development, stress response, etc. Materials and Reagents Falcon 25 cm2 rectangular canted neck cell culture flask with plug seal screw cap (Corning, catalog number: 353082) Nunc EasYDish dishes 100 mm (Thermo Fisher Scientific, catalog number: 150466) Primaria 25 cm2 rectangular canted neck cell culture flask with vented cap (Corning, catalog number: 353808) Safe lock tubes 2.0 mL (Eppendorf, catalog number: 3-7353-04) Tube, 15 mL, PP, 17/120 mm, conical bottom, cellstar, blue screw cap, natural, graduated, writing area, sterile, 5 psc./bag, triple packed (Greiner Bio-One, catalog number: 188271) Pipette, 10 mL, graduated 1/10 mL, sterile, paper-plastic packaging, single packed (Greiner Bio-One, catalog number: 607180) Pipette, 25 mL, graduated 2/10 mL, sterile, paper-plastic packaging, single packed (Greiner Bio-One, catalog number: 760180) 10 μL, long filter tip, graduated, system rack (PP), sterilized (WATSON, catalog number: 1252P-207CS) 20 μL, hyper filter tip, refill plate, sterilized (WATSON, catalog number: 127-20S) 200 μL, hyper filter tip, refill plate, sterilized (WATSON, catalog number: 0127-20S) 1,000 μL, long filter tip, graduated, refill plate, sterilized (WATSON, catalog number: 126-1000S) Falcon 5 mL round bottom polystyrene test tube, with snap cap, sterile (Corning, catalog number: 352058) Falcon 5 mL round bottom polystyrene test tube, with cell strainer snap cap (Corning, catalog number: 352235) Stericup quick release-GP sterile vacuum filtration system (Millipore, catalog number: S2GPU01RE) A375 [American Type Culture Collection (ATCC), catalog number: CRL-1619] C2C12 (ATCC, catalog number: CRL-1772) H1944 (ATCC, catalog number: CRL-5907) H2009 (ATCC, catalog number: CRL-5911) H2122 (ATCC, catalog number: CRL-5985) H441 (ATCC, catalog number: HTB-174) HEK293 (ATCC, catalog number: CRL-1573) HEK293T (ATCC, catalog number: CRL-3216) HeLa S3 (RIKEN BioResource Research Center, RCB1525) Alexa-Fluor 647 Anti-Tom20 antibody (Abcam, catalog number: ab209606, stored at 4°C) Anisomycin (from Streptomyces griseolus) (Chem-Impex International, catalog number: 00466, stored at -20°C) 10× Click-iT cell reaction buffer (Thermo Fisher Scientific, catalog number: C10269, stored at 4°C) Click-iT reaction buffer additive (Thermo Fisher Scientific, catalog number: C10269, stored at -20°C) Copper (II) sulfate (CuSO4) (Thermo Fisher Scientific, catalog number: C10269, stored at 4°C) Cy3-azide (Jena Bioscience, catalog number: CLK-046, stored at -20°C) Digitonin (Nacalai Tesque, catalog number: 19390-91, stored at room temperature) DMEM, high glucose, GlutaMAX supplement (Thermo Fisher Scientific, catalog number: 10566-016, stored at 4°C) DMEM, high glucose, no glutamine, no methionine, no cystine (Thermo Fisher Scientific, catalog number: 21013-024, stored at 4°C) Dimethyl sulfoxide (DMSO), nuclease and protease tested (Nacalai Tesque, catalog number: 09659-14, stored at room temperature) L-homopropargylglycine (HPG) (Jena Bioscience, catalog number: CLK-1067, stored at -20°C) Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524, stored at -20°C) 1 mol/L HEPES-KOH buffer solution (pH 7.5) (Nacalai Tesque, catalog number: 15639-84, stored at room temperature) Intercept (TBS) blocking buffer (LI-COR, catalog number: 927-60001, stored at 4°C) 200 mmol/L L-alanyl-L-glutamine solution (100×) (Nacalai Tesque, catalog number: 04260-64, stored at 4°C) L-cystine dihydrochloride, animal-free (Nacalai Tesque, catalog number: 13003-12, stored at room temperature) 1 mol/L magnesium chloride solution (MgCl2), sterile-filtered (Nacalai Tesque, catalog number: 20942-34, stored at room temperature) 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, catalog number: 09154-85, stored at 4°C) 5 mol/L sodium chloride solution (NaCl) (Nacalai Tesque, catalog number: 06900-14, stored at room temperature) Sucrose, ultra-pure (Wako, catalog number: 198-13525, stored at room temperature) Triton X-100 (Nacalai Tesque, catalog number: 12967-32, stored at room temperature) Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, catalog number: 25300-054, stored at 4°C) UltraPure DNase/RNase-Free distilled water (Thermo Fisher Scientific, catalog number: 10977-015, stored at room temperature) Bovine serum albumin (Sigma-Aldrich, catalog number: A4919) Met-free DMEM (500 mL) (see Recipes) 100 mg/mL anisomycin (250 µL) (see Recipes) 100 mM HPG (6.11 mL) (see Recipes) Met-free DMEM with anisomycin and HPG (10 mL) (see Recipes) Met-free DMEM with anisomycin (10 mL) (see Recipes) 0.5% digitonin (10 mL) (see Recipes) Mitochondria-protective buffer (5 mL) (see Recipes) Mitochondria-protective buffer with 0.0005% digitonin (5 mL) (see Recipes) 10% Triton X-100 (40 mL) (see Recipes) PBS with 0.1% Triton X-100 (0.5 mL) (see Recipes) 5 mM Cy3-azide (370 µL) (see Recipes) 50 µM Cy3-azide (100 µL) (see Recipes) Click-reaction mixture (1 mL) (see Recipes) Intercept (TBS) blocking buffer with Alexa-Fluor 647 Anti-Tom20 antibody (101 µL) (see Recipes) FACS buffer (see Recipes) Equipment High-speed micro centrifuge (himac, model: CF16RN) High-speed refrigerated micro centrifuge (TOMY, model: MDX-310) Swing rotor (himac, model: T4SS31) Swing rack (TOMY, model: SAR015-24) CO2 incubator (PHCbi, model: MAC-170AIC) Pipet-Aid XP2 (Drummond Scientific Company, catalog number: 4-040-501) Pipetman P-10 (Gilson, catalog number: F144802) Pipetman P-20 (Gilson, catalog number: F123600) Pipetman P-200 (Gilson, catalog number: F123601) Pipetman P-1000 (Gilson, catalog number: F123602) BD FACSMelody [BD, 3-laser (488, 640, and 561 nm), 8-color (2-2-4) configuration)] CellDrop BF (DeNovix, CellDrop BF-UNLTD) Software BD FACSChorus (BD FACSMelody operating software) FlowJo (BD, v10.8.1) R version 4.1.1 (R Development Core Team, https://www.r-project.org ) Procedure Note: When the cells are centrifuged, use swing rotors. Cell culture Culture cells to 70%–80% confluency with 5 mL of medium in a humidified incubator with 5% CO2 at 37°C. The media and flask used for each cell line in our experiments are described below. Medium and flasks: Cell type Media Flask C2C12 DMEM, high glucose, GlutaMAX supplement with 10% FBS Falcon T25 flask HeLa S3 Falcon T25 flask HEK293 Falcon T25 flask HEK293T Falcon T25 flask A375 Falcon T25 flask H2009 Falcon T25 flask H2122 RPMI 1640 medium with 10% FBS Primaria T25 flask H1944 Falcon T25 flask H441 Falcon T25 flask Aspirate the medium from the flask and add 3 mL of PBS slowly. Aspirate PBS from the flask and add 1 mL of trypsin-EDTA. Incubate the cells in a humidified incubator with 5% CO2 for 5 min at 37°C. Add 5 mL of the medium described in the table above and detach cells from the flask by pipetting. Transfer the cell suspension to a 15 mL tube. Centrifuge the cells at 800 × g for 3 min at 25°C. Aspirate the supernatant and resuspend the cell pellet with 5 mL of the medium described in the table above. Count the cell number by CellDrop. Note: The cell suspension should be at 0.5 × 106 –2 × 106 cells/mL. Dilute the cell suspension to 2 × 105 cells/mL with the medium described in the table above. Seed 10 mL of cell suspension (2 × 105 cells/mL) in a 10 cm dish. Note: The following cultures should be prepared: one culture for the tested sample and another for the negative control for FACS analysis. Incubate the cells in a humidified incubator with 5% CO2 overnight at 37°C. HPG labeling For the sample, aspirate the medium from one of the 10 cm dishes and add 10 mL of Met-free DMEM (see Recipes). Aspirate the medium and add 10 mL of Met-free DMEM with anisomycin and HPG (see Recipes) to the 10 cm dish. For the negative control, use DMEM with anisomycin (see Recipes). Note: Anisomycin is used to shut off cytosolic protein synthesis. Incubate the 10 cm dish in a humidified incubator with 5% CO2 for 3 h at 37°C. Fixation and permeabilization Aspirate the medium from the 10 cm dish and add 10 mL of PBS. Aspirate the PBS and add 1.5 mL of trypsin-EDTA. Incubate the cells in a humidified incubator with 5% CO2 for 5 min at 37°C. Transfer the cell suspension to a 2 mL tube. Centrifuge at 300 × g for 3 min at 25°C. Aspirate the supernatant and resuspend the cell pellet with 0.5 mL of cold PBS. Centrifuge the cells at 300 × g for 3 min at 25°C. Aspirate the supernatant and resuspend the cell pellet with 0.5 mL of mitochondria-protective buffer with 0.0005% digitonin (see Recipes) at room temperature. Incubate the cells for 5 min at 25°C. Centrifuge the cells at 300 × g for 3 min at 25°C. Discard the supernatant with a pipette and resuspend the cell pellet with 0.5 mL of ice-cold 4% paraformaldehyde phosphate buffer solution on ice. Incubate the cells for 15 min on ice. Centrifuge the cells at 300 × g for 3 min at 25°C. Discard the supernatant with a pipette and resuspend the cell pellet with 0.5 mL of PBS with 0.1% Triton X-100 (see Recipes) at room temperature. Incubate the cells for 5 min at 25°C. Centrifuge the cells at 1,000 × g for 3 min at 25°C. Discard the supernatant and proceed to the “Click reaction” immediately. Click reaction Resuspend the cell pellet with 250 µL of click-reaction mixture (see Recipes) at room temperature. Incubate the cells for 30 min at 25°C. Centrifuge at 1,000 × g for 3 min at 25°C. Discard the supernatant with a pipette and proceed to the “Tom20 immunostaining” immediately. Tom20 immunostaining Resuspend the cell pellet with 0.5 mL of intercept (TBS) blocking buffer at room temperature. Centrifuge at 1,000 × g for 3 min at 25°C. Discard the supernatant with a pipette and resuspend the cell pellet with 100 µL of ice-cold intercept (TBS) blocking buffer with Alexa-Fluor 647 Anti-Tom20 antibody (see Recipes) on ice. Note: For negative control of Tom20 immunostaining, use ice-cold intercept (TBS) blocking buffer (omitting Alexa-Fluor 647 Anti-Tom20 antibody). Incubate the cells for 1 h on ice. Centrifuge at 1,000 × g for 3 min at 2°C. Discard the supernatant with a pipette and resuspend the cell pellet in 0.5 mL of cold PBS. Repeat steps E41–42 twice (for a total of three washes). Resuspend the cell pellet in 0.5 mL of cold FACS buffer (see Recipes). Proceed to “FACS preparation and analysis” immediately. FACS preparation and analysis Filter the cell suspension with a cell strainer and collect the cells in a 5 mL round-bottom polystyrene test tube. Transfer the cells that passed through the filter to a new 5 mL round-bottom polystyrene test tube and preserve in ice until measurement. Place the 5 mL round-bottom polystyrene test tube containing the negative control (neither HPG-Cy3 nor Tom20-AF647 labeling) on the BD FACSMelody and start the flow. Set appropriate photomultiplier tube (PMT) voltages for forward scatters (FSCs) and side scatters (SSCs) with BD FACSChorus so that the signal lies within the detectable range. Then, gate a cell population as “Gate: Cells” in BD FACSChorus. To discriminate doublets, set “Gate: Singlet” according to the values of FSC-A/FSC-H and FSC-H/FSC-W in FlowJo. Set the PMT voltages for HPG-Cy3 labeling [561 nm laser, PE filter set (582/15 filter and 582 LP mirror); alternatively, 488 nm laser, PE filter set (586/42 filter and 560 LP)] and Tom20-AF647 labeling [640 nm laser, APC filter set (660/10 filter and 660/10 mirror)] in BD FACSChorus so that the signal is shown in approximately 100. Replace the 5 mL round-bottom polystyrene test tube containing the sample with the one containing the negative control in BD FACSMelody and start the flow. Record the data up to 10,000 events in “Gate: Singlet” and then export all the data at least including FSC-A, SSC-A, PE-A, and APC-A as an FCS file. Data analysis Load the FCS file in FlowJo. Readjust the gating to discriminate the dead cells, doublet, and triplet as a new population: “New singlet population.” Export “New singlet population” as a csv file containing APC-A (Tom20-AF647 signal) and PE-A (HPG-Cy3 signal). Note: The representative data shown inFigure 1are provided in representative csv files (“HEK293_WithoutHPG,” “HEK293_WithHPG.csv,” “H441#2.csv,” and “H2122#2.csv”) in the Supplemental material . Load the csv file to R and run “Mito-FUNCAT_FACS_script_Fig1AB.R” or “Mito-FUNCAT_FACS_script_Fig1CD.R” (found in Supplemental items) to conduct the following steps. Extract 10,000 rows from the top of the csv file. Remove rows containing fluorescence intensity less than 0. Normalize the HPG-Cy3 value by the Tom20-AF647 value for each row. Visualize the distribution of AF647-normalized Cy3 values in the density plot (seeFigure 1for representative data). Note: To determine statistical significance between samples, we typically used the MannWhitney U test. Figure 1. Distribution of Tom20-AF647 (mitochondrial mass)–normalized HPG-Cy3 (mitochondrial translation) signals. (A–D) The Cy3-conjugated nascent peptide in mitochondria was normalized to AF647-labeled Tom20 abundance for HEK293 (A and B), H411 (C), and H2122 (D). For A, HPG was omitted from the medium as the negative control. The vertical lines represent the median of the distributions. The original data (“HEK293_WithoutHPG,” “HEK293_WithHPG.csv,” “H441#2.csv,” and “H2122#2.csv”) and scripts (“Mito-FUNCAT_FACS_script_Fig1AB.R” and “Mito-FUNCAT_FACS_script_Fig1CD.R”) that were used to generate these graphs are provided in the Supplemental material . Recipes Met-free DMEM (500 mL) Reagent Final concentration Amount L-cystine dihydrochloride 48 µg/mL 24 mg 200 mM L-alanyl-L-glutamine solution 4.08 mM 10.2 mL FBS 10% 50 mL DMEM (4.5 g/L D-glucose, no L-glutamine, no sodium pyruvate, no L-methionine and L-cystine) n/a 439.8 mL 100 mg/mL anisomycin (250 µL) Reagent Final concentration Amount Anisomycin 100 mg/mL 25 mg DMSO n/a 250 µL 100 mM HPG (6.11 mL) Reagent Final concentration Amount HPG 100 mM 100 mg DMSO n/a 6.11 mL Met-free DMEM with anisomycin and HPG (10 mL) Reagent Final concentration Amount Met-free DMEM n/a 10 mL 100 mg/mL anisomycin 100 µg/mL 10 µL 100 mM HPG 100 µM 10 µL Note: Prepare before use. Met-free DMEM with anisomycin (10 mL) Reagent Final concentration Amount Met-free DMEM n/a 10 mL 100 mg/mL anisomycin 100 µg/mL 10 µL Note: Prepare before use. 0.5% digitonin (10 mL) Reagent Final concentration Amount Digitonin 0.5% 50 mg RNase-free water n/a 10 mL Mitochondria-protective buffer (5 mL) Reagent Final concentration Amount Sucrose 10% (w/v) 0.5 g (corresponds to 370 μL) 5 M NaCl 10 mM 10 µL 1 M MgCl2 5 mM 20 µL 1 M HEPES-KOH pH 7.5 10 mM 50 µL RNase-free water n/a 4.55 mL Mitochondria-protective buffer with 0.0005% digitonin (5 mL) Reagent Final concentration Amount Mitochondria-protective buffer n/a 5 mL 0.5% digitonin 0.0005% 5 µL 10% Triton X-100 (40 mL) Reagent Final concentration Amount Triton X-100 10% 4 mL RNase-free water n/a 36 mL PBS with 0.1% Triton X-100 (0.5 mL) Reagent Final concentration Amount PBS n/a 0.5 mL 10% Triton X-100 0.1% 5 µL 5 mM Cy3-azide (370 µL) Reagent Final concentration Amount Cy3-azide (MW: 539.35) 5 mM 1 mg DMSO n/a 370 µL 50 µM Cy3-azide (100 µL) Reagent Final concentration Amount 5 mM Cy3-azide 50 µM 1 µL RNase-free water n/a 99 µL Note: Prepare before use. Click-reaction mixture (1 mL) Reagent Final concentration Amount 10× Click-iT cell reaction buffer 1× 100 µL 100 mM CuSO4 2 mM 20 µL Click-iT reaction buffer additive n/a 100 µL 50 µM Cy3-azide 1 µM 20 µL RNase-free water n/a 760 µL Note: Prepare before use. Intercept (TBS) blocking buffer with Alexa-Fluor 647 Anti-Tom20 antibody (101 µL) Reagent Final concentration Amount Intercept (TBS) blocking buffer n/a 100 µL Alexa-Fluor 647 Anti-Tom20 antibody n/a 1 µL Note: Prepare before use and keep on ice. FACS buffer Reagent Final concentration Amount PBS n/a 100 mL Bovine serum albumin 3% 3 g Note: Prepare before use. Acknowledgments We are grateful to Yusuke Kimura, Taisei Wakigawa, Yasuhiro Ikegami, Kenji Ohtawa for their constructive advice on the experiments and to all the members of the Iwasaki laboratory for their critical reading of the manuscript. We also thank the Komaba Analysis Core, Institute of Industrial Science, The University of Tokyo, for FACS analysis. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (JP20H05784 to S.I.; JP20H05786 to Y.I.) and the Japan Agency for Medical Research and Development (AMED) (JP21gm1410001 to S.I. and Y.I.). H.S. was a RIKEN Junior Research Associate (JRA). This protocol was derived from an original paper (Kimura et al., 2022) and the manufacturer's protocol for the Click-it Cell Reaction Buffer Kit . Competing interests The authors declare no competing interests. Ethics No human or animal subjects were included in this study. References Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., et al. (1981). Sequence and organization of the human mitochondrial genome. Nature 290(5806): 457-465. Antonicka, H., Sasarman, F., Nishimura, T., Paupe, V. and Shoubridge, E. A. (2013). The mitochondrial RNA-binding protein GRSF1 localizes to RNA granules and is required for posttranscriptional mitochondrial gene expression. Cell Metab 17(3): 386-398. Apostolopoulos, A. and Iwasaki, S. (2022). Into the matrix: current methods for mitochondrial translation studies. J Biochem 171(4): 379-387. Chomyn, A. (1996). In vivo labeling and analysis of human mitochondrial translation products. Methods Enzymol 264: 197-211. Dieterich, D. C., Hodas, J. J., Gouzer, G., Shadrin, I. Y., Ngo, J. T., Triller, A., Tirrell, D. A. and Schuman, E. M. (2010). In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13(7): 897-905. Estell, C., Stamatidou, E., El-Messeiry, S. and Hamilton, A. (2017). In situ imaging of mitochondrial translation shows weak correlation with nucleoid DNA intensity and no suppression during mitosis. J Cell Sci 130(24): 4193-4199. Fung, S., Nishimura, T., Sasarman, F. and Shoubridge, E. A. (2013). The conserved interaction of C7orf30 with MRPL14 promotes biogenesis of the mitochondrial large ribosomal subunit and mitochondrial translation. Mol Biol Cell 24(3): 184-193. Gao, F., Wesolowska, M., Agami, R., Rooijers, K., Loayza-Puch, F., Lawless, C., Lightowlers, R. N. and Chrzanowska-Lightowlers, Z. M. A. (2017). Using mitoribosomal profiling to investigate human mitochondrial translation. Wellcome Open Res 2: 116. Gorman, G. S., Chinnery, P. F., DiMauro, S., Hirano, M., Koga, Y., McFarland, R., Suomalainen, A., Thorburn, D. R., Zeviani, M. and Turnbull, D. M. (2016). Mitochondrial diseases. Nat Rev Dis Primers 2: 16080. Imami, K., Selbach, M. and Ishihama, Y. (2021). Monitoring mitochondrial translation by pulse SILAC. bioRxiv. https://doi.org/10.1101/2021.01.31.428997 Iwasaki, S., Floor, S. N. and Ingolia, N. T. (2016). Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor. Nature 534(7608): 558-561. Kashiwagi, K., Shichino, Y., Osaki, T., Sakamoto, A., Nishimoto, M., Takahashi, M., Mito, M., Weber, F., Ikeuchi, Y., Iwasaki, S., et al. (2021). eIF2B-capturing viral protein NSs suppresses the integrated stress response. Nat Commun 12(1): 7102. Kimura, Y., Saito, H., Osaki, T., Ikegami, Y., Wakigawa, T., Ikeuchi, Y. and Iwasaki, S. (2022). Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation. RNA 28(6): 895-904. Li, S. H.-J., Nofal, M., Parsons, L. R., Rabinowitz, J. D. and Gitai, Z. (2021). Monitoring mammalian mitochondrial translation with MitoRiboSeq. Nat Protoc 16(6): 2802-2825. Morscher, R. J., Ducker, G. S., Li, S. H.-J., Mayer, J. A., Gitai, Z., Sperl, W. and Rabinowitz, J. D. (2018). Mitochondrial translation requires folate-dependent tRNA methylation. Nature 554(7690): 128-132. Pearce, S. F., Rorbach, J., Van Haute, L., D’Souza, A. R., Rebelo-Guiomar, P., Powell, C. A., Brierley, I., Firth, A. E. and Minczuk, M. (2017). Maturation of selected human mitochondrial tRNAs requires deadenylation. Elife 6: e27596. Rooijers, K., Loayza-Puch, F., Nijtmans, L. G. and Agami, R. (2013). Ribosome profiling reveals features of normal and disease-associated mitochondrial translation. Nat Commun 4: 2886. Sasarman, F. and Shoubridge, E. A. (2012). Radioactive labeling of mitochondrial translation products in cultured cells. Methods Mol Biol 837: 207-217. Schöller, E., Marks, J., Marchand, V., Bruckmann, A., Powell, C. A., Reichold, M., Mutti, C. D., Dettmer, K., Feederle, R., Hüttelmaier, S., et al. (2021). Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs. Mol Cell 81(23): 4810-4825.e12. Suzuki, T., Yashiro, Y., Kikuchi, I., Ishigami, Y., Saito, H., Matsuzawa, I., Okada, S., Mito, M., Iwasaki, S., Ma, D., et al. (2020). Complete chemical structures of human mitochondrial tRNAs. Nat Commun 11(1): 4269. Yousefi, R., Fornasiero, E. F., Cyganek, L., Montoya, J., Jakobs, S., Rizzoli, S. O., Rehling, P. and Pacheu-Grau, D. (2021). Monitoring mitochondrial translation in living cells. EMBO Rep 22(4): e51635. Zhang, X., Zuo, X., Yang, B., Li, Z., Xue, Y., Zhou, Y., Huang, J., Zhao, X., Zhou, J., Yan, Y., et al. (2014). MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell 158(3): 607-619. Zorkau, M., Albus, C. A., Berlinguer-Palmini, R., Chrzanowska-Lightowlers, Z. M. A. and Lightowlers, R. N. (2021). High-resolution imaging reveals compartmentalization of mitochondrial protein synthesis in cultured human cells. Proc Natl Acad Sci U S A 118(6). Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Molecular Biology > Protein > Flow Cytometry Cell Biology > Cell staining > Protein Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Immunohistochemistry of Immune Cells and Cells Bound to in vivo Administered Antibodies in Liver, Lung, Pancreas, and Colon of B6/lpr Mice Kieran Adam and Adam Mor Jul 20, 2022 2202 Views Novel Antibody-independent Method to Measure Complement Deposition on Bacteria Toska Wonfor [...] Maisem Laabei May 5, 2023 459 Views Immunofluorescent Staining Assay of 3D Cell Culture of Colonoids Isolated from Mice Colon Trisha Mehrotra [...] Didier Merlin Mar 5, 2024 940 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,603	https://bio-protocol.org/en/bpdetail?id=4603&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Generating Reproducing Anoxia Conditions for Plant Phenotyping IM Iny E. Mathew HR Hormat Shadgou Rhein AG Ardawna J. Green KH Kendal D. Hirschi Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4603 Views: 659 Reviewed by: Wenrong HeYao Xiao Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plant Physiology Nov 2022 Abstract Based on the availability of oxygen, plant growth environment can be normoxic (normal environment), hypoxic (reduced oxygen, <21%), or anoxic (complete depletion of oxygen). Hypoxic/anoxic environment is created when a plant is exposed to stresses such as submergence, flooding, or pathogen attack. Survival of the plants following stress conditions is in part dependent on their ability to overcome the stress induced by anoxia/hypoxia conditions. This shows the need for the development of strategies for understanding the mechanisms involved in plant tolerance to anoxia. Previous studies have employed different methods for establishing an anerobic environment. Here, we describe a simple method for creating anoxic environment using an anaerobic atmosphere generation bag. Anoxic conditions can be maintained in a cylindrical jar, a rectangular box, or a vacuum sealer bag, enabling the screening of a large number of samples. This protocol is particularly useful to screen plant mutants that are tolerant to anoxia. The method is simple, easy, cost-efficient, reproducible, and does not require any sophisticated instruments. Graphic abstract Keywords: Anoxia Arabidopsis Flooding Hypoxia Submergence Plants Background Anaerobic conditions are part of an ensemble of stresses plants incur during submergence. Flooding also imposes temperature changes and altered oxidative conditions (Sasidharan et al., 2018). While submergence assays for plants have been detailed, they are often hard to perform and produce variable results (Bui et al., 2020; Yang et al., 2022; Loreti and Perata, 2020). Meanwhile, multiple methods have been used to create anaerobic conditions (Clark, 2019). Some of these methods include the Hungate technique, which uses a roll-tube approach to culture medium through which anoxic gas is bubbled to remove the remaining oxygen (Hungate, 1969). Other techniques are the VPI (Virginia Polytech Institute) method, a modification to the Hungate technique for large-scale culture using pre-reduced medium (Moore, 1966), and Glove-box chambers, a sealed chamber with attached gloves that are filled with anoxic gases (Clark, 2019). Recently, glass desiccators have been used for creating a hypoxic environment for plants. The method involves flushing oxygen-depleted air (humidified 100% N2 gas) into air-tight closed glass desiccators under dark conditions to generate an anoxic environment (Hartman et al., 2019). Similarly, an enclosed anerobic workstation (Anaerobic System model 1025; Forma Scientific) has been used for generating an anoxic environment for plant experiments (Loreti et al., 2018). Some of the disadvantages of these older methods include accidental gas leaks that expose the chamber to oxygen, high cost of gas tanks, regular maintenance requirements, space limitation, single purpose application, and lower efficiency and complexity (Killgore et al., 1973). Here, we introduce a simple and robust anoxia assay. Compared to previous methods, this protocol is quick, easy to perform, highly reproducible, and economical, requires minimum space, and produces a controlled (0% oxygen) anoxic environment. Materials and Reagents Aluminum foil Anaerobic atmosphere generation bags, OxoidTM AnaeroGenTM 2.5 L sachet (Thermo Scientific; catalog number: OXAN0025A) AnaeroPackTM 2.5 L rectangular jar (Thermo Scientific, catalog number: R685025) FalconTM 15 mL conical tubes (CorningTM, catalog number: 14-959-53A) Protect laboratory bottles (DURAN, PSC-47-11619) Anaerobic system/cylindrical jar (BD GasPakTM 150 Systems, catalog number: 11-816) Microcentrifuge tubes, 1.5 mL (PierceTM, catalog number: 69715) Petri plates with clear lid (FisherbrandTM, catalog number: S33580A) Surgical tape (Micropore, available through Amazon, USA) Vacuum sealer bags (Wevac, available through Amazon, USA) Anaerobic indicator (Thermo Scientific, catalog number: BR0055B) Vaseline® (available through Amazon, USA) Half-strength MS media (see Recipes; modified from Cheng et al., 2003) Murashige and Skoog basal salt mixture (MS) media (Sigma-Aldrich, catalog number: M5524) Sucrose (Sigma-Aldrich, catalog number: S1888) Agar (Sigma-Aldrich, catalog number: A1296) 2-(N-Morpholino) ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: M3671) Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473) 20% (V/V) bleach (see Recipes) Commercially available bleach (Clorox concentrated household bleach; available through Amazon, USA) 80% (V/V) acetone (see Recipes) Acetone (Sigma-Aldrich, catalog number: 179124) Plant materials Recently, we have established that loss of a tonoplast-localized H+/Ca transporter, CAX1, imparts tolerance to anoxia. A mutation in another H+/Ca transporter, CAX3, is sensitive to anoxia in a manner indistinguishable from wildtype Colombia-0 (Col-0) (Yang et al., 2022). In the present study, we have used the same three mutants (two cax1 alleles and one cax3 mutant) along with Col-0 to standardize the anoxia assay conditions; the details are given below. Arabidopsis wild type (Col-0) Arabidopsis mutants: cax1-1 - CS25435 (Cheng et al., 2003) cax1-2- SALK_021486 (Catala et al., 2003) cax3-1- CS25429 (Cheng et al., 2003) Equipment Varian Cary 50 Bio UV-visible spectrophotometer Laminar air flow hood (SteriGard Hood, The Baker Company, Main, US) Pipettes (GILSON, PIPETMAN CLASSIC P1000, catalog number: F123602 and GILSON, PIPETMAN L P20L, 2–20 µL, catalog number: FA10003M) Nikon D80 Digital SLR camera (Nikon Corp., Tokyo, Japan) Plant growth chamber (Geneva Scientific, CU-36L6 tissue culture chamber) Heat poly bag sealer (Thomas Scientific, catalog number: 1147J64) Procedure Prepare half-strength MS media plates and 20% (V/V) bleach (see Recipes) Seed sterilization Transfer 100–200 seeds from each genotype to separate microcentrifuge tubes. Add 1 mL of 20% (V/V) bleach to the tubes. Mix the seeds thoroughly by brief vortexing. Incubate the seeds in bleach solution for 15 min with occasional vortexing. Give a short spin so that the seeds settle to the bottom of the tube. Do the remaining steps in a sterile environment. Decant the bleach solution carefully with a 1 mL pipette. It is normal to have some seeds (poorly developed/empty seeds) to float on the supernatant bleach solution. Add 1 mL of sterile water to the seeds and mix it thoroughly by inverting the tubes 3–4 times. Remove the water and add fresh sterile water to the seeds. Repeat steps B7–8 five more times to remove any trace of bleach. Add 1 mL of sterile water to the seeds. At this stage, you can store the seeds or immediately proceed for seed plating. The seeds can be stored at 4°C for a week without losing its germination frequency. Seed plating Bring the half-strength MS media plates and sterilized seeds to room temperature. Seed plating is done in a laminar flow hood. Using a 20 µL pipette, draw up the sterilized seeds such that they are individually lined up in the pipette tip (use cut tips). Carefully release the seeds onto the agar plate in the desired location (if two seeds are clumped together, add a small drop of water, separate the seeds, draw up one seed, and place it in another location). You can plate up to 50–60 seeds in a single plate. Make sure that the seeds are placed equidistantly. Leave the plate open in the flow hood surface for 2–3 min to dry out the water from the seeds. Cover the plates with surgical tape and keep them in a plant growth chamber maintained at 12 h light and 22°C, and 12 h night and 20°C for three weeks. Note: Depending on the plant species, the optimal plant size/growth stage for the anoxia assay may vary. We recommend modifying the protocol based on the plant species by performing the anoxia assay at different stages of plant growth to acquire optimal plant size. For Arabidopsis, the assay was performed at several stages of plant growth and 3-week-old plants showed the optimal phenotype. Preliminary data with tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) suggest that anoxia conditions for young seedlings will be in the range of conditions used for 3-week-old Arabidopsis; however, the initial tests with rice (Oryza sativa) and medicago (Medicago truncatula) lines suggest they are much more anoxia-tolerant and may require days to show sensitivity. Anoxia experiment Using a cylindrical jar (refer to Video 1): Remove the surgical tape from the Petri dishes. Stack the plates (nine plates/stack) into the anaerobic system/cylindrical jar (BD GasPakTM). Apply a light coat of Vaseline on the rims of the jar and ensure proper sealing of the box. Open a single sachet of AnaeroGenTM 2.5 L GasPakTM and place it in the jar on the side. Place 2–3 tissue papers between the GasPakTM and the plates to dissipate heat generated from the GasPakTM. Tighten the jar, cover with aluminum foil, and incubate in the dark at room temperature for required period of time. Note: Depending on the plant species, optimal anoxia treatment time may vary. We recommend modifying the protocol based on the plant species by performing the anoxia assay for multiple periods to acquire optimal duration. For Arabidopsis, the assay was performed for 4, 6, 8, and 10 h. At 8 h, tolerant and sensitive lines showed the most distinctive phenotype. Optional: Place an anaerobic indicator in the container to ensure the creation of anoxic environment. After the anoxia treatment, re-tape and incubate the plates in the plant growth chamber maintained at 12 h light and 22°C, and 12 h night and 20°C for 4–5 days. Photograph the plates once the phenotype is clear (after 4–5 days). Video 1. Anoxia treatment using “The Jar” Using a rectangular 2.5 L AnaeroPackTM system (refer to Video 2): Remove the surgical tape from the Petri dishes. Arrange the Petri plates containing plants in two stacks (four plates/stack) in the box. Apply a light coat of Vaseline on the rims of the box and ensure proper sealing of the box. Open a single sachet of AnaeroGenTM 2.5 L GasPakTM and place it inside the small compartment within the rectangular box; place 2–3 tissue papers between the GasPakTM and the plates to dissipate heat generated from the GasPakTM. Cover the jar with aluminum foil and incubate in the dark at room temperature for required period of time. After the anoxia treatment, return the plants to standard growth conditions. Photograph the plates after 4–5 days. Video 2. Anoxia treatment using “The Box” Using a vacuum sealer bag (refer to Video 3): Vacuum sealer bag is used for performing anoxia for a small number of plates (1–2). Remove the surgical tape from the Petri dishes. Cover both sides of the Petri dish with aluminum foil. Place the Petri dish on top of a layer of folded tissue paper inside the Wevac vacuum sealer bag. Open a single sachet of AnaeroGenTM 2.5 L GasPakTM and place it beneath the tissue paper inside the bag. Immediately seal the bag with a heat poly bag sealer and incubate at room temperature for required period of time. After the anoxia treatment, return the plates to standard growth conditions and photograph after 4–5 days. Video 3. Anoxia treatment using “The Bag” Chlorophyll estimation (modified from Liang et al., 2017) Grind 50 mg of tissue from both pre- and post-anoxia samples in liquid nitrogen. Add 1 mL of 80% (V/V) acetone to the ground tissue and mix thoroughly by vortexing. Incubate at room temperature for 30 min. Centrifuge at 17,900 × g for 1 min. Transfer the supernatant to a 15 mL centrifuge tube. Repeat steps E2–5 three more times or until the pellet becomes colorless. Add 80% (V/V) acetone to a final volume of 6 mL. Read the absorbance of the pooled supernatant at wavelengths of 645 and 663 nm with a spectrophotometer. Calculate the total chlorophyll in the sample using Arnon’s equation [total chlorophyll (µg/mL) = 20.2 (A645) + 8.02 (A663)]. Repeat the experiment at least two more times and represent the data as average ± SE (standard error). Data analysis Results from anoxia experiments were confirmed three times with 13–15 seeds tested each time. Chlorophyll estimation was performed for three biological replicates and represented as average ± SE (standard error). Notes While we do not see any advantage or disadvantage, we observed minor variations in the result of anoxia depending upon the chamber used, plant age, anoxia persistence, and diurnal conditions as given below. Comparison of three anoxia chambers suggests that the rectangular box and vacuum sealer bag produce a more consistent response than the cylindrical jar. The cylindrical jar sometimes produced more variability, as plants in closer proximity to the gas pack occasionally displayed enhanced sensitivity than those placed further from the gas pack in the same jar. Thus, here we used the rectangular box/vacuum sealer bags for subsequent experiments. The smaller volume containers may produce more rapid anoxic conditions; however, these differences were not significant enough to produce phenotypic changes in these assay conditions with Arabidopsis. Plant age: Using the mutants and control, we assessed the impact of plant age on anoxia tolerance (Boyes et al., 2001). Plants at the 9-rosette leaf stage (21 days old) and 6–7-rosette leaf stage (18 days old) were slightly more tolerant than plants at the 5-rosette leaf stage (14 days old; Figure 1). For all the remaining assays, plants at the 7-rosette leaf stage (18 days old) were used. Figure 1. Effect of plant age on anoxia response. Plants belonging to 5-rosette (A), 7-rosette (B), and 9-rosette (C) leaf stages were exposed to an anaerobic environment for 9 h during the night. The experiment was repeated three times, with 13–15 plants analyzed for each mutant in each replicate. Col-0 (wild type), cax1-1 and cax1-2 (two alleles of cax1 with T-DNA insertions at different locations), and cax3 (cax3 mutant). For further information including statistical comparisons and transcriptomic and proteomics data analyses refer to Yang et al. (2022). Duration of treatment: The effect of anoxia persistence was then assayed. A difference in the phenotype was observed as soon as at 6 h of treatment in the rectangular 2.5 L AnaeroPackTM system (Figure 2A). The phenotype became very clear with prolonged incubation time. At 8–9 h of anoxia treatment, there was a clear difference between the phenotype of sensitive and tolerant genotypes. The two sensitive genotypes (Col-0 and cax3) did not survive after anoxia (Figure 2B–E). However, when the incubation was extended for 10 h, the survival rate for the tolerant genotypes (cax1-1 and cax1-2) reduced significantly (Figure 2F). The survival rate following anoxia was indirectly measured by estimating the residual chlorophyll content five days after the anoxia treatment. As shown in figure 2G, cax1 exhibited less chlorophyll loss compared to Col-0 and cax3. Figure 2. Effect of the duration of treatment to anoxia response. Plants belonging to 7-rosette leaf stage (18 days old) were exposed to anaerobic environment for 6 (A), 7 (B), 8 (C), 8.5 (D), 9 (E), and 10 h (F) during the day. Differential responses of the genotype to anoxia represented in terms of their chlorophyll loss (G). cax1 exhibited less chlorophyll loss than Col-0 and cax3. For further information including chlorophyll measurements, statistical comparisons, and transcriptomic and proteomics data analyses refer to Yang et al. (2022). Diurnal variations: We then sought to better understand how the photoperiod influences anoxia tolerance. While the anoxia treatment was always performed in the dark, plants treated during the daytime were slightly more sensitive than those treated at night (Figure 3). Hence, anoxia treatment at night without disturbing the diurnal rhythm was consistently used in our experiments. Figure 3. Effect of diurnal phase of treatment on response to anoxia. Plants belonging to 7-rosette leaf stage (18 days old) were exposed to an anaerobic environment for 8.5 h during the night (A), 8.5 h during the day (B), 9 h during the night (C), and 9 h during the day (D). By limiting variations in the above-mentioned parameters, differences in the response of a particular genotype to anoxia can be minimized. Although there was a slight difference in the extent of chlorophyll loss within a genotype (Figure 4), the assay clearly distinguished the tolerant from the susceptible genotypes. We have used the assay for Arabidopsis thaliana in this study, but this approach should be applicable to other plant species as well. Figure 4. Variations in the anoxia response with similar external conditions. Plants belonging to 7-rosette leaf stage (18 days old) were exposed to anaerobic condition during the night. Recipes Half-strength MS media (Preparation of 1 L media; enough for preparing approximately 20 plates) Dissolve the following components in 800 mL of double distilled water: 2.15 g of MS media 5 g of sucrose 500 mg of MES Adjust the pH to 5.6–5.8 by adding KOH while stirring. Make up the volume to 1 L. Add 8% (W/V) agar into two 500 mL bottles. Distribute the media equally into each bottle containing agar (500 mL). Sterilize the media by autoclaving at 121°C for 20 min. Once the media has cooled, pour each bottle into 10 Petri plates in the laminar flow hood. Allow the media to solidify by leaving it on the laminar flow hood for 30 min. The media can be stored at 4°C for 2–3 weeks. Bring the media to room temperature and inspect the plate for any contamination prior to use. 20% bleach (V/V) Mix 10 mL of bleach with 40 mL of autoclaved double distilled water in a 50 mL centrifuge tube. Mix it thoroughly by inverting the tube. Bleach can be stored at room temperature for 2–3 weeks. 80% acetone (V/V) Mix 40 mL of acetone with 10 mL of double distilled water. Mix it thoroughly by inverting the tube or vortexing. The solution can be stored at room temperature in a flame-resistant area. Acknowledgments This work was supported by grants (to K.D.H) from the National Science Foundation (1557890), USDA (3092-51000-061-00D), and National Institute of Health (R03 AI149201-02). This protocol is derived from Yang et al. (2022). Competing interests The authors declare that there is no conflict of interest. References Boyes, D. C., Zayed, A. M., Ascenzi, R., McCaskill, A. J., Hoffman, N. E., Davis, K. R. and Gorlach, J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13(7): 1499-1510. Bui, L. T., Shukla, V., Giorgi, F. M., Trivellini, A., Perata, P., Licausi, F. and Giuntoli, B. (2020). Differential submergence tolerance between juvenile and adult Arabidopsis plants involves the ANAC017 transcription factor. Plant J 104(4): 979-994. Catala, R., Santos, E., Alonso, J. M., Ecker, J. R., Martinez-Zapater, J. M. and Salinas, J. (2003). Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis. Plant Cell 15(12): 2940-2951. Cheng, N. H., Pittman, J. K., Barkla, B. J., Shigaki, T. and Hirschi, K. D. (2003). The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters. Plant Cell 15(2): 347-364. Clark, H. (2019). Culturing anaerobes. Nature Portfolio. Hartman, S., Liu, Z., van Veen, H., Vicente, J., Reinen, E., Martopawiro, S., Zhang, H., van Dongen, N., Bosman, F., Bassel, G. W., et al. (2019). Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress. Nat Commun 10(1): 4020. Hungate, R. E. (1969). Chapter IV: A Roll Tube Method for Cultivation of Strict Anaerobes. In: Methods in Microbiology. Norris, J. R. and Ribbons, D. W. (Eds.). Academic Press. 3: 117-132. Killgore, G. E., Starr, S. E., Del Bene, V. E., Whaley, D. N. and Dowell, V. R., Jr. (1973). Comparison of three anaerobic systems for the isolation of anaerobic bacteria from clinical specimens. Am J Clin Pathol 59(4): 552-559. Liang, Y., Urano, D., Liao, K. L., Hedrick, T. L., Gao, Y. and Jones, A. M. (2017). A nondestructive method to estimate the chlorophyll content of Arabidopsis seedlings. Plant Methods 13: 26. Loreti, E. and Perata, P. (2020). The Many Facets of Hypoxia in Plants. Plants (Basel) 9(6): 745. Loreti, E., Valeri, M. C., Novi, G. and Perata, P. (2018). Gene Regulation and Survival under Hypoxia Requires Starch Availability and Metabolism. Plant Physiol 176(2): 1286-1298. Moore, W. E. C. (1966). Techniques for routine culture of fastidious anaerobes. Intern J Syst Bacteriol 16: 173-190. Sasidharan, R., Hartman, S., Liu, Z., Martopawiro, S., Sajeev, N., van Veen, H., Yeung, E. and Voesenek, L. (2018). Signal Dynamics and Interactions during Flooding Stress. Plant Physiol 176(2): 1106-1117. Yang, J., Mathew, I. E., Rhein, H., Barker, R., Guo, Q., Brunello, L., Loreti, E., Barkla, B. J., Gilroy, S., Perata, P. and Hirschi, K. D. (2022). The vacuolar H+/Ca transporter CAX1 participates in submergence and anoxia stress responses. Plant Physiol 190(4): 2617-2636. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Plant Science > Plant physiology > Phenotyping Plant Science > Plant physiology > Axenic cultivation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Quantification of Blumenol Derivatives as Leaf Biomarkers for Plant-AMF Association Elisabeth Mindt [...] Ian T. Baldwin Jul 20, 2019 5530 Views A Protocol for Flavonols, Kaempferol and Quercetin, Staining in Plant Root Tips Nguyen Hoai Nguyen Oct 5, 2020 4218 Views Micrografting in Arabidopsis Using a Silicone Chip Hiroki Tsutsui [...] Michitaka Notaguchi Jun 20, 2021 5559 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,604	https://bio-protocol.org/en/bpdetail?id=4604&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Continuous Measurement of Reactive Oxygen Species Formation in Bacteria-infected Bone Marrow–derived Macrophages Using a Fluorescence Plate Reader NB Natascha Brigo * PG Philipp Grubwieser * IT Igor Theurl MN Manfred Nairz GW Günter Weiss CP Christa Pfeifhofer-Obermair (*contributed equally to this work) Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4604 Views: 972 Reviewed by: Andrea PuharDhiman Sankar PalJunsik SungSaskia F. Erttmann Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Front. Cell. Infect. Microbiol. May 2022 Abstract Macrophages are at the center of innate immunity and are the main target cells of the intracellular pathogen Salmonella enterica serovar Typhi. The production of reactive oxygen and nitrogen species (ROS/RNS) is the host’s early response to invading microbes, as oxidative stress is highly toxic for bacteria. Adequate ROS/RNS production in infected macrophages is critical for the clearance of intracellular pathogens; this is achieved by several enzymes, including inducible NADPH phagocyte oxidase (NOX) and nitric oxide synthase (iNOS), respectively. The pro-inflammatory cytokine interferon gamma (IFNγ), primarily produced by activated natural killer cells and T-helper cells type 1, is a potent inducer of iNOS. Therefore, it is crucial for infection control through oxidative microbicidal activity. To characterize the early oxidative stress response via ROS formation, which is critical for the reduction of Salmonella proliferation within macrophages, we established an in vitro model of murine macrophages infected with Salmonella enterica serovar Typhimurium (S.tm). This serovar induces a systemic infection in mice that is frequently used as a model for typhoid fever, which, in human subjects, is caused by Salmonella Typhi. We generated bone marrow–derived macrophages (BMDM) from C57BL/6N wildtype mice using macrophage colony-stimulating factor (M-CSF) stimulation for six days. Thereafter, we infected BMDM with S.tm for one hour. Shortly before infection, cells were stained with CellROXTM Deep Red reagent. In its reduced form, CellROXTM is non-fluorescent. As a result of oxidation by ROS, this reagent exhibits strong fluorescence and persists within the cells. Subsequently, changes as a result of the oxidative stress response can be measured with a TECAN Spark microplate reader over time. We designed this protocol to measure oxidative stress in macrophages through the course of an infection with an intracellular bacterium. The protocol has several advantages over established techniques. First, it allows to continuously monitor and quantify ROS production in living cells from the very start of the infection to the final clearance of the intracellular pathogen. Second, this protocol enables efficient ROS detection without stressing the cells by detaching or staining procedures. Graphical abstract Keywords: Salmonella Typhimurium Macrophages Infection control Reactive oxygen species Background The Gram-negative enteric bacterium Salmonella enterica serovar Typhi causes typhoid fever in humans, which is a major cause of disability and death worldwide (Stanaway et al., 2019). The closely related Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, S.tm) induces self-limiting gastroenteritis in humans and systemic infection in mice—a commonly used typhoid fever model. As an intracellular pathogen, S.tm is capable of actively invading various cell types, but its virulence is dependent on its ability to selectively infect and proliferate inside macrophages (Fields et al., 1986; Haraga et al., 2008). Upon phagocytosis, S.tm establishes a replicative niche within a membrane-bound compartment termed the Salmonella-containing vacuole. There, the pathogen competes with various bactericidal mechanisms deployed by the host cell. One factor pivotal for initial inhibition of bacterial proliferation as well as pathogen clearance is the production of reactive oxygen and nitrogen species (ROS/RNS) (Vazquez-Torres et al., 2000; Herb and Schramm, 2021). In response to S.tm infection, macrophages activate several innate immune pathways that lead to ROS/RNS production and thus induction of bactericidal oxidative stress. A crucial component of the innate immune response identified is the membrane-bound enzyme NADPH oxidase (NOX), with the isoform NOX2 being at the center of antimicrobial ROS generation (Herb and Schramm, 2021). The importance of this enzyme is highlighted by the increased susceptibility of NOX2-deficient mice to infection with normally avirulent S.tm strains (Felmy et al., 2013). Another source of macrophage antimicrobial oxidative stress is mitochondria-augmented ROS production, which is directly linked to Toll-like receptor activation (West et al., 2011). Finally, the activity of inducible nitric oxide synthase (iNOS), crucial for pathogen control, is positively regulated by inflammatory cytokines, including interferon gamma (IFNγ) (Mastroeni et al., 2000). In multiple infection models, IFNγ has thus been implicated in adequate ROS/RNS production and enhancement of intracellular bacterial clearance in macrophages (Mastroeni et al., 2000; Nairz et al., 2008; Brigo et al., 2021). This antimicrobial effect of ROS/RNS is not only attributed to direct toxicity to the pathogen, but also to indirect effects, with oxidative stress being implicated in numerous cellular signaling pathways (Tan et al., 2016). Especially for the multi-faceted role of oxidative stress in innate immune defense, iNOS activation is deemed critical for the activation of nuclear factor erythroid 2–related factor-2 (Nrf2)-dependent pathways, which lead to a nutritional immune response aimed at limiting essential nutrients to the pathogen’s compartment (Nairz et al., 2013). The importance of ROS/RNS-mediated pathogen control is also evident in patients lacking functional ROS induction due to chronic granulomatous disease, as they are at high risk for invasive or recurrent forms of salmonellosis (Burniat et al., 1980;Mouy et al., 1989;Dinauer, 2005). Due to its central involvement in innate immunity, an accurate measurement of oxidative stress is vital to study host–pathogen interactions. Herein, we report a method that allows to continuously determine ROS formation during infections in vitro. Most of the already established methods, which include usage of ROS-sensitive probes or biosensors expressed in cells or bacteria, rely on flow cytometry or fluorescence imaging techniques to quantify ROS levels (van der Heijden et al., 2015; Grander et al., 2022; Leone et al., 2016). These methods typically allow measurements only at single time points, as samples must be fixed and/or mounted. Furthermore, extensive preparation procedures will likely stress cells and thus decrease the signal-to-noise ratio. Of note, our novel protocol enables efficient detection of oxidative stress over time without stressing the cells by detaching and/or staining procedures. Furthermore, as fluorescence is read in a plate reader, multiple controls and experimental conditions can be quantified simultaneously. The protocol presented herein allows the immediate and continuous monitoring and quantification of oxidative stress responses during infection of macrophages with intracellular microbes (Figure 1). Figure 1. Timeline and 24-well plate layout of the experimental procedure Materials and Reagents 250 mL Erlenmeyer flask (Stoelzle Medical, catalog number: 21226368000) 0.5 mL Eppendorf tubes (Eppendorf, catalog number: 0030121.023) 50 mL polypropylene tube (Falcon, catalog number: 352070) 24-well plate (Falcon, catalog number: 353047) Cell scraper (Sarstedt, catalog number: 83.3951) LunaTM cell counting slides (Biocat, catalog number: L201B1C3GB) Disposable cuvette (BRAND, catalog number: 759015) 15 cm cell culture dish (Falcon, catalog number: 353025) 5 mL disposable syringe (BD, catalog number: 309050) 10, 200, and 1,250 µL pipette tips (STARLAB, catalog number: S1110-3700, S1120-3810, and S1112-1720, respectively) 5, 10, and 25 mL disposable pipettes (Falcon, catalog number: 606180, 607180, and 357525, respectively) Disposable hypodermic needle 100 Sterican R (Braun, catalog number: 4657519) Cell strainer 40 µm (Falcon, catalog number: 352360) Salmonella enterica serovar Typhimurium ATCC14028 (ATCC) Lysogeny broth (LB) medium Lennox (Roth, catalog number: X964.2) Glycerol (Sigma, catalog number: G5516-100ML) Phosphate buffer saline (PBS) (Lonza, catalog number: 17-515 F) Disposable cuvette (BRAND, catalog number: 759015) Casy cup (OMNI Life Science, catalog number: 5651794) Casy Ton buffer (OMNI Life Science, catalog number: 5651808) Aqua bidest (Fresenius Kabi, catalog number: 16.231) Pen-Strep (Lonza, catalog number: DE17-602E) Gentamicin (Gibco, catalog number: 15750-037, stock: 50 mg/mL) L-glutamine (Lonza, catalog number: BE17-605E) Dulbecco′s modified Eagle′s medium (DMEM) (Pan BiotechTM, catalog number: P04-01500) Fetal bovine serum (FBS) (Pan BiotechTM, catalog number: P30-3031) CellROXTM Deep Red (Thermo Fisher, catalog number: C10422) N-acetylcysteine (NAC) (Sigma-Aldrich, catalog number: A7250) Acridine orange/propidium iodide stain (Biocat, catalog number: F23001) Recombinant murine interferon-gamma (Peprotech, catalog number: 315-05; stock 20 µg/mL) Recombinant murine colony-stimulating factor M-CSF (Peprotech, catalog number: 315-02) Ketamine (Livisto, catalog number: 6680219) Xylazine (Animedica, catalog number: 7630517) Omnican F syringes (Braun, catalog number: 91615025) M-Lyse buffer concentrate (10×) (erythrocyte lysis kit) (R&D, catalog number: WL2000) LB medium (see Recipes) LB medium with 30% glycerol (see Recipes) Cell culture medium (see Recipes) Cell culture medium for infection with S.tm (see Recipes) Mouse line Bone marrow cells were collected from female C57BL/6N mice, which were bred in the Animal facility of the Medical University of Innsbruck or ordered from Charles River Laboratories. Equipment Pipetman L Starter kit: 2, 20, 200, and 1,000 µL pipettes (GILSON, catalog number: F167370) Sartorius Midi Plus pipetting controller (Sartorius, catalog number: 710931) Biosafety level 2 laminar flow cabinet, EuroClone Sicherheitswerkbank Safe Mate Eco 1.2 (Politakis Laborgeräte, catalog number: EN 12 469) Shaking incubator (VWR, catalog number GFL 3031) Heraeus® HERAcell® CO2 Incubator (Thermo Fisher Scientific) Photometer (Eppendorf, catalog number: BioPhotometer D30) Centrifuge (Hettich Micro 200R and Rotanta 460R) Casy counting system (OMNI Life Science) Automated multimode microplate reader (TECAN Spark) LUNA automated cell counter (Biocat, catalog number: L10001-LG) Millivac-Maxi vacuum pump (Merck, catalog number: SD1P014M04) Software SparkControlTM (TECAN Trading, Ltd.) GraphPad Prism 9.1 (GraphPad Software) Procedure Precautions Note: Salmonella enterica serovar Typhimurium is a biosafety level 2 bacterium with low-to-moderate hazard potential to personnel and the environment. Make sure to follow all relevant national and institutional regulations. Contact the Institutional Biosafety Committee (IBC) before planning the work with Salmonella enterica serovar Typhimurium (Burnett et al., 2009; Byrd et al., 2019) Use appropriate personal protective equipment and disinfectants Work in a biosafety level 2 laminar flow cabinet Collect and autoclave waste Generate the bone marrow–derived macrophages (BMDM) as described in Brigo et al. (2022) Note: Perform next steps in a sterile laminar flow cabinet. Anesthetize a wildtype C57BL/6N mouse by injecting 50 µL of 100 mg/kg ketamine + 10 mg/kg xylazine intraperitoneally. Note: A video demonstrating the general procedure can be found at: Intraperitoneal Injection in the Mouse: https://researchanimaltraining.com/articles/intraperitoneal-injection-in-the-mouse/. Euthanize the deeply anesthetized mouse by cervical dislocation. Thereafter, place a large tweezer behind the base of the anesthetized mouse's skull and pull back sharply on the tail at a 45° angle. Fix the animal on a Styrofoam panel and spray the surface of the animal with 75% alcohol. Remove skin and muscle tissue from the leg by cutting upwards from the heel with sterile scissors. Cut around the femur head. Cut in the middle of the knee joint. Make sure that the bones are not damaged. Cut the ankle joint. Remove excess muscles with a tissue paper. Pull on the upper leg to remove the femur head from the hip joint. Place the bones into PBS containing 1% penicillin and 1% streptomycin (Pen-Strep) on ice. The next steps should be performed on ice. Open the ends of the bones by cutting with a pair of sterile scissors. Place a 40 µm cell strainer on a 50 mL polypropylene tube. Flush out the bone marrow: Use a disposable hypodermic needle and a 5 mL syringe. Fill the syringe with PBS containing 1% penicillin and 1% streptomycin. Place a needle on one end of the opened bone. Flush the bone marrow out onto the 40 µm cell strainer. Repeat flushing of the bone until it is completely white. Wash the cell strainer with 10 mL of PBS containing 1% penicillin and 1% streptomycin. Use the plunger of the syringe to strain the cells through the cell strainer. Wash the cell strainer with 10 mL of PBS containing 1% penicillin and 1% streptomycin. Centrifuge the 50 mL polypropylene tube containing the flushed bone marrow at 300 × g for 5 min at 4°C. Dilute the M-Lyse buffer concentrate (10×) from the mouse erythrocyte lysis kit 1:10 with aqua bidest. Discard the supernatant. Resuspend the pellet in 2 mL of diluted M-Lyse buffer concentrate (1×). Incubate at room temperature for 3 min. Add 15 mL of PBS containing 1% penicillin and 1% streptomycin on top of the M-Lyse buffer concentrate (1×). Centrifuge the 50 mL polypropylene tube containing the flushed bone marrow at 300 × g for 5 min at 4°C. Discard the supernatant. Resuspend the pellet in 15 mL of PBS containing 1% penicillin and 1% streptomycin. Centrifuge the 50 mL polypropylene tube containing the flushed bone marrow at 300 × g for 5 min at 4°C. Discard the supernatant. Resuspend the pellet in 15 mL of PBS containing 1% penicillin and 1% streptomycin. Centrifuge the 50 mL polypropylene tube containing the flushed bone marrow at 300 × g for 5 min at 4°C. Discard the supernatant. Resuspend the cell pellet in 45 mL of DMEM supplemented with 10 % FBS, 1% L-glutamine, 1% penicillin, 1% streptomycin, and 50 ng/mL of recombinant murine M-CSF. Pipette 15 mL of the cell suspension into three 15 cm dishes and place them into a cell incubator with the following growth conditions: 5% CO2, 37°C, and 95% humidity. Change the medium every second day. On day 5, cells can be harvested. Harvesting, counting, and seeding of cells Note: Perform next steps in a sterile laminar flow cabinet. Remove the culture medium from the 15 cm cell culture dish using a cell culture aspiration pump. Wash the cells twice with 10 mL of PBS. Add 8 mL of DMEM supplemented with 10% FBS and 1% L-glutamine. Scrape the cells using a disposable cell scraper. Transfer the cells into a 50 mL polypropylene tube. Wash the dish with another 2 mL of DMEM. Transfer the washing medium into the 50 mL polypropylene tube containing the cells. Close the tube and invert the cells 2–3 times. Take 9 µL of the cell suspension and place it in a 0.5 mL Eppendorf tube. Mix 1 µL of the acridine orange/propidium iodide stain solution to the cell aliquot. Pipette 10 µL of the mixture into a Luna cell counting slide. Measure the cell number using the LUNA automated cell counter. Seed the cells at a density of 1.5 × 105 cells/mL in a 24-well plate. Note: Approximately 4.5 × 107 cells are gained from one mouse after isolating and culturing the bone marrow of both hind legs. Preparation of Salmonella Typhimurium (S.tm) stock as described in Brigo et al. (2022) Take an aliquot of Salmonella enterica serovar Typhimurium ATCC14028 from the -20°C storage. Thaw the aliquot at room temperature. Note: Perform next steps in a sterile biosafety level 2 laminar flow cabinet. Pipette 10 µL of S.tm into 10 mL of LB medium in a 250 mL Erlenmeyer flask and cover the top of the flask using a tin foil. Incubate at 37°C overnight in a shaking incubator at 200 rpm. The following day, pipette 50 µL of the overnight culture into fresh 10 mL of LB medium in a 250 mL Erlenmeyer flask and cover the top with tin foil. Dispose of the remaining overnight culture of S.tm and wash and sterilize the 250 mL Erlenmeyer flask. Incubate the culture at 37°C for 1–2 h at 200 rpm in a shaking incubator. Calibrate a photometer using 500 µL of LB medium in a disposable cuvette as blank. Measure OD600 to check if S.tm reached 0.5. Note: S. tm reaches the optimal logarithmic growth phase when OD600 is between 0.5 and 0.7. If the OD600 value is below 0.5, continue the incubation of the culture in the 250 mL Erlenmeyer flask as described above until the OD600 value of 0.5 is reached. Of note , S. tm density duplicates every 20 min. If the OD600 value is above 0.7, dilute the culture 1:1 with LB medium and incubate the culture in the 250 mL Erlenmeyer flask until an OD600 value of 0.5. Transfer the culture into a 50 mL Falcon tube. Centrifuge the S.tm culture at 4,600 × g for 5 min at room temperature. Remove the supernatant by using a vacuum pump. Resuspend the pellet in 1 mL of freshly prepared LB medium with 30% glycerol. Prepare 50 µL aliquots in 0.5 mL Eppendorf tubes. Store the aliquots at -20°C. Culturing and counting of S.tm Notes: Perform next steps in a sterile biosafety level 2 laminar flow cabinet. The procedures of culturing and counting of S.tm have been recently described in Brigo et al. (2022). Thaw an aliquot of S.tm at room temperature. Pipette 10 µL of the S.tm aliquot to 10 mL of LB medium in a 250 mL Erlenmeyer flask overnight at 37°C in a shaking incubator at 200 rpm after covering the top with tin foil. The next day, prepare 10 mL of LB medium and pipette 50 µL of the overnight culture in a fresh 250 mL Erlenmeyer flask. Dispose of the remaining overnight culture and wash and sterilize the Erlenmeyer flask. Incubate at 37°C with shaking for 1–2 h. Measure OD600: Calibrate a photometer by measuring the blank with 500 µL of LB medium in a disposable cuvette. Pipette 500 µL of the S.tm culture in a new disposable cuvette and measure. OD600 should be between 0.5 and 0.7; in this state, S.tm are in their logarithmic growth phase. Pipette 5 µL of the S.tm culture to 10 mL Casy Ton buffer in a Casy cup and count living S.tm. Keep the remaining bacteria on ice until the infection of the cells is performed. Counting of viable S.tm using a Casy counting system Note: The procedure along with the employment of the specific programs for the Casy counting system have been recently described in Brigo et al. (2022). Use the 45 µm capillary. Measure the background by placing a new Casy cup with fresh 10 mL of Casy Ton buffer under the measuring unit. Select Program for the background measurement. Measure the background activity. It should be below 30 counts and 1 µm size. Otherwise, wash the Casy counting system. Prepare a new Casy cup with 10 mL of Casy Ton buffer and add 5 µL of S.tm OD600 0.5. Shake gently. Place the sample in the measuring unit. Select the program for measuring between 1 and 3 µm. Measure. Click Next to get the number of viable counts/mL = viable S.tm /mL. Note: Viable counts from a freshly prepared S.tm culture with an OD600 of 0.5 is between 2.5 × 108 and 3 × 108 viable counts/mL. Staining with CellROXTM and infection with S.tm Note: Perform the next steps in a sterile biosafety level 2 laminar flow cabinet. The antioxidant NAC is used as a negative control, as it drastically decreases ROS stress in cells. Importantly, NAC is added before the infection or other treatments, to increase the antioxidant capacity of cells. We recommend at least triplicate well replicates per each treatment condition (e.g., four wells each of uninfected control, uninfected + NAC, infected, and infected + NAC, as depicted in Figure 1). Add 5 µL of 500 mM antioxidant NAC (final concentration: 2.5 mM) to the negative control samples (Figure 1). Incubate the cells for 15 min at 37°C and 5% CO2 in a cell incubator. Pipette 1 µL of CellROXTM Deep Red reagent to each well (final concentration 2.5 µM). Incubate the cells for 15 min at 37°C and 5% CO2 in an incubator. Infect the cells with S.tm using a multiplicity of infection (MOI) of 10; therefore, 10 times more S.tm than cells are added to each well. Example for calculation of S.tm: To gain a MOI10, multiply the cell number by 10: 1.5 × 105 × 10 = 1.5 × 106 Divide the calculated cell number by the gained viable Salmonella count (e.g., 2.5 × 108): 1.5 × 106 viable counts/mL: 2.5 × 108 viable counts/mL = 0.006 mL = 6 µL Add the calculated amount of S.tm directly into the cell culture wells containing 1 mL of DMEM containing 1% L-glutamine and 10% FBS. Immediately after infection, place the plate into a pre-heated plate reader (TECAN Spark). Setup of the plate reader (TECAN Spark) and measurement of the infection phase Note: A captured image of the setup screen is displayed in Figure 2. Figure 2. Screenshot of the plate reader (TECAN Spark) method editor setup Open the plate reader method editor. Select a 24-well plate. Note: Select the correct model of the 24-well plate to avoid inaccurate measurements in “bottom read mode” and to avoid damaging the detector. Select measurement “Fluorescence Intensity.” Set the instruments temperature to 37°C. Set the CO2 flow to 5%. Change the mode to “Bottom” for fluorescence intensity bottom reading. Change the Fluorophore Setting to “other.” Set the fluorophore parameters to: Monochromator 625 nm excitation; bandwidth 20 nm Monochromator 670 nm emission; bandwidth 20 nm Set “Flashes” to 60. Select cycles: Repeat the measurement every 5 min. Cycles: 12 Change the gain to “manual” and use 130. Set the z-position to “manual.” Note: The z-position can be calibrated right before the measurement and changed if needed (see step G19). Select “multiple reads” per well: User defined Type: filled circles (4 × 4) Border: 800 µM Save the setup. Open the plate reader dashboard control. Load the CellROXTM measurement setup. Confirm that the temperature is at 37°C and CO2 is at 5%. Place the 24-well plate into the plate reader. Let the z-position be calibrated by the machine (Figure 3): Select “z-position” in the menu on the bottom left. In the Scan sub-menu, select wells for signal detection. Click the Scan button on the bottom left. In the resulting graph, signal strength for different z-positions is visible. Figure 3. Screenshots of the plate reader dashboard control Apply automatically suggested z-position or enter a manual value. Close the Scan sub-menu. Click “Start.” After the measurement is completed, an Excel file will open automatically. Save the Excel file and proceed to data analysis. Take the 24-well plate out of the plate reader and continue with the gentamicin neutralization assay. Long-term measurement of ROS in the presence of gentamicin (clearance phase) Note: Perform next steps in a sterile biosafety level 2 laminar flow cabinet. Use this assay to avoid uncontrolled proliferation of bacteria that were not phagocytosed by macrophages. Gentamicin is not able to penetrate eukaryotic cells and therefore cannot affect internalized bacteria. Treatments or positive control (IFNγ) is typically added during this bacterial clearance phase, as depicted in Figure 1. Pre-warm the medium and PBS in a water bath at 37°C to avoid additional stress to the cells. Remove the medium containing non-phagocytosed S.tm using a cell culture aspiration pump. Wash the cells twice with 1 mL PBS + 25 µg/mL gentamicin. Add 1 mL of DMEM supplemented with 10% FBS, 1% L-glutamine, and 25 µg/mL gentamicin. Add 100 ng/mL IFNγ to the positive control samples. Add 5 µL of 500 mM of the antioxidant NAC (final concentration 2.5 mM) to the negative control samples. During the bacterial clearance phase, incubate the cells for up to 24 h in a cell culture incubator (37°C, 5% CO2, 95% relative humidity). Remove the cell culture plate only for hourly measurements. Setup of the plate reader (TECAN Spark) and measurement of the clearance phase Note: During the bacterial clearance phase, we recommend single individual measurement in the plate reader. In between these measurements, the cell culture plate is kept in a cell culture incubator to ensure optimal environmental conditions for cells (37°C, 5% CO2, 95% relative humidity). Furthermore, it is advised to keep the plate reader’s temperature at 37°C and atmosphere at 5% CO2 all the time. Open the plate reader (TECAN Spark) method editor. Load the “Setup” from section G into the method editor. Remove “Select cycles” (step G10) to perform only single fluorescence measurements. Use the parameters of the previous “Setup” (Section G). Save the setup. Open the plate reader control. Load the CellROXTM measurement setup. Confirm that the temperature is at 37°C and CO2 is at 5%. Place the 24-well plate into the plate reader. Let the z-position be calibrated by the machine. Apply the new z-position if wanted. Click “Start.” After the measurement is completed, an Excel file will open automatically. Save this Excel file and proceed to data analysis. Note: Alternatively, the plate reader can also be set up for hourly measurements during the entire 24 h bacterial clearance phase with constant incubation of the 24-well plate in the plate reader, to minimize hands-on time. In this case, we recommend using a humidity cassette as well as the same environmental conditions as in the cell culture incubator (37°C, 5% CO2). Data analysis Open the generated Excel file from the infection phase. The Excel file shows all the performed actions in the experimental setup. Scroll down as far as the measured data show up. The Excel file depicts the results for each well (Table 1 shows an example). Table 1. Example of output data of one well during 1 h with consecutive measurements every 5 min Well A1 Time [s] 0 300 600 900 1,200 1,500 1,800 2,100 2,400 2,700 3,000 3,300 CO2% 4.9 4.9 4.9 5.2 4.9 4.8 5 4.9 5 5 4.9 4,9 Temp. [°C] 37.1 37 37 37 37 37 37 37 37 37 37.1 37 Mean 592 641 600 609 586 585 606 668 704 730 707 763 StDev 58 60 38 76 44 66 51 63 73 59 85 78 1;2 638 594 560 618 536 585 683 596 809 711 641 760 1;3 566 624 574 498 549 494 663 667 743 820 682 754 2;1 690 634 575 619 555 616 558 779 755 713 754 834 2;2 531 656 596 484 566 522 659 667 778 739 662 793 2;3 512 493 577 556 615 632 542 758 691 636 659 730 2;4 607 674 618 644 561 532 616 713 635 712 722 798 3;1 611 680 590 739 623 698 654 713 615 813 780 825 3;2 573 628 650 559 617 608 569 571 636 807 839 868 3;3 527 609 544 629 694 667 635 675 701 668 537 810 3;4 553 717 673 662 576 512 567 647 812 702 818 723 4;2 612 684 626 599 569 527 598 613 624 763 653 684 4;3 684 704 621 709 571 634 539 619 649 681 739 577 Each individual measurement of the 4×4 multiple reads per well is shown in the table. The position of each individual measurement is displayed in a map, also found in the Excel file (shown in Figure 4). Figure 4. Position of the multiple reads per well as found in the Excel table (left) and an illustration of multiple measurements in one well (right) In the table, beneath the time [sec], atmosphere [% CO2], and the temperature [°C], the mean and standard deviation of all individual measurements of one individual well are displayed. Open GraphPad Prism. Select a “XY” graph. Select “4” technical replicate values in side-by-side in sub-columns for the y-axis. Note: Adapt this number to the number of replicate wells used in the experimental design. Label the x-axis with analyzed time points 0–60 min. Label the columns with experimental groups. Add the means of the technical replicates for each experimental group in corresponding sub-columns. Note: If desired, data can be normalized to uninfected control conditions. Check the graph and assign the colors for different conditions. Label the y-axis as fluorescence intensity of CellROXTM. Label the x-axis as Time [min]. Note: An example graph for the 1 h infection phase is shown in Figure 5. Figure 5. Fluorescence intensity of CellROXTM during the infection phase with S.tm over the course of 1 h. Data are shown as mean fluorescence intensity ± SD, normalized to uninfected control conditions (dashed line). Analyze the data of the S.tm clearance phase in the same way as the S.tm infection phase. Change the label of the x-axis to Time [h]. Note: An example graph for 2–7 h S. tm clearance phase is shown in Figure 6. Figure 6. Fluorescence intensity of CellROXTM between 2 and 7 h after washing away unphagocytosed S.tm (clearance phase). Data are shown as mean fluorescence intensity ± SD, normalized to uninfected control conditions (dashed line). The left panel shows treatments of uninfected cells and right panel shows treatments of infected cells. To show differences at a single time point (e.g., 24 h), select a column graph in GraphPad prism. Label the columns according to the groups. Add the technical quadruplicates of each group into each column. Check the graph. Label the y-axis as fluorescence intensity of CellROXTM. Label the x-axis as 24 h stimulation. Note: An example graph of the measured 24 h time point is shown in Figure 7. Figure 7. Fluorescence intensity of CellROXTM after 24 h of stimulation with or without infection. Data are shown as mean fluorescence intensity ± SD, normalized to uninfected control conditions (dashed line). Recipes LB medium 200 mL sterile water 2 g LB medium powder Autoclave (20 min at 121°C and 10 min at 50°C) LB medium with 30% glycerol Add 300 µL of glycerol to 700 µL of LB medium Cell culture medium 500 mL of DMEM 50 mL of FBS 5 mL of L-glutamine 5 mL of penicillin/streptomycin Cell culture medium for infection with S.tm 500 mL of DMEM 50 mL of FBS 5 mL of L-glutamine Acknowledgments G.W. is supported by grants from the Christian Doppler Society and the Austrian research Funds (FWF doctoral program W1253 HOROS; and FWF-docfund-82-CBD). N.B. was supported by FWF doctoral program - W1253 HOROS. P.G. and I.T. were supported by the Austrian Science Fund (FWF docfound 82-CBD). M.N. was funded by the Austrian Science Fund (FWF, P33062). This protocol was adapted and modified after Wu et al. (2017). Competing interests The authors declare no conflicts of interest. References Brigo, N., Pfeifhofer-Obermair, C., Demetz, E., Tymoszuk, P. and Weiss, G. (2022). Flow Cytometric Characterization of Macrophages Infected in vitro with Salmonella enterica Serovar Typhimurium Expressing Red Fluorescent Protein. Bio Protoc 12(11): e4440. Brigo, N., Pfeifhofer-Obermair, C., Tymoszuk, P., Demetz, E., Engl, S., Barros-Pinkelnig, M., Dichtl, S., Fischer, C., Valente De Souza, L., Petzer, V., et al. (2021). Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection. Cells 10(7): 1823. Burnett, L. C., Lunn, G. and Coico, R. (2009). Biosafety: guidelines for working with pathogenic and infectious microorganisms. Curr Protoc Microbiol Chapter 1: Unit 1A 1. Burniat, W., Toppet, M. and De Mol, P. (1980). Acute and recurrent salmonella infections in three children with chronic granulomatous disease. J Infect 2(3): 263-268. Byrd, J. J., Emmert, E., Maxwell, R., Townsend, H. and ASM Task Committee on the Revision of the 2012 Laboratory Biosafety Guidelines. (2019). Guidelines for Biosafety in Teaching Laboratories Version 2.0: A Revised and Updated Manual for 2019. J Microbiol Biol Educ 20(3): 20.3.57. Dinauer, M. C. (2005). Chronic Granulomatous Disease and Other Disorders of Phagocyte Function. Hematology 2005(1): 89-95. Felmy, B., Songhet, P., Slack, E. M., Müller, A. J., Kremer, M., Van Maele, L., Cayet, D., Heikenwalder, M., Sirard, J. C. and Hardt, W. D. (2013). NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium. PLoS One 8(10): e77204. Fields, P. I., Swanson, R. V., Haidaris, C. G. and Heffron, F. (1986). Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci U S A 83(14): 5189-5193. Grander, M., Hoffmann, A., Seifert, M., Demetz, E., Grubwieser, P., Pfeifhofer-Obermair, C., Haschka, D. and Weiss, G. (2022). DMT1 Protects Macrophages from Salmonella Infection by Controlling Cellular Iron Turnover and Lipocalin 2 Expression. Int J Mol Sci 23(12). Haraga, A., Ohlson, M. B. and Miller, S. I. (2008). Salmonellae interplay with host cells. Nat Rev Microbiol 6(1): 53-66. Herb, M. and Schramm, M. (2021). Functions of ROS in Macrophages and Antimicrobial Immunity. Antioxidants (Basel) 10(2): 313. Leone, L., Mazzetta, F., Martinelli, D., Valente, S., Alimandi, M., Raffa, S. and Santino, I. (2016). Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells. PLOS ONE 11(1): e0146365. Mastroeni, P., Vazquez-Torres, A., Fang, F. C., Xu, Y., Khan, S., Hormaeche, C. E. and Dougan, G. (2000). Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo. J Exp Med 192(2): 237-248. Mouy, R., Fischer, A., Vilmer, E., Seger, R. and Griscelli, C. (1989). Incidence, severity, and prevention of infections in chronic granulomatous disease. J Pediatr 114(4 Pt 1): 555-560. Nairz, M., Fritsche, G., Brunner, P., Talasz, H., Hantke, K. and Weiss, G. (2008). Interferon-gamma limits the availability of iron for intramacrophage Salmonella typhimurium. Eur J Immunol 38(7): 1923-1936. Nairz, M., Schleicher, U., Schroll, A., Sonnweber, T., Theurl, I., Ludwiczek, S., Talasz, H., Brandacher, G., Moser, P. L., Muckenthaler, M. U., et al. (2013). Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection. J Exp Med 210(5): 855-873. Stanaway, J. D., Reiner, R. C., Blacker, B. F., Goldberg, E. M., Khalil, I. A., Troeger, C. E., Andrews, J. R., Bhutta, Z. A., Crump, J. A., Im, J., et al. (2019). The global burden of typhoid and paratyphoid fevers: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Infect Dis 19(4): 369-381. Tan, H.-Y., Wang, N., Li, S., Hong, M., Wang, X. and Feng, Y. (2016). The Reactive Oxygen Species in Macrophage Polarization: Reflecting Its Dual Role in Progression and Treatment of Human Diseases. Oxid Med Cell Longev 2016: 2795090. van der Heijden, J., Bosman, E. S., Reynolds, L. A. and Finlay, B. B. (2015). Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages. Proc Natl Acad Sci U S A 112(2): 560-565. Vazquez-Torres, A., Jones-Carson, J., Mastroeni, P., Ischiropoulos, H. and Fang, F. C. (2000). Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J Exp Med 192(2): 227-236. West, A. P., Brodsky, I. E., Rahner, C., Woo, D. K., Erdjument-Bromage, H., Tempst, P., Walsh, M. C., Choi, Y., Shadel, G. S. and Ghosh, S. (2011). TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472(7344): 476-480. Wu, A., Tymoszuk, P., Haschka, D., Heeke, S., Dichtl, S., Petzer, V., Seifert, M., Hilbe, R., Sopper, S., Talasz, H., et al. (2017). Salmonella Utilizes Zinc To Subvert Antimicrobial Host Defense of Macrophages via Modulation of NF-κB Signaling. Infect Immun 85(12). Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Immunology > Immune cell function > Macrophage Biochemistry > Other compound > Reactive oxygen species Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS Waraporn Suvannapruk [...] Morgan R. Alexander Aug 5, 2023 451 Views In vitro Assessment of Efferocytic Capacity of Human Macrophages Using Flow Cytometry Ana C.G. Salina [...] Larissa D. Cunha Dec 20, 2023 2818 Views Quantification of Macrophage Cellular Ferrous Iron (Fe2+) Content using a Highly Specific Fluorescent Probe in a Plate-Reader Philipp Grubwieser [...] Christa Pfeifhofer-Obermair Feb 5, 2024 758 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,605	https://bio-protocol.org/en/bpdetail?id=4605&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Dual-Color Live Imaging of Adult Muscle Stem Cells in the Embryonic Tissues of Drosophila melanogaster MZ Monika Zmojdzian BD Binoj Dhanarajan KJ Krzysztof Jagla RA Rajaguru Aradhya Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4605 Views: 620 Reviewed by: Gal HaimovichAmr Galal Abdelraheem IbrahimPradeep Kumar Bhaskar Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Dec 2015 Abstract Adult muscle stem cells (MuSCs) show remarkable capability in repairing injured tissues. Studying MuSCs in suitable model organisms, which show strong homology with vertebrate counterparts, helps in dissecting the mechanisms regulating their behavior. Additionally, ease of handling and access to technological tools make model organisms well suited for studying biological processes that are conserved across species. MuSCs quiescence, proliferation, and migration are regulated by various input of signals from the surrounding tissues that constitute the MuSCs niche. Observing MuSCs along with their niche in vivo through live imaging provides key information on how MuSCs behave in quiescent and activated states. Drosophila melanogaster is well known for its genetic tool arsenal and the similarity of its different biological processes with vertebrates. Hence, it is widely used to study different types of stem cells. Gained knowledge could then be extrapolated to the vertebrate/mammalian homologous systems to enhance our knowledge in stem cell fields. In this protocol, we discuss how to perform live cell imaging of Drosophila MuSCs, called adult muscle precursors (AMPs) at embryonic stages, using dual-color labelling to visualize both AMPs and the surrounding tissues. This dual-color fluorescent labelling enables the observation of in vivo behavior of two types of cells simultaneously and provides key information on their interactions. The originality of this protocol resides in its biological application to MuSCs and their niche. Keywords: Muscle stem cells Drosophila Live imaging Embryo Time-lapse microscopy Adult muscle precursors GFP mCherry Peripheral nervous system 3D image analysis Background Drosophila's adult muscle precursors (AMPs) are quiescent muscle stem cells specified from the mesodermal lineages during embryonic stage 12 (Bate et al., 1991; Figeac et al., 2010; Aradhya et al., 2015). They form as sibling cells of muscle progenitor cells, giving rise to differentiated skeletal muscle tissue for larval locomotion. However, AMPs remain quiescent and non-differentiated during the embryonic and first parts of the larval life (Aradhya et al., 2015). The ability to maintain their quiescent nature makes AMPs an attractive model to study mechanisms regulating their dormant state, which could be homologous to those that control quiescence of mammalian muscle stem cells (MuSCs) that ensure repair of damaged muscle tissue (Sambasivan and Tajbakhsh, 2015). Through live cell imaging, using a gap-GFP transgene to mark cell membranes, we have previously demonstrated that AMPs display dynamic cellular processes during their quiescent state (Figeac et al., 2010; Aradhya et al., 2015). In contrast to the dot-like pattern of AMPs revealed by antibody staining against the nuclear protein Twist (Bate et al., 1991;Broadie and Bate, 1991; Sellin et al., 2009), our time-lapse live imaging allowed to observe that quiescent AMPs send both filopodia and cellular projections to the neighboring cells (Figeac et al., 2010). This new finding encouraged us to look deeper into the cellular behavior of AMPs through further generation of molecular and genetic tools for dual-color live imaging. Using these new transgenic Drosophila lines and in-depth observations through confocal imaging, we showed that AMPs interact with the embryonic muscles and the peripheral nervous system (Aradhya et al., 2015). We were able to visualize how filopodia projected by AMPs find the surrounding muscles, which in turn serve as their niche (Figure 1). These fine cellular structures would not have been discovered in fixed tissue due to the harsh nature of fixative agents. Hence, studying the cellular nature of a given cell type during development through live imaging provides a better resolution of the tissue morphogenesis. Dual-color live imaging allows documenting the dynamic behavior of two types of cells/tissues over time (Video 1). In this article, we describe the detailed protocol for performing dual-color time-lapse live imaging in Drosophila embryos using our previously generated molecular tools (Aradhya et al., 2015). The readers can apply this protocol to their own transgene combinations to label other cells of interest. Though there are other methods to visualize multiple cell types with different colors in Drosophila, they require the construction of a fluorescent gene cassette combined with GAL4 drivers, each specific to the cells of interest or unable to label tissues that are different in origin. Additionally, the signal intensity and ability to observe fine cellular structures at embryonic stages are comparatively weaker in the method we have described in this protocol (Boulina et al., 2013, Hadjieconomou et al., 2011, Hampel et al., 2011). The strength of the genetic tool mentioned here lies in combining already known enhancer driver lines, which directly drive the expression of GFP cassette, instead of using a binary expression system such as UAS-GAL4 in the cells of interest. GFP cassette expression through binary systems tends to delay the temporal activity of a given enhancer. Also, restricting the expression of GFP cassette under the regulation of cell type–specific enhancers allows the manipulation of complementary cell types using other transgenes, such as RNA interference lines, against a specific gene by incorporating a separate binary expression system using a simple genetic cross. Materials and Reagents NuncTM ThermanoxTM coverslips (Fisher Scientific, catalog number: 174942) Flystuff embryo collection cage-mini, fits 35 mm Petri dishes (Genesee Scientific, catalog number: 59-105) FisherbrandTM dissecting needle wood (Fisher Scientific, catalog number: 13-820-024) Double-sided tape (Scientific Industries, catalog number: SI1616) NuncTM cell culture/Petri dishes (Fisher Scientific, catalog number: 12-565-90) NuncTM square BioAssay dishes (Fisher Scientific, catalog number: 166508) Flystuff mesh basket, small, 3/4 inch inside diameter (Genesee Scientific, catalog number: 46-101) Flystuff Nitex nylon mesh 630 μm, 45 inch wide roll, 1 foot/unit (Genesee Scientific, catalog number: 57-101) Dechorionation chamber, prepared by adding a suitable size of Flystuff nylon mesh to the Flystuff mesh basket Agar powder (Fisher Scientific, catalog number: A10752.22) Sucrose (crystalline/certified ACS) (Fisher Scientific, catalog number: S5-500) Apple/grape juice (any commercial product available from local sources) Yeast granules (any commercial product available from local sources) Methyl 4-hydroxybenzoate, 99% (Fisher Scientific, catalog number: AAA1428930) Sodium hypochlorite solution (commercial bleach), available chlorine 4% (Fisher Scientific, catalog number: Q27908) N-heptane, certified AR for analysis (Fisher Scientific, catalog number: H/0160/15) Halocarbon oil 27 (Sigma-Aldrich, catalog number: H8773 Ultra-soft tissues (Kleenex) Fly stocks Duf-Gal4 (a gift from K. Vijayraghavan, NCBS, India) M6-GapGFP [lines were created as part of a previous study (Aradhya et al., 2015)] UAS-mCD8-mCherry (Bloomington Drosophila Stock Center, stock number: BL27391) Note: All stocks should be maintained at 25°C on standard Drosophila food medium. Equipment Drosophila incubator (Percival, catalog number: DR-36VL) P10 micropipette (Eppendorf, catalog number: 3123000020) Stereo dissecting microscope (Olympus Stereo Microscope System, catalog number: SZX7) Leica TCS SP5 confocal microscope Software Imaris (BitPlane), https://imaris.oxinst.com/ Fiji (ImageJ), https://imagej.net/software/fiji/downloads Procedure Drosophila cage setup, embryo collection, and dechorionation Set up a cross between the M6-gapGFP, Duf-Gal4, and UAS-mCD8-mCherry transgenic flies with 40–60 flies in an embryo collection cage of suitable size (Figure 2A). Prepare apple/grape juice agar medium using methyl 4-hydroxybenzoate according to standard protocols (Cold Spring Harb Protoc, 2011), pour it into 35 mm cell culture dishes, and allow the food medium to solidify at room temperature. The plates with medium can be stored at 4°C for up to two weeks (Figure 2A). Add a layer of yeast paste to the center of the apple juice agar plate before setting up the cage to stimulate the egg production. Allow the flies to mate and start laying eggs in the egg-laying cage for two days in a 25°C incubator with a 12:12 h light/dark cycle. Once the flies are synchronized to the cage, the apple juice agar plates can be changed every 12 h to collect embryos with a developmental time point ranging from 0 to 12 h. Alternatively, collect the egg-laying plates by transferring flies to a new egg collection cage every 2 h and incubate separately in a 25°C Drosophila incubator to obtain age-synchronized embryos. Wash the embryos from the plates with a fine brush into a dechorionation chamber; rinse with water repeatedly to remove any traces of yeast paste (Figure 2B–D). Dechorionate embryos using 50% commercial bleach with continuous swirling of the dechorionation chamber for up to 2 min, until all embryos look like shiny rice granules. At this step, embryos can be observed under the dissection microscope to ensure proper removal of the chorion membrane (Figure 2E). Thoroughly rinse the dechorionation chamber with distilled water to remove any traces of bleach; pat dry the embryos by placing the nylon mesh from the dechorionation chamber on a pile of Kleenex tissues for a few minutes (Figure 2F). Dechorionated embryos are hygroscopic in nature and tend to stick to each other. After pat drying for 2–3 min, gently pick up the clusters of embryos with a fine brush or dissecting needle; place them on a rectangular block of apple juice agar cut from the square cell culture plates containing a uniform layer of apple/grape juice agar medium (Figure 2G–H). Embryo alignment and picking onto a coverslip Align the embryos in a linear fashion on the edge of the rectangular agar block. Maintain the orientation of the embryos in such a way that the dorsal side faces the edge of the agar block, and the lateral side faces up to visualize the abdominal AMPs (Figure 2I). For other specific tissues or cells, the embryos can be aligned accordingly so they can be visualized easily using an inverted microscope. Place approximately 10 strips of double-sided tape (10 × 1 cm) in a 100 mL glass bottle and fill it with 90 mL of N-heptane. Allow the tape and heptane mixture to sit at room temperature for 12 h for proper extraction of the glue. The heptane/glue solution can be kept at 4 °C and used for several experiments. Using a P10 micropipette, add a small volume of heptane/glue solution to the center of a rectangular coverslip in a thin line that covers the entire coverslip length (Figure 2J). Allow the glue to semidry for 5 min in a sterile empty plastic box. Gently press the side containing glue against the aligned embryos on the agar block; this way, the embryos are transferred to the coverslip in the same alignment of the agar block, with the lateral side facing towards the coverslip (Figure 2K). This side can be easily visualized with high magnification lenses, which require applying immersion oil to the coverslip. Cover the embryos with a thin layer of halocarbon oil 27 to prevent them from desiccation (Figure 2L). Halocarbon oil allows embryos to exchange gases with the surrounding air, keeping them in healthy conditions for a long period. Imaging the embryos with a confocal microscope using time-lapse series Visualize the embryos using an inverted microscope, in which the objectives can touch the bottom side of the coverslip without disturbing the embryos (Figure 2M). Place the coverslips in the microscope in the same manner as glass slides using suitable holders. Using the lower magnification eyepiece of a Leica SP5 confocal microscope with time series imaging, select suitable embryos with appropriate developmental stage and better visualization of muscle stem cells and surrounding tissues (Figure 2N). Use the 40× oil immersion objective for enlargement of particular hemisegments of the embryo and better observation of the tissues of interest. Select Z-stacks based on the depth of tissue needed to be imaged (Figure 2O). Using the Fiji (ImageJ) software, analyze the raw files from the confocal microscope and generate images with the Z-stack of the selected optical section. Using Imaris (BitPlane) software, perform 3D reconstruction of both single time-point image and time-series live imaging. Data analysis For maximum intensity projection of the single time-point image, select the desired optical sections in ImageJ and export them as a Z-stack picture. Similarly, use the Imaris software to generate a 3D rotating model of either a single time-point projection or images captured on a time-lapse series mode. Figure 1. Dual-color live imaging of muscle stem cells and surrounding tissues. (A) Lateral view of stage 15 Drosophila M6-GapGFP; Duf-Gal4 X UAS-mCD8-mCherry embryos; AMPs expressing GFP are in green and the differentiated muscles expressing mCherry are in red. Scale bar: 100 μm. (B) Two hemisegments around the lateral muscles and associated AMPs have been magnified to observe the ultrastructure of cytoplasmic extensions protruding from AMPs. Scale bar: 9 μm. Video 1. Time-lapse live imaging of M6-gapGFP embryos displaying dynamic behaviour of filopodia from the newly formed AMPs Figure 2. Illustrations of different steps described in this protocol Acknowledgments This protocol was adapted from the previously published paper (Aradhya et al., 2015). We thank Pierre Pouchin for helping in the image processing during the initial analysis. We also thank the Imaging platform at GReD institute, France for allowing us to use the Leica SP5 confocal microscope. Competing interests The authors declare no competing interests. References Aradhya, R., Zmojdzian, M., Da Ponte, J. P. and Jagla, K. (2015). Muscle niche-driven Insulin-Notch-Myc cascade reactivates dormant Adult Muscle Precursors in Drosophila. Elife 4: e08497. Bate, M., Rushton, E. and Currie, D. A. (1991). Cells with persistent twist expression are the embryonic precursors of adult muscles in Drosophila. Development 113(1): 79-89. Boulina, M., Samarajeewa, H., Baker, J. D., Kim, M. D. and Chiba, A. (2013). Live imaging of multicolor-labeled cells in Drosophila. Development 140(7): 1605-1613. Broadie, K. S. and Bate, M. (1991). The development of adult muscles in Drosophila: ablation of identified muscle precursor cells. Development 113(1): 103-118. Cold Spring Harb Protoc. (2011). Drosophila apple juice-agar plates recipe. doi:10.1101/pdb.rec065672 Figeac, N., Jagla, T., Aradhya, R., Da Ponte, J. P. and Jagla, K. (2010). Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway. Development 137(12): 1965-1973. Hadjieconomou, D., Rotkopf, S., Alexandre, C., Bell, D. M., Dickson, B. J. and Salecker, I. (2011). Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat Methods 8(3): 260-266. Hampel, S., Chung, P., McKellar, C. E., Hall, D., Looger, L. L. and Simpson, J. H. (2011). Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nat Methods 8(3): 253-259. Sambasivan, R. and Tajbakhsh, S. (2015). Adult skeletal muscle stem cells. Results Probl Cell Differ 56: 191-213. Sellin, J., Drechsler, M., Nguyen, H. T. and Paululat, A. (2009). Antagonistic function of Lmd and Zfh1 fine tunes cell fate decisions in the Twi and Tin positive mesoderm of Drosophila melanogaster. Dev Biol 326(2): 444-455. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). How to cite Category Developmental Biology > Cell growth and fate > Myofiber Cell Biology > Cell imaging > Confocal microscopy Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols In vivo Assessment of Lysosomal Stress in the Drosophila Brain Using Confocal Fluorescence Microscopy Felipe Martelli Jan 20, 2023 672 Views Live Imaging of Phagoptosis in ex vivo Drosophila Testis Diana Kanaan [...] Hila Toledano Mar 20, 2023 752 Views Protocol for Imaging the Same Class IV Neurons at Different Stages of Development Sonal Shree and Jonathon Howard Aug 20, 2024 460 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,606	https://bio-protocol.org/en/bpdetail?id=4606&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Protocol for the Splinted, Human-like Excisional Wound Model in Mice KF Katharina S. Fischer BL Ben Litmanovich DS Dharshan Sivaraj HK Hudson C. Kussie WH William W. Hahn AH Andrew C. Hostler KC Kellen Chen GG Geoffrey C. Gurtner Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4606 Views: 1599 Reviewed by: Alak MannaPriyanka Banerjee Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Dec 2021 Abstract While wound healing in humans occurs primarily through re-epithelization, in rodents it also occurs through contraction of the panniculus carnosus, an underlying muscle layer that humans do not possess. Murine experimental models are by far the most convenient and inexpensive research model to study wound healing, as they offer great variability in genetic alterations and disease models. To overcome the obstacle of contraction biasing wound healing kinetics, our group invented the splinted excisional wound model. While other rodent wound healing models have been used in the past, the splinted excisional wound model has persisted as the most used model in the field of wound healing. Here, we present a detailed protocol of updated and refined techniques necessary to utilize this model, generate results with high validity, and accurately analyze the collected data. This model is simple to conduct and provides an easy, standardizable, and replicable model of human-like wound healing. Keywords: Excisional wound model Splinted wound healing model Rodent Full thickness skin wound Murine Background The normal biological process of wound healing is a complex dynamic composition of four different phases: hemostasis, inflammation, proliferation, and remodeling. Proper interactions between these different phases are necessary for the wound to heal sufficiently and successfully. Impaired wound healing occurs when these phases do not integrate correctly with one another. Dysfunctional wound healing is currently a major burden in our healthcare system, and 6.5 million people per year suffer from this condition (Sen, 2021). Often, chronic diseases, such as diabetes mellitus, chronic venous insufficiency, or age-associated diseases, are underlying issues behind dysfunctional healing. The healthcare costs of non-healing wounds are high since there are no highly effective therapies currently available (Alexiadou and Doupis, 2012). For example, over the past fifteen years, the success rate for therapeutic trials in wound healing has been zero percent, resulting in no approved therapeutics for wound healing in that time frame (Thomas et al., 2016). Studying the molecular mechanisms involved in these processes is essential to discover new therapeutic applications to promote healing. Many different species are currently being studied in the field of wound healing research, such as pigs, rats, rabbits, and mice. Pigs have the most similar physiological, anatomical, and functional skin conditions to humans (Ibrahim et al., 2017), but are exceedingly expensive to house and hard to maintain. Rat and rabbit models have been used extensively in the past but with decreasing frequency in recent times. The evolution of various knock-out lineages has made the use of murine models more attractive for studying the effects of various gene targets on healing. The most significant difference between murine and human wound healing is the presence of the panniculus carnosus, an underlying muscle layer that causes wound healing to occur not only through re-epithelization but also through contraction. In rodents, contraction can account for up to 40%–60% of wound closure (Chen et al., 2015), so rodent models are often critiqued for not being translationally applicable to human wound healing kinetics. To overcome this issue, our group invented the splinted excisional wound model in 2004 (Galiano et al., 2004). In this model, silicone splints are sutured around excisional wounds to prevent contraction and instead promote healing through re-epithelialization. This model has become a commonly used wound healing model due to its simplicity and feasibility (Michaels et al., 2007; Nauta et al., 2013; Cho et al., 2016; Chong et al., 2017; Cogan et al., 2018; Hu et al., 2018; Kurt et al., 2018; Dallas et al., 2019; Rhea and Dunnwald, 2020; Liu et al., 2020; Kosaric et al., 2020; Hu et al., 2021; Lintel et al., 2022; Maschalidi et al., 2022; K. Chen et al., 2022b). Specifically, this wound healing model has been established to test multiple different therapies for wound healing. Several studies have used different wound dressings incorporating various hydrogel compositions. Our group has used the excisional wound model to compare the ability to promote wound healing of four different types of wound dressings made from biocompatible hydrogels (K. Chen et al., 2022b). Casado-Diaz et al. (2022) have used the excisional wound model to evaluate the efficacy of a novel Olea europaea–based hydrogel matrix as a wound dressing. Cell-based therapies can also be studied with the excisional wound model. Our group has shown that treating excisional wounds with Trem2+ macrophages in a hydrogel-based wound dressing enhances angiogenesis and accelerates wound healing (Henn et al., 2021). Our group has also applied an adipose-derived stromal cell-seeded hydrogel wound dressing to excisional burn wounds to evaluate its effect on wound closure (Barrera, 2021). Another application of this model is to evaluate the effect of drug-based therapies on wound healing. For example, adding a recombinant Agrin fragment on excisional wounds has been shown to promote healing (Chakraborty et al., 2021). By utilizing the excisional wound model as well as a burn wound model, our group has shown that wounds treated with the small molecule focal adhesion kinase inhibitor (FAKI) imbued with pullulan hydrogel heal significantly faster than untreated wounds (Ma et al., 2018), establishing the relevance of FAKI therapy to reduce inflammation and fibrosis in rodent models (K. Chen et al., 2021,2022a). Other wound healing models have been described in mice. One is the dorsal skin fold chamber (Michael et al., 2013), which has been previously used to study burn wounds in rodents. This model also prevents wound healing by contracture by having a titanium frame fixated around the wound. In comparison to that model, our excisional wound model utilizes commercially available silicone sheets as splints to prevent contraction, and the splints necessary to prevent wound contraction are easily manufactured. These important steps allow this model to be easily incorporated at low cost for any research group across the world. In this protocol, we describe the exact methods of the excisional wound model and how they can be applied to different mouse strains. The wound healing in normal wildtype mice is compared to wound healing in a diabetic mouse strain with a mutation on the leptin gene (db/db). While previous papers have used this model, their methods are usually described vaguely and do not comment on the exact methods necessary to perform this surgery, hindering the reproducibility of the model. Furthermore, methods have become outdated due to improvements in materials and documentation techniques, and our lab has significantly improved our methods compared to our first publication on the model in Galiano et al. (2004). Here, we provide a detailed protocol of this important humanized model to promote reproducibility and present several common troubleshooting situations while performing the surgeries and during post-operative wound care. Additionally, we provide a precise guide on how to perform the main analysis of wound healing curve analysis. Materials and Reagents CorningTM FalconTM 50 mL conical centrifuge tubes (Fisher Scientific, catalog number: 14-432-22) Covidien sterile gauze (Fisher Scientific, catalog number: 2187) BD brand isopropyl alcohol swabs (Fisher Scientific, catalog number: 13-680-63) Betadine solution swab stick (Fisher Scientific, catalog number: 19065534) Covidien TelfaTM non-adherent pads (Fisher Scientific, Covidien, 1961) Tegaderm, 3 M, 1626W (VWR, catalog number: 56222-191) Dental surgical ruler (DoWell Dental Products, catalog number: S1070) C57/BL6 females (6–8 weeks old) (The Jackson Laboratory, catalog number: 000664) B6.BKS(D)-Leprdb/J females (6–8 weeks old) (The Jackson Laboratory, catalog number: 000697) Buprenorphine SR (0.5 mg/mL) (Buprenex, Indivior Inc., catalog number: 12496-0757-1) Isoflurane, USP (Dechra Veterinary Products, catalog number: 17033-094-25) Puralube® ophthalmic ointment (Dechra, NDC, catalog number: 17033-211-38) Vetbond (3 M) (Saint Paul, MN, catalog number: 1469SB) Depilatory cream (Nair Hair Remover Lotion, Church&Dwight, CVS, catalog number: 339823) Ethanol 70% solution (Fisher Scientific, catalog number: 64-17-5) Medequip Depot Silk Black Braided Sutr 6-0 Rx (Medequip Depot D707N, Fisher Scientific, catalog number: NCO835822) Equipment Tissue forceps 4¾ in. stainless 1 × 2 teeth (Mckesson, catalog number: 43-2-775) Iris scissors 4½ in. stainless (McKesson, catalog number: 43-2-104) Disposable biopsy punch 8 mm (Integra Miltex, catalog number: 33-37) Needle holder 5 in. with serrated jaws (McKesson, catalog number: 43-2-842) Keyes cutaneous punch 16 mm (Delasco, catalog number: KP-16) Keyes cutaneous punch 10 mm (Delasco, catalog number: KP-10) Silicone sheet 0.5 mm, no PSA (Sigma-Aldrich, Grace Bio-Labs CultureWell, GBL664581-5EA) Liquid repellent drape 75 × 90 cm with adhesive hole 6 × 9 cm (Omnia S.p.A., catalog number: 12.T4362) Inhalation anesthesia system (VetEquip, catalog number: 922130) Aesculap Exacta mini trimmer (Aesculap) Thermo-peep heating pad (K&H, Amazon) Surgical skin marker (McKesson, 19-1451_BX) Software ImageJ (ImageJ, Wayne Rasband, imagej.net) Prism 9 (GraphPad Holdings, LLC, graphpad.com) R studio Desktop (RStudio PBC, rstudio.com, open-source software) Excel (Microsoft Cooperation, Microsoft.com) Procedure Making splints (equipment shown in Figure 1A) (Table 1) Time: Each splint takes approximately 30 s to make. Remove the sticky coverings from both sides of the silicone sheets. Place the silicone sheet on a soft base (e.g., folded cloth). First, punch out the outer circle with the 16 mm cutaneous punch using circular movements and applying pressure on the silicone sheet. Punch out the inner circle with the 10 mm cutaneous punch, also using circular movements and applying pressure on the silicone sheet. Place splints in a 70% ethanol bath in a conical tube for disinfection (leave in the 70% ethanol solution until use). Figure 1. Splints and Excisional Wounds. A. From left to right: biopsy punches used to make splints [10 mm (left) and 16 mm cutaneous punch (right)]; splints; all material necessary to make splints, with the silicone sheet on top. B. From left to right: shaved and depilated dorsum of mouse; excisional wounds on mouse dorsum; excisional wounds with splints; wound dressing (Telfa + Tegaderm). Table 1. Making splints Step Procedure Troubleshooting 1 Remove sticky coverings from both sides of the silicone sheets. 2 Place the silicone sheet on a soft base. Folded cloth works best, as it has high resistance to punching forces. 3 First, punch out the outer circle with a 16 mm punch using circular movements and applying pressure on the silicone sheet. 4 Punch out the inner circle with a 10 mm punch, also using circular movements and applying pressure on the silicone sheet. 5 Place splints in a 70% ethanol bath in a conical tube for disinfection (leave in the 70% ethanol solution until use). Replace ethanol bath regularly. Excisional wounding (Table 2) Time: 12–30 min per mouse (after sufficient practice), time varies depending on operator’s skills and mouse line. Diabetic mice (high BMI) tend to be easier to handle during surgery than thin wildtype mice. Prepare surgical field with heating pad set at 40°C, surgical drape, and anesthesia system. Anesthetize mouse with isoflurane at a flow rate of 5% in 100% oxygen (flow rate 1 L/min). Inject buprenorphine SR 0.5 mg/kg subcutaneously 5 min prior to incision and apply ophthalmic ointment on both eyes. Maintain anesthetized state with 1%–1.5% isoflurane; monitor mouse every 90 s for changes in breathing rate. Place mouse in a prone position on the prepared surgical table. Shave complete dorsum of mouse from the neck to the root of the tail and to the sides; shave until the beginning of all four extremities. Apply depilatory cream on the shaved area for 30 s. Use wet gauze swabs to remove all remaining cream and fur. Disinfect skin with three alternating betadine and alcohol wipes in a circular fashion. Place sterile drape over the mouse, leaving the surgical area free. Use the biopsy punch to outline the pattern for excision of skin on the dorsum of the mice (Figure 1B). Create two wounds on the dorsum of the mice with the biopsy punch. Push the biopsy punch firmly onto the skin and then twist fairly quickly in swift circulating motions to cut through the dermis. Notes: Fixate skin between fingers to create tension on the skin in order to excise biopsy. Do not push too hard or you will wound the underlying muscle. Wet the skin with an alcohol swab to ease friction force from the biopsy punch. Excise skin (with the aid of scissors if necessary). Take out the splint from ethanol bath and let it dry on sterile surgical area. Apply minimal amounts of Vetbond to the splint and place it carefully around the wound. Notes: Leave an area of the splint without Vetbond for forceps to grab. Otherwise, the splint will be glued to the forceps. Make sure to apply enough Vetbond on the silicone splints so that the splint sticks well to the skin, but care must be taken to not apply Vetbond onto the wound. Use 6-0 sutures to apply eight interrupted sutures fixating the splint to the surrounding skin (Figure 1B). Take a picture of the wound with a ruler for documentation and baseline for analysis of wound closure. Note: Be sure to try to always take the images from the same angle, orientation, and distance from each wound to promote consistency. Table 2. Excisional wounding Step Procedure Troubleshooting 1 Prepare surgical field with heating pad set at 40 °C, surgical drape, and anesthesia system. To limit anesthesia time for mice, accurate preparation is essential. 2 Anesthetize mouse with isoflurane at a flow rate of 5% in 100% oxygen (flow rate 1 L/min). Monitor mouse carefully and take out of anesthesia box immediately when sufficient level of anesthesia has been reached. Exposure to high flow rates of isoflurane for too long can cause death. 3 Inject buprenorphine SR 0.5 mg/kg subcutaneously 5 min prior to incision and apply ophthalmic ointment on both eyes. Do not start surgery immediately after injecting buprenorphine, as activation period needs to be accounted for. 4 Maintain anesthetized state with 1%–1.5% isoflurane; monitor mouse every 90 s for changes in breathing rate. Maintaining flow rate as low as possible, but as high as necessary is imperative. An insufficient flow rate will cause peri-surgical awakening; however, an excessive flow rate will cause death during anesthesia. 5 Place mouse in a prone position on prepared surgical table. 6 Shave complete dorsum of mouse from the neck to the root of the tail and to the sides; shave until the beginning of all four extremities. Make sure to shave a sufficient area, as Vetbond will not stick to unshaved skin. 7 Apply depilatory cream on the shaved area for 30 s. Use wet gauze swabs to remove all remaining cream and fur. Do not leave depilatory cream on for longer than 30 s. Leaving it on too long will cause skin irritations that will likely result in wound dehiscence in a later stage. 8 Disinfect skin with three alternating betadine and alcohol wipes in a circular fashion. 9 Place sterile drape over the mouse, leaving the surgical area free. 10 Use biopsy punch to outline pattern for excision of skin on the dorsum of the mice (Figure 1B). 11 Create two wounds on the dorsum of the mice with biopsy punch. 12 Push the biopsy punch firmly onto the skin and then twist fairly quickly in swift circulating motions to cut through the dermis. Fixate skin between fingers to create tension on the skin in order to excise biopsy. Do not push too hard or you will wound the underlying muscle. Wet the skin with an alcohol swab to ease friction forces from the biopsy punch. 13 Excise skin. Scissors can be used to aid excision of skin if necessary. 14 Take out the splint from ethanol bath and let it dry on sterile surgical area. Let splint fully dry before applying Vetbond. 15 Apply minimal amounts of Vetbond to the splint and place it carefully around the wound. Leave an area of the split without Vetbond for forceps to grab. Otherwise, the splint will be glued to the forceps. Make sure to apply enough Vetbond on the silicone splints, so the splint sticks well to skin, but care must be taken to not apply Vetbond onto the wound. 16 Use 6-0 sutures to apply eight interrupted sutures fixating the splint to the surrounding skin (Figure 1B). 17 Take a picture of the wound with a ruler for documentation and baseline for analysis of wound closure. Be sure to try and always take the images at the same angle, orientation, and distance from each wound to promote consistency. Therapeutics and dressings (Table 3) Time: Varies on applied treatment (approximately 2–15 min per mouse). Injecting therapeutic (e.g., cells, pharmacologic) or no treatment (if using hydrogels, skip to step C2) Inject therapeutic or vehicle control around the wound edge. Wrap the wounds with an initial layer of Telfa. Skip to step C3. Hydrogel therapy (Figure 2A) Apply piece of circular hydrogel onto the wound (with or without therapeutic imbued) (Figure 2B). Note: Hydrogel should be cut out so that it has the same measurements as the excisional wound. Wrap the wounds with an initial layer of Tegaderm. Note: Initial layer keeps hydrogel protected, moist, and pressed against wound to maximize delivery. Next, cover the wound with Telfa dressing. Proceed to step C3. Cut Tegaderm sheets in half and use the halves to wrap around the mice (Figure 1B). Monitor mouse until it is fully awake. Place in cage—single housing is necessary now. Figure 2. Wound healing analysis. A. Pictures of wound healing over time in wildtype (WT) and diabetic mice (db/db). B. ImageJ settings for analysis of wound closure. Blue boxes demonstrate settings and wound area, green boxes demonstrate settings and measurement of 1 cm as scale bar. Table 3. Therapeutics and dressings Step Procedure Troubleshooting 1 Injecting therapeutic (e.g., cells, pharmacologic) or no treatment (if using hydrogels, skip to step 2) 1a Inject therapeutic or vehicle control around the wound edge. 1b Wrap the wounds with an initial layer of Telfa. 1c Skip to step 3. 2 Hydrogel therapy (Figure 2A) 2a Apply piece of circular hydrogel onto the wound (with or without therapeutic imbued) (Figure 2B). Hydrogel should be cut out so that it has the same measurements as the excisional wound. 2b Wrap the wounds with an initial layer of Tegaderm. Initial layer keeps hydrogel protected, moist, and pressed against wound to maximize delivery. 2c Next, cover the wound with Telfa dressing. 3 Cut Tegaderm sheets in half and use the halves to wrap around the mice (Figure 1B). 4 Monitor mouse until it is fully awake. 5 Place in cage—single housing is necessary now. Monitoring and wound measurements (Table 4) Time: Approximately 2–10 min per mouse (depending on status of splint; time increases if additional sutures have to be placed or treatment has to be applied). Monitor mouse daily. Every other day: measure wound using the following steps. Anesthetize mouse as described in section B. Carefully remove wound dressing (Tegaderm and Telfa). Note: Be sure to remove the dressings carefully so as to not disturb the wound bed or tear off splints and sutures. Take picture of wound for analysis as described in Step B17. Check if splints are securely fixed to skin; otherwise, the wound will contract and cannot be used for analysis anymore. If splint is not securely fixated to skin, reapply Vetbond and 6-0 interrupted suture(s). Note: Examples of damaged splints and explanation on how to deal with them are shown in Figure 4A–F. Re-apply dressings and/or therapeutics as described in section C. Table 4. Monitoring and wound measurements Step Procedure Troubleshooting 1 Monitor mouse daily. 2 Every other day: measure wound using the following steps. 3 Anesthetize mouse as described in section B. Monitor mouse carefully and take out of anesthesia box immediately when sufficient level of anesthesia has been reached. Exposure to high flow rates of isoflurane for too long can cause death. 4 Carefully remove wound dressing (Tegaderm and Telfa). Be sure to remove the dressings carefully so as to not disturb the wound bed or tear off splints and sutures. 5 Take a picture of wound for analysis as described in Step B17. 6 Check if splints are securely fixed to skin; otherwise, the wound will contract and cannot be used for analysis anymore. 7 If splint is not securely fixated to skin, reapply Vetbond and 6-0 interrupted suture(s). Examples of damaged splints and explanation on how to deal with them are shown in Figure 4A–F. 8 Re-apply dressings and/or therapeutics as described in section C. Explant and analysis (Table 5) Time: Approximately 10 min should be allocated for each mouse to excise the wounded skin (steps E1–7). Time for consecutive analyses varies according to analysis technique. Anesthetize mouse as described in section B. Take off wound dressing. Take a picture of wound for analysis as described in Step B17. Mark area of excisional wound with a surgical skin marker. Remove splints from underlying skin by carefully cutting sutures and peeling off. Euthanize mouse using neck dislocation while still under anesthesia. Note: Other approved methods of mouse euthanasia may also be used, depending on institutional rules. Immediately after euthanizing, excise skin within marked area of the wound. Excised tissue can now be further processed for desired analysis techniques. Further data analysis techniques of the tissue can include: Histological staining (H&E, Trichrome, etc.) Immunofluorescent staining for proteins Single cell RNA-sequencing Mechanical testing Table 5.Explant and analysis Step Procedure Troubleshooting 1 Anesthetize mouse as described in section B. 2 Take off wound dressing. 3 Take a picture of wound for analysis as described in Step B17. 4 Mark area of excisional wound with a surgical skin marker. 5 Remove splints from underlying skin by carefully cutting sutures and peeling off. Take care not to disturb wounded skin as this can lead to tissue damage. 6 Euthanize mouse using neck dislocation while still under anesthesia. 7 Immediately after euthanizing, excise skin within marked area of the wound. 8 Excised tissue can now be further processed for desired analysis techniques. Further data analysis techniques of the tissue can include: Histological staining (H&E, Trichrome, etc.) Immunofluorescent staining for proteins Single-cell RNA sequencing Mechanical testing Data analysis N = 5 per experimental group is needed to obtain statistically significant results. Note: Exclusion criteria for the wounds are severe displacement of the splint, infection, or auto-mutilation of the wound (Figure 4). Upload images into ImageJ in a TIFF format. Note: Use images until full re-epithelization has occurred. Create table in Microsoft Excel. Take measurement of 1 cm by tracing along the ruler with the fixed drawing tool in ImageJ. Trace the outer linings of the wound with the freestyle or polygon drawing tool in ImageJ. Transfer the two values to the excel sheet. Convert area into cm2 with the following equation: x cm2=(Wound Pixel Area)/(pixel length of 1 cm)2 Transfer data to GraphPad Prism 9. Use an unpaired Student’s t-test for two experimental groups. Use a 2-way ANOVA test for multiple (> two) groups. Consider p < 0.05 as statistically significant. Use a standard XY graph to create a wound healing curve [seen in figure 6H of the original publication (Henn et al., 2021) and Figure 3A of this protocol]. Multiple other comparisons can be performed using the same tests described in step 8: Comparison of days until wound closure (Figure 3B). Wound closure at specific post-operative days (POD) (Figure 3C and 3D). Figure 3. Statistical analysis of wound healing. A. Graph of wound size (closure) over time. Post-operative day (POD). x-axis represents the POD and y-axis the percentage of wound closure. B. Bar graph of days until wound closure. x-axis represents the experimental group and y-axis the days until wound closure. C. Bar graph of wound size at POD 8. x-axis represents the experimental group and y-axis the percentage of wound size to the initial wound size at POD 0. D. Bar graph of wound size at POD 10. x-axis represents the experimental group and y-axis the percentage of wound size to the initial wound size at POD 0. Figure 4. Troubleshooting images. A. Splint is partially broken. If the rest of the splint is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be included in experimental analyses. B. Sutures have come off. If splint is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be included. C. Splint is partially ripped. If the rest is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be used. D. Splint is entirely broken on one side; wound has to be excluded from further analysis. E. Splint has ripped through the ring. If splint is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be used. (F) Splint is entirely broken on one side; wound has to be excluded from further analysis. Acknowledgments We thank Theresa Carlomagno for administrative support. This work was supported by the National Institute of Health RO1 grant (DK074095). Schematic figures were created with BioRender.com. The original research paper to this protocol is found here (DOI: 10.1126/sciadv.abi4528) (Henn et al., 2021). Competing interests The authors declare no competing interests. Ethics All animal experiments were performed in accordance with both Stanford University and University of Arizona Institutional Animal Care and Use Committees (IACUC) and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The study was approved by the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University (APLAC protocol number: 12080), as well at the University of Arizona under IACUC protocol 2021-0828. References Alexiadou, K. and Doupis, J. (2012). Management of diabetic foot ulcers. Diabetes Ther 3(1): 4. Barrera, J. A. (2021). Adipose-Derived Stromal Cells Seeded in Pullulan-Collagen Hydrogels Improve Healing in Murine Burns. Tissue Engineering Part A 27(11-12): 844-856. Casado-Diaz, A., Moreno-Rojas, J. M., Verdú-Soriano, J., Lázaro-Martínez, J. L., Rodríguez-Mañas, L., Tunez, I., La Torre, M., Berenguer Pérez, M., Priego-Capote, F. and Pereira-Caro, G. (2022). Evaluation of Antioxidant and Wound-Healing Properties of EHO-85, a Novel Multifunctional Amorphous Hydrogel Containing Olea europaea Leaf Extract. Pharmaceutics 14(2). Chakraborty, S., Sampath, D., Yu Lin, M. O., Bilton, M., Huang, C. K., Nai, M. H., Njah, K., Goy, P. A., Wang, C. C., Guccione, E., et al. (2021). Agrin-Matrix Metalloproteinase-12 axis confers a mechanically competent microenvironment in skin wound healing. Nat Commun 12(1): 6349. Chen, K., Henn, D., Januszyk, M., Barrera, J. A., Noishiki, C., Bonham, C. A., Griffin, M., Tevlin, R., Carlomagno, T., Shannon, T. et al. (2022a). Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting. Sci Transl Med 14(645): eabj9152. Chen, K., Kwon, S. H., Henn, D., Kuehlmann, B. A., Tevlin, R., Bonham, C. A., Griffin, M., Trotsyuk, A. A., Borrelli, M. R., Noishiki, C. et al. (2021). Disrupting biological sensors of force promotes tissue regeneration in large organisms. Nature Communications 12(1): 5256. Chen, K., Sivaraj, D., Davitt, M. F., Leeolou, M. C., Henn, D., Steele, S. R., Huskins, S. L., Trotsyuk, A. A., Kussie, H. C., Greco, A. H., et al. (2022b). Pullulan-Collagen hydrogel wound dressing promotes dermal remodelling and wound healing compared to commercially available collagen dressings. Wound Repair Regen 30(3): 397-408. Chen, L., Mirza, R., Kwon, Y., DiPietro, L. A. and Koh, T. J. (2015). The murine excisional wound model: Contraction revisited.Wound Repair Regen 23(6): 874-877. Cho, H., Balaji, S., Hone, N. L., Moles, C. M., Sheikh, A. Q., Crombleholme, T. M., Keswani, S. G. and Narmoneva, D. A. (2016). Diabetic wound healing in a MMP9-/- mouse model. Wound Repair Regen 24(5): 829-840. Chong, K. K. L., Tay, W. H., Janela, B., Yong, A. M. H., Liew, T. H., Madden, L., Keogh, D., Barkham, T. M. S., Ginhoux, F., Becker, D. L., et al. (2017). Enterococcus faecalis Modulates Immune Activation and Slows Healing During Wound Infection. J Infect Dis 216(12): 1644-1654. Cogan, N. G., Mellers, A. P., Patel, B. N., Powell, B. D., Aggarwal, M., Harper, K. M. and Blaber, M. (2018). A mathematical model for the determination of mouse excisional wound healing parameters from photographic data. Wound Repair Regen 26(2): 136-143. Dallas, A., Trotsyuk, A., Ilves, H., Bonham, C. A., Rodrigues, M., Engel, K., Barrera, J. A., Kosaric, N., Stern-Buchbinder, Z. A., White, A., et al. (2019). Acceleration of Diabetic Wound Healing with PHD2- and miR-210-Targeting Oligonucleotides. Tissue Eng Part A 25(1-2): 44-54. Galiano, R. D., Michaels, J. t., Dobryansky, M., Levine, J. P. and Gurtner, G. C. (2004). Quantitative and reproducible murine model of excisional wound healing. Wound Repair Regen 485-492. Henn, D., Chen, K., Fehlmann, T., Trotsyuk, A. A., Sivaraj, D., Maan, Z. N., Bonham, C. A., Barrera, J. A., Mays, C. J., Greco, A. H., et al. (2021). Xenogeneic skin transplantation promotes angiogenesis and tissue regeneration through activated Trem2+ macrophages. Science Advances 7(49): eabi4528. Hu, M. S., Cheng, J., Borrelli, M. R., Leavitt, T., Walmsley, G. G., Zielins, E. R., Hong, W. X., Cheung, A. T. M., Duscher, D., Maan, Z. N., et al. (2018). An Improved Humanized Mouse Model for Excisional Wound Healing Using Double Transgenic Mice. Adv Wound Care (New Rochelle) 7(1): 11-17. Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W. X., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., et al. (2021). Wounds Inhibit Tumor Growth In Vivo. Ann Surg 273(1): 173-180. Ibrahim, M., Bond, J., Medina, M. A., Chen, L., Quiles, C., Kokosis, G., Bashirov, L., Klitzman, B. and Levinson, H. (2017). Characterization of the Foreign Body Response to Common Surgical Biomaterials in a Murine Model. Eur J Plast Surg 40(5): 383-392. Kosaric, N., Srifa, W., Bonham, C. A., Kiwanuka, H., Chen, K., Kuehlmann, B. A., Maan, Z. N., Noishiki, C., Porteus, M. H., Longaker, M. T., et al. (2020). Macrophage Subpopulation Dynamics Shift following Intravenous Infusion of Mesenchymal Stromal Cells. Mol Ther 28(9): 2007-2022. Kurt, B., Bilge, N., Sözmen, M., Aydın, U., Önyay, T. and Özaydın, I. (2018). Effects of Plantago lanceolata L. extract on full-thickness excisional wound healing in a mouse model. Biotech Histochem 93(4): 249-257. Lintel, H., Abbas, D. B., Lavin, C. V., Griffin, M., Guo, J. L., Guardino, N., Churukian, A., Gurtner, G. C., Momeni, A., Longaker, M. T., et al. (2022). Transdermal deferoxamine administration improves excisional wound healing in chronically irradiated murine skin. J Transl Med 20(1): 274. Liu, P., Choi, J. W., Lee, M. K., Choi, Y. H. and Nam, T. J. (2020). Spirulina protein promotes skin wound repair in a mouse model of full-thickness dermal excisional wound. Int J Mol Med 46(1): 351-359. Ma, K., Kwon, S. H., Padmanabhan, J., Duscher, D., Trotsyuk, A. A., Dong, Y., Inayathullah, M., Rajadas, J. and Gurtner, G. C. (2018). Controlled Delivery of a Focal Adhesion Kinase Inhibitor Results in Accelerated Wound Closure with Decreased Scar Formation. J Invest Dermatol 138(11): 2452-2460. Maschalidi, S., Mehrotra, P., Keçeli, B. N., De Cleene, H. K. L., Lecomte, K., Van der Cruyssen, R., Janssen, P., Pinney, J., van Loo, G., Elewaut, D., et al. (2022). Targeting SLC7A11 improves efferocytosis by dendritic cells and wound healing in diabetes. Nature 606(7915): 776-784. Michael, S., Sorg, H., Peck, C. T., Reimers, K. and Vogt, P. M. (2013). The mouse dorsal skin fold chamber as a means for the analysis of tissue engineered skin. Burns 39(1): 82-88. Michaels, J. t., Churgin, S. S., Blechman, K. M., Greives, M. R., Aarabi, S., Galiano, R. D. and Gurtner, G. C. (2007). db/db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. Wound Repair Regen 15(5): 665-670. Nauta, A., Seidel, C., Deveza, L., Montoro, D., Grova, M., Ko, S. H., Hyun, J., Gurtner, G. C., Longaker, M. T. and Yang, F. (2013). Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing. Mol Ther 21(2): 445-455. Rhea, L. and Dunnwald, M. (2020). Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis. J Vis Exp (162): 10.3791/61616. Sen, C. K. (2021). Human Wound and Its Burden: Updated 2020 Compendium of Estimates. Advances in Wound Care 10(5): 281-292. Thomas, D., Burns, J., Audette, J., Carroll, A., Dow-Hygelund, C. and Hay, M. (2016). Clinical Development Success Rates 2006-2015. Biomedtracker, Amplion, Biotechnology Innovation Organization (Accessed 15 June 2022). Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cell Biology > Cell Transplantation > Xenograft Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 1 Q&A Hello, How can I overcome the self grooming? my mice keep chewing the sutures and the ring falls down after three days from the surgery. 2 Answers 47 Views Apr 4, 2023 Related protocols Determining Bone-forming Ability and Frequency of Skeletal Stem Cells by Kidney Capsule Transplantation and Limiting Dilution Assay Hitoshi Uchida [...] Wei Hsu Mar 20, 2023 371 Views The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo Maryna Pavlova [...] Ganna Bilousova Jan 20, 2024 953 Views Mesenteric Parametrial Fat Pad Surgery for in vivo Implantation of Hepatocytes in Nude Mice Saloni Sinha [...] Robert E. Schwartz Jan 20, 2024 606 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,607	https://bio-protocol.org/en/bpdetail?id=4607&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A CRISPR-based Strategy for Temporally Controlled Site-Specific Editing of RNA Modifications YX Ying Xu * YW Yufan Wang * FL Fu-Sen Liang (contributed equally to this work) Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4607 Views: 639 Reviewed by: David PaulKenji Sugiyama Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Communications Apr 2022 Abstract Chemical modifications on RNA play important roles in regulating its fate and various biological activities. However, the impact of RNA modifications varies depending on their locations on different transcripts and cells/tissues contexts; available tools to dissect context-specific RNA modifications are still limited. Herein, we report the detailed protocol for using a chemically inducible and reversible platform to achieve site-specific editing of the chosen RNA modification in a temporally controlled manner by integrating the clustered regularly interspaced short palindromic repeats (CRISPR) technology and the abscisic acid (ABA)-based chemically induced proximity (CIP) system. The procedures were demonstrated using the example of inducible and reversible N6-methyladenosine (m6A) editing and the evaluation of its impact on RNA properties with ABA addition and reversal with the control of ABA or light. Keywords: CRISPR Chemically induced proximity Abscisic acid RNA modification m6A Temporal control Background More than 170 chemical modifications on RNA have been discovered, which contribute to the regulation of genetic information (Licht and Jantsch, 2016; Ontiveros et al., 2019; Boccaletto et al., 2022). Among all RNA modifications, some are known to be dynamic and reversible, fine-tuned by several endogenous writers, erasers, and reader proteins in cells, which are responsible to generate, remove, and interpret these RNA modifications, respectively (Wang and He, 2014; Xu and Liang, 2022). The most prevalent dynamic RNA modification, N6-methyladenosine (m6A), is deposited by methyltransferase-like 3 (METTL3) and other auxiliary proteins, such as methyltransferase-like 14 (METTL14) and Wilms’ tumor 1associated protein (WTAP) (Wang and He, 2014; Yang et al., 2018). The m6A erasers consist of the fat mass and obesity–associated protein and ALKB homologue 5 (ALKBH5), while heterogenous nuclear ribonucleoproteins and the YTH protein family recognize m6A and facilitate its function (N. Liu and Pan, 2016; Yang et al., 2018). With the rapid advance in the field of RNA epigenetics, consensus has been reached that RNA modifications play important roles in regulating its fate and orchestrating various associated biological processes. Nevertheless, the relationships between a particular RNA modification in specific contexts and the observed phenotypes are still elusive, due to limitations in the current strategies to dissect the functions of RNA modifications in a context-dependent and temporally controlled manner. Strategies to investigate RNA modifications relying on the genetic overexpression and knockdown or knockout of the corresponding enzymes/proteins generate global and irreversible changes, which complicate the functional studies of specific RNA modifications and the understanding of their direct biological consequences. The last decade has witnessed the flourish of clustered regularly interspaced short palindromic repeats (CRIPSR), which can be reprogrammed for various biological and biomedical applications (Pickar-Oliver and Gersbach, 2019;Lo et al., 2022). There have been different studies using the nuclease inactive Cas (dCas) protein to recruit m6A writers, erasers, and readers to specific sites on RNA that are accessible for m6A editing (Rauch et al., 2018; X. M. Liu et al., 2019; Wilson et al., 2020). These methods achieved site-specific m6A editing, taking the advantage of the targeting ability from the combination of dCas protein and single guide RNA (sgRNA). Unfortunately, the temporal control in these RNA modifications editing processes remains lacking. To address this critical issue, we recently reported a new strategy by integrating CRISPR with the abscisic acid (ABA)-based chemically induced proximity (CIP) system (Shi et al., 2022). The CRISPR-CIP integrated methods have been used to provide inducible and reversible control of epigenome editing (Chen et al., 2017) and site-specific m6A modification editing (Shi et al., 2022), under the regulation of small molecule ABA. The ABA-controlled inducible platform for m6A writing was designed via individually fusing the dCas13b protein and the m6A writer, METTL3, with PYL and ABI (two inducer-binding domains of ABA). The easy reprogramming nature of this platform makes it a promising tool to target various RNAs of interest by switching the target sequences of sgRNA to achieve site-specific m6A writing. In addition, the m6A writers can be replaced by erasers to achieve site-specific m6A removal, which implies a great potential for achieving editing of other RNA modifications by employing corresponding enzymes. We expect that this versatile inducible tool can be adapted for editing other RNA modifications, which opens a new door for targeted RNA modification editing in a temporally controlled manner. In this protocol, we describe the experimental details for developing and evaluating the inducible and reversible m6A modification editing platform (Shi et al., 2022). The design of this platform is in the scheme below (Figure 1). Figure 1. Reversible and inducible m6A editing platform. The targeted m6A editing is triggered by the addition of ABA, which can be reversed by ABA removal. The figure was created with Biorender.com. Materials and Reagents Universal pipet tips, 10 µL (VWR, catalog number: 76323-396) Universal pipet tips, 200 µL (VWR, catalog number: 76323-390) Universal pipet tips, 1,250 µL (VWR, catalog number: 76323-456) Microcentrifuge tubes (VWR, catalog number: 87003-294) 1.7 mL culture tubes (VWR, catalog number: 60818-689) 50 mL centrifuge tube (Celltreat, catalog number: 229421) BioDot pure8-strip PCR tubes (Dot Scientific, catalog number: 415-8PCR) 100 mm TC-treated cell culture dish (Falcon, catalog number: 353003) Tissue culture plates, 6 well, sterile (VWR, catalog number: 10062-892) Tissue culture plates, 12 well, sterile (VWR, catalog number: 10062-894) Tissue culture plates, 24 well, sterile (VWR, catalog number: 10062-896) 500 mL solution bottle, sterile (Celltreat, catalog number: 229784) 500 mL bottle top filter, sterile (Celltreat, catalog number: 229717) Universal pipet tips, 10 µL, filtered, pre-sterile (VWR, catalog number: 76322-528) Universal pipet tips, 20 µL, filtered, pre-sterile (VWR, catalog number: 76322-134) Universal pipet tips, 200 µL, filtered, pre-sterile (VWR, catalog number: 76322-150) Universal pipet tips, 1,250 µL, filtered, pre-sterile (VWR, catalog number: 76322-528) Disposable serological pipets, 2 mL (VWR, catalog number: 75816-104) Disposable serological pipets, 5 mL (VWR, catalog number: 89130-896) Disposable serological pipets, 10 mL (VWR, catalog number: 89130-898) Disposable serological pipets, 25 mL (VWR, catalog number: 89130-900) HEK293T cell (gift from Dr. Gerald R Crabtree at Stanford University Medical School) HeLa cell (gift from Dr. Gerald R Crabtree at Stanford University Medical School) Q5 High-Fidelity 2× Master Mix (New England BioLabs, catalog number: M0492S) Monarch® PCR & DNA Cleanup kit (New England Biolabs, catalog number: T1030S) NotI-HF restriction enzyme (New England BioLabs, catalog number: R3189) EcoRI-HF restriction enzyme (New England BioLabs, catalog number: R3101S) PmeI restriction enzyme (New England BioLabs, catalog number: R0560S) BbsI restriction enzyme (New England BioLabs, catalog number: R0539S) HindIII-HF restriction enzyme (New England BioLabs, catalog number: R3104S) KpnI-HF (New England BioLabs, catalog number: R3142) CutSmart buffer (New England BioLabs, catalog number: B7204) Bst 2.0 DNA polymerases (New England BioLabs, catalog number: M0537S) SplintR ligase (New England BioLabs, catalog number: M0375S) ATP solution (100 mM) (Thermo Scientific, catalog number: R0441) pCMV-dCas13-M3nls (Addgene, catalog number: 155366) pC0043-PspCas13b-crRNA (Addgene, catalog number: 103854) QIAquick Gel Extraction kit (QIAGEN, catalog number: 28704) Agarose I (Thermo Fisher Scientific, catalog number: 17852) T4 DNA ligase reaction buffer (New England BioLabs, catalog number: B0202A) T4 DNA ligase (New England Biolabs, catalog number: M0202L) In-Fusion Snap Assembly bundles for seamless DNA cloning kit (Takara, catalog number: 638952) 1 kb plus DNA ladder (New England BioLabs, catalog number: N3200) T4 polynucleotide kinase (New England BioLabs, catalog number:M0201S) TAE buffer (Tris-acetate-EDTA) (50×) (Thermo Fisher Scientific, catalog number: B49) Ampicillin sodium salt (Sigma, catalog number: A9518) Nuclease-free water (Sigma, catalog number: BCCG1748) Dulbecco’s modified Eagle’s medium, high glucose (Sigma, catalog number: RNBK9791) Opti-MEM reduced serum medium (OMEM), GlutaMax supplement (Gibco, catalog number: 51985034) Fetal bovine serum, premium selected (R&D Systems, catalog number: S11550) Lipofectamine 2000 transfection reagent (Invitrogen, catalog number: 11668019) TRIzol reagent (Invitrogen, catalog number: 15596026) Choloroform:isoamyl alcohol 24:1 (Sigma, catalog number: MKCM1807) 2-propanol (Sigma, catalog number: 19516) Ethanol (Fisher Bioreagents, catalog number: BP28184) RIPA buffer (Thermo Fisher Scientific, catalog number: 89901) Protease inhibitor cocktail (Thermo Fisher Scientific, catalog number: 78430) Dulbecco’s phosphate buffered saline (PBS) (Sigma, catalog number: D8537) PierceTM 660 nm protein assay reagent (Thermo Fisher Scientific, catalog number: 22660) 2-mercaptoethanol (Sigma, catalog number: M6250) 4× Laemmli sample buffer (Bio-Rad, catalog number: 1610747) EZ-Run prestained protein (Fisher Bioreagents, catalog number: BP36011) 4%–15% precast polyacrylamide gel (Bio-Rad, catalog number: 4561084) Methanol (Fisher Scientific, catalog number: A412-4) Non-fat milk (Bio-Rad, catalog number: 1705016) Tween-20 Surfact-AmpsTM detergent solution (Thermo Scientific, catalog number: PI85113) Anti-HA Tag monoclonal antibody (2-2.2.14) (Invitrogen, catalog number: 26183) Anti-Flag Tag monoclonal antibody (M2) (Sigma, catalog number: F1804) Anti-Gapdh antibody GAPDH (D16H11) XP® Rabbit mAb (Cell Signaling Technology, catalog number: 5174) Anti-FOXM1(A-11) (Santa Cruz, catalog number: sc-271746) Anti-SOX2 (E-4) (Santa Cruz, catalog number: sc-365823) Anti-vinculin (E1E9V, 1:1,000) (Cell Signaling Technology, catalog number: 13901) Anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, catalog number: 7074) Anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, catalog number: 7076) ClarityTM Western ECL substrate (Bio-Rad, catalog number: 1705060) Immuno-Blot PVDF membrane (Bio-Rad, catalog number: 1620177) 4% paraformaldehyde (Thermo Fisher Scientific, catalog number: J19943.K2) TritonTM X-100 surfact-AmpTM detergent solution (Thermo Fisher Scientific, catalog number: 85111) Alexa Fluor Plus 488 (Thermo Fisher Scientific, catalog number: 32723) DAPI (Fisher Scientific, catalog number: NBP2311561) Actinomycin D (Thermo Fisher Scientific, catalog number: 11805017) RNA clean & concentrator-5 (Zymo Research, catalog number: R1014) N6-Methyladenosine (m6A) (D9D9W) rabbit mAb (Cell Signaling Technology, catalog number: 56593) PierceTM Protein G magnetic beads (Thermo Fisher Scientific, catalog number: 88848) N6-Methyladenosine 5′-monophosphate sodium salt (m6A salt) (Sigma, catalog number: M2780-10MG) Protease K solution, RNA grade (Invitrogen, catalog number: 25530049) RNasin Plus ribonuclease inhibitor (Promega, catalog number: N2611) Ultrapure bovine serum albumin (BSA), 50 mg/mL (Invitrogen, catalog number: AM2616) Zinc chloride (ZnCl2) (Sigma, catalog number: Z0152) 0.5 M EDTA pH 8.0 (Invitrogen, catalog number: AM9260G) Formaldehyde solution (Sigma, catalog number: F8775-500ML) Anti-METTL3 antibody (Invitrogen, catalog number: 15073-1-AP) Glycine (Fisher bioreagents, catalog number: BP181-1) Magnesium chloride solution, 1 M (MgCl2) (Sigma, catalog number: M1028-100ML) Sodium dodecyl sulfate (SDS) (Sigma, catalog number: L3771-500G) 5 M sodium chloride (NaCl) (Invitrogen, catalog number: AM9759) 1 M Tris-HCl, pH 7.0 (Sigma, catalog number: T1819-1L) 1 M Tris-HCl, pH 7.4 (Sigma, catalog number: T2663-1L) IGEPAL-CA-630 (Sigma, catalog number: I8896) Sodium acetate (3 M), pH 5.5, RNase-free (Invitrogen, catalog number: AM9740) Pierce RIPA buffer (Thermo Fisher Scientific, catalog number: 89900) Halt protease inhibitor cocktail (Thermo Scientific, catalog number: 1861279) PowerUpTM SYBRTM Green Master Mix (Thermo Fisher, catalog number: A25778) iScript cDNA Synthesis kit (Bio-Rad, catalog number: 1708891) Abscisic acid (ABA) (Gold Biotechnology, catalog number: A-050-5) Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418-100 mL) Tris base (Sigma, catalog number: T1503-500 g) Agar (Sigma, catalog number: A6686) LB media (see Recipes) LB plate with ampicillin (see Recipes) 1× gel running buffer (see Recipes) 1× membrane transfer buffer (see Recipes) 1× TBST buffer (see Recipes) Fragmentation buffer, 10× (see Recipes) 5× IP buffer for m6A-IP study (see Recipes) m6A elution buffer (see Recipes) Wash buffer for RNA-crosslink immunoprecipitation (see Recipes) NT-2 buffer, 5× (see Recipes) Protease K digestion buffer (see Recipes) Equipment Centrifuge 5424/5424R (Eppendorf, catalog number: EP5404000537) New Brunswick I26/I26R stackable incubator shakers (Eppendorf, catalog number: VWR-89173-938) Sorvall LYNX 6000 superspeed centrifuge (Thermo Scientific, catalog number: 75006590) 7900HT Fast Real-Time PCR system (Applied Biosystems, catalog number: 4329001) ChemiDoc MP imaging system (Bio-Rad, catalog number: 17001402) C1000 touch thermal cycler (Bio-Rad, catalog number: 1851148) NanoDrop One spectrophotometer (Thermo Scientific, catalog number: ND-ONE-W4) Bioruptor Pico sonication device (Diagenode, catalog number: B01060010) Fluorescence microscopy (Agilent BioTek LFXSN, catalog number: BTFLX) CriterionTM Blotter with wire electrodes (Bio-Rad, catalog number: 1704071) Procedure Cloning of dCas13b-PYL-HA plasmid Amplify the dCas13b fragment with the template of pCMV-dCas13b-M3nls, using the designed primers via Q5 High-Fidelity 2× Master Mix according to the manufacturer’s protocol. Amplify the PYL [pyrabactin resistance (PYR)/PYR1-like] fragment with the template of dCas9-PYL (a plasmid cloned by our former group member), using the designed primers for In-Fusion cloning via Q5 High-Fidelity 2× Master Mix according to the manufacturer’s protocol. Run a 0.8% DNA agarose gel to purify the DNA fragments amplified above with the QIAquick Gel Extraction kit. The size of amplified fragment dCas13b is 3,147 bp and the size of PYL fragment is 560 bp. Linearize 1 µg backbone plasmids dCas13b-ALKBH5-HA with the HindIII-HF and KpnI-HF restriction enzymes by incubating at 37°C for 2 h and purify the linearized vector by running a 0.8% DNA agarose gel. The size of the linearized backbone is 5,452 bp. Use the In-Fusion master mix from the In-Fusion Snap Assembly bundles for seamless DNA cloning kit to insert the dCas13b and PYL fragments into the linearized vector in the step A4 to get the dCas13b-PYL-HA plasmids. The primers used for PCR and sequencing are listed in Table 1 and the scheme of cloning is shown in Figure 2. Notes: Set up the PCR reaction in 50 µL of reaction. Briefly, gently mix 10 µL of 5× Q5 reaction buffer, 1 µL of 10 mM dNTPs, 2.5 µL of forward primer (10 µM), 2.5 of µL reverse primer, 100 ng of template (1 µL), 0.5 µL of forward primer Q5 high-fidelity DNA polymerase, and 32.5 µL of nuclease-free water. Run the PCR reaction at 98°C for 30 s, 34 cycles of 98°C for 10 s + 60 °C for 30 s + 72°C for 90 s, and finally 72°C for 120 s. There are many plasmids containing PYL fragments that can be obtained from Addgene for the purpose of amplification. For example: PYL1-nTEVp (catalog number: 119213). Table 1. Information on primers used to clone and sequence the dCas13b-PYL-HA plasmid Primers used for cloning and sequencing Sequences (5′-3′) Forward primer to amplify the dCas13b fragment cgtttaaacttaagcttGCCACCatgaaacggacagccgacgga Reverse primer to amplify the dCas13b fragment cttgagtagcggccgcgctctctggtgttgctgactc Forward primer to amplify the PYL fragment gagcgcggccgctactcaagacgaattcacccaa Reverse primer to amplify the PYL fragment CGTATGGGTAGGTACCgttcatagcttcagtgatcga Sequencing primer for the dCas13b-PYL-HA aagcagagctctctggctaac Sequencing primer for the dCas13b-PYL-HA tgcgagttcctgaccagcaca Sequencing primer for the dCas13b-PYL-HA gattacggcaagctgttcgac Sequencing primer for the dCas13b-PYL-HA agcggcaatgcccacggcaag Sequencing primer for the dCas13b-PYL-HA cccagacagatgttcgacaat Sequencing primer for the dCas13b-PYL-HA atcaccagcgagggcatgaag Figure 2. Scheme of cloning of the dCas13b-PYL-HA plasmid. The dCas13b-ALKBH5-HA plasmid was linearized to give the backbone of target plasmid. Amplified dCas13b and PYL fragments, as well as the linear backbone, were mixed together with In-fusion assembly master mix from the In-Fusion Snap Assembly bundles for seamless DNA cloning kit to generate dCas13b-PYL-HA plasmid. Cloning of ABI (abscisic acid insensitive 1)-M3nls plasmid Amplify the ABI (abscisic acid insensitive 1) fragments with the designed primers and template of ABI-P300 plasmid (P300 is a lysine acetyltransferase and ABI-P300 plasmid is constructed by former members in our lab). Amplify SV40 NLS (nuclear localization sequence) fragment with the template of dCas13b-M3nls using designed primers listed in Table 2 for In-Fusion cloning method. Purify the amplified ABI fragment and SV40 NLS fragments by running a 0.8% DNA agarose gel. The size of the ABI fragment is 1,013 bp and the SV40 NLS is 122 bp. Linearize the 1 µg backbone plasmids pCMV-dCas13-M3nls with EcoRI and NotI restriction enzymes by incubating at 37°C for 2 h to get the linearized vector containing M3nls fragment. Purify the linearized vector by 0.8% DNA agarose gel electrophoresis. The linearized vector size is 4,365 bp. Use 1 kb plus DNA ladder as the size marker. Use the In-Fusion master mix from the In-Fusion Snap Assembly bundles for seamless DNA cloning kit to insert the SV40 NLS and ABI fragments into the linearized vector according to the manufacturer’s protocol, to generate ABI-M3nls plasmid construct. The primers used for PCR and sequencing are listed in Table 2 and the scheme of cloning is shown in Figure 3. Notes: The PCR setup condition is the same as described in section A. There are many plasmids containing ABI fragments that can be obtained from Addgene for the purpose of amplification. For example: ABI-cTEVp (catalog number: 19214). Table 2. Information of primers used to clone and sequence the ABI-M3nls plasmid Primers used for cloning and sequencing Sequences (5′-3′) Forward primer to amplify the SV40 NLS fragment tagagatccgcggccgctaatacgactcactataggga Reverse primer to amplify the SV40 NLS fragment ctttgtagtcgcgcgcgactttccgcttcttctttgg Forward primer to amplify the ABI fragment Agtcgcgcgcgactacaaagaccatgacggtgattataaagatcatgacatcg attacaaggatgacgatgacaaggtccccctgtatgggttcacc Forward primer to amplify the ABI fragment tcttgggctcgaattcgctgccgtcggcggttcttttggatcccttcaggtccacgacgacgac Sequencing primer for the ABI-M3nls caactccgccccattgacgca Sequencing primer for the ABI-M3nls ctcctgagaccgtgggctcta Figure 3. Scheme of cloning of the ABI-M3nls plasmid. The pCMV-dCas13b-M3nls plasmid was linearized to give the backbone of target plasmid. Amplified ABI and NLS fragments as well as the linear backbone were mixed together with In-fusion master mix from the In-Fusion Snap Assembly bundles for seamless DNA cloning kit to generate ABI-M3nls plasmid. Cloning of sgRNA For sgRNA cloning, anneal the forward and reverse spacer sequences with overhang to obtain the sgRNA spacer DNA fragments in an 18 µL reaction. Then, phosphorylate the annealed insert with T4 polynucleotide kinase according to the manufacturer’s protocol. Linearize 1 µg of pC0043-PspCas13b-crRNA backbone vector with BbsI restriction enzyme by incubating at 55°C for 2 h and purify the linearized vector with Monarch® PCR & DNA Cleanup kit. Ligate the sgRNA spacer inserts with the linearized sgRNA backbone with T4 ligase according to the manufacturer’s protocol to obtain the sgRNA targeting the RNA of interest. The spacer sequences of sgRNA are listed in Table 3 and the scheme of sgRNA cloning is shown in Figure 4. Note: The distance of sgRNA spacer from the targeting site can impact the editing efficiency; we chose the sgRNA with its’ 3’-end two nucleotides (nts) away from the targeting site after screening. We suggest the audience to screen the optimal sgRNA for different targets. Table 3. Information of spacers of targeting sgRNA Spacer of sgRNA targeting RNA of interest Sequence (5′-3′) Actb gRNA-2 TCCATCGTCCACCGCAAATGCTTCTAGGCG Actb gRNA-8 GGCCCCTCCATCGTCCACCGCAAATGCTTC Actb gRNA-22 AGTATGACGAGTCCGGCCCCTCCATCGTCC Gapdh gRNA-8 GGGAAACTGTGGCGTGATGGCCGCGGGGCT Foxm1 gRNA ATGTTTCTCTGATAATGTCCCCAATCATAC Sox2 gRNA AACGGCACACTGCCCCTCTCACACATGTGA MALAT gRNA-1 AACGGAAGTAATTCAAGATCAAGAGTAATT MALAT gRNA-50 GAAGGCCTTAAATATAGTAGCTTAGTTTGA MALAT gRNA-100 ATTTAAAAAAAACTAAGGCAGAAGGCTTTT Figure 4. Scheme of sgRNA cloning. The pC0043-PspCas13b-crRNA plasmid was linearized to give the backbone of target plasmid. Annealed and phosphorylated spacer inserts and the linear backbone were mixed together with In-fusion master mix from the In-Fusion Snap Assembly bundles for seamless DNA cloning kit to generate sgRNA plasmid. Study the expression of plasmids by western blot Seed 250,000 HEK293T or HeLa cells in each well of a 6-well plate and incubate for 16 h to reach a confluency of 75%–80%. Transfect cells with 1.5 µg of each plasmid (dCas13b-PYL-HA, Flag-ABI-METTL3, or Flag-ABI-ALKBH5) with 3 µL of lipofectamine 2000 and incubate for 24 h. Remove the cell culture media and wash cells with DPBS. Lyse cells with 200 µL of ice-cold RIPA buffer with 2 µL of protease inhibitor cocktail for 30 min on rotation at 4°C. Spin down the cell debris via centrifugation at 12,000 × g for 15 min at 4°C. Save the supernatant and measure the protein concentration with the PierceTM 660 nm protein assay reagent according to the manufacturer’s protocol. Denature 20 µg of protein extracted from cells transfected with the different plasmids described in step D2 by incubation at 95°C for 5 min with the addition of 4× Laemmli sample buffer supplemented with 2-mercaptoethanol according to the manufacturer’s protocol. Prepare the gel electrophoresis setup and load the samples or EZ-Run prestained protein (marker) into the wells of a 4%–15% precast polyacrylamide gel. Run the gel electrophoresis in 1× gel running buffer at 60 V for 5 min, then change the voltage to 120 V and run for another 60 min. Prepare the Immuno-Blot PVDF membrane transfer setup and run the transfer process with 10 mA current for 16 h. Block the membrane with 5% non-fat milk in TBST buffer for 1 h. Dilute the primary antibodies (anti-HA, anti-Flag, anti-GAPDH, anti-FOXM1, anti-SOX2, and anti-vinculin) in 1% TBST buffer (1:1,000 dilution) and incubate with the membrane at room temperature for 2 h. Wash the membrane three times with TBST buffer. Dilute the secondary antibody in 1% TBST buffer (anti-rabbit, anti-mouse) (1:2,000 dilution) and incubate with the membrane at room temperature for 1 h. Wash the membrane three times with TBST buffer. Prepare the ECL substrate by combining the two components in a 1:1 ratio. Image each membrane with 200 µL of prepared ECL substrate using the ChemidocTM touching imaging system. Study the localization of inducible m6A editors in cells via immunofluorescence imaging Seed 250,000 HEK293T or HeLa cells in a 6-well plate and incubate for approximately 16 h to reach 75%–80% confluency. Transfect cells with 1.5 µg of dCas13b-PYL and ABI-METTL3 or 1.5 µg of dCas13b-PYL and ABI-ALKBH5 and incubate for 24 h. The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Fix cells with 4% paraformaldehyde for 10 min. Treat the above fixed cells with 0.1% TritonTM X-100 for 15 min for permeabilization. Block the cells with 1% ultrapure bovine serum albumin for 1 h at room temperature. Incubate cells with anti-HA or anti-Flag antibody (1:1,000 dilution) at 4°C overnight. Incubate cells with Alexa Fluor Plus 488 for 1 h at room temperature. In addition, add DAPI into fixed cells and incubate for 30 min. Wash the cells three times with PBS and obtain fluorescence images using the fluorescence microscope with the objective lens (10×). RNA extraction for m6A-immunoprecipitation Seed 2,000,000 cells into 100 mm dishes and incubate for approximately 16 h to reach 75%–80% confluency. Transfect cells with 6 µg of dCas13b-PYL plasmid and 6 µg of ABI-X (X refers to M3, M3, ALK, or ALK) plasmid and 4 µg of PspCas13b guide RNA plasmid using lipofectamine 2000. (Set condition of 6 µg of dCas13b-PYL, dCas13b-M3, or dCas13b-ALK plasmid with 4 µg of sgRNA as negative and positive control.) The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Add 100 µM ABA or DMSO into cells transfected with dCas13b-PYL+ABI-M3+gRNA and incubate for 24 h. Remove cell culture media in each group with different transfection and treatment and add 2 mL of TRIzol reagent to the 100 mm culture dish to lyse the cells. Pipette the lysate up and down several times to homogenize. Incubate for 5 min on ice to allow complete dissociation of nucleoprotein complexes. Add 0.2 mL of choloroform:isoamyl alcohol 24:1 per 1 mL of TRIzol reagent used for lysis, thoroughly mix by shaking, and incubate for 2–3 min. Centrifuge the sample for 15 min at 12,000 × g and 4°C (the mixture separates into a lower red phenol-chloroform, a white interphase, and a colorless upper aqueous phase) Transfer the aqueous phase containing RNA by angling the tube 45o and gently transferring the upper layer solution into a new centrifuge tube. Add 0.5 mL of 2-propanol to the aqueous phase per 1 mL of TRIzol Reagent used for lysis and incubate on ice for 10 min. Centrifuge for 10 min at 12,000 × g and 4°C. (Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.) Discard the supernatant with a pipette. Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol reagent used for lysis. Centrifuge for 10 min at 10,000 × g and 4°C. Discard the supernatant with a pipette. Vacuum or air dry the RNA pellet for 5–10 min. Resuspend the pellet in 200 µL of nuclease-free water by pipetting up and down and incubate for 5–10 min at room temperature for RNA to dissolve. Investigation of m6A level by m6A-immunoprecipitation Seed 2,000,000 cells into 100 mm dishes and incubate for approximately 16 h to reach 75%–80% confluency. Transfect cells with 6 µg of dCas13b-PYL plasmid and 6 µg of ABI-X (X refers to M3, M3, ALK, or ALK) plasmid and 4 µg of PspCas13b guide RNA plasmid using lipofectamine 2000. (Set condition of 6 µg of dCas13b-PYL, dCas13b-M3, or dCas13b-ALK plasmid with 4 µg sgRNA as negative and positive control.) The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Incubate cells for 24 h and replace cell culture media with ABA at different concentrations for another 24 h of incubation before harvesting for RNA extraction with TRIzol reagent. (For studying the reversible m6A writing, this step was done after gently washing the cell once with 10 mL of PBS, replacing the ABA-containing media with ABA-free fresh media, and incubating for another 24 h.) Extract RNA following the procedures described above. Dispense RNA into 1 µg/µL concentration for fragmentation. Fragment 180 µg RNA with 20 µL of fragmentation buffer (see Recipes) in 10 PCR tubes equally at 94°C for 1 min. (Proceed as quickly as possible.) Add 2 µL of 0.5 M EDTA to each reaction (18 µL) immediately to terminate fragmentation on ice. (The addition of EDTA should be quick to avoid excessive fragmentation, which will generate improper size of the RNA fragments.) Collect fragmented RNA into a new 1.7 mL tube. Add 22 µL of sodium acetate (3 M, pH 5.5) and 550 µL of 100% ethanol, then incubate at -80°C overnight. Pellet RNA with centrifugation at 15,000 × g for 30 min at 4°C and discard the supernatant. Wash the RNA pellet with 1 mL of 75% ethanol. Centrifuge again at 15,000 × g for 15 min at 4°C. Carefully remove the supernatant and let the RNA air-dry; use 150 µL of nuclease-free water to dissolve RNA. Prepare the m6A-immunoprecipitation (m6A-IP) reaction as below. Component Volume (µL) Fragmented RNA 120 RNasin Plus ribonuclease inhibitor (40 U/µL) 4 IP buffer, 5× 80 m6A-specific antibody (~0.5 mg/mL) Nuclease-free water 10 186 Total volume (µL) 400 Incubate the m6A-IP reaction on a rotator at 4°C for 2 h. While the samples are incubating, wash 40 µL of recombinant protein G beads twice with 1× IP buffer (diluted five times from the 5× IP buffer, see Recipes) for each IP reaction. Resuspend the beads in 1 mL of 1× IP buffer supplemented with BSA (0.5 mg/mL) at 4°C for 2 h. Incubate the IP reaction with beads at 4°C overnight on rotation. Wash beads with 1 mL of 1× IP buffer three times and remove the buffer. Add 100 µL of m6A elution buffer (with RNasin, see Recipes) to the sedimented beads and incubate the mixture for 1 h with continuous shaking at 4°C. Spin down the beads carefully and save the supernatant containing eluted RNA fragments. Repeat the elution step again and combine the 200 µL eluted RNA together. Purify RNA with RNA clean & concentrator-5 and dissolve RNA in 15 µL of nuclease-free water according to the manufacturer’s protocol. Perform reverse-transcription PCR (RT-PCR) with 12 μL of RNA after IP or 2 μL of input RNA (RNA before IP) to synthesize the cDNA using the iScript cDNA Synthesis kit according to the manufacturer’s protocol. Analyze the RNA level with PowerUpTM SYBRTM Green Master Mix according to the manufacturer’s protocol. The primers used for qPCR are listed in Table 4 and the scheme for the investigation of m6A level on target RNA is in Figure 5. Table 4. Information of primers used in the RT-qPCR assay to study the m6A enrichment and enzyme recruitment Primers used for RT-qPCR in this study Sequences (5′-3′) Actb-m6A-Foward AGATGTGGATCAGCAAGC Actb-m6A-Reverse TCATCTTGTTTTCTGCGC Gapdh-m6A-Forward CATCACTGCCACCCAGAAGA Gapdh-m6A-Reverse CAGTAGAGGCAGGGATGATGTT Foxm1-m6A-Forward TGCCCAGATGTGCGCTATTA Foxm1-m6A- Reverse CTTCTCAAGCCTCCACCTGA Sox2-m6A-Forward GGCCATTAACGGCACACTG Sox2-m6A-Reverse TCTTTTGCACCCCTCCCATT MALAT1-m6A- Forward CGTAACGGAAGTAATTCAAG MALAT1-m6A- Reverse GTCAATTAATGCTAGTCCTC CYB5A-m6A- Forward GTTTTAAGGGAACAAGCTGGAG CYB5A-m6A- Reverse TCCACCAACTGGAACTAGAATC CTNNB1-m6A- Forward TGGATTGATTCGAAATCTTGCC CTNNB1-m6A- Reverse GAACAAGCAACTGAACTAGTCG MYC-m6A- Forward CAGCTGCTTAGACGCTGGATT MYC-m6A- Reverse GTAGAAATACGGCTGCACCGA Figure 5. Scheme of the study of m6A investigation assay. The cartoon was created with Biorender.com. mRNA stability assay Seed 50,000 HEK293T or HeLa cells into a 24-well plate and incubate for approximately 16 h to reach 80% confluency. Transfect the cells with DNA plasmids expressing different components of ABA-induced m6A modification system with lipofectamine 2000 for 24 h (0.9 μg of dCas13b-PYL + 0.6 μg of gRNA, 0.9 μg of dCas13b-M3 + 0.6 μg of gRNA, 0.9 μg of dCas13b-PYL + 0.9 μg of ABI-M3 + 0.6 μg of gRNA). The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Add 100 µM ABA or DMSO into cells transfected with dCas13b-PYL+ABI-M3+gRNA and incubate for 24 h. Treat cells with 5 µg/mL actinomycin D and harvest cells at specific time points (e.g., 0, 1, 3, and 6 h). (For studying the reversible effect on RNA stability, this step was done after gently washing the cell once with 5 mL of PBS, replacing the ABA-containing media with ABA-free fresh media, and incubating for another 24 h.) Extract total RNA with TRIzol reagent and purify according to the manufacturer’s protocol. Reverse-transcribe the RNA into cDNA with the iScript cDNA Synthesis kit. Perform qPCR assays to study the mRNA levels of target transcripts (ACTB, GAPDH, SOX2, and FOXM1) with the PowerUpTM SYBRTM Green Master Mix according to the manufacturer’s protocol. The primers used for qPCR are listed in Table 5. Table 5. Information of primers used in RT-qPCR to study mRNA level Primers used in this study Sequences (5′-3′) Actb- Forward TCCCAAGTCCACACAGG Actb- Reverse CACGAAGGCTCATCATTCAAA Gapdh- Forward GGTGTGAACCATGAGAAGTATGA Gapdh- Reverse GAGTCCTTCCACGATACCAAAG Foxm1- Forward CAATGGCAAGGTCTCCTTCT Foxm1- Reverse GGTAGCAGTGGCTTCATCTT Sox2- Forward AGACGCTCATGAAGAAGGATAAG Sox2- Reverse TCATGTGCGCGTAACTGT Note: The impact of installed m6A modification is in a context-dependent manner, which means that different results of stabilization or destabilization can be observed depending on the target RNA. In the cases studied in Shi’s work, the deposited m6A destabilize the ACTB mRNA, FOXM1 mRNA, and SOX2 mRNA, but no destabilization was found of GAPDH. Enrichment of METTL3 on targeted A1216 of ACTB mRNA Seed 250,000 cells into a 6-well plate and incubate for approximately 16 h to reach 75% confluency. Transfect cells with 1.5 µg of dCas13b-PYL plasmid and 1.5 µg of ABI-M3 plasmid and 1 µg of PspCas13b guide RNA plasmid using lipofectamine 2000. (Set condition of 1.5 µg of dCas13b-PYL with 1 µg sgRNA as negative control.) The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Incubate cells for 24 h and replace cell culture media with 100 µM of ABA for another 24 h incubation. Add 35 µL formaldehyde to each well and incubate for 10 min for crosslinking. Add 167 µL glycine (0.9 mg/mL) to each well and incubate for 5 min. Remove the media in each well and wash cell with 1 mL of ice-cold DPBS twice. Add another 167 µL of DPBS supplemented with protease inhibitor cocktail and collect all the cells into a new tube using a scraper. Centrifuge cells at 5,000 × g for 5 min at 4°C. Discard supernatant, resuspend cells with 220 µL of RIPA buffer (with protease inhibitor cocktail and RNasin) and incubate for 10 min on ice. Sonicate cells in RIPA buffer for 2 min (30 s on/30 s off for one repeat) with the Bioruptor Pico sonication device. Centrifuge at 16, 000 × g for 20 min at 4°C. Save supernatant for immunoprecipitation or input and store at -80°C. Wash 10 µL of Protein G magnetic beads with 1 mL of wash buffer (see Recipes) twice for each IP reaction in a 1.7 mL centrifuge tube. Resuspended the beads in 200 µL of wash buffer with 1 µg of anti-METTL3 antibody and 3 µL of RNasin Plus ribonuclease inhibitor and incubate at room temperature for 2 h on rotation for the RNA-protein immunoprecipitation. Remove the supernatant on a magnetic rack and wash the beads three times with 200 µL of wash buffer. Add 200 µL of the supernatant saved in step I12 into the beads and incubate at 4°C overnight on rotation. Add the remaining lysate supernatant to each antibody bead reaction. Incubate all tubes on a rotating wheel overnight at 4°C on rotation. Remove the supernatant on a magnetic rack and wash beads with 200 µL of wash buffer. Add 100 µL of protease K digestion buffer to the beads after IP or 10 µL input. Incubate tubes at 65°C for 3 h with shaking to digest the proteins. Spin down all tubes and collect the supernatants on a magnetic rack. Use RNA clean & concentrator-5 kit for RNA purification and dissolve RNA in 15 µL of nuclease-free water according to the manufacturer’s protocol. Reverse-transcribe 12 μL of RNA after IP or input into cDNA and analyze the RNA level with PowerUpTM SYBRTM Green Master Mix according to the manufacturer’s protocol. The primers used in the qPCR assay are listed in Table 4 and the scheme for the study of enrichment of the METTL3 enzyme is in Figure 6. Figure 6. Scheme of investigation of enrichment of METTL3 on target RNA of interest. The cartoon was created with Biorender.com Specificity of targeted m6A editing by SELECT Seed 100,000 cells in a 12-well plate and incubate for approximately 16 h to reach 80% confluency. Transfect cells with different components of ABA-induced m6A modification system for 24 h (1.2 µg of dCas13b-PYL + 0.8 µg of gRNA, 1.2 µg of dCas13b-M3 + 0.8 µg of gRNA, 1.2 µg of dCas13b-PYL + 1.2 µg of ABI-M3 + 0.8 µg of gRNA). The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Add 100 µM ABA or DMSO into cells transfected with dCas13b-PYL+ABI-M3+gRNA and incubate for 24 h. Harvest cells for RNA extraction as described above. Mix 1.5 µg of total RNA with 40 nM Up primer, 40 nM Down primer, and 5 µM dNTP in 17 µL of 1× CutSmart buffer. Run the following PCR program to anneal the RNA mixture: 90°C (1 min), 80°C (1 min), 70°C (1 min), 60°C (1 min), 50°C (1 min), and 40°C (6 min). Incubate 17 µL of annealing products with 3 µL of enzyme mixture containing 0.01 U Bst 2.0 DNA polymerases, 0.5 U SplintR ligase, and 10 nmol ATP. Incubate the final mixture from above (total 20 µL) at 40°C for 20 min and denature at 80°C for 20 min. Analyze the DNA level via qPCR with PowerUpTM SYBRTM Green Master Mix. Briefly, mix 2 µL of the product from step J8, 5 µL of master mix, 1 µL of forward primer, and 1 µL of reverse primer, as well as 1 µL of nuclease-free water to prepare the qPCR reaction. Run qPCR with the setup of 95°C for 2 min and 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by a dissociation step of 95°C for 15 s, then 60°C for 1 min and 95 °C for 15 s. Light-inducible m6A writing Seed 2,000,000 HEK293T cells in 100 mm dishes and incubate for approximately 16 h to reach 75%–80% confluency. Transfect cells with different components of ABA-induced m6A modification system for 24 h (6 µg of dCas13b-PYL + 4 µg of gRNA, 6 µg of dCas13b-M3 + 4 µg of gRNA, 6 µg of dCas13b-PYL + 6 µg of ABI-M3 + 4 µg of gRNA). The ratio of plasmids to lipofectamine 2000 is 100 ng:0.2 µL. Treat cells with 100 µM ABA or ABA-DMNB (see note below) with and without exposure to 405 nm light for 2 min. Incubate cells for another 24 h. Harvest cells for RNA extraction, m6A-IP, and RT-PCR as described above. Note: The photo-caged ABA, ABA-DMNB, was synthesized by lab members by caging the ABA with 4,5-Dimethoxy-2-nitrobenzyl (DMNB), which can be removed by 405 nm light. The light was emitted from a fluorescence microscopy (model information provided in the Equipment section). Additionally, the primer information can be found in section G. Investigation of m6A level by m6A-immunoprecipitation. Exemplary results Here are some exemplary results (Figure 7) adapted from the original article “Inducible and reversible RNA N6-methyladenosine editing” (Shi et al., 2022). Figure 7. Exemplary results of main assays. A) Protein expression of the constructs of dCas13b-PYL with HA tag and ABI-M3nls with Flag tag. B) Localization of the expressed dCas13b-PYL and ABI-M3nls. Both proteins were localized in the nucleus. C) Targeted m6A deposition by the inducible m6A writing system on the targeted ACTB mRNA. M3 refers to the m6A writer, METTL3, and M3 refers to the mutant version of METTL3 without the catalytic function. The m6A level can only be elevated by the inducible m6A writing platform (dCas13b-PYL + ABI-M3 + gRNA) in the presence of ABA to a comparable level as the positive control (dCas13b-M3 + gRNA), but no increase can be observed without ABA or by using the catalytic inactive M3. Results are normalized to the group of dCas13b-PYL + sgRNA + ABA. D) Impact of installed m6A on the stability of target ACTB mRNA. The installed m6A on the ACTB mRNA by the inducible m6A writing platform destabilized the mRNA just like the reported dCas13b-M3 + sgRNA. Results are normalized to the group of dCas13b-PYL + ABI-M3 + NT-sgRNA + ABA. E) Targeted m6A level removal by the inducible m6A erasing system on the target Malat1 lncRNA by SELECT assay. The smaller Ct value reflects the higher amounts of full-length product of SELECT-PCR, indicating the lower enrichment of m6A on the target. ALK refers to the m6A eraser, ALKBH5, and ALK refers to the mutant version of ALKBH5 without the catalytic function. The m6A level can only be reduced by the inducible m6A erasing platform (dCas13b-PYL + ABI-ALK + gRNA) in the presence of ABA to a comparable level as the positive control (dCas13b-ALK + gRNA), but no increase can be observed without ABA or by using the catalytic inactive ALK. Results are normalized to the group of dCas13b-PYL + sgRNA + ABA. F) Recruitment of METTL3 on the target ACTB mRNA after ABA removal. The METTL3 enzyme of inducible m6A writing platform can be recruited to target site by the treatment of ABA, which will be released after the ABA removal by wash. Results are normalized to the group of dCas13b-PYL + sgRNA + ABA. Data analysis For each assay, three independent trials were performed to obtain data and no exclusion of data was performed. Data and statistics were analyzed with one-way ANOVA using GraphPad Prism 8.0. The p value lower than 0.05 was marked as , lower than 0.01 as , lower than 0.001 as , and lower than 0.0001 as ***. Notes RNA extraction and other RNA-related experiments should be performed on ice or at 4°C, unless specifically noted otherwise, to avoid RNA degradation, as RNA integrity is important. We suggest investigators to optimize the time of the fragmentation process in the m6A-IP by themselves, as the speed of taking the PCR tubes out of the thermo-cycler and addition of EDTA will impact the size of fragmented RNA. Usually, a size between 100 and 300 nt is optimal. For all transfection procedures mentioned in this protocol, mix the plasmid and Lipofectamine 2000 transfection reagent separately in OMEM, then combine and mix them according to the manufacturer’s protocol. For the amplification of the plasmids that we cloned, LB media and LB plates with ampicillin sodium salt are needed, because all plasmids have ampicillin resistance. Recipes LB plates with ampicillin Reagents Final concentration Amount LB broth 25 g/L 25 g Agar 15 g/L 15 g 100 mg/mL ampicillin sodium salt 100 μg/mL 1 mL ddH2O N/A 980 mL Total volume N/A 1,000 mL Dissolve 25 g of LB broth and 15 g of agar in 980 mL of ddH2O in a glass bottle of 1 L volume. Autoclave the LB broth liquid at 121°C for 15 min. Add 1 mL of 100 mg/mL ampicillin solution to the autoclaved liquid when it cools down to 30–40°C. Transfer 15 mL of the prepared solution to 10 cm Petri dishes using a serological pipette. Allow the agar plates to cool and dry in a laminar flow hood for 20 min. Store the plates at 4°C in a plastic bag. LB media Reagents Final concentration Amount LB broth 25 g/L 25 g ddH2O N/A 985 mL Total volume N/A 1,000 mL Dissolve 25 g of LB broth in 985 mL of ddH2O in a glass bottle of 1 L volume. Autoclave the LB broth liquid at 121°C for 15 min. Store the LB media at 4°C when it cools down. The shelf life for LB media is three months at 4°C. 1× TBST buffer Reagents Final concentration Amount Tris base 2.42 g/L 2.42 g NaCl 8 g/L 8 g Tween-20 0.1% 1 mL ddH2O N/A 995 mL Total volume N/A 1,000 mL Dissolve 2.42 g of Tris base and 8 g of NaCl in 995 mL of ddH2O in a glass bottle of 1 L volume. Add 1 mL of Tween-20 into the bottle and mix well. Store the buffer at room temperature before use. 1× membrane transfer buffer Reagents Final concentration Amount Glycine 1.44 g/L 1.44 g Tris base 3.03 g/L 3.03 g Methanol 20% 200 mL ddH2O N/A 800 mL Total volume N/A 1,000 mL Dissolve 1.44 g of glycine and 3.03 g of Tris base in 800 mL of ddH2O in a glass bottle of 1 L volume. Add 200 mL of methanol into the bottle and mix well. Store at 4°C before use. 1× gel running buffer Reagents Final concentration Amount Tris base 3.04 g/L 3.04 g Glycine 1.44 g/L 1.44 g SDS 1 g/L 1 g ddH2O N/A 995 mL Total volume N/A 1,000 mL Dissolve 1.44 g of glycine, 3.04 g of Tris base, and 1 g of SDS in 995 mL of ddH2O in a glass bottle of 1 L volume. Store the buffer at room temperature before use. Fragmentation buffer, 10× Reagents Final concentration Amount Tris-HCl, pH 7.0 (1 M) 100 mM 100 µL ZnCl2 (1 M) 100 mM 100 µL ddH2O N/A 800 µL Total volume N/A 1,000 µL Mix 800 µL of nuclease-free water with 100 µL (1 M stock) of Tris-HCl (pH 7.0) and 100 µL of ZnCl2 (1 M stock). Freshly prepare the buffer. 5× IP buffer for m6A-IP study Reagents Final concentration Amount Tric-HCl, pH 7.4 (1 M) 50 mM 0.5 mL NaCl (5 M) 750 mM 1.5 mL IGEPAL-CA-630 (10% vol/vol) 0.5% vol/vol 0.5 mL ddH2O N/A 10 mL Total volume N/A 10 mL Mix 0.5 mL of Tris-HCl (1 M, pH 7.4), 1.5 mL of NaCl (5 M), and 0.5 mL of IGEPAL-CA-630 (10% vol/vol) in a total volume of 10 mL nuclease-free water. Freshly prepare the buffer for use. m6A elution buffer Reagents Final concentration Amount 5× IP buffer 1× 90 µL 20 mM m6A salt 2.67 mM 60 µL RNasin Plus ribonuclease inhibitor N/A 7 µL Nuclease-free water N/A 293 µL Total volume N/A 450 µL Wash buffer for RNA-crosslink immunoprecipitation Reagents Final concentration Amount DPBS N/A 50 mL Tween-20 0.02% 10 μL NT-2 buffer, 5× Reagents Final concentration Amount Tric-HCl, pH 7.4 (1 M) 250 mM 12.5 mL MgCl2, 1 M 5 mM 0.25 mL NaCl (5 M) 750 mM 7.6 mL IGEPAL-CA-630 (10% vol/vol) 0.25% vol/vol 1.25 mL ddH2O N/A 50 mL Total volume N/A 50 mL Protease K digestion buffer Reagents Final concentration Amount Protease K N/A 5 µL SDS (10%) 1% 15 µL NT-2 buffer, 5× N/A 30 µL Nuclease-free water N/A 100 µL Total volume N/A 150 µL Acknowledgments This work was supported by National Institutes of Health (R21CA247638). We acknowledge our previous work “Inducible and reversible RNA N6-methyladenosine editing” published in Nature Communication. DOI: https://doi.org/10.1038/s41467-022-29665-y. Competing interests The authors declare no competing interests. References Boccaletto, P., Stefaniak, F., Ray, A., Cappannini, A., Mukherjee, S., Purta, E., Kurkowska, M., Shirvanizadeh, N., Destefanis, E. and Groza, P. (2022). MODOMICS: a database of RNA modification pathways. 2021 update. Nucleic Acids Res 50(D1): D231-D235. Chen, T., Gao, D., Zhang, R., Zeng, G., Yan, H., Lim, E. and Liang, F.-S. (2017). Chemically controlled epigenome editing through an inducible dCas9 system. J Am Chem Soc 139(33): 11337-11340. Licht, K. and Jantsch, M. F. (2016). Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications. J Cell Biol 213(1): 15-22. Liu, N. and Pan, T. (2016). N6-methyladenosine-encoded epitranscriptomics. Nat Struct Mol Biol 23(2): 98-102. Liu, X. M., Zhou, J., Mao, Y., Ji, Q. and Qian, S. B. (2019). Programmable RNA N(6)-methyladenosine editing by CRISPR-Cas9 conjugates. Nat Chem Biol 15(9): 865-871. Lo, N., Xu, X., Soares, F. and He, H. H. (2022). The Basis and Promise of Programmable RNA Editing and Modification. Front Genet 13: 834413. Ontiveros, R. J., Stoute, J. and Liu, K. F. (2019). The chemical diversity of RNA modifications. Biochem J 476(8): 1227-1245. Pickar-Oliver, A. and Gersbach, C. A. (2019). The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Biol 20(8): 490-507. Rauch, S., He, C. and Dickinson, B. C. (2018). Targeted m6A Reader Proteins To Study Epitranscriptomic Regulation of Single RNAs. J Am Chem Soc 140(38): 11974-11981. Shi, H., Xu, Y., Tian, N., Yang, M. and Liang, F.-S. (2022). Inducible and reversible RNA N6-methyladenosine editing. Nature Commun 13(1): 1958. Wang, X. and He, C. (2014). Dynamic RNA modifications in posttranscriptional regulation. Mol Cell 56(1): 5-12. Wilson, C., Chen, P. J., Miao, Z. and Liu, D. R. (2020). Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nat Biotechnol 38(12): 1431-1440. Xu, Y. and Liang, F.-S. (2022). On demand CRISPR-mediated RNA N6-methyladenosine editing. Genes Dis 9(6): 1389-1390. Yang, Y., Hsu, P. J., Chen, Y. S. and Yang, Y. G. (2018). Dynamic transcriptomic m(6)A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res 28(6): 616-624. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Cell Biology > Cell engineering > CRISPR-cas9 Molecular Biology > RNA > Epitranscriptome Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing Beverly Naigles [...] Nan Hao Mar 20, 2024 2172 Views Expansion and Precise CRISPR-Cas9 Gene Repair of Autologous T-Memory Stem Cells from Patients with T-Cell Immunodeficiencies Xun Li [...] Klaus Rajewsky Oct 20, 2024 520 Views Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1 Haiting Ma Nov 20, 2024 461 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,608	https://bio-protocol.org/en/bpdetail?id=4608&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates SR Sven Reichardt AS Annick Stintzi AS Andreas Schaller Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4608 Views: 465 Reviewed by: Giusy TornilloBilly Tasker-BrownSimab Kanwal Download PDF Ask a question Favorite Cited by Version history Bio-protocol journal peer-reviewed Feb 05, 2023 \| This version Preprint Mar 04, 2022 Original Research Article: The authors used this protocol in Science Mar 2020 Abstract Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor–processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage. Graphical abstract Keywords: Enzyme assay Nicotiana benthamiana Protein purification Phytaspase Protease Substrate specificity Synthetic peptide substrate Transient expression Background Phtytaspases are a subgroup of plant subtilases that are characterized by their specificity for aspartic acid immediately upstream of the cleavage site (in position P1) of their oligopeptide and protein substrates (Chichkova et al., 2014; Schaller et al., 2018). Phytaspases were originally characterized in tobacco and rice (Chichkova et al., 2010) and more recently in Arabidopsis and tomato (Beloshistov et al., 2018; Chichkova et al., 2018; Reichardt et al., 2018 and 2020). In addition to the canonical Asp residue at the scissile bond, several amino acids upstream of the critical Asp were found to contribute to substrate recognition, resulting in high selectivity of individual phytaspases for limited proteolysis at specific sites of their protein targets (Chichkova et al., 2018; Reichardt et al., 2018). Phytaspases in particular, and plant subtilases in general, are synthesized as pre-pro-proteins that are directed to the secretory pathway for proteolytic maturation, glycosylation, and secretion (Schaller et al., 2018). While there is a single report on the successful expression of phytaspase in E. coli (Narayanan et al., 2017), the complex maturation pattern of subtilases rather calls for an eukaryotic host for recombinant protein expression (Meyer et al., 2016). The baculovirus–insect cell system and stably transformed plant cell lines have been used to produce post-translationally modified and fully active plant subtilases (Janzik et al., 2000; Cedzich et al., 2009; Ottmann et al., 2009). Here, we used Nicotiana benthamiana plants for the expression of C-terminally His-tagged phytaspases by agroinfiltration. This transient expression system may not yield as much recombinant protein as insect and plant cell culture systems but is much more rapid and allows for simple extraction of the secreted subtilases from extracellular (apoplastic) wash fluids. Subsequent purification by metal-chelate affinity chromatography followed by gel filtration results in near homogeneity of the recombinant enzymes for in vitro cleavage assays. Cleavage assays include synthetic oligopeptides that comprise several precursor-derived amino acids upstream and/or downstream of the cleavage sites as protease substrates. In this experiment, we used a decamer peptide consisting of five amino acids of the precursor followed by the di-sulfated PSK pentapeptide as a substrate for tomato phytaspases. To demonstrate specificity of cleavage, we included a second peptide, in which Ala substituted the critical Asp residue at the cleavage site. Cleavage products were then identified and quantified by mass spectrometry (Reichardt et al., 2020). We provide a protocol for (A) the transient expression of His-tagged phytaspases by agroinfiltration of N. benthamiana plants, (B) the purification of the recombinant proteins from apoplastic leaf extracts, (C) the cleavage assay using synthetic peptide substrates, and (D) sample preparation for mass spectrometry (MS). Not included is a protocol for the cloning of the protease of interest. In the experiment described here, we used expression constructs for phytaspases from tomato that were generated by conventional cloning techniques in the binary vector pART27 and transformed into Agrobacterium tumefaciens strain C58C1 as described (Reichardt et al., 2018). A protocol for the mass spectrometric analysis of cleavage products is also not included, as this part of the analysis is usually either performed by a central facility of the respective institution or provided as a commercial service. For SDS-PAGE analysis, we followed standard procedures (Stintzi et al., 2022). The protocols we provide here can easily be adapted to other secreted proteases that tolerate the addition of a C-terminal His tag. Peptide sequences will have to be chosen according to the specific substrate requirements of the protease of interest. Materials and Reagents 50 mL culture tubes (Corning/Falcon, catalog number: 352070) 15 mL culture tubes (Corning/Falcon, catalog number: 352196) 1 mL PE/PP syringes (avantor/VWR, catalog number: 613-2001) 100 mL PE/PP syringes (Th.Geyer/Becton Dickinson, catalog number: 6287774) Oak Ridge High-Speed PPCO centrifuge tubes (Thermo Fisher Scientific/Nalgene, catalog number 3139-0030) 250 or 500 mL centrifuge bottles (Thermo Fisher Scientific/Nalgene, catalog number: 3141-0250 or 3141-0500) Scalpel or razor blades 17 or 18 gauge blunt-tipped syringe needle (e.g., B. Braun Sterican®, 18G / 1.2 × 40 mm, catalog number: 4038088) 300 or 500 mL Pyrex beakers Vivaspin 20 centrifugal concentrator (30 k molecular weight cut-off) (Sartorius Stedim, catalog number: VS2021) A. tumefaciens C58C1 (RifR, TetR) (community resource; NCBI:txid176299) Expression construct for the protease of interest under control of the CaMV 35S promoter in a binary vector for plant transformation. Here, we used expression constructs for His-tagged tomato phytaspases in pART27 (SpecR) transformed into A. tumefaciens, strain C58C1 (Reichardt et al., 2018). Agrobacteria transformed with the empty expression vector (here pART27) were used as control A. tumefaciens C58C1 with p19 silencing suppressor in pBin61 (KanR, RifR, TetR) (Voinnet et al., 2003) Nicotiana benthamiana seeds (Agroscience GmbH, Neustadt, Germany) N. benthamiana plants, grown on seeding substrate at 25°C and 16:8 h day/night cycle. Plants older than three weeks are watered with 1.48 g/L N (20%), P (20%), and K (20%) universal fertilizer including micronutrients. Note: Expression levels are much reduced in flowering plants. Therefore, use the plants before they start to bolt, usually when they are between four and five weeks old Spectinomycin (Duchefa, catalog number: S0188); 100 mg/mL stock solution in H2O, store at -20°C Rifampicin (Duchefa, catalog number: R0146); 100 mg/mL stock solution in DMSO, store at -20°C Tetracycline (Duchefa, catalog number: T0150); 25 mg/mL stock solution in 70 % (v/v) ethanol, store at -20°C Kanamycin sulphate (Duchefa, catalog number: K0126); 50 mg/mL stock solution in H2O, store at -20°C Tryptone (Duchefa, catalog number: T1332) Nickel (Ni)-NTA agarose (Qiagen, catalog number: 30210), store at 4°C Bio-Rad Protein Assay kit II, with bovine serum albumin as the standard protein (Bio-Rad, catalog number: 5000002) Coomassie protein stain (e.g., InstantBlue, abcam, catalog number: ISB1L) Custom-synthesized synthetic substrate peptides at >90% purity, EAHLD[sY]I[sY]TQM and EAHLA[sY]I[sY]TQM (sY = sulfotyrosine), (PepMic, Suzhou, China). The lyophilized peptides are stored at -20°C. Working solutions can be stored at 4°C for one month, or at -20°C. Avoid repeated freeze/thaw cycles MgCl2·6H2O (Carl Roth, catalog number: 3532.1) NaCl (Carl Roth, catalog number: 9265.2) KCl (Carl Roth, catalog number: 6781.1) NaH2PO4·2H2O (Carl Roth, catalog number: T879.2) Na2HPO4·2H2O (Carl Roth, catalog number: T877.1) NaOH (Carl Roth, catalog number: P031.3) Acetosyringone (Carl Roth, catalog number: 6003.2), store at -20°C Imidazole (Carl Roth, catalog number: 3899.4) 2-(N-Morpholino)-ethanesulfonic acid (MES·H2O) (Carl Roth, catalog number: 6066.2) Yeast extract (Carl Roth, catalog number: 2904.3) Glacial acetic acid (Carl Roth, catalog number: 7332.1) Glycerol (Carl Roth, catalog number: 4043.1) Acetonitrile (Carl Roth, catalog number: 7330.2) Trifluoroacetic acid (Carl Roth, catalog number: P088.1) H2O, HPLC grade (Carl Roth, catalog number: A511.2) LB medium (lysogeny broth) (see Recipes) Infiltration buffer (see Recipes) Extraction buffer, reaction buffer (see Recipes) Binding buffer (see Recipes) Elution buffer (see Recipes) Gel filtration buffer (see Recipes) Solvent A (see Recipes) Solvent B (see Recipes) Solvent C (see Recipes) Equipment Microcentrifuge (Eppendorf, model: 5418 R) Tabletop centrifuge with swing-out rotor for 15/50 mL tubes (Eppendorf, model: 5810 R) RC-3B refrigerated centrifuge (Sorvall Instruments) for 250/500 mL bottles RC6+ refrigerated centrifuge (Sorvall Instruments) for 30 mL tubes UV/Vis spectrophotometer (e.g., Eppendorf Biophotometer, catalog number: 634-0839) Desiccator (5 L) with vacuum pump Rotating wheel (e.g., Steinberg Systems, model: SBS-LBM-200) Microbiological incubators for plate cultures at 28 and 37°C Enrich SEC 650 10 × 300 gel filtration column (Bio-Rad, catalog number: 7801650) Fast protein liquid chromatography system. We used the NGC Quest system (Bio-Rad, catalog number: 788-0003) Note: Other chromatography systems, e.g., ÄKTA Pure (Cytiva, catalog number: 29018226) can be used as well. Vacuum concentrator, e.g., Savant SpeedVac (Thermo Fisher Scientific, catalog number: SPD1030A) Standard SDS-PAGE equipment (e.g., Mini-PROTEAN Tetra Cell and casting module from Bio-Rad, catalog numbers: 1658000EDU and 1658015EDU) Electrophoresis power supply (e.g., PowerPac Basic from Bio-Rad, catalog number: 1645050) PTFE (polytetrafluorethylene; TeflonTM) membranes with embedded C18 beads, e.g., Supelco EmporeTM solid phase extraction discs (Millipore Sigma/Supelco, catalog number: 66887U) Procedure Transient expression of phytaspases in N. benthamiana leaves Pick single colonies of A. tumefaciens C58C1 containing the phytaspase expression vector or the empty-vector control and resuspend them in 500 µL of LB medium. Streak the resuspended colonies evenly on LB plates with appropriate antibiotics (rifampicin, 100 µg/mL; tetracycline, 25 µg/mL; spectinomycin, 100 µg/mL). Note: The goal is to get a bacterial lawn. Similarly, streak a single colony of agrobacteria carrying the P19 expression construct resuspended in 500 µL of LB on LB plates containing kanamycin (50 µg/mL) instead of spectinomycin. Incubate for 48 h at 28°C. Use 6 mL of infiltration buffer (see Recipes) to wash off the bacteria from the respective plates and transfer the suspensions into 15 mL culture tubes. Collect cells by centrifugation for 10 min at 1,000 × g; discard supernatant. Resuspend cells in 6 mL of infiltration buffer each. Determine optical density (OD) at 600 nm. Mix the two suspensions in a 50 mL culture tube and dilute with infiltration buffer, to obtain 50 mL of infiltration solution with a final OD600 of 0.7 for the phytaspase expression construct and 1:0 for the P19 silencing suppressor. Do the same for the empty-vector control. Use a 1 mL plastic syringe without the needle to inject the suspensions into the abaxial side of leaves of N. benthamiana plants (Figure 1A, Video 1). Note: Support the leaf on the opposite site with your index finger. Use gentle pressure and be careful not to injure the leaves. Infiltrated areas are water-soaked; they become much darker than the remainder of the leaf. Try to infiltrate as much of the leaf area using as few infiltration sites as possible. Video 1. Agroinfiltration of N. benthamiana plants Continue to grow the plants at 25°C and 16:8 h day/night cycle. Harvest the leaves at five days after infiltration. Note: The optimum time for transient protein expression in N. benthamiana depends on the protein of interest and needs to be determined experimentally. If degradation of the expressed protease or tissue necrosis as a result of protease overexpression are observed, earlier time points may have to be chosen for maximum yield. Use a razor blade or scalpel to remove the central vein. Place the remaining leaf material, upper side down, into a beaker (one for the expression construct and one for the empty-vector control) containing approximately 100 mL of extraction buffer (see Recipes) on ice (Figure 1B). Place the beakers into a desiccator and vacuum infiltrate the extraction buffer at 75 mbar for 2 min; then, slowly release the vacuum. Blot the leaves dry (Figure 1C). Stack them on top of each other, roll them up (Figure 1D), and place them into the barrel of a 100 mL plastic syringe (Figure 1E). Figure 1. Transient protein expression in N. benthamiana leaves. A. Infiltration of agrobacterial suspension into the abaxial side of N. benthamiana leaves. B. Harvesting of leaves into extraction buffer. C. Leaves on blotting paper after vacuum infiltration. D, E. Leaves are piled one on top of the other and rolled up (D), in order to place them into the barrel of a 100 mL plastic syringe (E). (F) Diagram of a syringe barrel placed into a centrifuge bottle. Place the syringe barrel into a 250/500 mL centrifuge bottle (Figure 1E, F) and spin at 1,500 × g for 7 min at 4°C, to collect the intercellular fluid (apoplastic wash). Transfer the wash fluid to Oak Ridge centrifuge tubes and clear by centrifugation at 20,000 × g at 4°C. Transfer the supernatant to a new tube Add imidazole to a final concentration of 4 mM. Purification of phytaspases His-tagged phytaspases are purified from apoplastic washes by metal-chelate affinity chromatography on Ni-NTA agarose in a batch procedure, followed (optionally) by size exclusion chromatography on an Enrich SEC 650 10 × 300 gel filtration column. As a control, apoplastic washes from empty-vector-infiltrated plants are subjected to the same purification procedure. Pipette 500 µL of Ni-NTA agarose (50% slurry of agarose beads in storage buffer) into a 15 mL culture tube and spin for 2 min at 500 × g to sediment the matrix. Prepare two tubes, one for the recombinant protease and one for the mock purification from empty-vector-infiltrated plants. To wash the matrix, use a pipette to remove the buffer. Note: Be careful not to aspirate the settled matrix. Add 10 mL of binding buffer (see Recipes), mix gently and spin down, and remove the buffer as before. Repeat two times. Resuspend in 1 mL of binding buffer after the final wash. Add the apoplastic wash from step A19 to the matrix and incubate for 1 h at 4°C on a rotating wheel. Collect the matrix by centrifugation as above and wash three times with 12 mL of binding buffer, as described in step B2. Carefully remove the buffer after the last washing step. Add 600 µL of elution buffer (see Recipes), sediment by centrifugation, and recover the supernatant. Repeat twice. Combine the three 600 µL eluates and reduce the volume to approximately 100 µL by ultrafiltration using a Vivaspin centrifugal concentrator (30 k molecular weight cut-off) according to the manufacturer’s instructions. For buffer exchange, add 500 µL of elution buffer without imidazole. Reduce the volume to 100 µL as before (step B7). Repeat three times. Assess purity by SDS-PAGE and Coomassie-Brilliant Blue staining following standards protocols (Stintzi et al., 2022) (Figure 2A). Note: Many proteases that are expressed at high levels are sufficiently pure for further characterization at this stage (see for example P69A in Figure 2A). However, for applications that require the highest purity (e.g., the PICS assay for analysis of substrate specificity, as reported in Reichardt et al., 2018), we recommend further purification by gel filtration as detailed in the following steps B10–B14. Apply the sample to an Enrich SEC 650 10 × 300 gel filtration column (or similar), equilibrated in 50 mM sodium phosphate buffer pH 7.0, 300 mM NaCl, at 0.5 mL/min on an NGC Quest (Bio-Rad) chromatography system. Monitor UV absorbance at 280 nm; collect the column eluate in 200 µL fractions. Results are shown for tomato phytaspase 2 (Phyt2) in Figure 2B. For the highest purity, use only the fraction at the peak maximum in further experiments. Alternatively, pool all peak fractions, concentrate by ultrafiltration as above (step B7), add glycerol to 50% final concentration, and store at -20°C. Determine protein concentration using a commercial Bradford assay system (e.g., Bio-Rad Protein Assay kit II) following the manufacturer’s instructions. Figure 2. Purification of tomato phytaspases from apoplastic extracts of agroinfiltrated N. benthamiana plants. A. SDS-PAGE analysis of phytaspases purified from apoplastic extracts by metal-chelate affinity chromatography on Ni-NTA agarose. Approximately 2 (Phyt4), 5 (Phyt5), or 6 µg (Phyt1, Phyt2, P69A) of protein were loaded onto the gel. A Coomassie-stained 10% gel is shown; the molecular mass of marker proteins is indicated in kDa. B. Further purification of Phyt2 by gel permeation chromatography. The elution volume is shown in milliliters. Protein elution was monitored at 280 nm and is shown as m(illi) A(bsorbance) U(nits) in blue; 200 µL fractions were collected and assayed for Phyt2 activity using a fluorogenic peptide substrate. Activity is shown in arbitrary units in red, as relative fluorescence increase per minute. Modified from Reichardt et al. (2018), Figures 3a and 4a (Reichardt et al., 2018). In vitro cleavage assay for phytaspase specificity Activity and cleavage specificity of the purified protease of interest is tested with synthetic oligopeptides as substrates. Lyophilized custom-synthesized peptides can be obtained at >90% purity from commercial suppliers. The amino acid sequence has to be chosen to match the substrate requirements of the protease of interest. To analyze the cleavage specificity of tomato phytaspases, we used a decamer peptide comprising the five residues of PSK ([sY]I[sY]TQM; sY = sulfotyrosine) with five additional precursor-derived amino acids at the N-terminus (EAHLD[sY]I[sY]TQM) and a second peptide in which the critical Asp residue at the cleavage site was replaced by Ala (EAHLA[sY]I[sY]TQM). Resuspend the lyophilized peptide in ddH2O; determine the concentration spectrophotometrically at 260 nm, based on the molar extinction coefficient for two sulfotyrosine residues (ϵ260 = 566 M-1 cm-1). Set up the in vitro digest in 100 µL of reaction buffer (see Recipes). Prepare two tubes, one containing 140 nM of the purified phytaspase, and the second an equal volume of the mock-purified fraction. Note: Use high-quality microfuge tubes (e.g., the original Eppendorf tubes) and tips at this and subsequent steps of protocol C. Low quality tubes may leak plasticizer into the sample, which is detrimental to MS/MS analysis. Start the reaction by adding 5 µM substrate peptide and incubate at 30°C. Note: The peptide substrates were used in a 3,000-fold molar excess over the protease. However, this cannot be generalized. The quantity of protease in the assay and the required substrate concentration depend on the properties of the protease under study and have to be adjusted accordingly. Prepare six microfuge tubes, each containing 90 µL of 0.1% trifluoroacetic acid (TFA). Stop the reaction by taking 10 µL aliquots at 0 min, 10 min, 30 min, 1 h, 5 h, 24 h. Add aliquots to the tubes containing 90 µL of 0.1% TFA. Sample preparation for mass spectrometry Prior to mass spectrometry (nanoLC-ESI-MS/MS) analysis, samples need to be desalted and concentrated on C18 ZIP tips or StageTips (Rappsilber et al., 2003). In order to prepare StageTips, use a hypodermic needle to punch out small disks from PTFE membranes with embedded C18 beads, and place them into 200 µL (yellow) pipet tips. Use two discs per tip. Full details for StageTip preparation are given in Rappsilber et al. (2003). To condition the StageTips, add 50 µL of solvent A (see Recipes) and spin in a microfuge tube at 2,300 × g for 1 min at 4°C. Wash twice by adding 100 µL of solvent B (see Recipes) and spin as above. Apply the sample from step C5 and spin at 800 × g for 1 min at 4°C. Wash twice by adding 150 µL of solvent B and spin at 2,300 × g for 1 min at 4°C. Transfer the StageTips to new microfuge tubes and elute twice by adding 20 µL of solvent C (see Recipes); centrifuge at 800 × g for 1 min at 4°C. Dry samples in a vacuum concentrator and continue with nanoLC-ESI-MS/MS analysis or store at -20°C until further analysis. Data analysis Phytaspases are Asp-specific proteases. They cleave their substrate proteins on the carboxy side of aspartic acid residues. The peptide derived from the PSK precursor, EAHLD[sY]I[sY]TQM, that was tested here as a phytaspase substrate is thus expected to be cleaved between aspartate (D) and sulfotyrosine (sY). To confirm Asp-specificity of cleavage, we used a second peptide, in which the critical Asp residue was replaced by Ala. The Ala-substituted peptide, EAHLA[sY]I[sY]TQM, is expected not to be cleaved by phytaspases or to be much less efficiently cleaved as compared to the original peptide. Search the MS/MS data for any cleavage products generated from the two substrate peptides. As search parameters, do not specify a specific enzyme and set mass tolerance at 5 ppm for peptide precursors and 0.02 Da for fragment ions (Note: The mass tolerance settings may vary depending on the resolution and mass accuracy of the mass spectrometer used). The sulfotyrosine side chain is not very stable, resulting in the loss of sulfate, and methionine is frequently oxidized during MS analysis. Therefore, allow for methionine oxidation and tyrosine sulfation as variable modifications during the MS search. Scrutinize the search results for peptides diagnostic for cleavage at the D-sY bond. These are the N-terminal cleavage products EAHLA and EAHLD for the Ala-substituted and the original peptide, respectively, and the C-terminal cleavage product [sY]I[sY]TQM that is the same for both substrate peptides. Be aware that the C-terminal cleavage product contains two sulfotyrosine and one methionine residue, resulting in many possible variants. In our analysis of Phyt2 cleavage specificity, we therefore used the N-terminal peptide for quantification. We quantified the EAHLD and EAHLA cleavage products as the sum of ion intensities for the MS/MS fragment ions of the b- and y-series. Results shown by Reichardt et al. (2020) in Figure 4 and in Supplementary Figure S11 indicated that the PSK-derived peptide is cleaved more efficiently compared to its Ala-substituted variant. EAHLA-derived fragment ions amounted to 20% ± 8% of the corresponding EAHLD-derived ions, which is significantly different from the 1-to-1 ratio expected if both precursor ions were cleaved with equal efficiency. Recipes LB medium (lysogeny broth) Reagent Final concentration Amount Tryptone 10 g/L 10 g Yeast extract 5 g/L 5 g NaCl Agar (only for solid media) H2O 5 g/L 15 g/L n/a 5 g 15 g up to 1 L Autoclave for 20 min. Cool down to 60°C and then add antibiotics from 1,000× stock solutions. Use for liquid culture or pour plates if solid media are needed. Infiltration buffer Reagent Final concentration Amount MgCl2 (1 M) 10 mM 1 mL MES-NaOH (0.5 M, pH 5.6) 10 mM 2 mL Acetosyringone (100 mM) H2O 0.15 mM n/a 1.5 mL 95.5 mL Total n/a 100 mL Extraction buffer, reaction buffer Reagent Final concentration Amount NaH2PO4/Na2HPO4 (1 M, pH 6.5) 50 mM 10 mL KCl (1 M) H2O 200 mM n/a 40 mL 150 mL Total n/a 200 mL Binding buffer Reagent Final concentration Amount NaH2PO4/Na2HPO4 (1 M, pH 7.0) 50 mM 10 mL KCl (1 M) Imidazole (1 M) H2O 200 mM 4 mM n/a 40 mL 0.8 mL 149.2 mL Total n/a 200 mL Elution buffer Reagent Final concentration Amount NaH2PO4/Na2HPO4 (1 M, pH 7.0) 50 mM 5 mL KCl (1 M) Imidazole (1 M) H2O 200 mM 200 mM n/a 20 mL 20 mL 55 mL Total n/a 100 mL Gel filtration buffer Reagent Final concentration Amount NaH2PO4/Na2HPO4 (1 M, pH 7.0) 50 mM 50 mL NaCl (1 M) 300 mM 300 mL H2O Total n/a n/a 650 mL 1,000 mL Solvent A Reagent Final concentration Amount Glacial acetic acid 0.5% (v/v) 0.5 mL Acetonitrile 80% (v/v) 80 mL H2O, HPLC-grade Total n/a n/a 19.5 mL 100 mL Solvent B Reagent Final concentration Amount Glacial acetic acid 0.5% (v/v) 0.5 mL H2O, HPLC-grade Total n/a n/a 99.5 mL 100 mL Solvent C Reagent Final concentration Amount Glacial acetic acid 0.5% (v/v) 0.5 mL Acetonitrile 50% (v/v) 50 mL H2O, HPLC-grade Total n/a n/a 49.5 mL 100 mL Acknowledgments The original research papers in which these procedures were described (Reichardt et al., 2018 and 2020); Research in our laboratory is supported by the German Research Foundation (DFG). Competing interests No financial or non-financial competing interests are declared. References Beloshistov, R. E., Dreizler, K., Galiullina, R. A., Tuzhikov, A. I., Serebryakova, M. V., Reichardt, S., Shaw, J., Taliansky, M. E., Pfannstiel, J., Chichkova, N. V., et al. (2018). Phytaspase-mediated precursor processing and maturation of the wound hormone systemin. New Phytol 218(3): 1167-1178. Cedzich, A., Huttenlocher, F., Kuhn, B. M., Pfannstiel, J., Gabler, L., Stintzi, A. and Schaller, A. (2009). The protease-associated (PA) domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3). J Biol Chem 284(21): 14068-14078. Chichkova, N. V., Galiullina, R. A., Beloshistov, R. E., Balakireva, A. V. and Vartapetian, A. B. (2014). Phytaspases: Aspartate-specific proteases involved in plant cell death. Russ J Bioorg Chem 40(6): 606-611. Chichkova, N. V., Galiullina, R. A., Mochalova, L. V., Trusova, S. V., Sobri, Z. M., Gallois, P. and Vartapetian, A. B. (2018). Arabidopsis thaliana phytaspase: identification and peculiar properties. Funct Plant Biol 45(2): 171-179. Chichkova, N. V., Shaw, J., Galiullina, R. A., Drury, G. E., Tuzhikov, A. I., Kim, S. H., Kalkum, M., Hong, T. B., Gorshkova, E. N., Torrance, L., et al. (2010). Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity. EMBO J 29(6): 1149-1161. Janzik, I., Macheroux, P., Amrhein, N. and Schaller, A. (2000). LeSBT1, a subtilase from tomato plants. Overexpression in insect cells, purification and characterization. J Biol Chem 275(7): 5193-5199. Meyer, M., Leptihn, S., Welz, M. and Schaller, A. (2016). Functional characterization of propeptides in plant subtilases as intramolecular chaperones and inhibitors of the mature protease. J Biol Chem 291(37): 19449-19461. Narayanan, S., Sanpui, P., Sahoo, L. and Ghosh, S. S. (2017). Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease. Int J Biol Macromol 95: 288-293. Ottmann, C., Rose, R., Huttenlocher, F., Cedzich, A., Hauske, P., Kaiser, M., Huber, R. and Schaller, A. (2009). Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3. Proc Natl Acad Sci U S A 106(40): 17223-17228. Rappsilber, J., Ishihama, Y. and Mann, M. (2003). Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75(3): 663-670. Reichardt, S., Piepho, H.-P., Stintzi, A. and Schaller, A. (2020). Peptide signaling for drought-induced tomato flower drop. Science 367(6485): 1482-1485. Reichardt, S., Repper, D., Tuzhikov, A. I., Galiullina, R. A., Planas-Marques, M., Chichkova, N. V., Vartapetian, A. B., Stintzi, A. and Schaller, A. (2018). The tomato subtilase family includes several cell death-related proteinases with caspase specificity. Sci Rep 8(1): 10531. Schaller, A., Stintzi, A., Rivas, S., Serrano, I., Chichkova, N. V., Vartapetian, A. B., Martínez, D., Guiamét, J. J., Sueldo, D. J., van der Hoorn, R. A. L., et al. (2018). From structure to function – a family portrait of plant subtilases. New Phytol 218(3): 901-915. Stintzi, A., Stührwohldt, N., Royek, S., Schaller, A. (2022). Identification of cognate protease/substrate pairs by use of class-specific inhibitors. In: Klemenčič, M., Stael, S., Huesgen, P. F. (Eds.). Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_6. Voinnet, O., Rivas, S., Mestre, P. and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5): 949-956. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Plant Science > Plant biochemistry > Protein Biochemistry > Protein > Synthesis Biochemistry > Protein > Isolation and purification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols An in vitro Assay to Probe the Formation of Biomolecular Condensates Yu Zhang and Shen Lisha Sep 5, 2023 1442 Views Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome Dione Gentry-Torfer [...] Federico Martinez-Seidel May 5, 2024 700 Views Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms Ana P. Lando [...] Giselle M. A. Martínez-Noël Dec 20, 2024 315 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,609	https://bio-protocol.org/en/bpdetail?id=4609&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Unscheduled DNA Synthesis at Sites of Local UV-induced DNA Damage to Quantify Global Genome Nucleotide Excision Repair Activity in Human Cells PM Paula J. van der Meer DH Diana van den Heuvel ML Martijn S. Luijsterburg Published: Vol 13, Iss 3, Feb 5, 2023 DOI: 10.21769/BioProtoc.4609 Views: 705 Reviewed by: Alak Manna Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Cell Biology Jun 2021 Abstract Nucleotide excision repair (NER) removes a wide variety of structurally unrelated lesions from the genome, including UV-induced photolesions such as 6–4 pyrimidine–pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). NER removes lesions by excising a short stretch of single-stranded DNA containing the damaged DNA, leaving a single-stranded gap that is resynthesized in a process called unscheduled DNA synthesis (UDS). Measuring UDS after UV irradiation in non-dividing cells provides a measure of the overall NER activity, of which approximately 90% is carried out by the global genome repair (GGR) sub pathway. Here, we present a protocol for the microscopy-based analysis and quantification of UDS as a measurement for GGR activity. Following local UV-C irradiation, serum-starved human cells are supplemented with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into repair patches following NER-dependent dual incision. The incorporated nucleotide analogue is coupled to a fluorophore using Click-iT chemistry, followed by immunodetection of CPD photolesions to simultaneously visualize both signals by fluorescence microscopy. Accompanying this protocol is a custom-built ImageJ plug-in to analyze and quantify UDS signals at sites of CPD-marked local damage. The local UDS assay enables an effective and sensitive fluorescence-based quantification of GGR activity in single cells with application in basic research to better understand the regulatory mechanism in NER, as well as in diagnostics to characterize fibroblasts from individuals with NER-deficiency disorder. Graphical abstract Keywords: Nucleotide excision repair DNA damage DNA repair Unscheduled DNA synthesis Background Cells are continuously exposed to different sources of DNA damage. The nucleotide excision repair (NER) pathway comprises two sub pathways, transcription-coupled repair (TCR) and global genome repair (GGR), which remove helix-distorting lesions including UV-induced photoproducts from genomic DNA. While TCR preferentially removes DNA lesions from the transcribed strand of active genes (van den Heuvel et al., 2021), GGR removes lesions throughout the genome and is the dominant contributor to overall cellular NER activity (Marteijn et al., 2014). Both pathways funnel into a common molecular mechanism involving the same set of core NER proteins that mediate double incision by endonucleases ERCC1-XPF and XPG, resulting in the removal of a 30-nucleotide stretch of single-stranded DNA containing the lesion (van Toorn et al., 2022). After dual incision, the resulting gap is filled by DNA polymerases and sealed by DNA ligases (Ogi et al., 2010). DNA repair–associated DNA synthesis is distinct from synthesis during the S-phase to duplicate the genome, and is therefore referred to as unscheduled DNA synthesis (UDS) (Lehmann and Stevens, 1980). Irradiation with UV-C light generates two main types of genomic lesions: 6–4 pyrimidine–pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). The 6-4PPs are repaired considerably faster (within ∼5 h) than the CPDs, which are still present 24 h after UV irradiation (van Hoffen et al., 1995; Luijsterburg et al., 2010; Verbruggen et al., 2014). UDS after UV irradiation can be directly measured via nucleotide incorporation in non-dividing cells and mainly reflects GGR activity, which is responsible for approximately 90% of the overall cellular NER activity (Limsirichaikul et al., 2009). Although radioactive nucleotide analogues were initially used to measure UDS (Cleaver, 1968; Friedberg, 2004), the use of alkyne-linked thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) is more common nowadays (Limsirichaikul et al., 2009). The incorporated EdU can be visualized by its coupling to a fluorescent azide via Click-iT chemistry (Salic and Mitchison, 2008). Measuring UDS can offer insight in the potential involvement of GGR regulators or assess the functionality of mutated NER proteins, as recently reported for chromatin remodeler ALC1 or the ERCC1R156W variant found in patients with liver and kidney dysfunction, respectively (Blessing et al., 2022;Apelt et al., 2021). In this protocol, we describe a straightforward and accessible method for measuring UDS at sites of local UV irradiation. In brief, cells are exposed to local UV-C irradiation through a micropore filter, causing DNA damage induction and NER-dependent UDS in sub-nuclear regions. The UDS signal at sites of local UV damage can be captured by pulse-labeling with EdU within the first few hours after UV irradiation, followed by fixation and visualization of the incorporated EdU by fluorescent Click-iT chemistry. NER-dependent DNA synthesis mainly reflects rapid 6-4PP repair within the first hours after UV irradiation, during which CPD repair is negligible (van Hoffen et al., 1995; Luijsterburg et al., 2010; Verbruggen et al., 2014). The simultaneous immunodetection of CPD photolesions, which are largely unrepaired within the first hours after UV irradiation, can be used to define sites of local damage. The use of local UV irradiation enables a per-cell correction for unspecific EdU incorporation and accurate exclusion of replicating cells with scheduled EdU incorporation throughout the nucleus. Moreover, we provide a custom-built ImageJ plug-in to quantify UDS signals and apply a correction for potential differences in DNA damage induction between experimental conditions based on the CPD signal. We show that local UDS at sites of UV-induced DNA damage is robustly detected in RPE1-hTERT human cells, while the UDS signal is nearly abolished in an isogenic knockout cell-line for essential NER geneXPG. Materials and Reagents 12-well cell culture plate (Sigma, catalog number: CLS3512) 18 mm cover glasses, round (VWR, catalog number: 631-1580) 5 µm polycarbonate filters (Millipore, catalog number: TMTP04700) Parafilm (Sigma, catalog number: P7543) Microscope slides (VWR, catalog number: 12362098) DMEM, high glucose, GlutaMAXTM supplement, pyruvate (Gibco, catalog number: 31966-047) Fetal bovine serum (FBS) (Capricorn Scientific, catalog number: FBS-12A) 5-Ethynyl-2'-deoxyuridine (5-EdU) (Jena Bioscience, catalog number: CLK-N001-100); 50 mM stock in PBS [100 mg in 7.9 mL; to dissolve put in water bath (70°C) for 1 min (until dissolved); aliquot and store at -20°C] 5-Fluoro-2’-deoxyuridine (FuDR) (Sigma, catalog number: F0503); 10 mM stock in MilliQ water (MQ) [100 mg in 40.6 mL; aliquot and store at -20°C] Thymidine (Sigma, catalog number: T1895); 100 mM stock in MQ [1 g in 41.3 mL; aliquot and store at -20°C] Formaldehyde 37% (Sigma, catalog number: 252549) Triton-X100 (Sigma, catalog number: X100) Bovine serum albumin fraction V (BSA) (Sigma, catalog number: 10735086001) Tris (Sigma, catalog number: 10708976001) Atto azide Alexa 647 (Atto Tec, catalog number: 647N-101); 6 mM stock in DMSO [1 mg in 173 µL; aliquot and store at -20°C] CuSO4 (VWR, catalog number: 23174.233); 40 mM stock in MQ [store at room temperature (RT)] Ascorbic acid (Sigma, catalog number: A0278); 20 mg/mL stock in MQ [prepare fresh before use] Glycine (Sigma, catalog number: G7126) NaOH (Sigma, catalog number: 567530) Tween20 (Sigma, catalog number: P1379) CPD antibody (Cosmo Bio, CAC-NM-DND-001) Goat anti-mouse Alexa 555 (Thermo Fisher Scientific, catalog number: A-21424) DAPI (Thermo Fisher Scientific, catalog number: D1306) Aqua poly/mount (Brunschwig, catalog number: 18606) Sterile PBS (Thermo Fisher Scientific, catalog number: 10010023) Click reaction mix (see Recipes) Wash buffer (WB) (see Recipes) Equipment Tweezers (stainless steel forceps curved, Sigma, Z168785) TUV PL-S 9 W UV-C lamp (Philips) International Light NIST traceable radiometer photometer (Model IL1400BL SEL240/NS254/W) Fluorescence microscope (Zeiss Axio Imager M2) Software Imaging software: Zen (2012 Blue edition) Data analyses software: ImageJ (version 1.48), with the following requirements: ImageJ Plugin “bioformats_package.jar” at https://www.openmicroscopy.org/bio-formats/downloads In-house generated ImageJ macro “GGR-UDS_DataExtraction_ImageJ.txt” available at https://git.lumc.nl/dvandenheuvel/ggr-uds-protocol_lumc_luijsterburglab In-house generated data quantification file GGR-UDS_analyses_template.xls available at https://git.lumc.nl/dvandenheuvel/ggr-uds-protocol_lumc_luijsterburglab Procedure Cell culture (Day 0) For each condition, seed 100,000 RPE1 cells per well on 18 mm sterile glass coverslips in a 12-well plate in DMEM supplemented with 10% FBS. Let the cells grow to confluency for two days. Cell culture (Day 2) Replace the medium with DMEM without FBS to remove the available thymidine and to minimize the number of dividing cells. Incubate for 24 h. Local UV-C treatment, EdU incorporation, and fixation (Day 3) Prepare a new 12-well plate with 1 mL of DMEM (without FBS), supplemented with 20 µM EdU and 1 µM FuDR (FuDR inhibits thymidine synthesis and maximizes EdU incorporation). Wash the cells with PBS. Rinse the 5 µm polycarbonate filters in PBS. Cover a tray (e.g., a 15 cm plate) with parafilm and transfer the coverslips (cells facing up) to the parafilm. Make sure different conditions stay separated. Drain excess PBS from the 5 µm filters and carefully place the filter on top of the coverslips. Duplicates or triplicates can be combined per filter; make sure to not move the filter after placing. Irradiate the cells through the filter with 30 J/m2. Note: To prevent drying of the coverslips, irradiate up to three covered sets of coverslips per run and keep the irradiation time as short as possible by reducing the distance to the lamp. Our UV lamp = 0.33 J/m2/s = 91 s for 30 J/m2. Carefully add PBS on top of the filter to slightly lift it from the coverslips and remove the filter with tweezers. Drain the excess PBS from the coverslips, place the coverslips in the prepared 12-well plate with 20 µM EdU and 1 µM FuDR, and incubate for 1 h at 37°C. Note: A longer incubation up to 4 h may be helpful to assess potential residual repair activity in (partially) NER-deficient cells (Apelt et al., 2021). Wash the cells with DMEM (without FBS) and incubate with DMEM (without FBS) supplemented with 10 µM thymidine for 15 minutes at 37°C, to dilute the non-incorporated EdU. Wash with PBS and fix the cells in 3.7% formaldehyde (diluted in PBS) for 15 min at RT. Wash the coverslips twice with PBS and store in PBS at 4°C until click reaction and staining. Note: Coverslips are stable at 4°C. However, continuation with fluorescent EdU labeling and CPD staining is recommended within approximately one week. Fluorescent labeling of incorporated EdU by Click-iT chemistry (preferably within one week after fixation in step C) Remove PBS from the coverslips. Permeabilize the cells with 0.5% Triton-X100 in PBS for 20 min at RT. Wash twice for 5 min with 3% BSA in PBS. Wash once with PBS. Prepare 100 µL of click reaction mix per coverslip (see Recipes). Place 100 µL droplets of the click reaction mix on parafilm, place the coverslips (cells facing down) on the droplets, and incubate for 30 min at RT in the dark. Place the coverslips back in the 12-well plate (cells facing up). Wash with PBS. Keep the coverslips in the dark during the subsequent steps of the protocol. Note: Continue immediately with antibody-mediated labeling of remaining CPDs to detect sites of local damage. Antibody-mediated detection of remaining CPDs Fix cells again in 2% formaldehyde in PBS for 10 min at RT to prevent any loss of Click-iT signal. Wash twice with PBS. Block unreacted aldehydes with 100 mM glycine in PBS for 10 min at RT. Wash with PBS. Denature the DNA with 0.5 M NaOH in PBS for 5 min at RT (the CPD antibody only recognizes CPDs in a single-stranded DNA configuration). Wash three times with PBS. Block with 10% BSA in PBS for 15 min at RT. Wash with PBS. Equilibrate with WB (see Recipes). Place the coverslips on parafilm (cells facing up), add 100 µL per coverslip of the primary CPD antibody (diluted 1/1,000 in WB), and incubate for 2 h at RT or overnight at 4°C. Remove the antibody and wash the coverslips while on parafilm four times for 5 min with WB. Add 100 µL per coverslip of the secondary antibody (goat anti-mouse Alexa 555) diluted 1/1,000 in WB and incubate for 1 h at RT. Wash once for 5 min in WB. Add 100 µL per coverslip of DAPI diluted to 1 ug/mL (typically 1/1,000) in WB and incubate for 5 min at RT. Wash twice for 5 min with PBS. Mount the coverslips (cells facing down) in aqua poly/mount on glass microscopy slides marked for each condition. Let dry horizontally overnight at RT. Image the slides with a fluorescence microscope using 63× magnification and store images in Carl Zeiss Image Data Format (.czi files). Data analysis For efficient data extraction from the obtained .czi image files, we developed a custom ImageJ macro that can be combined with downstream data selection in Excel. The related macro-script and Excel template are available on https://git.lumc.nl/dvandenheuvel/ggr-uds-protocol_lumc_luijsterburglab . In brief, ImageJ is used to quantify the signal of the CPD staining and to define nuclei with damaged areas. Subsequently, these nuclei are separated into undamaged and damaged regions of interest (ROIs) and CPD and EdU signals are quantified in both regions. In Excel, cells with similar sizes of damaged areas and levels of CPD signal in their damaged regions are selected to prevent signal bias by large differences in damage induction (Figure 1a, b). Cells undergoing replication, and therefore scheduled DNA synthesis, are excluded from downstream analyses based on the mean EdU signal and its standard deviation in the undamaged region of the nucleus (Figure 1c). Early stages of replication usually start with foci of EdU signal throughout the nucleus, which continues into pan-nuclear EdU signal in later stages of replication. Cells in late-stage replication are excluded based on high EdU signal in undamaged regions of their nuclei. Even though cells in early stages of replication might still have a low mean EdU signal in undamaged regions of their nucleus, the standard deviation of this EdU signal is high, still enabling their exclusion from downstream analyses. After cell selection, for each nucleus, the EdU signal in the damaged region is corrected for its corresponding background signal in the undamaged region. These background-subtracted EdU intensities are subsequently normalized to their corresponding CPD signal and calculated relative to one condition [usually the wild-type (WT) condition] to allow combining multiple experiments. A detailed data extraction and analyses protocol is provided below. Extracting the CPD and EdU intensities in damaged and undamaged regions of nuclei CPD and EdU intensities in damaged and undamaged regions of nuclei can be easily extracted using the custom ImageJ macro available at https://git.lumc.nl/dvandenheuvel/ggr-uds-protocol_lumc_luijsterburglab . Combine all .czi files of an experiment in a separate folder (make sure no other files or folders are present) Run the custom ImageJ macro “GGR-UDS_DataExtraction_ImageJ.txt” to quantify the CPD signal and define damaged and undamaged regions in the nuclei. Click Plugins>Macros>Run… and select the location of the macro. When asked, select the folder where the .czi files are stored. Open one of the pictures so the macro can extract file details. In the dialog popping up, fill in the following details: Total number of channels included per picture. Number of the channel in which DAPI is detected (usually channel 1). Number of the channel you want to quantify (CPD channel). Number of the channel in which you detected the CPD signal for damaged area detection. Cut-off values for nucleus diameter (values depend on image capture settings; adjust by trial and error). Preferred minimum size of the damaged area to be included (put to 0 to include all and manually select later). Select one of the built-in thresholding methods of ImageJ for defining the damaged areas (usually either the “Yen” or “Li” thresholding method from ImageJ works well, but some other options are available; optimize by trial and error). Select one of the built-in thresholding methods of ImageJ for defining the nuclei (usually either the “Yen” or “Li” thresholding method from ImageJ also works well, but some other options are available; optimize by trial and error). Click “yes” to analyze all pictures in the folder. Important: Running the macro in step 2 generates a new data folder at the location of the pictures, containing the results. Move this generated data folder to another location of choice, away from the pictures. Again, run the custom ImageJ macro “GGR-UDS_DataExtraction_ImageJ.txt,” this time to quantify the EdU signal. Use the same settings as in step 2, except for the channel to measure in step 2.d.iii. Again, move the generated data folder to a location of choice. The data folders extracted from ImageJ contain three types of output. First, the data folder contains a log file with your submitted macro input. Second, it contains a zip-file per picture with the ROIs of all selected nuclei and damage areas, which can be opened in ImageJ. Third, it contains a results file with quantifications that can be opened in Excel. The quantifications are grouped per damaged nucleus, with data of damaged and undamaged ROIs within the same nucleus in two consecutive rows. Data selection and normalization Downstream data selection and normalization can be performed using the Excel template available at https://git.lumc.nl/dvandenheuvel/ggr-uds-protocol_lumc_luijsterburglab . This template contains predefined calculations and graphs and therefore is partially protected from editing. Fill in all the cells in yellow, also detailed below. Copy to columns C–F the extracted data for the EdU channel. Copy to column G the extracted data for the CPD channel. Note: Make sure to have the same row order in both CPD and EdU files, which is automatically correct when the same pictures are analyzed with the exact same settings. The area column for both the CPD and EdU data should be the same and can be used to check the files if needed. To be able to calculate with the data, give all cells per condition a number in column H. Note: If you want to regroup data rows in the file, always make sure to keep the consecutive rows of damaged and undamaged ROIs per nuclei together. To compare duplicates within an experiment, give all cells per duplicate a number in column I. Fill in the condition numbers and names in columns Y and Z. Use the basic graphs in the gray frame to include or exclude individual cells (seeFigure 1). Define minimum and maximum sizes of damage areas in cells AK4 and AL4. Note: This can be used to exclude very small random CPD speckles or large CPD positive regions that are larger than the pore size of the filter. Define the maximum intensity and standard deviation that is allowed for undamaged background ROIs in cells AM4 and AN4. Note: Nonreplicating cells show low EdU intensity and standard deviation in their background ROI; early-stage replicating cells show low EdU intensity but high standard deviation in their background ROI; late-stage replicating cells show high EdU intensity but low standard deviation in their background ROI. Define the minimum and maximum CPD intensities in damaged ROIs in cells AO4 and AP4. Note: Select cells with equal CPD intensities throughout the conditions to prevent EdU signal bias by biased damage induction. Figure 1. Defining selection criteria for cell inclusion. (a) CPD intensity plotted against area size of both damaged (small area and high CPD signal) and undamaged regions (large area and low CPD signal) of the nuclei, quantified in ImageJ. This plot can be used to define settings for minimum and maximum sizes of damaged regions to include in downstream analyses, indicated in the blue square. (b) CPD intensity in damaged regions separated per condition. This plot can be used to define settings for minimum and maximum CPD intensity in damaged regions to include in downstream analyses, indicated in the blue square. (c) EdU intensity plotted against standard deviation of all undamaged areas. This plot can be used to select nonreplicating cells having low EdU signal and standard deviation in undamaged regions, indicated in the blue square. The inset figures show microscopy examples of EdU signal in cells at different stages of replication. The Excel file provides the data of background-subtracted and normalized CPD/EdU intensities of individual cells in columns S–V. Overview plots of non-normalized CPD/EdU intensities can be found in the green/orange frames. Overview plots of EdU intensities after normalization to CPD intensity and conditions can be found in the blue frame. To generate violin-dotplot figures from the individual normalized datapoints, we suggest PlotsOfData, a simple freely available online tool (Postma and Goedhart, 2019). As an example of representative results from this protocol, we provide local UDS data of RPE1-hTERT human cells and an isogenic knockout (KO) cell-line for essential NER gene XPG . The parental WT cells show a strong UDS signal at sites of local damage, which is nearly abolished in XPG -KO cells (Figure 2a), as quantified in a violin-dotplot in Figure 2b. Figure 2. UDS results. (a) Representative immunofluorescence images generated with the unscheduled DNA synthesis protocol in RPE1-hTERT wild-type (WT) or XPG-KO cells. Scalebar represents 10 µm. (b) Quantification of the EdU levels in the damaged areas of the cells. Each datapoint represents the UDS signal from a single cell from three independent replicates. The mean of each biological replicate is depicted as a colored point with black line, while the bar represents the mean of all data points. Plots were generated using plots of data. Recipes Click reaction mix (prepare 100 µL per coverslip, within 15 min before use) Reagent Final concentration Amount Tris (50 mM pH 8) ~50 mM 79 µL Atto azide Alexa 647 (6 mM) 60 µM 1 µL CuSO4 (40 mM) 4 mM 10 µL Ascorbic acid (20 mg/mL)* 2 mg/mL 10 µL Total n/a 100 µL * Prepare stock ascorbic acid fresh before use Wash buffer (WB) (for 50 mL) Reagent Final concentration Amount PBS 1× 50 mL BSA 0.5% 0.25 g Tween 20 0.05% 25 μL Total n/a 50 mL Acknowledgments This research was supported by an LUMC research fellowship, an ALW-VIDI (ALW.016.161.320) grant from the Dutch Research Council (NWO), and an ERC consolidator grant (101001144) to MSL. Competing interests There are no conflicts of interest or competing interests. References Apelt, K., White, S. M., Kim, H. S., Yeo, J. E., Kragten, A., Wondergem, A. P., Rooimans, M. A., Gonzalez-Prieto, R., Wiegant, W. W., Lunke, S., et al. (2021). ERCC1 mutations impede DNA damage repair and cause liver and kidney dysfunction in patients. J Exp Med 218(3). Blessing, C., Apelt, K., van den Heuvel, D., Gonzalez-Leal, C., Rother, M. B., van der Woude, M., Gonzalez-Prieto, R., Yifrach, A., Parnas, A., Shah, R. G., et al. (2022). XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair. Nat Commun 13(1): 4762. Cleaver, J. E. (1968). Defective repair replication of DNA in xeroderma pigmentosum. Nature 218(5142): 652-656. Friedberg, E. C. (2004). The discovery that xeroderma pigmentosum (XP) results from defective nucleotide excision repair. DNA Repair (Amst) 3(2): 183, 195. Lehmann, A. R. and Stevens, S. (1980). A rapid procedure for measurement of DNA repair in human fibroblasts and for complementation analysis of xeroderma pigmentosum cells. Mutat Res 69(1): 177-190. Limsirichaikul, S., Niimi, A., Fawcett, H., Lehmann, A., Yamashita, S. and Ogi, T. (2009). A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU). Nucleic Acids Res 37(4): e31. Luijsterburg, M. S., von Bornstaedt, G., Gourdin, A. M., Politi, A. Z., Mone, M. J., Warmerdam, D. O., Goedhart, J., Vermeulen, W., van Driel, R. and Hofer, T. (2010). Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair. J Cell Biol 189(3): 445-463. Marteijn, J. A., Lans, H., Vermeulen, W. and Hoeijmakers, J. H. (2014). Understanding nucleotide excision repair and its roles in cancer and ageing. Nat Rev Mol Cell Biol 15(7): 465-481. Ogi, T., Limsirichaikul, S., Overmeer, R. M., Volker, M., Takenaka, K., Cloney, R., Nakazawa, Y., Niimi, A., Miki, Y., Jaspers, N. G., Mullenders, L. H., Yamashita, S., Fousteri, M. I. and Lehmann, A. R. (2010). Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells. Mol Cell 37(5): 714-727. Postma, M. and Goedhart, J. (2019). PlotsOfData-A web app for visualizing data together with their summaries. PLoS Biol 17(3): e3000202. Salic, A. and Mitchison, T. J. (2008). A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105(7): 2415-2420. van den Heuvel, D., van der Weegen, Y., Boer, D. E. C., Ogi, T. and Luijsterburg, M. S. (2021). Transcription-Coupled DNA Repair: From Mechanism to Human Disorder. Trends Cell Biol 31(5): 359-371. van Hoffen, A., Venema, J., Meschini, R., van Zeeland, A. A. and Mullenders, L. H. (1995). Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts. EMBO J 14(2): 360-367. van Toorn, M., Turkyilmaz, Y., Han, S., Zhou, D., Kim, H. S., Salas-Armenteros, I., Kim, M., Akita, M., Wienholz, F., Raams, A., et al. (2022). Active DNA damage eviction by HLTF stimulates nucleotide excision repair. Mol Cell 82(7): 1343-1358 e1348. Verbruggen, P., Heinemann, T., Manders, E., von Bornstaedt, G., van Driel, R. and Hofer, T. (2014). Robustness of DNA repair through collective rate control. PLoS Comput Biol 10(1): e1003438. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Molecular Biology > DNA > DNA damage and repair Cell Biology > Cell imaging > Fluorescence Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy Stephan Tetenborg [...] John O`Brien Jul 20, 2024 341 Views Calibrating Fluorescence Microscopy With 3D-Speckler (3D Fluorescence Speckle Analyzer) Chieh-Chang Lin and Aussie Suzuki Aug 20, 2024 404 Views Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca2+ Imaging Sergei G. Gaidin [...] Sultan T. Tuleukhanov Feb 5, 2025 46 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,610	https://bio-protocol.org/en/bpdetail?id=4610&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource This is an update notice. See the updated protocol. Peer-reviewed Update Notice: Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis Alexandra R Crowe Wei Yue Published: Jan 20, 2023 DOI: 10.21769/BioProtoc.4610 Views: 1440 Download PDF Ask a question Favorite Cited by After official publication in Bio-protocol (https://bio-protocol.org/e3465), we made some improvements to our protocol. All changes are highlighted in blue in the updated version linked here. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,611	https://bio-protocol.org/en/bpdetail?id=4611&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Imaging of Chloroplast Movement Responses to Light Stimulation in Different Intensities in Rice HW Hui Wu * ZW Ziyi Wang * YY Yanchun Yu YZ Yanli Zhang (*contributed equally to this work) Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4611 Views: 557 Reviewed by: Wenrong HeYao Xiao Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plant Physiology Jan 2023 Abstract Chloroplast movement has been observed and analyzed since the 19th century. Subsequently, the phenomenon is widely observed in various plant species such as fern, moss, Marchantia polymorpha, and Arabidopsis. However, chloroplast movement in rice is less investigated, presumably due to the thick wax layer on its leaf surface, which reduces light sensitivity to the point that it was previously believed that there was no light-induced movement in rice. In this study, we present a convenient protocol suitable for observing chloroplast movement in rice only by optical microscopy without using special equipment. It will allow researchers to explore other signaling components involved in chloroplast movement in rice. Keywords: Imaging Chloroplast movement Rice Optical microscope Light Background The chloroplast, as a plant-specific photosynthetic organelle, has different responses to different light intensities (Kong and Wada, 2016). Under weak light, chloroplasts accumulate at the front face of the cells to absorb enough light for photosynthesis; under strong light, chloroplasts move to the anticlinal cell wall to avoid photo-damage (Wada, 2013). In model systems such as Arabidopsis thaliana, a number of components and downstream signaling proteins have been shown to mediate chloroplasts movement signaling in response to different light conditions (Kong and Wada, 2014). However, the molecular mechanism of chloroplast movement in rice still remains unclear, due to limitations on experimental methods and detection tools. The main methods used for detecting chloroplast movement include: (i) green and white band assay, (ii) optical microscope, (iii) measurement of red-light transmittance, (iv) confocal laser scanning microscope, and (v) leaf cross section. Due to the complexity of rice’s leaf structure, the existing methods are not suitable (Suetsugu et al., 2017). In this protocol, we describe a method to detect chloroplast movement using rice leaf sheath. The method is based on a protocol established for Arabidopsis and rice (Zhang et al., 2022), which we have adapted and optimized for convenient and accurate analysis of rice and other gramineous plants. There is no doubt that the system will be beneficial for further understanding of the molecular mechanisms and evolutionary history of chloroplast movement in green plants. Materials and Reagents 9 cm Petri dish (Sangon Biotech, catalog number: F611001) Hydroponic tank (Koraba, catalog number: 53506733434) Razor blade and forceps 1.5 mL tube (Nantong Haidi Experimental Equipment Co., Ltd, catalog number: B030003) Sealing film (Parafilm, catalog number: pm996) Microscope slides, 76 × 26 mm (Sangon Biotech, catalog number: F518101) Cover slides, 24 × 60 mm (Sangon Biotech, catalog number: F518118) Rice seeds (Shuhui 527, indica) Glycerol (Sangon Biotech Co., Ltd, catalog number: A501745-0500) Mounting medium (Thermo Fisher, catalog number: 008030) NH4Cl (Sigma, catalog number: A9434-500G) Ca(NO3)2·4H2O (Sigma, catalog number: C1396-500G) MgSO4·7H2O (Sigma, catalog number: M2773-500G) K2SO4 (Sigma, catalog number: P0772-250G) NaH2PO4·2H2O (Sigma, catalog number: 71500-250G) FeSO4·7H2O (Sigma, catalog number: F7002-500G) Na2EDTA·2H2O (Sigma, catalog number: 03685-500G) H3BO3 (Sigma, catalog number: B0394-100G) ZnSO4·7H2O (Sigma, catalog number: Z0251-500G) CuSO4·5H2O (Sigma, catalog number: 209198-250G) MnCl2·4H2O (Sigma, catalog number: M3634-100G) (NH4)6·Mo7O24·4H2O (Sangon Biotech, catalog number: A600067-0100) Formaldehyde (Sigma-Aldrich, catalog number: F8775) Acetic acid (Sigma-Aldrich, catalog number: A6283) Na2HPO4·12H2O (Sangon Biotech, catalog number: A607793) Ethanol (any manufacturer) Culture solution (see Recipes) FAA stationary fluid (see Recipes) 0.2 M PBS (pH 7.0) (see Recipes) Equipment Constant-temperature incubator (Shanghai Jing Hong Laboratory Instrument Co. Ltd., catalog number: GNP-9080) LED illumination (Fujian Jiupo Biotechnology Co. Ltd., catalog number: WL50) Spectrum analyzer (EVERFINE, catalog number: PLA-30) Eppendorf Research® plus Pipette (100–1,000 μL pipette, catalog number: 3120000062) Vacuum (LABCONCO, CentriVap DNA vacuum concentrator, catalog number: 7970037) Optical microscope (Nikon, model: ECLIPSE Ni-U) Software ImageJ (National Institutes of Health, https://imagej.nih.gov/ij/index.html) Procedure Rice cultivation Select plump rice seeds, put them in the Petri dish without sterilization, and add 20 mL of distilled water to submerge the seeds. Put the Petri dish into the constant-temperature incubator at 37 °C for two days. During this time, change the water every 12 h (Figure 1A). Select the germinated seeds with an identical radical length. Put them into the wells of the hydroponic tank one by one with the embryo upward. Fill the hydroponic tank with tap water; the liquid level should reach the seed radicle. Put the hydroponic tank in a growth chamber under white light at approximately 50 μmol m–2 s–1 in a 14:10 h dark/light cycle at 30 °C (Figure 1A). After approximately two days of growth, change the water to culture solution and grow for ten additional days. During growth, replenish with some culture solution when the liquid level gets too low (Figure 1B). Light treatment Turn on the light set at 100%. Use the spectrum analyzer to detect light intensity at different distances from the light plate and find the positions of 50 and 300 μmol m–2 s–1. Prepare three Petri dishes and fill them with culture solution. Select nine healthy seedlings (three for each Petri dish) and place them horizontally in the Petri dish in parallel. Cover them with a big box for 3 h (Figure 1C). After dark treatment for 3 h, take out two of the Petri dishes and place them at the position where the light intensity is at 50 μmol m–2 s–1 (weak light) and 300 μmol m–2 s–1 (strong light); the remaining one remains in the dark (Figure 1D). Keep the seedlings under the light treatment for 2 h. Sampling Prepare three 1.5 mL tubes and add 1 mL of FAA fixative into each tube with a pipette. Mark them as dark, weak light, and strong light. Make a small hole over each tube with a sharp forceps. After the light treatment, cut out small squares (2 × 2 mm, two for each) on the upper side of the leaf sheath with a razor blade and put them into the corresponding tubes (Figure 1E). Samples must be cut in the same environment as the light treatment. Specifically, samples treated with 50 μmol m–2 s–1 weak light must be cut under 50 μmol m–2 s–1 weak light; samples treated with 300 μmol m–2 s–1 strong light must be cut under 300 μmol m–2 s–1 strong light. Such operation ensures that the distribution of chloroplasts in the sample will not be changed during sample cutting. Take dark-treated samples under darkness or dim light conditions. Cut a small triangle at the upper-right corner of each sample as a mark to prevent the front and back of the sample from being unclear during observation (Figure 1F). Vacuum the FAA tubes with a vacuum equipment until no air bubble comes out and exhaust slowly to let the fix solution go into the tissue (keeping the tubes under -0.1 MPa for 5 min is enough). Replenish some FAA fixative into the tube if there is any loss in the vacuum process (Figure 1G). Seal the tubes’ orifice with sealing film and fix in FAA fixative for 24 h at 4 °C. Observation Wash the samples with 0.2 M PBS three times, each time for 5 min. Place the sample face-up on the slide with a drop of 50% glycerol and cover it gently with a cover slide. Seal the cover slide with mounting medium. Observe the samples on an optical microscope with 100× oil lens and take photos (Figure 1H). Figure 1. Illustration of the experimental procedure. (A) Seed germination. (B) Cultivate the seedlings for 10 days, select healthy seedlings, and place them in the Petri dishes. (C) Dark treatment for 3 h. (D) Light treatment for 2 h. Take out two of the Petri dishes and place them at the position where the light intensity is 50 μmol m–2 s–1 (weak light) and 300 μmol m–2 s–1 (strong light). (E) Cut out small squares (2 × 2 mm) on the illuminated side of the leaf sheath with a razor blade. (F) Cut a small triangle at the upper right corner of each sample and put them into the FAA fixative. (G) Vacuum the FAA tubes with a vacuum equipment and fix in FAA fixative for 24 h at 4 °C. (H) Observe the samples on an optical microscope with 100× oil lens and take photos. Data analysis As shown in Figure 2, different distribution patterns of chloroplasts in wild-type plant cells were visualized. In cells of dark-adapted plants, most chloroplasts were positioned at the bottoms of cells. In the weak white light treatment, most chloroplasts moved to the upper side of the cells. However, the chloroplasts moved along the anticlinal cell walls under strong white light stimulus. Figure 2. Upper view of wild-type cells. (A) Upper view of wild-type cells under dark. (B) Upper view of wild-type cells under weak light. (C) Upper view of wild-type cells under strong light. Scale bar = 5 μm. Recipes Culture solution For 10 L working culture solution, add 10 mL of nutrient solution I, II, III, IV, and VI, 50 mL of nutrient solution V, and 9.9 L of H2O. Adjust the pH value to 5.8–6.0 with HCl before use. Component Reagent Amount (g/L) Nutrient solution I NH4Cl 58 Ca(NO3)2·4H2O 142 Nutrient solution II MgSO4·7H2O 324 Nutrient solution III K2SO4 71.4 Nutrient solution IV NaH2PO4·2H2O 40.3 Nutrient solution V FeSO4·7H2O 6.965 Na2EDTA·2H2O 9.315 Nutrient solution VI H3BO3 0.934 ZnSO4·7H2O 0.035 CuSO4·5H2O 0.031 MnCl2·4H2O 1.5 (NH4)6·Mo7O24·4H2O 0.074 FAA stationary fluid Reagent Stock concentration Amount per 100 mL Final concentration Formaldehyde 38% 5 mL 1.9% Acetic acid 100% 5 mL 5% Ethanol 70% 90 mL 63% Glycerol 100% 5 mL 5% 0.2 M PBS (pH 7.0) Solution A: 0.2 M Na2HPO4, 7.164 g of Na2HPO4·12H2O in 100 mL distilled water Solution B: 0.2 M NaH2PO4, 3.121 g of NaH2PO4·2H2O in 100 mL distilled water Put 61 mL of solution A and 39 mL of solution B together and mix well to make 0.2 M PBS at pH 7.0. HCl solution is used for adjusting the pH value. Acknowledgments This study was supported by Funds from the Natural Science Foundation of Zhejiang (LY20C130004, LGN19C130006) and the National Natural Science Foundation of China (91335103). This protocol was modified from Zhang et al. (2022). Competing interests The authors declare no competing interest. References Kong, S. G. and Wada, M. (2014). Recent advances in understanding the molecular mechanism of chloroplast photorelocation movement. Biochim Biophys Acta 1837(4): 522-530. Kong, S. G. and Wada, M. (2016). Molecular basis of chloroplast photorelocation movement. J Plant Res 129(2): 159-166. Suetsugu, N., Higa, T. and Wada, M. (2017). Ferns, mosses and liverworts as model systems for light-mediated chloroplast movements. Plant Cell Environ 40(11): 2447-2456. Wada, M., (2013). Chloroplast movement. Plant Science 210177-182. Zhang, Y., Dong, G., Wu, L., Wang, X., Chen, F., Xiong, E., Xiong, G., Zhou, Y., Kong, Z. and Fu, Y. (2022). Formin protein DRT1 affects gross morphology and chloroplast relocation in rice. Plant Physiol. doi: 10.1093/plphys/kiac427. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Plant Science > Plant cell biology > Cell imaging Biological Sciences > Biological techniques Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Fluorescent Staining and Quantification of Starch Granules in Chloroplasts of Live Plant Cells Using Fluorescein Shintaro Ichikawa and Yutaka Kodama Nov 5, 2024 461 Views Fast and High-Resolution Imaging of Pollinated Stigmatic Cells by Tabletop Scanning Electron Microscopy Lucie Riglet and Isabelle Fobis-Loisy Nov 20, 2024 409 Views Confocal Live Imaging of Reproductive Organs Development in Arabidopsis Binghan Wang [...] Daniel Kierzkowski Feb 5, 2025 297 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,612	https://bio-protocol.org/en/bpdetail?id=4612&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Flow Cytometry-based Method for Efficient Sorting of Senescent Cells EG Erwan Goy NM Nathalie Martin CD Claire Drullion LS Laure Saas OM Olivier Molendi-Coste LP Laurent Pineau DD David Dombrowicz ED Emeric Deruy HB Hélène Bauderlique-Leroy OS Olivier Samyn JN Joe Nassour YL Yvan de Launoit CA Corinne Abbadie Published: Vol 13, Iss 7, Apr 5, 2023 DOI: 10.21769/BioProtoc.4612 Views: 1981 Reviewed by: Chiara AmbrogioSoumya Moonjely Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Mar 2022 Abstract Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-β-galactosidase (SA-β-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-β-D-galactopyranoside (C12FDG), a fluorigenic substrate of β-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents. Graphical abstract Keywords: Senescence Flow cytometry Sorting FSC SSC SA-β-Gal C12FDG Nozzle Background Cellular senescence can be defined as a reprogrammed cell state triggered to adapt to a variety of stresses, most often those affecting the genome integrity. The term cellular senescence thus encompasses i) replicative senescence, which is associated with shortened and dysfunctional telomeres, ii) stress-induced premature senescence (SIPS) in response to oxidative stress, iii) oncogene-induced senescence (OIS) in response to the activation of some oncogenes (mostly components of the RAS pathway), and iv) therapy-induced senescence (TIS) triggered in response to several anticancer chemotherapeutics or to radiotherapy. There is ample evidence that senescent cells accumulate in most tissues with age and also at sites of many pathologies, and that they contribute to the development of several age-associated dysfunctions and pathologies (for reviews see Abbadie et al., 2017; Di Micco et al., 2021; Tripathi et al., 2021). Senescent cells undergo multiple molecular and phenotypic changes based on both epigenetic and genetic reprogramming. They become larger and more spread than their proliferating counterparts. They also stop dividing, most often arrested at the G1 phase of the cell cycle. Their secretomes are quantitatively and qualitatively modified, with an enrichment in pro-inflammatory cytokines and chemokines, growth factors, and matrix-remodeling enzymes. They suffer from chronic oxidative stress and accumulate oxidized cell components, such as lipofuscin. They are also subject to chronic endoplasmic reticulum stress, which activates the unfolded protein response pathway. Moreover, they accumulate unrepaired DNA damage, constantly activating some signaling and repair pathways, such as the DNA damage response and/or the single-strand break response pathways, leading to the activation of the p21 and/or p16 cyclin-dependent kinase inhibitors (encoded by CDKN1A and CDKN2A, respectively). Some leakage of nuclear chromatin in the cytosol may occur, leading to the activation of the cGAS-STING pathway. Under such stress conditions, senescent cells show an increased autophagic activity reflected by an increased mass of phagosomes, phagolysosomes, and lysosomes, and an increased activity of lysosomal enzymes, such as β-galactosidase (for reviews see Malaquin et al., 2016; Abbadie et al., 2017; Hernandez-Segura et al., 2018; Li and Chen, 2018; Abbadie and Pluquet, 2020; Young et al., 2021). Several of these molecular and phenotypic changes are used as markers to identify senescent cells in mixed cell populations using in vitro culture assays, as well as in tissue sections. The most common among these markers are the following: the senescence-associated-β-galactosidase (SA-β-Gal) activity, with reflects the lysosomal mass; the p16 expression, which is a marker of the cell cycle arrest; and the expression of some cytokines, such as IL-6, which reflect the secretome change. However, since none of these molecular and phenotypic alterations are strictly specific to cellular senescence, experts agree that more than one marker must be recorded to assert that a cell is indeed senescent (Hernandez-Segura et al., 2018). Although considerable progress has been made in the past 20 years, not all of the molecular pathways contributing to senescence induction, maintenance, and escape have been entirely elucidated. To fill this knowledge gap, assays on in vitro cultures of primary cells or cell lines induced into senescence have been commonly used. While this is still relevant, their use has some limitations. According to our experience, a population of senescent cells is never homogenous. It can comprise senescent cells featuring senescent markers to different extents. To make matters worse, senescent cells can be mixed either with still proliferating pre-senescent cells or with post-senescent ones, which reproliferate after senescence escape. Facing these limitations, we have set up a protocol for sorting senescent cells by flow cytometry. In our previous studies, we sorted different subpopulations of senescent normal human epidermal keratinocytes (NHEKs) in order to get insights on their outcomes, i.e., stability, cell death, or neoplastic escape. To this end, we sorted the cells according to their forward and side scatter (FSC and SSC) factors, which reflect cell size and granularity, respectively. We observed a global increase in these two parameters in a population of NHEKs at the senescence plateau compared with an early passage of proliferating NHEKs. However, we found a continuous spectrum of FSC and SSC values within the population at the senescence plateau, evidencing a great variability in the extent of the senescent phenotype. We then sorted different subpopulations according to their FSC and SSC values. We showed that senescent cells with the highest FSC and SSC values were most likely to undergo cell death by autophagy, whereas those with values just below the highest were all alive and had a higher potential to escape senescence and give rise to pre-transformed cells (Deruy et al., 2010, 2014; Gosselin et al., 2009a, 2009b; Nassour et al., 2016). Very recently (Goy et al., 2022), we have improved our sorting protocol by adding a third parameter to better discriminate senescent cells: the SA-β-Gal activity. This enzymatic activity can be detected using C12FDG, as initially described by Debacq-Chainiaux et al. (2009). C12FDG is a cell-permeant fluorogenic substrate of β-galactosidase. It is composed of a di-β-D-galactopyranoside (FDG) and a 12-carbon lipophilic moiety (C12). The β-Gal-mediated hydrolysis of FDG generates the fluorescent signal. According to the manufacturer, the C12 moiety helps to retain the fluorescent product in the cells, probably by insertion into cellular membranes. It is noteworthy that we applied this technique to normal human fibroblasts. These cells are the most commonly used model in the senescence field, but they pose an additional problem regarding flow cytometry sorting: their very large sizes at senescence, larger than those of senescent NHEKs. Because of this, we did not succeed in sorting senescent fibroblasts on cytometers equipped with a standard nozzle of 100–130 µm in diameter. Herein, we present evidence that normal human dermal fibroblasts (NHDFs) at replicative senescence can be efficiently sorted using a sorter equipped with a 200 µm nozzle, based on three senescence markers: size, granularity, and SA-β-Gal activity. Our technique is sufficiently sensitive to separate senescent sub-populations of different sizes, granularity, and/or SA-β-Gal activity, which may have different properties, behaviors, and/or outcomes. It could also be used for a pseudo-time reconstruction, to discriminate and/or sort cells that are in deep senescence from those having just acquired the senescent phenotype. Materials and Reagents Pre-sorting tubes and post-sorting collecting tubes: polypropylene round-bottom tubes with snap caps (BD, catalog number: BD352063) Polypropylene centrifuge tubes (Greiner, catalog number: 188271) Cover slides 5, 10, and 25 mL pipettes Micropipettes with tips T75 cell culture flasks (Falcon, catalog number: 353136) Normal human dermal fibroblasts (NHDFs) (Promocell, catalog number: C-12300) Fibroblast growth medium-2 BulletKitTM (Lonza, catalog number: cc-3132) Trypsin/EDTA (TE) (Gibco, catalog number: R001-100) Trypan blue (Gibco, catalog number: 15250-061) Trypsin neutralizer solution (TN) (Gibco, catalog number: R002-100) 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG) (Invitrogen, catalog number: D2893); prepare a C12FDG 20 mM stock solution in DMSO Pre-sort buffer (BD, catalog number: BD 563503); the pre-sort buffer must be cold: put it at 4 °C one day before the experiment Fetal bovine serum (FBS) (Eurobio, catalog number: CVFSVF00-0U), heat-inactivated at 56 °C for 30 min Sterile DPBS (no calcium, no magnesium) (Gibco, catalog number: 14190-144) Equipment Refrigerated centrifuge (Eppendorf, centrifuge 5810R or equivalent) CO2 incubator (Thermo Scientific, HeraCell 150i, or equivalent) Class II biosafety cabinet (Thermo Scientific, HeraSafe KS12, or equivalent) Inverted phase contrast microscope (Zeiss, Axio Vert.A1, or equivalent) Cell sorter: INFLUX V7 sorter (Becton Dickinson, ref: 646500M8 – 3B3R5V5YG SD AE) equipped with a 200 µm nozzle or equivalent. If your objective is to culture the sorted cells, wash the cell sorter before sorting, carefully applying all the steps recommended by the cytometer’s manufacturer. Sorting senescent cells is challenging due to their large sizes, and even more so when using fibroblasts, which are large compared with other cell types. Therefore, sorters equipped with a standard 100–130 µm nozzle are inadequate. Indeed, we have measured the diameter of suspended NHDFs at the replicative senescence plateau (Figure 1A) and in SIPS (Figure 1B). The mean diameter of cells in both senescence states was approximately 50 µm, representing approximately a two-fold increase compared with exponentially growing cells, with some senescent cells reaching a diameter of up to 100 µm (Figure 1C–F). Usually, it is recommended by sorter manufacturers that the size of the nozzle be five-fold that of the cell diameter, to avoid generating a hydrodynamic rebound when the cell goes through the nozzle. This would induce parasite vibrations of the flux leading to the instability of the breakoff point and, consequently, to spreading of drop deflection. However, practice shows that the nozzle diameter can usually be reduced to two-fold the diameter of cells before drop deflection spreading is observed. Noteworthy, very few sorters are provided with or even designed to run with nozzles larger than 130 µm in diameter. The INFLUX v7 cell sorter (Becton Dickinson) we chose to use is designed to run with a 200 µm nozzle, which was efficient in providing stable deflection of senescent fibroblasts in our hands. The cell sorter is equipped with a supplemental circulating water–cooling device supplied with the instrument (Thermo Scientific HAAKE A10 – 152510101) Figure 1. Characteristics of senescent normal human dermal fibroblasts (NHDFs). A. Growth curve of NHDFs up to the replicative senescence plateau. The time at which the cells were collected for sorting is indicated. B. Growth curves of control NHDFs and NHDFs induced into stress-induced premature senescence (SIPS) by a 60 µM H2O2 daily treatment. C–D. Cell diameters of NHDFs in suspension by their population doubling number (C) or by the H2O2 treatment duration (the day after the beginning of the treatment; D). Each dot represents the diameter of one cell. The bars represent the mean ± SD. A Kruskal-Wallis analysis was done. NS: non-significant; : p < 0.01; : p < 0.0001. Right panels: representative phase contrast microscopy images used to measure cell diameters. Malassez chambers (Marienfeld, catalog number: 0640610, or equivalent); if you use a cell counter device, check if it can count large cells Software BD FACS Software Ink (developed and supplied by Becton Dickinson®) FlowJo V10.8.1 (developed by FlowJo LLC and supplied by Becton Dickinson®) Procedure The protocol described below is for sorting out fully senescent NHDFs from among NHDFs at the replicative senescence plateau. These cells are sorted as C12FDGhigh/FSChigh/SSChigh. We also used the same protocol to sort NHDFs induced into senescence by X-ray irradiation (Goy et al., 2022). Cell culture To be able to objectively delineate the sorting gates corresponding to senescent cells, it is mandatory to use a negative control and advisable to have a positive one. The best negative control is to use cells from the same cell culture at the earliest possible passage. There is no obvious positive control for replicative senescence. However, cells at replicative senescence can be used as a positive control for SIPS, OIS, or TIS. Of course, classical cytometry controls must also be included (i.e., unstained cells in our protocol) to take into consideration the level of autofluorescence. Note that this autofluorescence level can be higher in senescent cells than in proliferating ones because of autofluorescent oxidized component aggregates, like lipofuscin, that accumulate in senescent cells (Evangelou et al., 2017). NHDFs are primary cells. Cultivate them at 37 °C in an atmosphere of 5% CO2 and at atmospheric O2 pressure, in fibroblast growth medium-2 (160 µL/cm2). Always proceed to subculturing before the cells reach 70% confluency. For this, rinse them with TE preheated at 37 °C, add fresh TE (40 µL/cm2), and incubate at 37 °C for 5 min in case of exponentially growing cells or 10 min for cells having reached the senescence plateau. Then, add the same volume of TN preheated at 37 °C. Collect the cells and pellet them by centrifugation at 90 × g for 5 min at room temperature. Replate at 3,500 cells/cm2 in case of exponentially growing cells, and at 2,000 cells/cm2 for cells at replicative senescence. Under these conditions, exponentially growing NHDFs will reach the replicative senescence growth plateau after 50–70 population doublings, i.e., in approximately five months (Figure 1A). At this plateau, senescent cells can be easily recognized by their increased sizes observable under a phase contrast microscope. Freeze early passage cells and put them again in culture a few days before the sorting to use them as a negative control. C12FDG staining The incubation time and concentration of C12FDG should be adapted to the cell type. For NHDFs, we incubate the cells with 33 µM C12FDG at 37 °C for 2 h. For NHEKs, we use a 16 µM C12FDG concentration and incubate the cells at 37 °C for 16 h. Mind that high concentrations of C12FDG or long incubation times may be toxic, depending on the cell type. The efficacy and non-toxicity of the C12FDG staining protocol can be checked under an inverted epifluorescence microscope. Dilute C12FDG from the stock solution in the culture medium preheated at 37 °C to obtain a 33 µM concentration. For unstained controls, dilute the same volume of DMSO (the C12FDG vehicle) in the culture medium preheated at 37 °C and put it on the cells at 100 µL/cm2. Incubate for 2 h at 37 °C. Cell harvesting All the steps must be performed in the dark to avoid photobleaching of C12FDG. Remove the C12FDG- or DMSO-containing culture medium. Rinse the cells twice with 30 µL/cm2 of sterile DPBS at room temperature. Remove the DPBS and harvest the cells by incubating them at 37 °C with TE preheated at 37 °C (40 µL/cm2) until the cells are detached and separated from each other (check under an inverted phase contrast microscope). Note that the incubation time may be increased by approximately 50% for senescent cells compared with that for proliferating cells. Neutralize TE by adding TN preheated at 37 °C (a volume of TN for a volume of TE). Count the cells (to be able to resuspend them at the desired concentration afterwards). Centrifuge the cells at 90 × g for 5 min at 4 °C. Rinse the pellets with 500 µL of cold pre-sort buffer. Centrifuge the cells at 90 × g for 5 min at 4 °C. Resuspend the cells in cold pre-sort buffer to the concentration of 4 × 106 cells/mL. Keep the tubes on ice until sorting to protect the fluorescent SA-β-Gal product from degradation. Cell sorting We propose to sort senescent cells using the following three parameters: size, granularity, and SA-β-Gal activity. Four gates will have to be designed (Figure 2). The first one is used, as usually in flow cytometry sorting, to separate live cells from the debris (Figure 2A), the second to exclude doublets (Figure 2B), and the third to define the C12FDGhigh cells. This gate is delineated by comparing the dot-plot of C12FDG-stained senescent cells to those of both unstained senescent cells and C12FDG-stained non-senescent cells (Figure 2C). The fourth gate serves to delineate the largest and most granular cells within the C12FDGhigh population, using their FSC and SSC values (Figure 2D). The stringency of the two last gates can be adapted, depending on your objectives. Moreover, these two last gates can be subdivided to sort different senescent subpopulations featuring the three senescence markers to different extents. In that case, we recommend delineating the gates such that they are not strictly adjacent, in order to obtain sufficiently different subpopulations. Proceed to cell sorting as soon as possible after cell harvesting. Use a BD INFLUX V7 or an equivalent cell sorter, equipped with a 200 µm nozzle. Sorting parameters must be tuned adequately to the nozzle size, according to the cytometer’s manufacturer recommendations. Pre-sorting tubes and post-sorting collecting tubes must be in polypropylene and coated with heat-inactivated FBS to avoid cell adhesion to the tube walls. Moreover, we recommend regularly shaking the pre-sorting tubes gently during the sorting process to resuspend the cells that could have sedimented. Figure 2. Gating strategy for the sorting of senescent normal human dermal fibroblasts at the replicative senescence plateau. A. The first gate is set to eliminate debris using the forward scatter (FSC) and side scatter (SSC) values. Note that large and granular cells are much more numerous in the population at the replicative senescence plateau than in the population of exponentially growing cells. B. The second gate serves to exclude doublets using the forward scatter–width (FSC-W) and forward scatter–area (FSC-A) values. C. The third gate is set to define the C12FDGhigh population. It is delineated by considering the autofluorescence level of unstained cells and the fluorescence level of C12FDG-stained exponentially growing cells. D. The fourth gate is used to delineate the FSChigh/SSChigh cells within the C12FDGhigh population that will be sorted. Prepare the pre-sorting polypropylene tubes and post-sorting polypropylene collecting tubes: fill the tubes with FBS previously inactivated by a 30 min incubation at 56 °C. Let the tubes be coated during 30 min at 37 °C. Prepare the cell sorter for acquisition of FSC, SSC, and C12FDG fluorescence [the 530/40 filter–5/6-fluorescein isothiocyanate (FITC) equivalent, 488 nm laser], according to the manufacturer’s instructions. Cool the pre- and post-sorting tubes to 4 °C using the circulating water–cooling device. Analyze the FSC vs. SSC parameters of the unstained batches of NHDFs at early passages and those at the senescence plateau. Optimize the amplification parameters and scales to visualize the cells of all FSC and SSC values. Given very large sizes of senescent cells, instrument signal processing is particularly challenged and the linearity of the scale for FSC may be questionable. However, in contrast to the classical digital display on most cell sorters, the INFLUX V7 cell sorter displays analogic signals, theoretically ensuring that the linearity of the scales is preserved despite extreme parameters adjustments. Delineate the first gate to exclude debris (Figure 2A). Analyze the FSC-W vs. FSC-A parameters to delineate a singlet cell gate. The heterogeneity of cell sizes requires particular attention to delineate doublets’ exclusion on FSC-W/FSC-A parameters, with an unusual necessity to draw a large gate not to exclude the cells with high FSC-W values (Figure 2B). Indeed, these cells may be doublets, but also singlet large cells, i.e., senescent cells. In order to determine whether the cells with the highest FSC-W values were doublets or singlet large cells, we analyzed their C12FDG, FSC, and SSC values in comparison with those of cells with the lowest FSC-W values. Regarding exponentially growing cells, we found that those with the highest FSC-W values had a higher C12FDG signal than the cells with the lowest FSC-W values. A majority of these cells had low FSC and SSC values, indicating that these were indeed mainly doublets (Figure 3). Regarding cells at the senescence and deep senescence plateau, the cells with the highest FSC-W values had the same C12FDG signal than those with lower FSC-W values. Compared with exponentially growing cells, a higher proportion of them had high FSC and SSC values, suggesting that a portion of them were doublets of still small cells and the others were large singlet cells, i.e., senescent cells (Figure 3). Figure 3. Checking the accuracy of the doublet exclusion strategy. Cells were analyzed by their forward scatter–width (FSC-W) and forward scatter–area (FSC-A) values. Gates of assumed singlets (blue) and doublets (red) were delineated. Cells in these gates were further analyzed for their C12FDG, forward scatter (FSC), and side scatter (SSC) values. Analyze the green fluorescence of all stained and unstained batches in a Log scale histogram. Delineate the third gate of C12FDGhigh cells by comparing the fluorescence intensity of C12FDG-stained senescent cells to that of (i) unstained senescent cells (to evaluate autofluorescence) and (ii) C12FDG-stained early passage cells (Figure 2C). Then, analyze the FSC vs. SSC parameters of the C12FDGhigh cells only. Delineate the gate of FSChigh/SSChigh cells (Figure 2D). Sort the C12FDGhigh/FSChigh/SSChigh cells using the parameters indicated in Table 1, at 500–1,000 events/s (it is possible to go up to 2,000 events/s, but a lower sorting speed is better to avoid the deflection stream spreading). Begin by checking (using the unstained cells at the senescence plateau) that the deflection stream is stable and that cells are properly deflected to the collecting tube. To do that, cover the collecting tube with a cover slide and check that the drop falls at its center. Table 1. Sorting parameters Nozzle diameter Sheath pressure Sort device Piezo amplitude Drop delay Sort mode Drop envelope Sort objective Phase mask Extra coincidence bits Drop frequency (kHz) 200 µm 3.7 psi 2 tube holder—2-way sort 4.01 15.6711 1.0 Drop Pure 1.0 Drop Purify 16/16 4 6.30 Collect the cells at 4 °C in the circulating water–cooling device and proceed to any further treatment (e.g., protein extraction or plating for follow-up). Data analysis Here, we sorted a bit more than 60% of the cells with the highest FSC and SSC values amongst approximately 60% of the cells with the highest C12FDG signal in a population of NHDFs at the replicative senescence plateau, either at the beginning of the plateau or several weeks after the plateau had been established (which is called deep senescence). These gates can be adapted to be more stringent. For example, in an experiment shown in the graphical abstract, we sorted only 30% of the cells with the highest FCS and SSC values within 30% of the cells with the highest C12FDG signal. Since the sorting of large cells can be challenging, we checked the sorting efficiency of our protocol. The sorting efficiency provided in the sort report (Table 2) was very high, between 86.5% and 91.3%. To validate this sorting efficiency, we manually counted the number of cells in the collecting tubes (using a Malassez chamber) and calculated the percentage of cells that were actually sorted (Table 3). Our results were very similar to those provided by the cell sorter. To challenge the sorter’s ability to sort rare senescent cells, we mixed 50% of exponentially growing cells with 50% of cells at the senescence plateau or at the deep senescence plateau. We found that the sorting efficiencies were similar (Tables 2 and 3). Table 2. Sort reports Table 3. Sorting efficiency Number of cells in the gate Number of cells in the collecting tube Sorting efficiency (%) Cells at the senescence plateau 21,852 20,000 91.52 Cells at the deep senescence plateau 33,859 32,000 94.51 Mix (50/50) of exponentially growing cells and cells at the senescence plateau 6,936 6,000 86.51 Mix (50/50) of exponentially growing cells and cells at the deep senescence plateau 9,403 8,000 85.08 Mean 89.40 SD 4.38 Another delicate issue could have been the viability of the sorted senescent cells. To address this question, we assessed the viability of the sorted cells compared with the pre-sorted total population using trypan blue staining. We observed an about 2-fold increase in the percentage of trypan-blue-positive cells in the sorted population compared to the pre-sorted one, however this difference was not statistically significant, and the level of cells with an altered membrane permeability revealed by the trypan-blue staining remained low, at about 15% (Figure 4A). Moreover, sorted senescent cells, and even deep senescent ones, were able to replate (Figure 4B). Finally, we checked that the sorted cells were indeed senescent. We first examined their morphology after plating using phase contrast microscopy. Sorted cells were very large and spread, with refringent vesicles in the cytoplasm and often big nucleoli (Figure 4B). Second, we searched for the expression of p21, the main cyclin-dependent kinase inhibitor involved in the cell cycle arrest of replicative senescent cells, using immunofluorescence. We found an increase of approximately 2.6-fold in the nuclear expression of p21 in sorted cells compared with pre-sorted cells at the senescence plateau (Figure 4C). Finally, we investigated the telomere status of the sorted cells by enumerating 53BP1 foci, which mainly form at the shortened telomeres. The number of 53BP1 foci per nucleus increased by approximately 2.8-fold in sorted cells compared with pre-sorted cells at the senescence plateau (Figure 4C). Altogether, these results demonstrate that our sorting strategy makes it possible to efficiently sort senescent fibroblasts, which remain viable and able to replate, and whose senescent nature is confirmed by the presence of molecular senescent canonical markers. The protocol we developed enables the sorting of senescent cells using three parameters. We presume that it is possible to include additional markers, provided that they can be detected using a vital fluorochrome. Figure 4. Characteristics of sorted senescent normal human dermal fibroblasts. A. Cell death levels were determined by manually counting trypan blue (15250-061, Gibco) positive cells amongst a mean of 57 cells from pre-sort cell populations compared to a mean of 15 cells from sorted cells, in two independent sorts, one with cells at the beginning of the senescence plateau, the other with cells in deep senescence. The bars represent the means ± SD of these counts. A t-test was performed to compare the sorted populations to the pre-sort ones. NS: non-significant difference. B. Phase contrast images of pre-sorted cells or cells plated after sorting. Scale bars: 50 µm. C. Immunostaining of p21 and 53BP1 in the sorted population compared with the pre-sorted population. Left: representative immunostaining images. Upper right: quantification of the p21 nuclear staining intensity. Each dot represents the nuclear fluorescence intensity of one cell. The bars represent the means ± SD of at least 37 cells. A Mann-Whitney test was done to compare the two populations. indicates p < 0.001. Lower right: quantification of 53BP1 foci. Each dot represents the number of 53BP1 foci in the nuclear plane of one cell. The bars represent the means ± SD of at least 28 cells. A Mann-Whitney test was done to compare the two populations. * indicates p < 0.001. Acknowledgments This work was supported by le Centre National de la Recherche Scientifique, l’Université de Lille, la Ligue contre le Cancer (Comité du Pas-de-Calais, Comité de la Somme, Comité du Nord), le Cancéropôle Nord-Ouest, l’Institut Pasteur de Lille, le SIRIC OncoLille (Grant INCa-DGOS-Inserm 6041), le Contrat de Plan Etat Région CPER Cancer 2015-2020, and l’Institut National du Cancer (INCa_16078). E.G received a fellowship from l’Institut Pasteur de Lille and la Région Hauts-de-France. C.D. and L.S. received fellowships from la Région Hauts-de-France. J.N. received fellowships from l’Université de Lille and from l’Association pour la Recherche sur le Cancer. We thank the Bioimaging Center Lille-Nord de France (Campus Calmette) for the services of their imaging and cytometry facilities. We acknowledge the authors of the article entitled “The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion” in which the use of this protocol was first described (Goy et al., 2022). Competing interests The authors have no conflicting financial or non-financial interests. References Abbadie, C. and Pluquet, O. (2020). Unfolded Protein Response (UPR) Controls Major Senescence Hallmarks. Trends Biochem Sci 45(5): 371-374. Abbadie, C., Pluquet, O. and Pourtier, A. (2017). Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses? Cell Mol Life Sci 74(24): 4471-4509. Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J. and Toussaint, O. (2009). Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4(12): 1798-1806. Deruy, E., Gosselin, K., Vercamer, C., Martien, S., Bouali, F., Slomianny, C., Bertout, J., Bernard, D., Pourtier, A. and Abbadie, C. (2010). MnSOD upregulation induces autophagic programmed cell death in senescent keratinocytes. PLoS One 5(9): e12712. Deruy, E., Nassour, J., Martin, N., Vercamer, C., Malaquin, N., Bertout, J., Chelli, F., Pourtier, A., Pluquet, O. and Abbadie, C. (2014). Level of macroautophagy drives senescent keratinocytes into cell death or neoplastic evasion. Cell Death Dis 5(12): e1577. Di Micco, R., Krizhanovsky, V., Baker, D. and d'Adda di Fagagna, F. (2021). Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat Rev Mol Cell Biol 22(2): 75-95. Evangelou, K., Lougiakis, N., Rizou, S. V., Kotsinas, A., Kletsas, D., Munoz-Espin, D., Kastrinakis, N. G., Pouli, N., Marakos, P., Townsend, P., et al. (2017). Robust, universal biomarker assay to detect senescent cells in biological specimens. Aging Cell 16(1): 192-197. Gosselin, K., Deruy, E., Martien, S., Vercamer, C., Bouali, F., Dujardin, T., Slomianny, C., Houel-Renault, L., Chelli, F., De Launoit, Y., et al. (2009a). Senescent keratinocytes die by autophagic programmed cell death. Am J Pathol 174(2): 423-435. Gosselin, K., Martien, S., Pourtier, A., Vercamer, C., Ostoich, P., Morat, L., Sabatier, L., Duprez, L., T'Kint de Roodenbeke, C., Gilson, E., et al. (2009b). Senescence-associated oxidative DNA damage promotes the generation of neoplastic cells. Cancer Res 69(20): 7917-7925. Goy, E., Tomezak, M., Facchin, C., Martin, N., Bouchaert, E., Benoit, J., de Schutter, C., Nassour, J., Saas, L., Drullion, C., et al. (2022). The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion. Elife 11: e67190. Hernandez-Segura, A., Nehme, J. and Demaria, M. (2018). Hallmarks of Cellular Senescence. Trends Cell Biol 28(6): 436-453. Li, T. and Chen, Z. J. (2018). The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer. J Exp Med 215(5): 1287-1299. Malaquin, N., Martinez, A. and Rodier, F. (2016). Keeping the senescence secretome under control: Molecular reins on the senescence-associated secretory phenotype. Exp Gerontol 82: 39-49. Nassour, J., Martien, S., Martin, N., Deruy, E., Tomellini, E., Malaquin, N., Bouali, F., Sabatier, L., Wernert, N., Pinte, S., et al. (2016). Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells. Nat Commun 7: 10399. Tripathi, U., Misra, A., Tchkonia, T. and Kirkland, J. L. (2021). Impact of Senescent Cell Subtypes on Tissue Dysfunction and Repair: Importance and Research Questions. Mech Ageing Dev 198: 111548. Young, A. R. J., Cassidy, L. D. and Narita, M. (2021). Autophagy and senescence, converging roles in pathophysiology as seen through mouse models. Adv Cancer Res 150: 113-145. Article Information Copyright Goy et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). How to cite Category Cancer Biology > Genome instability & mutation > Tumor formation Cell Biology > Cell isolation and culture > Cell isolation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems Ye Li [...] Annie C. Bowles-Welch Dec 20, 2023 1826 Views Dissociation and Culture of Adult Mouse Satellite Glial Cells Raquel Tonello [...] Temugin Berta Dec 20, 2023 859 Views Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing Hongzhe Li [...] Stefan Scheding Jun 20, 2024 645 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,613	https://bio-protocol.org/en/bpdetail?id=4613&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed CRISPR/Cas9-based Engineering of Immunoglobulin Loci in Hybridoma Cells CG Camille M. Le Gall * FF Felix L. Fennemann * JS Johan M.S. van der Schoot * FS Ferenc A. Scheeren MV Martijn Verdoes (contributed equally to this work) Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4613 Views: 995 Reviewed by: Luis Alberto Sánchez VargasDay-Yu Chao Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Aug 2019 Abstract Development of the hybridoma technology by Köhler and Milstein (1975) has revolutionized the immunological field by enabling routine use of monoclonal antibodies (mAbs) in research and development efforts, resulting in their successful application in the clinic today. While recombinant good manufacturing practices production technologies are required to produce clinical grade mAbs, academic laboratories and biotechnology companies still rely on the original hybridoma lines to stably and effortlessly produce high antibody yields at a modest price. In our own work, we were confronted with a major issue when using hybridoma-derived mAbs: there was no control over the antibody format that was produced, a flexibility that recombinant production does allow. We set out to remove this hurdle by genetically engineering antibodies directly in the immunoglobulin (Ig) locus of hybridoma cells. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and homology-directed repair (HDR) to modify antibody’s format [mAb or antigen-binding fragment (Fab’)] and isotype. This protocol describes a straightforward approach, with little hands-on time, leading to stable cell lines secreting high levels of engineered antibodies. Parental hybridoma cells are maintained in culture, transfected with a guide RNA (gRNA) targeting the site of interest in the Ig locus and an HDR template to knock in the desired insert and an antibiotic resistance gene. By applying antibiotic pressure, resistant clones are expanded and characterized at the genetic and protein level for their ability to produce modified mAbs instead of the parental protein. Finally, the modified antibody is characterized in functional assays. To demonstrate the versatility of our strategy, we illustrate this protocol with examples where we have (i) exchanged the constant heavy region of the antibody, creating chimeric mAb of a novel isotype, (ii) truncated the antibody to create an antigenic peptide-fused Fab’ fragment to produce a dendritic cell–targeted vaccine, and (iii) modified both the constant heavy (CH)1 domain of the heavy chain (HC) and the constant kappa (Cκ) light chain (LC) to introduce site-selective modification tags for further derivatization of the purified protein. Only standard laboratory equipment is required, which facilitates its application across various labs. We hope that this protocol will further disseminate our technology and help other researchers. Graphical abstract Keywords: Hybridoma CRISPR/Cas9 Antibody engineering Immunoglobulin Immunology Immunotherapy Background Production of antibodies using transient recombinant systems is laborious and confronted with reproducibility issues due to variable transfection efficacy and requires knowledge of the sequence of the variable domain of the antibodies. While this is routine for specialized companies and laboratories equipped with dedicated biofactories, typical academic laboratories are usually not equipped with these infrastructures. For most laboratories, hybridoma represent the cheapest and least labor-intensive way of producing monoclonal antibodies (mAbs). As immunized animals, from which the original cell line was generated, underwent multiple rounds of immunization and class-switch recombination, there is minor diversity among the isotypes, which are almost exclusively immunoglobulin (Ig) G subtypes. While the isotype might not always be relevant, it is becoming increasingly evident that it can be crucial for the efficacy of therapies (Waldor et al., 1987; Beers et al., 2016; Evers et al., 2021); isotype engineering could strongly increase the efficacy of mAbs used in cancer treatment, for instance (Sharma et al., 2019). Antigen-binding fragments (Fab’) are valuable tools in research due to their small size, but require post-isolation modification of the mAb using papain (Zhao et al., 2009). Lastly, chemical modification of mAbs is usually achieved by random labeling of lysine residues (Nanda and Lorsch, 2014), which is not site-specific and leads to undesirable heterogeneous products, or by engineering of the sequence to introduce unpaired cysteine residues (Adhikari et al., 2020), although novel methods are being developed (Yamada et al., 2019). To collectively address these issues, we developed a technique that reproducibly allows the modification of the Ig constant locus in hybridoma using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). This nowadays routine method of gene editing enables precise targeting of the constant regions of the Ig loci regardless of the variable region and enables the insertion of virtually any sequence to produce an antibody with any switched or mutant isotype, format, or tag. The low diversity in isotypes among hybridoma is, in this case, an advantage, as homology-directed repair (HDR) templates can be reused easily on many cell lines. While we focused on editing the constant heavy chain (CH)1, hinge (H), and light chain (LC) regions, feasibility of CH3 targeting was demonstrated by others (Khoshnejad et al., 2018). Collectively, this illustrates the versatility of the strategy. We expect that this protocol can be applied to benefit any field, as mAbs are a backbone of biological research, and hope to see peers successfully applying this method for applications we would not be able to envision. Materials and Reagents 875 cm2 5-layer culture flask (Corning, Falcon®, catalog number: 353144) 75 cm2 culture flask (Greiner Bio-one, Cellstar, catalog number: 658170) 25 cm2 culture flask (Greiner-Bio one, Cellstar, catalog number: 690175) 10 cm Petri dish, culture-treated (Greiner Bio-one, Cellstar, catalog number: 664161) 6-well plates (Corning, Costar, catalog number: 3516) 24-well plates (Corning, Costar, catalog number: 3524) 96-well plates (Corning, Costar, catalog number: 3799) 50 mL Falcon (Thermo Fisher, BrandTM 114821, catalog number: 10420602) 15 mL Falcon (Thermo Fisher, BrandTM 114818, catalog number: 10345521) 1.5 mL tube (Eppendorf, catalog number: 0030120086) PCR tubes (Eppendorf, catalog number: 0030124820) Disposable chromatography column (Bio-Rad, catalog number: 7321010) pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, catalog number: 42230) pSMART-HCKan, CloneSmart Blunt Cloning kit (Lucigen, catalog number: 40704-2) pHybr_r2a>mA-srt-his (Addgene, catalog number: 124806) pHybr_r2a>m2a(silent)-srt-his (Addgene, catalog number: 124807) PX458-gR2A_ISO (Addgene, catalog number: 124808) BbsI-HF® (New England Biolabs, catalog number: R3539) rSAP (New England Biolabs, catalog number: M0371) T4 DNA ligase (New England Biolabs, catalog number: M0202) T4 PNK (New England Biolabs, catalog number: M0201) Q5 polymerase (New England Biolabs, catalog number: M0491) dNTP (Thermo Fisher, catalog number: R0192) ddH2O (Thermo Fisher, Invitrogen, catalog number: AM9935) Agarose (Sigma-Aldrich, catalog number: A4718) Nancy-520 (Sigma-Aldrich, catalog number: 01494) NucleoBond Xtra Midi Plus EF kit (Macherey-Nagel, catalog number: 740422.50) NucleoSpin Gel and PCR cleanup (Macherey-Nagel, catalog number: 740609.50) ISOLATE II Genomic DNA isolation kit (Bioline, catalog number: BIO-52067) Hybridoma: rat IgG2a,λ anti mouse PD-L1 [MIH5] (in house) Hybridoma: rat IgG2a,κ anti mouse DEC-205 [NLDC-145] (ATCC, catalog number: HB-290TM) Hybridoma: mouse IgG1,κ anti human CD20 [NKI.B20/1] (in house) SF Cell Line 4D-NucleofectorTM X kit L (Lonza Biosciences, catalog number: V4XC-2024) PBS (Fresnius Kabi, catalog number: M087312/01) RPMI-1640 (Thermo Fisher, GibcoTM, catalog number: 42401018) CD hybridoma medium (Thermo Fisher, GibcoTM, catalog number: 11279023) Heat-inactivated fetal bovine serum (FBS) (HyClone, catalog number: SV35959) Ultraglutamine-1 (Lonza Biosciences, catalog number: BE17-605E/U1) Antibiotic-antimycotic (Thermo Fisher, GibcoTM, catalog number: 15240062) β-mercaptoethanol (β-ME) (Sigma-Aldrich, catalog number: 444203) Trypan blue (Thermo Fisher, GibcoTM, catalog number: 15250061) CELLine CL 1000 bioreactors (INTEGRA Biosciences AG, catalog number: 90005) Blasticidin (Invivogen, catalog number: ant-bl-05) Puromycin (Invivogen, catalog number: ant-pr-5) BSA (Sigma-Aldrich, catalog number: A9418) NaN3 (Sigma-Aldrich, catalog number: 26628-22-8) Recombinant Protein G (Thermo Fisher, PierceTM, catalog number: 21193) Ni-NTA agarose (Quiagen, catalog number: 30230) Anti-6×His-tag (clone J095G46) (PE, Biolegend, catalog number: 362603) Anti-rat IgG2a HC (clone MRG2a-83) (PE, Biolegend, catalog number: 407507) Anti-mouse IgG2a (clone m2a-15F8) (PE, eBioscience, catalog number: 12-4817-82) Rabbit anti-6×His-tag (clone RM146, unconjugated, Abcam, catalog number: AB14923) Goat anti-rabbit IgG (H+L) (polyclonal, IRD800, LI-COR, catalog number: 926-32211) Goat anti-rat IgG (H+L) (polyclonal, AF680, Thermo Fisher, catalog number: A-21096) Wash buffer (see Recipes) PBA (see Recipes) Culture medium (see Recipes) 1× selection medium (see Recipes) Equipment Electroporation device, e.g., AMAXA 4D Nucleofector (Lonza BioSciences, AAF-1002X/AAF-1002B) Humidified incubator, 5% CO2 Thermocycler (e.g., Bio-Rad, catalog number: 1861096EDU) Flow cytometer, e.g., FACSLyric (BD Biosciences, catalog number: 663518) Fluorescent scanner, e.g., Amersham TyphoonTM 5 (Cytiva, catalog number: 29187191) Sanger Sequencing Service Software Snapgene (from Insightful Science; available at snapgene.com) FlowJoTM Software (Ashland, OR; Becton, Dickinson and Company; 2021) Procedure General considerations Before starting hybridoma modification, knowledge on the host species and antibody isotype is required. We have used cut sites to knock in inserts upstream or downstream of CH1 heavy chain (HC), upstream of the hinge, and downstream of Cκ light chain (LC), but virtually any other cut site can be used to tailor the antibody to the intended use. It takes 4–6 weeks from the transfection of the target cell lines to the isolation of high levels of modified antibodies (Figure 1). Figure 1. Workflow overview. Hybridoma are transfected on day 0 with a plasmid encoding the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and guide ribonucleic acid (gRNA), and a plasmid encoding a homology-directed repair (HDR) template containing an antibiotic resistance gene. At day 3, selection pressure is applied, and cells start to grow out. Resistant clones are expanded until reaching confluency, and single-cell clones (SCC) are prepared in 96-well plates. By day 30, positive clones are screened for production of the modified antibody and expanded for production. For the purpose of this protocol and to illustrate the diversity of potential applications, we describe the full characterization of three successfully edited hybridoma: isotype switching of the constant heavy chain (HC) of rat IgG2a (r2a) Ig to a mouse IgG2a (m2a) constant HC and produce a chimeric r2a(V):m2a(C) mAb, introduction of an antigen in the hinge region of r2a HC to produce a Fab’ fragment–antigen fusion, and modification of the mouse IgG1 (m1) HC and mouse kappa (mκ) LC to introduce two site-specific labeling tags. Design of CRISPR/HDR template and gRNA For designing an HDR template and gRNA from scratch, the Addgene CRISPR guide or the CRISPR 101 book are good starting points. In Figures 2–6, the process for designing a CRISPR/HDR template and gRNAs is illustrated step by step. We take the example of targeting the C-terminus of m1 CH1 to engineer a mAb-secreting cell line to produce Fab’ fragments bearing a site-specific labeling site at the C-terminus of the CH1. Figure 2. Annotating the target sequence. A. Access the genomic sequence of the locus of interest (e.g., on NCBI Gene). Using “sequence text” view, visualize exons and introns in the target locus. B. Remove annotations to visualize only the nucleotide sequence. For a given locus, exons are marked in red and introns in green. Here are highlighted constant heavy chain (CH)1, hinge, CH2, and CH3 in the Ighg1 gene (mIgG1 constant region). C. Copy the sequence in Snapgene and annotate exons to identify the target region. This typically depends on the intended modification. Here, we target the C-terminus of CH1 of mIgG1. Figure 3. Identification of the target locus. A. Identify the region to target for the knock in. Here, we selected the C-terminus of the CH1 domain to insert part of the hinge region, a short linker, a site-specific labeling site, and a polyhistidine tag. This effectively transforms a mAb into a Fab’ fragment bearing a site-specific labelling site at the C-terminus of its heavy chain (HC). B. Copy the selected sequence to CRISPOR. C. Choose the appropriate host (here, Mus musculus) and protospacer adjacent motif (PAM) (typically, -NGG) and submit. Figure 4. Selection of gRNAs. A. Protospacer adjacent motif (PAM) sequences and corresponding gRNA candidates are identified by CRISPOR. All possible PAM sequences (-NGG here) are identified below the input sequence. B. Sequences and characteristics of potential gRNAs are displayed as a table, ordered by predicted efficacy. The insertion site should be less than 10 bp away from the double strand break if possible. When targeting a new region, select at least two or three gRNAs based on their proximity to the intended site of insertion and precited score. Here, we selected gRNA_m1_HC (5′-CTTGGTGCTGCTGGCCGGGT-3′). By clicking on “cloning,” a new window opens with sequences for cloning into a variety of Cas9-containing vectors. C. We used a U6 expression system (U6 promoter, pX330 vectors, and derivatives) (Cong et al., 2013). Here, we used a 20 nt gRNA, but it was recently proposed to use a 19 nt gRNA for improved efficiency (Kim et al., 2020). Select the appropriate primers and follow the instructions to clone the gRNAs into the pX330 vector at the BbsI site. Figure 5. nserting the desired sequence at the knock-in site. A. Copy the gRNA sequence(s) into Snapgene and B. visualize the protospacer adjacent motif (PAM) sequence(s). Insert the sequence to knock in as close as possible to the PAM sequence. C. Insert the predetermined nucleotides containing the desired insert. Here, we inserted part of the hinge region (VPRDC) allowing association with the light chain (LC) downstream of CH1, followed by a multiple cloning site (MCS), a flexible linker (GGGGS), a sortag motif (LAETGG), and a polyhistidine tag (HHHHHH), followed by a stop codon. The insert is followed by an internal ribosomal entry site (IRES) to allow for transcription of blasticidin resistance gene (BSD) and a simian vacuolating virus 40 (SV40) poly A tail to dissociate ribosomes. Each domain is surrounded by a MCS to allow easy exchange of any part of the plasmid. Here, we additionally optimized part of the CH1 to improve association with the LC (PACSTKVDKKI, S>C mutation). Figure 6. Homology arms (HA) and final HDR template design. The HAs span ca. 0.6 kb on each side of the insert. Their large size allows a few mismatches between the HAs and the target locus without affecting HA annealing to the genomic locus during HDR. Therefore, small adjustments can be done in the HA sequence compared to the germline sequence initially obtained from the genome browser without affecting HDR efficacy. A. It is necessary to mutate the “NGG” protospacer adjacent motif (PAM) sequence(s). To inactivate the PAM sequences, mutate “NGG” to “NGC,” “NGA,” or “NGT.” If the PAM sequence is located within the coding region, ensure that it is a silent mutation. Alternatively, remove the PAM sequences all together. Cells having undergone successful HDR will not contain PAM sequence(s) and the Cas9 will be inactivated. B. To define 5’HA and 3’HA, select ~0.6 kb on each side of the insert. Sequences of the constant immunoglobulin loci can be accessed on NCBI Gene; protospacer adjacent motifs (PAM) sequences and gRNA can be determined using CRISPOR or alternative tools. The HDR template should contain 5′ and 3′ homology arms (HAs) flanking the desired insert, a stop codon, an internal ribosomal entry site (IRES), an antibiotic resistance gene (blasticidin or puromycin), and a poly A tail (Figure 8A–C). Inserts are typically 1–2 kb in length. While longer inserts are possible, HDR efficacy greatly decreases beyond 2–3 kb. The length of HAs depends on the length of the insert (~0.5 kb HAs per 1 kb insert), and typically ranges between 0.3 kb and 1 kb, although shorter HAs are possible. Here, we used 0.6 kb HAs. Recommended: Sequence the target region to ensure that no derivation from the germline is present and ensure that PAM sequences and splice acceptor sequences in downstream exons are removed from HAs in the HDR template. General sequencing primers are provided in Table 2 and a general PCR protocol is provided in Supplementary file. Determine gRNA efficacy for the region of interest through a T7 assay. The HDR template is ordered as double-stranded DNA gBlock and cloned in a pSmart vector (e.g., pSMART-HCKan, CloneSmart Blunt Cloning kit). Candidate gRNAs are ordered as single-stranded DNA oligos that are annealed and cloned in vectors from the pX330 family (Cong et al., 2013). A basic cloning protocol is available in Supplementary file. After design and cloning, perform midipreps of the plasmid DNA encoding the HDR and gRNA templates using the NucleoBond Xtra Midi Plus EF kit and sequence using standard primers. The A260:A280 ratio should be at least 1.8. We have developed plasmids enabling modification of the HC of rat IgG2a hybridoma to replace the complete constant region by a new isotype, cleave in the hinge and insert any tag of interest (Fab’ fragment) (van der Schoot et al., 2019), and to modify mIgG1 CH1 (depicted in Figures 2–6 and recorded in the video) and Cκ kappa LC of murine hybridoma (Le Gall et al., 2021). Our published plasmids are available on Addgene or upon request. Inserts between the HAs can easily be swapped using standard molecular cloning techniques and dedicated restriction sites. gRNAs are reported in Table 1 and HAs in Table 5 (notes). Table 1. gRNA used or mentioned in this protocol. HA sequences for the corresponding HDR templates are reported in Table 5. gRNA Target Sequence gRNA_iso_r2a Upstream of rat IgG2a CH1 exon TGTAGACAGCCACAGACTTG gRNA_r2a_H Upstream of rat IgG2a hinge exon GACTTACCTGTACATCCACA gRNA_m1_HC C-terminus of mouse IgG1 CTTGGTGCTGCTGGCCGGGT gRNA_mκ_LC C-terminus of mouse κ LC GGAATGAGTGTTAGAGACAA Culture of hybridoma cells and titration of antibiotic ahead of transfection Selection of cells that have successfully undergone HDR recombination is performed under antibiotic pressure. Ensuring quality of the parental cell line ahead of the procedure is critical (see Notes). The target hybridoma should be tested for antibiotic (blasticidin/puromycin) sensitivity prior to starting the experiment. Culture target hybridoma cells, either generated in house or obtained from the American Type Culture Collection, in complete culture media. Make sure to maintain cells at a high viability (> 90%). When confluent, seed 1 × 106 cells per well in a 6-well plate in 3 mL of culture medium and supplement with 0, 0.5, 1, 2, 4, 8, 10, or 20 μg/mL of the appropriate antibiotic. Incubate cells at 37 °C for 3–7 days depending on the selected antibiotic (three days for puromycin, seven days for blasticidin). Determine the lowest concentration at which no viable cells remain in the culture by using Trypan blue and use the determined concentration to perform the selection procedure (Figure 7A–B). Figure 7. Antibiotic titration, transfection, and expansion of resistant cells. A. Titration of puromycin on wild-type (WT) anti-human CD20 [NKI.B20/1]. Serial dilution of 0–12.5 μg/mL puromycin. After 48 h, cells start dying in the treated conditions. Healthy hybridoma cells are round and regular and grow in clusters. Dead or dying cells lose their round shape and become irregular. Dead cells can be spotted at 0.5 μg/mL, and some live cells remain up to 4 μg/mL. From 8 μg/mL on, cells are clearly dead. A trained eye can easily recognize how viable the cells are. B. To quantify the viability, Trypan blue staining allows to discern live (white) from dead (blue) cells. Here, we selected 8 μg/mL puromycin to conduct the procedure. C. AMAXA cuvette and program (Cell line SF, CQ-104). Carefully pipette the suspension containing cells and DNA between the electrodes (arrow head) without introducing air bubbles. D. After selection, small clusters start to grow out in HDR-transfected plates among a majority of dying or dead cells (day 6). These resistant cells have successfully undergone HDR. Passage the plates until larger clusters start to grow out (day 10) and grow towards confluency (day 13). In contrast, green fluorescent protein (GFP)-transfected hybridoma remain dead and no cluster can be seen (day 13, right). When the plate is full, perform a limiting dilution. E. After limiting dilution, monoclonal cell lines can be easily identified at the bottom of their respective well (arrow heads). Expand and characterize these cell lines. Transfection of hybridoma cells This section describes the electroporation of 1 × 106 wild-type hybridoma cells using gRNA/Cas9 + HDR template or green fluorescent protein (GFP) as a mock transfection control. Having cells resuspended for an extended amount of time in SF transfection medium from the SF Cell Line 4D-NucleofectorTM X kit Lis detrimental: work fast and organized and perform transfections sequentially. On day 0, use confluent wild-type hybridoma cells at a high viability (>90%) to perform the electroporation. Add 3 mL of culture medium to a 6-well plate and incubate at 37 °C. Pre-warm culture medium in a 50 mL Falcon. Spin down hybridoma cells from your desired cell line (1,500 rpm, 5 min, 4 °C). Resuspend cell pellet in an appropriate amount of wash buffer (aim for 1 × 106–2 × 106 cells/mL) Count the cells using Trypan blue and transfer 1 × 106 cells to a 1.5 mL Eppendorf tube. Spin down cell suspensions in a tabletop centrifuge [1,500 × g, 5 min, room temperature (RT)]. During this time, prepare two or any desired numbers of DNA mixes in 0.2 mL Eppendorf tubes, by pipetting 1 μg of Cas9/gRNA + 1 μg of HDR template per condition, and 2 μg of pMAX-GFP as mock transfection control (DNA mix tube). Prepare the 4D-Nucleofector with the parameters: Program CQ-104, Cell line SF. Carefully aspirate supernatant of the cell pellet. Add 100 μL of SF transfection medium to the cell pellet and resuspend cells before transferring into DNA mix tube containing HDR template + gRNA or pMAX-GFP. Resuspend and carefully transfer the suspension to the bottom of a 100 μL electroporation cuvette provided with the Lonza nucleofection kit (Figure 7C). Gently tap the cuvette to ensure that the sample covers the electrodes and the bottom of the cuvette and check that there are no air bubbles. Close the lid and insert the cuvette in AMAXA nucleofector. Press Start on the display to perform electroporation with the parameters: Program CQ-104, Cell line SF (Figure 7C). Carefully add 1 mL of pre-warmed medium to the cuvette and transfer cells immediately to the 6-well plate with pre-warmed medium (total volume in well is now ca. 4 mL). Avoid repeated aspiration of the cells. Wash cuvette with medium to make sure that all cells are transferred. Proceed to the next transfection (repeat from step 8). Incubate the 6-well plate at 37 °C in a humidified CO2 incubator. Selection of engineered hybridoma cells and generation of monoclonal cell lines This section describes the selection of successfully engineered hybridoma cells to identify HDR-edited clones. This is done by antibiotic selection pressure of HDR- and GFP-transfected hybridoma from day 3 post transfection. Resistant cells are grown to confluency and monoclonal cell lines can be generated. Transfected cells are cultured for two days in selection-free medium to allow electroporated cells to grow. On day 1, check transfected cells for viability (by eye) and the transfection control cells for GFP expression using a fluorescent microscope. Handle cells carefully to preserve viability. On day 3, either scrape the cells loose or resuspend until the cells detach and transfer all 4 mL of cell suspension to a 10 cm Petri dish containing 5 mL of pre-warmed culture medium supplemented with the appropriate selection drug at twice the predetermined concentration (2× selection medium). Wash the well with 1 mL of culture medium to collect all the cells and transfer to the Petri dish. After transferring the cells, the final volume in the Petri dish should be adjusted with culture medium to 10 mL to ensure that cells are in 1× selection medium. Starting on day 5, and two times per week, passage transfected cells. Briefly, scrape cells loose and transfer to 15 mL Falcon tubes. Place 5 mL of new pre-warmed 1× selection medium in the 10 cm Petri dish. Spin down (1,500 rpm, 5 min, RT) and discard supernatant. Resuspend cell pellets in 2 mL of pre-warmed 1× selection medium and transfer suspension back to the Petri dish. Wash the 15 mL Falcon with 3 mL of 1× selection medium and transfer back to the Petri dish. By day 5–10 depending on the antibiotic, it should be obvious that small clusters are forming among a majority of dead cells in the HDR/gRNA-transfected plate, while GFP-transfected cells are all dead (Figure 7D). If no cell death is observed, antibiotic concentration should be adjusted (see Notes). If no live cells grow out following antibiotic pressure, refer to the listed troubleshooting solutions (see Notes). Passage cells until these clusters grow out to reach higher confluency and viability (Figure 7D, >90%, typically around day 15–20). When the HDR-transfected cells are full, cells should still be all dead in the GFP plate. At this point, discard the GFP-transfected plate, and proceed to limiting dilution of HDR-transfected cells. After this step, antibiotic pressure is not required anymore. In case few clusters grow out very slowly or some cells are still alive in the GFP-transfected plate, it is best to keep the selected antibiotic in the medium until after identification of monoclonal cell lines. Pre-plate 100 μL per well of culture medium in five round bottom 96-well plates and incubate at 37 °C. Scrape confluent HDR-transfected cells loose and spin down the cell suspension (1,500 rpm, 5 min, RT). Keep a 1 mL sample of the supernatant and discard the rest. Resuspend cells in 5 mL of culture medium and count using Trypan blue. Dilute the cells in culture medium in two steps to obtain a 50 mL suspension at the concentration of 3 cells/mL. [Example: Initial suspension is at 1.5 × 106 cells/mL → dilution factor: 500,000 (1.5 × 106/3). First dilute 500×, 100 μL of cell suspension in 50 mL of culture medium (3,000 cells/mL). Then take 50 μL of cell suspension (3,000 cells/mL) and dilute 1,000× in 50 mL culture medium (concentration 3 cells/mL).] Plate 100 μL/well of the suspension in the round bottom 96-well plates. The final concentration is 0.3 cells/well in 200 μL or 33 cells/plate. From the original cell suspension keep ~500,000 cells for genomic DNA (gDNA) isolation (ISOLATE II Genomic DNA kit isolation kit). Spin down and follow manufacturer's instructions. Cryo-preserve leftover cells of the bulk population by following a standard cryo-preservation protocol. While the single-cell clones (SCC) are growing, the bulk population and its supernatant should be characterized immediately for integration of the HDR template. For this, follow section 1–3 in the Data analysis section. Expansion of modified hybridoma cells Incubate plates for 10–15 days at 37 °C without changing the medium. When SCC are visible at the bottom of the wells (Figure 7E), transfer them to a new flat bottom 96-well plate and add 100 μL of new culture medium. When cells are confluent, collect and freeze supernatant and expand the clones to a 24-well plate and then a T75 flask. Collect ~500,000 cells for gDNA isolation and cryo-preserve the expanded clones. Data analysis Characterization of selected clones (from bulk and/or SCC populations) Validation that the cell line is producing the right antibody (fragment) is crucial. Screening approaches should be conducted on the bulk population first to ensure success of the procedure and afterwards on the SCC. We typically combine three or more approaches to ensure insert integration and correct protein production: at the genetic, protein, and functional levels. Several examples are provided below to illustrate different methods. This straightforward protocol does not require statistical analysis up until functional characterization of the proteins. If resistant cells do not produce the modified antibodies, refer to the listed troubleshooting solutions (see Notes). To illustrate this section, we present the full characterization of three distinct cell lines modified using this technique. MIH5 is a rat IgG2a, λ hybridoma producing monoclonal antibodies binding to murine PD-L1, a protein expressed by cancer cells and myeloid cells. PD-L1 is a therapeutic target in the mAb-based treatment of cancers (checkpoint inhibitors). We engineered MIH5 cell lines that produce chimeric anti-PD-L1 antibodies with the mouse IgG2a (m2a) and mouse IgG2a L234A/L235A/N297A (m2a(silent)) isotypes (Figure 8A) (van der Schoot et al., 2019). NLDC-145 is a rat IgG2a, κ hybridoma producing monoclonal antibodies binding to murine DEC-205, a C-type lectin expressed by a subset of cross-presenting dendritic cells. Due to its restricted expression, DEC-205 is a promising target for developing targeted vaccines that efficiently deliver antigens to a restricted cell population able to best elicit a T cell response. We engineered NLDC-145 to produce truncated antibodies (Fab’ fragments) bearing CD8 T or CD4 T cell epitopes from the model antigen ovalbumin (OVA) (respectively Fab’OTI and Fab’OTII) (Figure 8B) (unpublished data by Fennemann et al., 2021). NKI.B20/1 is a mouse IgG1,κ hybridoma producing monoclonal antibodies binding to human CD20. CD20 is the target of Rituximab, a B cell–depleting mAb used to treat B-cell lymphoma, rheumatoid arthritis, or lupus. We generated an NKI.B20/1 cell line that produces a Fab’ fragment bearing two orthogonal site-selective modification sites on the HC and LC (Figure 8C): LAETGG-His-tag on the HC and a LPESGG-Myc-tag motif on the LC. This construct can be used to generate homogenous antibody–drug conjugates bearing two distinct payloads (Le Gall et al., 2021). Figure 8. Validation of three engineered cell lines at the genetic, protein, and functional level. A. HDR template and gRNA for isotype switching of rat IgG2a hybridoma to mouse IgG2a. B. HDR template structure and gRNA for rat IgG2a hybridoma to Fab’ antigen (Fab’Ag). Ag presented here, OTI and OTII peptide. C. HDR template and gRNA for editing of the LC of mouse IgG1 hybridoma. In this strategy, HC and LC are both edited to form a Fab’ fragment bearing two site-selective modification sites (LC editing shown here). D. PCR amplification of gDNA isolated from a m2a and m2a(silent) resistant clone using a location specific (1) and HDR specific (2) primer set. E. PCR amplification of gDNA isolated from Fab’OTI and Fab’OTII resistant clones using a location-specific (1) and HDR-specific (2) primer set. F. PCR amplification of gDNA isolated from a mκ resistant clone using a location-specific (1) and HDR-specific (2) primer set. G. After selecting monoclonal hybridoma for each isotype, supernatant was incubated with PD-L1–expressing target cells (CT26) and cells were stained with anti r2a, anti m2a, or anti-His-tag. Displayed plots demonstrate that supernatants exclusively contain the engineered isotype variant with a C-terminal His-tag, while the original rIgG2a mAbs is absent. H. Western blot of Fab’-OTI and -OTII, stained for His-tag (green) and rat IgG2a (blue). Protein G–isolated NLDC-145 antibodies were used as negative control (WT). I. SDS-PAGE in-gel fluorescence (Sypro staining) visualization of WT mAb CD20, Fab’HC, and Fab’HC+LC proteins shows a change in molecular weight after editing of the HC and LC. J. Representative sensograms display interactions of MIH5 WT and MIH5-engineered mAbs (WT r2a, m2a, m2asilent) for immobilized murine FcγR (FcγRI, FcγRIIb, and FcγRIV) at increasing concentrations (0.49–1000 nM). Binding to FcγR is expressed in resonance units (RU). K. In vitro antibody-dependent cellular cytotoxicity (ADCC) assay to compare effector function of MIH5 isotype variants. MC38 cells were 51Cr-labeled, opsonized with m2a or m2a(silent) MIH5 isotype variants, and exposed to whole blood from C57BL/6 mice for 4 h. Specific lysis was quantified by measuring 51Cr release (n = 3, mean ± SEM, p < 0.05) and shows that m2a induces specific lysis of PD-L1-expressing cells, with no activity of m2a(silent) in vitro. L. In vivo depletion assay. Splenic B cells labeled with Violet and Red tracer dye were used as target cells for in vivo depletion and opsonized with either m2a(silent) variant or m2a. Subsequently, B cells were mixed 1:1 (m2a:m2a(silent) or m2a(silent):m2a(silent)) and injected intravenously into C57BL/6 mice. Twenty-four hours later, spleens were isolated, and ratios between fluorescently labeled cell populations were determined via flow cytometry to quantify the isotype-specific depletion of target cells, showing that m2a and m2a(silent) are both functional in vivo (n = 3, mean ± SEM, * p < 0.05). M. DEC-205-expressing murine dendritic cells were generated from bone marrow using Flt3L and OP9-DL-1 feeder layer (Kirkling et al., 2018). At day 8, CD11c+ cells were isolated using CD11c microbeads and incubated with 1 μM Fab’OTI, 5 nM OVA257-263 (SIINFEKL, positive control) or 10 μM OVA. After 2 h, cells were washed and exposed to freshly isolated fluorescently labeled OTI CD8 T cells in media supplemented with 0.3 μg/mL lipopolysaccharide (LPS). After 72 h, cells were analyzed by flow cytometry for proliferation (cell dye dilution) and activation markers (not shown), and supernatant was analyzed by ELISA for production of pro-inflammatory IFNγ and IL-2 cytokines. Fab’OTI-treated dendritic cells induced CD8 T cell proliferation and activation, showing that it was taken up by DEC-205-expressing dendritic cells and processed, and that the CD8 T cell epitope was presented on class I major histocompatibility complex (MHC) (n = 3, mean ± SD, ns: not significant, **** p < 0.0001, one-way ANOVA with Tukey’s correction for multiple testing). N. Day 8 DEC-205-expressing murine dendritic cells were incubated with 1 μM Fab’OTII, 1 μM OVA323-339 (ISQAVHAAHAEINEAGR, positive control), or 10 μM OVA. After 2 h, cells were washed and exposed to freshly isolated fluorescently labeled OTII CD4 T cells in media supplemented with 0.3 μg/mL LPS. After 72 h, cells were analyzed by flow cytometry for proliferation and activation marker expression (not shown) and supernatant was analyzed by ELISA for production of pro-inflammatory IFNγ and IL-2 cytokines. Fab’OTII-treated dendritic cells induced CD4 T cell proliferation and activation, showing that it was taken up by DEC-205-expressing dendritic cells and processed, and that the CD4 T cell epitope was presented on class II MHC (n = 3, mean ± SD, ns: not significant, * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s correction for multiple testing). O. SDS-PAGE analysis of Fab′CD20 sequentially site-specifically labeled with H-GGG-C-K(FITC)-NH2 on the HC and H-GGG-K(N3)-NH2 on the LC confirms introduction of two distinct cargos site-specifically onto the engineered Fab′ and functionality of the introduced motifs. P. Antigen binding competition assay of each engineered protein against mAbNKI.B20/1-AF647 reveals that proteins do not lose binding affinity to their target following CRISPR/Cas9 editing and sequential dual site-specific labeling. D, G, J, K, L. Adapted from van der Schoot et al. (2019) E, H, M, N. Adapted from Fennemann et al. (2021) F, I, O, P. Adapted from Le Gall et al. (2021). B: blasticidin resistance gene, FITC: Fluorescein isothiocyanate, H: hinge, H6: hexahistidine tag (HHHHHH), HA: homology arm, HC: heavy chain, HDR: homology directed repair, I: internal ribosomal entry site (IRES), IFNγ: interferon gamma, IL-2: interleukin-2, LC: light chain, myc: myc-tag (EQKLISEEDL), OVA: ovalbumin, P: puromycin resistance gene, pA: poly A tail, PAM: protospacer adjacent motif, PD-L1: programmed death ligand 1, PEG: polyethylene glycol, srt: sortag motif, WT: wild type. PCR amplification of target locus to determine HDR integration PCR screens should be performed on both bulk and SCC populations. For this, use the isolated gDNA from ~500,000 cells and perform amplification of the targeted locus using a primer that only anneals to the inserted sequence in combination with a location-specific primer that anneals around the insertion site outside the HA region. Use ~100 ng of gDNA to amplify the target locus using a standard PCR program (provided in Supplementary file). Table 2 provides sequences of primers that can be used for amplification of murine IgG1 HC, murine kappa LC, and rat IgG2a CH1 HC upon insertion of our HDR templates. Table 2. Locus-specific and HDR-specific for PCR amplification of target region Primer Target 5′-3′ Sequence r2a_iso_fw rat IgG2a upstream of CH1 (locus-specific) GGCGACCTGTAACAACTTGG r2a_CH1_fw rat IgG2a CH1 exon (locus-specific) TGTAGGAGCTTGGGTCCAGA m1_CH1_fw mouse IgG1 CH1 (locus-specific) GTGCCGACTTCAATGTGCTT mκ_LC_fw mouse kappa light chain (locus-specific) GTGCTTGTGTTCAGACTCCC blast_rv blasticidin resistance gene (HDR-specific) ATACATTGACACCAGTGAAGATGC ires_rv IRES (HDR-specific) GGCTTCGGCCAGTAACGTTA polyA_rv poly A tail (HDR-specific) CATAGAGCCCACCGCATCCC Run PCR products on a 1% (w/v) agarose gel and visualize DNA using Nancy-520 (1:15,000). Integration of the insert in the target region is confirmed by the presence of a single band at the expected height for the engineered MIH5 [r2a>m2a and r2a>m2a(silent) (r2a_iso_fw + blast_rv, Figure 8D)], NLDC-145 (r2a>Fab’OTI and r2a>Fab’OTII (r2a_HC_fw + blast_rv, Figure 8E)), and NKI.B20/1 [mκ> mκ-srt-myc (mκ_LC_fw + ires_rv, Figure 8F)] cell lines. The band at the correct height should be excised and gel-purified to perform Sanger sequencing and confirm correct integration. Flow cytometry analysis of hybridoma supernatant A flow cytometry screening can be performed to investigate the expression of e.g., a His-tag/Myc-tag, HC, LC, or a certain isotype. For a first screen, incubate 50 μL of supernatant of the bulk population or the SCC with 50,000 target-expressing cells for 20 min at 4 °C. After two washes with PBA, proceed with secondary staining using the antibodies indicated in Table 3 to stain the desired part of the produced antibodies (isotype, tag, etc.). Table 3. Secondary antibodies used to analyze hybridoma production by flow cytometry Target Clone Fluorophore Company Cat. number Dilution 6×His-tag J095G46 PE Biolegend 362603 25 rat IgG2a HC MRG2a-83 PE Biolegend 407507 400 mouse IgG2a m2a-15F8 PE eBioscience 12-4817-82 500 For a second screen aimed at selecting high-producing clones, use a similar workflow but include serial dilutions of the supernatants of positive clones. It is advised to take along a dilution of known concentrations of the parental antibody to calculate the amount of antibody in the samples. We modified r2a-producing MIH5 cell lines to produce m2a and m2a(silent) isotypes and incubated undiluted supernatant of SCC with PD-L1-expressing cells (Figure 8G). Flow cytometry analysis shows production of the original isotype by the initial cell line but not selected clones, and production of a m2a isotype with a His-tag, attesting for proper integration. Western blot analysis of hybridoma supernatant A flow cytometry screening can be performed to investigate the expression of e.g., His-tag/Myc-tag, heavy chain, light chain, or a certain isotype without requiring high numbers of target-expressing cells. Binding to the target needs to be separately confirmed on selected clones. We typically conduct a first screen using a dot blot. For this, take 3 μL of the supernatant of each single cell clone and spot on a nitrocellulose membrane using a 96-well plate as a guide. Let the nitrocellulose membrane dry for 30 min and proceed with the blocking step and secondary staining of a general western blot protocol, using the antibodies indicated in Table 4 to stain the desired part of the produced antibodies (isotype, tag, etc.). For a second screen, use diluted supernatant of positive clones to confirm production of the correct protein using a traditional western blot. Here, we analyzed the supernatant of NLDC-145 engineered cell lines to produce Fab’OTI and Fab’OTII using anti-rIgG2a (H+L) and anti-His-tag (Figure 8H). The results show production of a HC of 25 kDa bearing a His-tag over the wild-type (WT) HC of 50 kDa, confirming HDR insertion. Alternatively, SDS-PAGE analysis can confirm production of the right product (Figure 8I). Here, we sequentially modified NKI.B20/1 parental cell line into a cell line producing a Fab’ fragment bearing two site-specific modification sites, on its heavy and light chains. Following the first and second step of modification, we respectively detected a shift downwards of the HC from 50 to 25 kDa (HC WT > HC Fab’-srt-his), and a shift upwards of the LC (LC WT > LC-srt-myc) (Figure 8I). Table 4. Antibodies used to characterize presence of the right insert Target Clone Fluorophore Company Cat. number Dilution rabbit anti 6×His-tag RM146 unconjugated Abcam AB14923 1,000 goat anti-rabbit IgG (H + L) polyclonal IRD800 LI-COR 926-32211 5,000 goat anti-rat IgG (H + L) polyclonal AF680 Thermo Fisher A-21096 5,000 Confirming production of a functional protein Confirmation that the selected clone produces functional antibodies is paramount to validate the selected cell line. Production of high levels of modified antibody Based on genomic screen and confirmation at the protein level, one clone can be selected based on its production and growth rate and used to produce high levels of the modified antibody. For this, expand the selected clone to a 5-layer flask or CELLineCL 1000 bioreactors, following the manufacturer’s instructions. Isolation of modified antibody Isolation methods depend on the nature of the insert. The HDR template can be designed to include a purification tag such as His-tag or Myc-tag, for which affinity resins are available (e.g., Ni-NTA agarose). Antibodies that do not contain such tags can be isolated using e.g., protein G resin, in which case the media needs to be serum-free (CD hybridoma medium). Functional assays Assays needed to confirm antibody functionality typically depend on the nature of the modification, target, and intended use. Here, we provide examples of functional assays we conducted to validate the cell lines presented. These results illustrate that CRISPR/Cas9 knock in in the immunoglobulin locus conserves the specificity of the antibodies, and that the inserts are functional. We generated MIH5 cell lines that produce mAbs of the m2a and m2a(silent) isotypes. To confirm functionality of the isotype, we conducted surface plasmon resonance analysis of purified mAbs on immobilized murine fragment crystallizable gamma receptors (FcyR) (Figure 8J). As expected, m2a antibodies bind to murine FcyR, and binding is abrogated with the mutation introduced in m2a(silent). In addition, we performed in vitro killing of PD-L1-expressing cancer cells opsonized with the different isotypes (Figure 8K). Incubation with whole blood from C57BL6/J mice showed specific lysis of target cells for m2a, but not r2a (WT) and m2a(silent). Similarly, in vivo killing of splenocytes opsonized with m2a or m2a(silent) (Figure 8L) reveals specific killing of m2a-treated cells over m2a(silent). Together, these results validate the functionality of the novel chimeric antibodies (van der Schoot et al., 2019). The engineered NLDC-145 cell lines produce Fab’OTI and Fab’OTII that can be used to specifically deliver antigens to DEC-205-expressing dendritic cells (DCs). OTI and OTII antigens are respectively recognized by CD8 OTI and CD4 OTII primary T cells upon presentation on MHC molecules by DCs. We incubated DEC-205-expressing primary murine dendritic cells for 2 h with the constructs, short peptides (positive control, OVA257-264 or OVA323-339) that do not need processing, or OVA protein. We then exposed DCs to antigen-specific CD8 or CD4 T cells (Figure 8M-N) that were labeled with Cell Trace Violet (CTV). CD8 (Figure 8M) and CD4 (Figure 8N) T-cell activation was measured by means of proliferation (CTV dilution by flow cytometry), activation maker expression, and pro-inflammatory cytokine production in the supernatant [interleukin-2 (IL-2), interferon gamma (IFNγ) by ELISA]. CD8 and CD4 T cells proliferated and produced pro-inflammatory cytokines upon recognition of their cognate antigen by DCs exposed to short peptide and to the Fab’ antigen fusions. This confirmed that the Fab’ antigen fusion proteins retain their ability to be processed and presented by DCs, and potently induce T cell activation (unpublished data by Fennemann et al). Lastly, we generated a NKI.B20/1 cell line that produces a Fab’ fragment bearing orthogonal site-selective modification sites on both HC and LC at their C-terminus (Le Gall et al., 2021). To confirm functionality of the orthogonal sites, we sequentially modified the HC to introduce fluorescein isothiocyanate and the LC to introduce an azide group, as confirmed on fluorescent SDS-PAGE (Figure 8O). We finally confirmed that site-specific modification did not alter binding to the target by performing a competitive binding assay on CD20-expressing target cells (Figure 8P). Notes We have successfully applied this protocol to various hybridoma cell lines. We advise operators to get a good sense of the growth rate, production levels, and viability of the target cell lines before starting genetic engineering. We found that the following details can help refining the procedure: Ensuring quality of the parental cell line Hybridoma may lose production capacity over time or have a low viability, which severely impairs the efficacy of the procedure. To address this, we advise operators to subclone the parental line by performing a limiting dilution (as described in section E) and to quantify viability and WT antibody production of SCCs to select a new monoclonal line. This ensures selection of a highly viable parental clone with high production. Performing subcloning on the parental cell line also confirms that SCC can grow. If viability of the parental line does not improve, adjust culture conditions, for instance by removing β-ME (ensure that antibody production is not impaired) or increasing FBS concentration in the medium, spinning down cells at 800 rpm for 10 min instead of 1,500 rpm for 5 min, or removing dead cells using a density gradient such as Lymphoprep. Confirm sensitivity to antibiotic pressure of the parental line Normally, hybridoma lines do not have resistance to selection antibiotics, and we do not need to perform antibiotic titration of mock-transfected hybridoma. It might not be the case for every cell line, and in some situations, titration needs to be performed on mock-transfected hybridoma. If this is the case, follow section D to mock-transfect the cell line, then perform titration as indicated in section C. Adjusting concentration of the selection drug during the procedure Cell death starts around day 2–3. For puromycin, cells should be dead by day 7, and for blasticidin by day 10. Upon antibiotic pressure, we typically observe massive cell death in both GFP and HDR-transfected plates. If this is not the case, the concentration of the selection drug is too low. This might happen even after titration, and operators should increase the concentration. If cells do not grow out following antibiotic pressure If transfected cells do not sustain antibiotic pressure, try the following solutions. Check hybridoma viability prior to transfection and GFP transfection efficacy. Sequence the target region to ensure that no mutations are present that could affect the gRNA/HDR efficiency. Perform a T7 assay to determine gRNA ability to cut the target locus and try other gRNAs if necessary. Test for mycoplasma contamination. Synchronize the cells prior to transfection by starving (low/no FBS). Incorporate a non-homologous end joining inhibitor such as nocodazole to favor HDR. If SCCs do not grow out after limiting dilution While we have never encountered this issue, some cell lines might not grow out after limiting dilution. To solve this, doubling the FBS concentration in the culture medium can rescue single cells. Additionally, using conditioned medium to perform limiting dilution can help single cells to survive. To generate conditioned medium, plate ~1 × 106 cells from the parental line in 10 cm Petri dishes. Twenty-four or forty-eight hours later, depending on the growth rate, centrifuge the supernatant and pass it through a 0.45 µm filter. If resistant cells do not produce (only) modified antibodies Among resistant clones, we usually find a high fraction of correctly edited clones producing high amounts of antibodies (50%–90%). We have found sporadic SCC that resist antibiotic pressure and show correct integration of the insert, but produce WT antibodies, which we attribute to exceptions in allelic exclusion. Typically, other SCCs will produce the right protein and should be selected instead. However, if all SCCs surviving antibiotic pressure show integration of the insert in the target region by PCR but do not secrete modified antibodies, the HDR template might require adjustments. For instance, due to alternative splicing, SCCs can resist antibiotic pressure but produce both modified and WT antibodies, or only WT antibodies. To address this, remove splice acceptor sequences for exons downstream of the insert in 3′ Has, and insert a synthetic splice acceptor sequence (such as GCTAGCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAG) upstream of the synthetic exon. Especially if knocked in upstream of an exon, make sure that the insert is in frame with the variable sequence after splicing. Table 5. Homology arms used in this protocol. Bold: mutated PAM sequences to inactivate Cas9 after successful HDR recombination. Alternatively, complete excision in the donor sequence is possible (e.g., Fab r2a_HC). Underlined: original splice acceptor site upstream of rIgG2a CH1 exon: remove or mutate in HDR template to prevent alternative splicing of variable region with the original isotype. Homology arms 5′-3′ Sequence 5′ HA r2a_iso (rIgG2a WT > mIgG2a) AGAAAGATCTGAGTAGAACCAAGGTAAAAAGTGTGGGTAAAAACACATGTTCACAGGCCTGGCTGACATGATGCTGGGCACGTATGGAGGCAAAGTCAAGAGGGCAGTGTAAGGGCCAGAAGTGAATCCTGACCCAAGAATAGAGAGTGCTAAACCTACGTAGATCGAAGCCAACTAAAAAGACAAGCTACAAAACGAAGCTAAGGCCAGAGATCTTGGACTGTGAAGAGTTCAGAGAACCTAGGATCAGGAACCATTAGTAACAGGCCAAGGAAGATAGAAGCTGCCTAGGACTTGGCAAGAGCCAACATGGTTGGACTGGAAAAGAAAGGAGGAGACAGAAGACAGGAGAGATGTGCCAACTTGATTTTGGGCTTCACTGTTGTCCATACTGTGTGCAGCCATATGGCCCACAGATAACAGGTTTAGCCGAGGAACACAGATACCCACATTGGACAATGGTGGGGGAACACAGATACCCATACTACAGGGCTCTTTAGGGCATTTCCTGAAAGTGTACTAGGAGTGGGACTGGGCTCAAAGGGATTAGGTGTGATCTGGCCTGGTGAGGCTGACATTGGCAAGCCCAATGGTTGGGTGTTGCCTCCTCCATGT 3′ HA r2a_iso (rIgG2a WT > mIgG2a) TGTACAACTTGGGGAGGGTACAAAATGGAGGACTTGTAGGAGCTTGGGTCCAGACCTGTCAGACAAAATGATCACGCATACTTATTCTTGTAGCTGAAACAACAGCCCCATCTGTCTATCCACTGGCTCCTGGAACTGCTCTCAAAAGTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTCACCGTGACCTGGAACTCTGGAGCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGGACTCTACACTCTCACCAGCTCAGTGACTGTACCCTCCAGCACCTGGTCCAGCCAGGCCGTCACCTGCAACGTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGGTGAGAGAACAACCAGGGGATGAGGGGCTCACTAGAGGTGAGGATAAGGCATTAGATTGCCTACACCAACCAGGGTGGGCAGACATCACCAGGGAGGGGGCCTCAGCCCAGGAGACCAAAAATTCTCCTTTGTCTCCCTTCTGGAGATTTCTATGTCCTTTACACCCATTTATTAATATTCT 5′ HA r2a_HC (rIgG2a WT > Fab’ fragment) CCTGGAACTCTGGAGCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGGACTCTACACTCTCACCAGCTCAGTGACTGTACCCTCCAGCACCTGGTCCAGCCAGGCCGTCACCTGCAACGTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGGTGAGAGAACAACCAGGGGATGAGGGGCTCACTAGAGGTGAGGATAAGGCATTAGATTGCCTACACCAACCAGGGTGGGCAGACATCACCAGGGAGGGGGCCTCAGCCCAGGAGACCAAAAATTCTCCTTTGTCTCCCTTCTGGAGATTTCTATGTCCTTTACACCCATTTATTAATATTCTGGGTAAGATGCCCTTGCATCATGACATACAGAGGCAGACTAGAGTATCAACCTGCAAAAGGTCATACCCAGGAAGAGCCTGCCATGATCCCACACCAGAACCAACCTGGGGCCTTCTCACCTATAGACCATACTAACACACAGCCTTCTCTCTGCA 3′ HA r2a_HC (rIgG2a WT > Fab’ fragment) GGTAAGTCACTAGGACTATTACTCCAGCCCCAGATTCAAAAAATATCCTCAGAGGCCCATGTTAGAGGATGACACAGCTATTGACCTATTTCTACCTTTCTTCTTCATCTACAGGCTCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGACCAAAGATGTGCTCACCATCACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATTAGCCAGAATGATCCCGAGGTCCGGTTCAGCTGGTTTATAGATGACGTGGAAGTCCACACAGCTCAGACTCATGCCCCGGAGAAGCAGTCCAACAGCACTTTACGCTCAGTCAGTGAACTCCCCATCGTGCACCGGGACTGGCTCAATGGCAAGACGTTCAAATGCAAAGTCAACAGTGGAGCATTCCCTGCCCCCATCGAGAAAAGCATCTCCAAACCCGAAGGTGGGAGCAGCAGGGTGTGTGGTGTAGAAGCTGCAGTAGGCCATAGACAGAGCTTGACTTAACTAGACTT 5′ HA m1_HC (mIgG1 WT > Fab’ fragment) CAGTATTGTCCAGATTGTGTGCAGCCATATGGCCCAGGTATAAGAGGTTTAACAGTGGAACACAGATGCCCACATCAGACAGCTGGGGGGCGGGGGTGAACACAGATACCCATACTGGAAAGCAGGTGGGGCATTTTCCTAGGAACGGGACTGGGCTCAATGGCCTCAGGTCTCATCTGGTCTGGTGATCCTGACATTGATAGGCCCAAATGTTGGATATCACCTACTCCATGTAGAGAGTCGGGGACATGGGAAGGGTGCAAAAGAGCGGCCTTCTAGAAGGTTTGGTCCTGTCCTGTCCTGTCTGACAGTGTAATCACATATACTTTTTCTTGTAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCCAGACCGTCACCTGCAACGTTGCTCATCCGGCATGTAGCACCAAGGTAGACAAAAAGATA 3′ HA m1_HC (mIgG1 WT> Fab’ fragment) GGTGAGAGGACGTATAGGGAGGAGGGGTTCACTAGAGGTGAGGCTCAAGCCATTAGCCTGCCTAAACCAACCAGGCTGGACAGCCATCACCAGGAAATGGATCTCAGCCCAGAAGATCGAAAGTTGTTCTTCTCCCTTCTGGAGATTTCTATGTCCTTTACACTCATTGGTTAATATCCTGGGTTGGATTCCCACACATCTTGACAAACAGAGACAATTGAGTATCACCAGCCAAAAGTCATACCCAAAAACAGCCTGGCATGACCTCACACCAGACTCAAACTTACCCTACCTTTATCCTGGTGGCTTCTCATCTCCAGACCCCAGTAACACATAGCTTTCTCTCCACAGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGGTAAGTCAGTAGGCCTTTCACCCTGACCCCAGATGCAACAAGTGGCCATGTTAGAGGGTGGCCCAGGTATTGACCTATTTCCACCTTTCTTCTTCATCCTTAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGT 5′ HA mκ_LC (mκ LC > mκLC-tag) TCTGTCTGAAGCATGGAACTGAAAAGAATGTAGTTTCAGGGAAGAAAGGCAATAGAAGGAAGCCTGAGAATATCTTCAAAGGGTCAGACTCAATTTACTTTCTAAAGAAGTAGCTAGGAACTAGGGAATAACTTAGAAACAACAAGATTGTATATATGTGCATCCTGGCCCCATTGTTCCTTATCTGTAGGGATAAGCGTGCTTTTTTGTGTGTCTGTATATAACATAACTGTTTACACATAATACACTGAAATGGAGCCCTTCCTTGTTACTTCATACCATCCTCTGTGCTTCCTTCCTCAGGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCGATTGTAAAGAGTTTTAATAGCAATGAATGCGGATCC 3′ HA mκ_LC (mκ LC > mκLC-tag) TGAAGACAAAGATCCTGAGACGCCACCACCAGCTCCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTTTATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCACTTGAGATCTCTGTCTTTCTTACTAAATGGTAGTAATCAGTTGTTTTTCCAGTTACCTGGGTTTCTCTTCTAAAGAAGTTAAATGTTTAGTTGCCCTGAAATCCACCACACTTAAAGGATAAATAAAACCCTCCACTTGCCCTGGTTGGCTGTCCACTACATGGCAGTCCTTTCTAAGGTTCACGAGTACTATTCATGGCTTATTTCTCTGGGCCATGGTAGGTTTGAGGAGGCATACTTCCTAGTTTTCTTCCCCTAAGTCGTCAAAGTCCTGAAGGGGGACAGTCTTTACAAGCACATGTTCTGTAATCTGATTCAACCTACCCAGTAAACTTGGCGAAGCAAAGTAGAATCATTATCACAGGAAGCAAAGGCAACCTAAATGTGCA Recipes Wash buffer PBS + 0.5% BSA PBA PBS + 5% BSA + 0.03% NaN3 Culture medium RPMI-1640 (formulation with HEPES) + 10% heat-inactivated FBS + 2 mM ultraglutamine-1 + 1% (v/v) antibiotic-antimycotic + freshly added 50 μM β-ME. 1× selection medium RPMI-1640 (formulation with HEPES) + 10% heat-inactivated FBS + 2 mM ultraglutamine + 1% (v/v) antibiotic-antimycotic + freshly added 50 μM β-ME + freshly added selection antibiotic at pre-determined concentration. Acknowledgments M.V. was the recipient of ERC Starting Grant CHEMCHECK (679921) and a Gravity Program Institute for Chemical Immunology tenure track grant by NWO. F.A.S. was the recipient of an LUMC Strategic Fund (#049–19). This work was derived from two papers from our research group previously published by van der Schoot et al. (2019) and Le Gall et al. (2021). We thank Arthur E. H. Bentlage and Gestur Vidarsson (Sanquin, Amsterdam, The Netherlands) for collecting the surface plasmon resonance data (van der Schoot et al., 2019). Competing interests The authors declare no competing interest. Ethics Work described in this protocol was partially performed in animals, which was approved by the local authority for the Ethical Evaluation of Animal Experiments and Animal Welfare (Instantie voor Dierenwelzijn Radboudumc). Mice were kept in accordance with federal and state policies on animal research, and Annex III of the EU Directive (Directive 2010-63-EU). References Adhikari, P., Zacharias, N., Ohri, R. and Sadowsky, J. (2020). Site-Specific Conjugation to Cys-Engineered THIOMABTM Antibodies. In: Tumey, L. (Ed.). Antibody-Drug Conjugates. Methods in Molecular Biology. pp. 51-69. Beers, S. A., Glennie, M. J. and White, A. L. (2016). Influence of immunoglobulin isotype on therapeutic antibody function. Blood 127(9): 1097-1101. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339(6121): 819-823. Evers, M., Stip, M., Keller, K., Willemen, H., Nederend, M., Jansen, M., Chan, C., Budding, K., Nierkens, S., Valerius, T., et al. (2021). Anti-GD2 IgA kills tumors by neutrophils without antibody-associated pain in the preclinical treatment of high-risk neuroblastoma. J Immunother Cancer 9(10): e003163. Fennemann, F. L., Le Gall, C. M., van der Schoot, J. M. S., Ramos-Tomillero, I., Figdor, C. G., Scheeren, F. A., and Verdoes, M. (2021). Generation of dendritic cell targeted anti-cancer vaccines by CRISPR- Cas9-editing of the hybridoma immunoglobulin locus. In: Molecularly Defined Dendritic Cell-Targeted Vaccines for Cancer Immunotherapy. pp. 42-63. Le Gall, C. M., van der Schoot, J. M. S., Ramos-Tomillero, I., Khalily, M. P., van Dalen, F. J., Wijfjes, Z., Smeding, L., van Dalen, D., Cammarata, A., Bonger, K. M., et al. (2021). Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering. Bioconjug Chem 32(2): 301-310. Khoshnejad, M., Brenner, J. S., Motley, W., Parhiz, H., Greineder, C. F., Villa, C. H., Marcos-Contreras, O. A., Tsourkas, A. and Muzykantov, V. R. (2018). Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9. Sci Rep 8(1): 1760. Kim, H. K., Lee, S., Kim, Y., Park, J., Min, S., Choi, J. W., Huang, T. P., Yoon, S., Liu, D. R. and Kim, H. H. (2020). High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells. Nat Biomed Eng 4(1): 111-124. Kirkling, M. E., Cytlak, U., Lau, C. M., Lewis, K. L., Resteu, A., Khodadadi-Jamayran, A., Siebel, C. W., Salmon, H., Merad, M., Tsirigos, A., et al. (2018). Notch Signaling Facilitates In Vitro Generation of Cross-Presenting Classical Dendritic Cells. Cell Rep 23(12): 3658-3672 e3656. Köhler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517): 495-497. Nanda, J.S. and Lorsch, J.R. (2014). Labeling a protein with fluorophores using NHS ester derivitization. Methods Enzymol 536: 87-94. van der Schoot, J. M. S., Fennemann, F. L., Valente, M., Dolen, Y., Hagemans, I. M., Becker, A. M. D., Le Gall, C. M., van Dalen, D., Cevirgel, A., van Bruggen, J. A. C., et al. (2019). Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering. Sci Adv 5(8): eaaw1822. Sharma, A., Subudhi, S. K., Blando, J., Scutti, J., Vence, L., Wargo, J., Allison, J. P., Ribas, A., and Sharma, P. (2019). Anti-CTLA-4 immunotherapy does not deplete FOXP3+ regulatory T cells (Tregs) in human cancers.Clin Cancer Res 25(4): 1233-1238. Waldor, M. K., Mitchell, D., Kipps, T. J., Herzenberg, L. A., and Steinman, L. (1987). Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody. J Immunol 139(11): 360-3664. Yamada, K., Shikida, N., Shimbo, K., Ito, Y., Khedri, Z., Matsuda, Y. and Mendelsohn, B. A. (2019). AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Angew Chem Int Ed Engl 58(17): 5592-5597. Zhao, Y., Gutshall, L., Jiang, H., Baker, A., Beil, E., Obmolova, G., Carton, J., Taudte, S. and Amegadzie, B. (2009). Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells. Protein Expr Purif 67(2): 182-189. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Immunology > Antibody analysis > Antibody modification Cell Biology > Cell engineering > CRISPR-cas9 Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Efficient Large DNA Fragment Knock-in by Long dsDNA with 3′-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells Wenjie Han [...] Jianqiang Bao Oct 20, 2023 1161 Views Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing Beverly Naigles [...] Nan Hao Mar 20, 2024 2172 Views Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1 Haiting Ma Nov 20, 2024 461 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,614	https://bio-protocol.org/en/bpdetail?id=4614&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells KG Kirin D. Gada JK Jordie M. Kamuene TK Takeharu Kawano LP Leigh D. Plant Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4614 Views: 768 Reviewed by: Chiara AmbrogioSoumya Moonjely Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry May 2022 Abstract The cell surfaceome is of vital importance across physiology, developmental biology, and disease states alike. The precise identification of proteins and their regulatory mechanisms at the cell membrane has been challenging and is typically determined using confocal microscopy, two-photon microscopy, or total internal reflection fluorescence microscopy (TIRFM). Of these, TIRFM is the most precise, as it harnesses the generation of a spatially delimited evanescent wave at the interface of two surfaces with distinct refractive indices. The limited penetration of the evanescent wave illuminates a narrow specimen field, which facilitates the localization of fluorescently tagged proteins at the cell membrane but not inside of the cell. In addition to constraining the depth of the image, TIRFM also significantly enhances the signal-to-noise ratio, which is particularly valuable in the study of live cells. Here, we detail a protocol for micromirror TIRFM analysis of optogenetically activated protein kinase C-ϵ in HEK293-T cells, as well as data analysis to demonstrate the translocation of this construct to the cell-surface following optogenetic activation. Graphic abstract Keywords: Total internal reflection fluorescence microscopy TIRFM Fluorescence Membrane proteins Optogenetics Background Total internal reflection (TIR) occurs when the angle of incidence of a beam of light upon a refractive surface exceeds a certain threshold, called the critical angle, resulting in reflection of the incident beam of light. While this implies an absence of transmission of light across the interface, there is an escape of electromagnetic energy in the form of an evanescent wave. Total internal reflection fluorescence microscopy (TIRFM) relies on this exponentially decaying evanescent wave that penetrates to a depth of ~100 nm into the sample area. Given that TIR occurs at the interface of two materials with distinct refractive indices, in cell biology experiments the result is that the evanescent wave will only excite fluorescence capable moieties in the evanescent field at or near the cell surface. Thus, TIRFM illuminates fluorescent particles at the cell membrane below the Rayleigh limit for resolution, making the technique well suited for the study of membrane proteins and membrane-delimited signaling events, in fixed or live cell samples. Several types of microscopy are utilized in the literature to achieve optical sectioning with the intent to localize fluorescent biological moieties to various cellular fractions. Confocal microscopy is a common and versatile technique that has the potential to target any plane of the sample rather than only the glass/liquid interface, as in TIRFM. As with any microscopy, the resolution is determined by the lateral (X-Y) and axial dimensions of the imaging voxel (the Z-plane of a 3-dimensional pixel), each of which is dependent upon numerous factors. Thus, the resolution of the image is determined by the hardware (lens and detector), software, and sample preparation, as well as the fluorophore (protein or dye) and the light source in use. Commonplace confocal microscopes typically operate with a lateral dimension above the Abbe limit of ~250 nm. However, adaptations such as Airy disc scanners and stimulated emission dyes that form the basis of many super resolution techniques can reduce the lateral dimension to below 100 nm. Technologies such as Airy disc confocal, stimulated emission depletion, photoactivated localization microscopy, and stochastic optical reconstruction microscopy have yielded unprecedented information about subcellular architecture and protein localization (Vangindertael et al., 2018). However, they typically require a fixed sample (particularly where specific emission dyes must be employed) and have lower axial resolution (equivalent to the depth of the slice of up to ~500 nm) (Axelrod, 2001) and imaging speeds that are below the biological timescale. The issue of low axial resolution can be improved with two-photon localization microscopy, which, in some cases, can be combined with super resolution in the X-Y plane (Zong et al., 2017). However, this suite of tools is typically expensive and requires specialized hardware, software, and sample preparation. In contrast, TIRFM provides precise localization of fluorescence moieties, at or close to the cell membrane (axial resolution below ~100 nm). In addition to being commercially available, TIRFM systems can be homemade and constructed to image a range of user-preferred fluorophores, including genetically encoded fluorescent proteins, in live cells at relatively high speed in a cost-effective manner. Therefore, TIRFM is the imaging modality of choice to study membrane-delimited proteins with a high signal-to-noise ratio in fixed and live samples. Materials and Reagents Lens cleaning tissue (Thor Labs, catalog number: MC-50E) #1.5 coverslip (Warner Instruments, catalog number: CS-15R) 35 mm tissue culture-treated dishes (Fisherbrand, Fisher Scientific, catalog number: FB012920) 60 mm tissue culture-treated dishes (Fisherbrand, Fisher Scientific, catalog number: FB012921) Immersion oil (Cargille Laboratories, catalog number: 16241) Dulbecco’s modified Eagle medium (ATCC, catalog number: 30-2002) OptiMEM (Gibco, catalog number: 02634) 0.05% trypsin (Cytiva, catalog number: SH30236.01) Polyethylenimine (PEI) (Polysciences, catalog number: 24765-1) Human embryonic kidney 293-T cells (ATCC, catalog number: CRL-3216) Fetal bovine serum (R&D Systems, catalog number: S12450) Penicillin/streptomycin (Cytiva, catalog number: SV30010) CIBN-CAAX (Dr. Pietro De Camilli, Addgene ID# 79574) mCherry-CRY2-5PtaseOCRL (Dr. Pietro De Camilli, Addgene ID# 66836) mCherry-CRY2–mPKCϵ-CAT-HA (Gada et al., 2022, Addgene ID# 190483) NaCl (Fisher, catalog number: S271) KCl (Fisher, catalog number: P217) MgCl2 (Fisher, catalog number: M33) CaCl2 (Fisher, catalog number: C79) HEPES (Oakwood Chemical, catalog number: 047861) TIRF imaging solution (see Recipes) Equipment Lasers (Coherent, OBIS LX445 1185051, OBIS LS 561 1253301) Camera (Teledyne Photometrics, Prime95B) Stage (Mad City Labs, X-Y-Z nanopositioning, model: Nano-LPS) Any in-house or commercial microscope frame that can be modified to hold micromirrors below the objective lens. We use an RM21 frame from Mad City Labs Inc. Micromirrors are available from Mad City Labs High numerical aperture (NA) objective lens (e.g., Olympus 60×, 1.5 NA from Olympus) Quick release magnetic sample holder (Warner Instruments, model: QR-42LP) Software Micromanager (University of California at San Francisco, https://micro-manager.org) ImageJ (National Institute of Health/Wayne Rasband, https://imagej.nih.gov/ij/). Alternatively, use FIJI (ImageJ) software; a build of ImageJ with several prepopulated Plugins and Macros for data analysis. Procedure In this protocol, experimental analyses of mCherry-CRY2–mPKCϵ-CAT-HA, CIBN-CAAX, and mCherry-CRY2-5PtaseOCRL are used as an example. mCherry-CRY2–mPKCϵ-CAT-HA was developed and utilized in Gada et al. (2022); CIBN-CAAX and mCherry-CRY2-5PtaseOCRL were developed and demonstrated in Idevall-Hagren et al. (2012). Briefly, CRY2-CIBN are protein partners that form a light-controlled dimerization system consisting of cryptochrome 2 (CRY2) and CIBN, the N-terminal region of the transcription factor, CIB1. CIBN is anchored to the membrane with a C-terminal CAAX box (Liu et al., 2008; Idevall-Hagren et al., 2012). Following blue-light illumination, CRY2 absorbs a FAD molecule, undergoing a conformational change that promotes binding to CIBN, resulting in CRY2 and its cargo (in this case, mPKCϵ-CAT-HA) being targeted to the membrane. Equipment setup The assembly of a micromirror TIRF microscope was described by Larson et al. (2014) and this formed the basis of our equipment setup. Lasers for TIRFM are selected based on their ability to provide a continuous beam centered on the appropriate excitation wavelength(s) required for the study. We typically employ lasers that can be modulated to vary the output power according to the experimental conditions [e.g., Coherent OBIS solid-state diode lasers that operate with an output between 5–150 mW (wavelength dependent)]. Laser beams are conditioned for coherence with custom-built Keplerian beam expanders upstream of laser cleanup filters. Here, we excite monomeric Cherry (mCherry), and so employ a 561 nm cleanup filter with a bandwidth of 10 nm (561/10 nm). Laser alignment and independent verification of the output are obtained using an optical power meter that can be placed in the beam path. Multiple laser lines can be added to the system with appropriate cleanup filters. For example, we also use 445 and 514 nm lasers with 445/10 nm and 514/10nm cleanup filters, respectively, downstream of the 561 laser. Such arrangement allows for a broad range of applications with multicolor imaging. The 445 nm laser can be used to activate the optogenetic probes described in this protocol. Following expansion to ~8 mm, the laser line(s) are combined into a single coherent incident beam that is focused onto the micromirror positioned below the TIRF lens (Figure 1). We use a high numerical aperture apochromat objective [60×, 1.5 numerical aperture (NA); Olympus] mounted on an open-microscope frame equipped with a piezo-driven nanopositioning stage (Mad City Labs, Inc). Figure 1. A schematic representation of micromirror TIRF. A. Schematic showing a 514 nm laser line entering the microscope frame horizontally, below the lens. The coherent beam is reflected 90° by a micromirror held on an arm (a), into the TIRF objective lens (b). The lens is held in a threaded mount (c), and a second micromirror (d) bounces the exit beam away from the sample area. The system is adjusted to create an evanescent wave (e) that penetrates ~100 nm from the interface between the glass coverslip and the sample. B. A closeup showing that the evanescent wave (e) does not penetrate the cytoplasm of the cell of interest. Fluorophores excited by the evanescent wave formed by the 514 nm excitation laser beam (i.e., yellow fluorescent proteins such as eYFP and mVenus) emit a yellow light that penetrates the central optical axis of the TIRF lens (f) and is ultimately collected by a detector. C. Example image showing individual YFP-tagged membrane proteins captured at the surface of a cell studied in TIRF mode using the setup described. D. Photograph of the micromirrors (a, d) positioned below the TIRF objective. The excitation beam is reflected 90° by the first micromirror (a), into the lens. The lens is held in a threaded mount (c) and is reflected 90° away from the sample area. The fluorescence signal, not visible, passes between the micromirrors along the central axis of the lens. E, Photograph of the micromirrors from above with the lens removed. The entry and exit micromirrors (a, d) are visible, with the 514 nm excitation beam shown reflecting off the entry micromirror (a). Each micromirror is mounted on an arm below the threaded mount (c) that holds the lens. Fluorescence signal from the sample will pass between the micromirrors along the central axis of the lens through a ring diaphragm that is positioned below the micromirrors (g). We use an RM21 microscope frame from Mad City Labs Inc; however, any microscope frame (including older commercial systems) that can be modified to accommodate micromirrors below the objective lens is suitable. One micromirror is placed in the beam path, directly below the leading edge of the objective lens. Micromirrors are also available from Mad City Labs Inc. Adjusting the position of the mirror beneath the lens controls the angle at which the excitation beam enters the lens and thereby the depth of the evanescent wave and the exit position of the beam from the lens (see the graphical abstract for a visualization of the setup). The relative position of the micromirror and lens is adjusted to modify how light refracts at the interface with the cells. For experiments, the lens is paired with very low autofluorescence immersion oil that has a similar refractive index [e.g., Cargille Type LDF (refractive index of 1.5)]. For our studies, the emission of mCherry is isolated from the excitation beam by an exit micromirror placed below the lens, opposite from the entry mirror (graphical abstract). A ring diaphragm positioned below the micromirror assembly further reduces crosstalk between the laser and the emission signal from the fluorophores. When the emission signal contains fluorescence from two fluorophores, the beam is separated into two parallel tracks using a beam splitter comprised of two 510 nm long pass filters. The peak emission of mCherry is above 510 nm. Downstream of this, mCherry is imaged through a 620/60 nm bandpass filter and the signal from the fluor is then captured on a back-illuminated sCMOS camera (Teledyne Photometrics). In studies using a second fluorophore that emits below 510 nm, the emission is directed to an appropriate bandpass filter on the other arm of the beam splitter. In each case, the laser output and the camera are controlled by Micro-Manager freeware (University of California San Francisco). All filters and mirrors are from Chroma. Lenses, pinholes, diaphragms, and the power meter are from Thor-Labs. TetraSpeck beads (Thermo) are routinely imaged to map the sCMOS chip and calibrate the evanescent field depth to 100 nm. Cell seeding Culture HEK-293 cells in 10 cm tissue culture-treated dishes in growth media containing phenol red–free Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum and maintained at 5% CO2 at 37 °C. After cells are ~70% confluent, remove growth media and replace with 1 mL of 0.05% trypsin for 1–2 min. When cells have detached from the dish, place a few isolated drops (~5–8 µL total) of trypsinized cell suspension on a 15 mm #1.5 coverslip in a 35 mm dish (Figure 2). Figure 2. Cell seeding procedure for glass coverslips to obtain single cells in certain areas After ~1 min, add 2 mL of growth media (as in step B1) to the 35 mm dish and incubate overnight. Cell transfection Ensure that cells cover 30%–50% of the area of the coverslip prior to transfection. Mix plasmid DNA of constructs for study in 50 µL of OptiMEM or any low serum, antibiotic-free media in a centrifuge tube. In another tube, add the transfection reagent PEI in a 1:4 ratio of DNA:PEI and combine the contents of both tubes. We transfected 0.75 μg each of mCherry-CRY2–mPKCϵ-CAT-HA and CIBN–CAAX or mCherry-CRY2-5PtaseOCRL and CIBN-CAAX. Cells for experiments that are performed to estimate background can be transfected with 0.75 μg of CIBN-CAAX only. Incubate the transfection solution for 20 min. Remove growth media from cells and replace with 1 mL of OptiMEM. Add the transfection solution to the seeded cells and incubate for 2–3 h. Remove transfection media and replace with 2 mL of growth media per 35 mm culture dish. Incubate overnight at 37 °C and 5% CO2. Experimental setup and data acquisition To prepare the TIRF setup, gently wipe off the lens with lens cleaning tissue and place a drop of immersion oil on the lens. Set the exposure time to 100 ms to best capture the fluorescence without causing photobleaching of the fluorophore. Set the movie parameters under the data acquisition function in Micromanager to acquire data every 2–5 s for 500 frames. Enter the data file path for the storage disc location on which the data will be stored and set the storage mode to data stacks. Mount a coverslip on the sample stage by placing it within the holder (Figure 3), rotating the top to align the magnetic lock, and seal the coverslip into place. Figure 3. Sample holder assembly. A quick release magnetic sample holder for circular, 15 mm coverslips of 1.5 mm thickness from Warner Instruments (left). Assembly of sample holder (right). Gently add 150 µL of TIRF imaging solution on top of the coverslip, ensuring that cells are not sheared off the glass in the process. Place the sample holder on the stage and adjust the stage height using Micromanager so that the cells on the coverslip are visible under white transmitted light. Cells appear close together in some regions while remaining well-spaced in some areas. Under dim ambient light, set the 561 nm laser to a low power (~20%) to visualize the mCherry in cells that have been successfully transfected and select suitable cells for analysis. Initiate data acquisition with the 561 nm laser set at 50% power (or less), to prevent photobleaching of the fluorophore. After two to three frames have been captured, turn on the 445 nm laser at full power to trigger mCherry-CRY2-mPKCϵCAT-HA or mCherry-CRY2-5PtaseOCRL recruitment to the cell surface, macroscopically assessed by an increase in mCherry fluorescence at the cell surface through the course of data acquisition. A common 488 nm laser line can also be used here. Once data acquisition is complete, save the file in TIF format, place both lasers on standby mode, and replace the coverslip to begin data acquisition from another cell. Image cells transfected only with CIBN-CAAX for background signal using the same experimental protocol and lasers as the mCherry-tagged constructs. The mean fluorescence signal from the background is subtracted from the mCherry fluorescence measurements. Data analysis Launch ImageJ (or FIJI) computer program (Figure 4). In ImageJ, open the TIF file (Figure 4). Figure 4. Launching TIF file in ImageJ. A–C depict how to launch (open) a TIF file in ImageJ. Click Image > Stacks > Tools > Make Substack (Figure 5) In the area called “Slices,” number your slice. This will be your background slice from which all the slices are subtracted against. In the figure below, the slice is labeled “1.” Save slice 1 as “tiff.” Figure 5. Creating and saving background/baseline slice. A and B. Depicts how to create a substack slice. C. The resulting file. D. Depicts how to save the substack file as a tiff. Next click Process > Image Calculator (Figure 6) Under “Image 1.” Use the pull-down arrow to select the name of the file you are currently working on. Under “Operation.” Use the pull-down arrow to select the operation, “Subtract.” Under “Image 2.” Use the pull-down arrow to select the slice tiff image that you saved in the previous step. Click OK. A message will pop up asking if you want all the images to be processed; click “YES.” A new window (results window) containing the processed images will pop up. Use this window moving forward. Figure 6. How to subtract background/baseline from all images/frames. A. Depicts how to launch image calculator to process images. B. Shows how image 2 (the slice file) is subtracted from the original file (Image 1). C. Depicts that all the images/frames are processed. D. The resulting file. Click Image > Adjust > Brightness/contrast (Figure 7) Scroll beyond the first few frames and then click auto. You should now be able to visualize your cell(s). If you do not see your cell(s), scroll through a few more images and then click auto again. Do this until you can see the cell(s). Select the appropriate brightness and contrast that makes it possible to differentiate the cell(s) from the background. Click apply. Click OK when warned about pixel values changing. When asked whether to apply to all slices, click “YES.” Figure 7. How to adjust brightness and contrast. A–D. Depicts how to adjust the brightness and contrast of the images. Next click Analyze > Tools > ROI manager > Show all (Figure 8) Select ROI on the image; then, click Add[t] (do this for each ROI added). Once all your ROIs have been added, click More > Multi-Measure > OK. Figure 8. How to select and analyze region of interests (ROIs). A. Indicates how to launch region of interest (ROI) manager. B. Depicts five ROIs on the cell. Selected regions are indicated in the window to the right. C and D. Depicts how to launch multi-measure to measure ROIs across all images/frames. E. The resulting window with all the measurements. Plot the mean fluorescence intensity vs. time to create a time course or to create summary bar graphs for various time points/treatments. Include five or more cells in the analyses of each condition to create a summary graph with representative images and a time course of mean intensities, as well as a summary (Figure 9A and 8B respectively). Figure 9. Final data representation. A. Representative images demonstrating fluorescence intensity before and after blue light activation of mCherry-CRY2-PKCϵCAT-HA. Scale bar = 10 µm. B. Time course of the experiment; blue arrow indicates the time point at which the 445λ laser was turned on. C. Summary data demonstrating the mean fluorescence intensity ± S.E. of 12 cells (Gada et al., 2022). Recipes TIRF imaging Solution NaCl 130 mM KCl 4 mM MgCl2 1.2 mM CaCl2 2 mM HEPES 10 mM Adjust pH to 7.4 with NaOH. Acknowledgments We thank Dr. Pietro De Camilli for providing CIBN-CAAX and CRY2-5’ptaseOCRL (Idevall-Hagren et al., 2012). This work was funded by National Institutes of Health Heart, Lung and Blood Institute grant R01HL144615 to L.D.P. This protocol was validated in Gada et al. (2022). Competing interests The authors declare that they have no competing interests with the contents of this article. References Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2(11): 764-774. Gada, K. D., Kawano, T., Plant, L. D. and Logothetis, D. E. (2022). An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins. J Biol Chem 298(5): 101893. Idevall-Hagren, O., Dickson, E. J., Hille, B., Toomre, D. K. and De Camilli, P. (2012). Optogenetic control of phosphoinositide metabolism. Proc Natl Acad Sci U S A 109(35): E2316-2323. Larson, J., Kirk, M., Drier, E. A., O'Brien, W., MacKay, J. F., Friedman, L. J. and Hoskins, A. A. (2014). Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9(10): 2317-2328. Liu, H., Yu, X., Li, K., Klejnot, J., Yang, H., Lisiero, D. and Lin, C. (2008). Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322(5907): 1535-1539. Vangindertael, J., Camacho, R., Sempels, W., Mizuno, H., Dedecker, P. and Janssen, K. P. F. (2018). An introduction to optical super-resolution microscopy for the adventurous biologist. Methods Appl Fluoresc 6(2): 022003. Zong, W., Wu, R., Li, M., Hu, Y., Li, Y., Li, J., Rong, H., Wu, H., Xu, Y., Lu, Y., et al. (2017). Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat Methods 14(7): 713-719. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Biophysics > Microscopy Cell Biology > Cell signaling > Phosphorylation Cell Biology > Cell imaging > Fluorescence Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Calibrating Fluorescence Microscopy With 3D-Speckler (3D Fluorescence Speckle Analyzer) Chieh-Chang Lin and Aussie Suzuki Aug 20, 2024 404 Views Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays Tom Snelling Nov 20, 2024 257 Views Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca2+ Imaging Sergei G. Gaidin [...] Sultan T. Tuleukhanov Feb 5, 2025 46 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,615	https://bio-protocol.org/en/bpdetail?id=4615&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Novel Method to Isolate RNase MRP Using RNA Streptavidin Aptamer Tags VC Violette Charteau * MD Merel Derksen * GP Ger J. M. Pruijn (*contributed equally to this work) Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4615 Views: 1010 Reviewed by: Alessandro DidonnaKirsten A. Copren Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in RNA Biology Feb 2022 Abstract Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for mitochondrial RNA processing (MRP) and RNase P are highly similar RNPs that play distinct roles at the cellular level; as a consequence, the specific isolation of either of these complexes is essential to study their biochemical function. Since their protein components are nearly identical, purification of these endoribonucleases using protein-centric methods is not feasible. Here, we describe a procedure employing an optimized high-affinity streptavidin-binding RNA aptamer, termed S1m, to purify RNase MRP free of RNase P. This report details all steps from the RNA tagging to the characterization of the purified material. We show that using the S1m tag allows efficient isolation of active RNase MRP. Keywords: Ribonucleoprotein RNA aptamer RNA affinity purification RNase MRP Streptavidin Background In order to maintain RNA functions or defend the organism, most RNAs can be the substrate of ribonucleases (RNases), enzymes specialized in RNA cleavage and degradation. Ribonucleases are generally composed of a catalytic RNA-binding protein, but the catalyst can also be an associated RNA molecule, which is called a ribozyme. In the latter, the catalytic RNA is generally associated with one or more proteins, together forming a ribonucleoprotein particle (RNP). Two RNP-type ribonucleases in humans are RNase for mitochondrial RNA processing (MRP) and RNase P (Guerrier-Takada et al., 1983), complexes with a highly similar structure. These endoribonucleases, essential to cell survival, are ubiquitously expressed RNPs and are mostly located in the nucleolus of eukaryotic cells. RNase MRP and RNase P are evolutionarily related; RNase P is present in all domains of life, while RNase MRP is only found in eukaryotes (Randau et al., 2008). RNase MRP and RNase P both contain a unique long non-coding RNA, but share most of their protein subunits. Ten protein subunits (hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38, and Rpp40) have been demonstrated to interact with both particles, although hPop4, Rpp14, and Rpp21 might be more strongly associated with RNase P (Welting et al., 2006). The RNA components of RNase MRP and RNase P differ in size and sequence, but they share the same conserved domains and fold into highly similar secondary structures. Despite their similarity, RNase MRP and RNase P have very distinct functions. While RNase MRP, named after its first discovered role in Mitochondrial RNA Processing (Chang and Clayton, 1987), is also involved in ribosomal RNA (rRNA) (Lygerou et al., 1996; Lan et al., 2020) and messenger RNA (mRNA) maturation (Gill et al., 2004; Mattijssen et al., 2011), RNase P is best known for its function as a processor of transfer RNA (tRNA) precursors (Robertson et al., 1972). In addition to the maturation of pre-tRNA, RNase P has been reported to be involved in transcription by RNA polymerase I and III (Reiner et al., 2006; 2008), as well as in the maturation of small nucleolar RNAs (snoRNAs) in yeast (Coughlin et al., 2008). Mutations in the RNase MRP RNA (RMRP) cause a spectrum of disorders characterized by similar symptoms, the major condition being the autosomal recessive disease cartilage-hair hypoplasia (CHH, OMIM #250250). This pathology is mostly characterized by severe skeletal dysplasia and multiple pleiotropic symptoms (Hermanns et al., 2006; Mattijssen et al., 2010). CHH-causing mutations, mainly single-nucleotide substitutions, are hypothesized to affect RMRP function by altering its secondary structure or its interaction with other molecules (Welting et al., 2008). Today, the molecular mechanisms of CHH pathology remain elusive. In order to better comprehend the cellular function of RNase MRP and the disorders linked to mutations in its RNA subunit, it is necessary to gain knowledge about the composition of this RNP and its interactome. A ribonucleoprotein complex is commonly purified by either a protein-centric or an RNA-centric method, depending on which molecule is being targeted (Ramanathan et al., 2019). Protein-centric approaches, such as immunoprecipitation or the use of protein affinity tags, allow specific purification of some major RNA–protein cellular complexes. However, this is only possible if they contain a unique protein component, e.g., a protein subunit absent from any other RNP complex. No protein component unique to the human RNase MRP has been discovered to date and, as a consequence, any protein-centric method employed to purify the RNase MRP complex will also co-purify RNase P. This will result in a mixture of both enzymes in the purified samples, and the inability to differentiate between the two RNPs leads to uncertainties on their biochemical function. For instance, it was demonstrated that RNase MRP or RNase P is responsible for the endoribonucleolytic cleavage of m6A-modified RNAs (Park et al., 2019), but differentiation between these two endoribonucleases was not possible, since the purification was based on a shared protein subunit. When RNP complexes cannot be isolated by techniques targeting their protein components with sufficient specificity, RNA-centric methods can be employed. However, the non-coding RNA subunits of both RNase MRP and RNase P are relatively short, highly structured, and shielded with proteins. This makes it quite challenging to purify them with RNA-specific probes, such as biotin-labeled antisense oligonucleotides (Hou et al., 2016). As an alternative to antisense oligonucleotides, an RNA aptamer can be inserted in the RNA molecule of interest to serve as a tag for purification. Several RNA aptamers that bind ligands with high affinity have been developed in the last decades, making them powerful tools for RNA-specific purification (Walker et al., 2008). Examples include the StrepTag (Bachler et al., 1999), the MS2-TRAP (MS2-tagged RNA affinity purification) procedure (Yoon et al., 2012), and streptavidin aptamer (S1) tagging. Insertion of MS2 hairpins has been proven useful for the characterization of complexes containing a non-coding or messenger RNA of interest, but it requires co-expression of a tagged MS2-binding protein (Yoon et al., 2012; Yoon and Gorospe, 2016). The S1 aptamer was identified by in vitro selection as a streptavidin-binding short sequence with a high affinity and appeared to be effectively eluted with biotin for the successful recovery of the tagged molecule (Srisawat and Engelke, 2001). Widely available streptavidin-agarose beads can be used, and this tag has been shown to allow efficient purification of RNase P from yeast (Srisawat and Engelke, 2002) and human cells (Li and Altman, 2002). In this report, we describe a novel method to specifically isolate RNase MRP using the S1m RNA aptamer (Derksen et al., 2022), an optimized version of the original S1 aptamer (Leppek and Stoecklin, 2014). In addition to the increase in purification efficiency compared to the S1 tag, the S1m aptamer has a relatively short size. This lowers the risk of undesirable effects on the secondary and tertiary structure of the RNA of interest, known to be crucial for the function of many non-coding RNAs, including ribozymes. Briefly, the S1m aptamer is inserted in the RNA of interest by cloning and the purified plasmid is used to transfect human cells. The complex containing the tagged RNA is purified from the lysate using streptavidin-coated beads and all proteins and RNAs are extracted from the purified material. The composition of the bound material is then identified by northern and western blotting methods (Figure 1). Figure 1. Schematic illustration of the isolation of RNase MRP using the S1m tag. The S1m aptamer is introduced at the desired location of the gene of interest (5′, 3′, or internal) and human cells are transfected with this construct. The tagged RNA is transcribed in the nucleus and will assemble into ribonucleoprotein particles, leading to accumulation in the nucleoli. The cells expressing the tagged RNA are lysed, and the RNA–protein complex is purified with immobilized streptavidin, which binds to the S1m tag. The purified complex is eluted from the beads to extract the proteins and RNA separately. The RNA sample can be used to analyze the specificity of purification by northern blot hybridization and the protein sample can be used to analyze the associated proteins. Materials and Reagents Cell lines Human embryonic kidney HEK293T cells Disposables For all RNA work, use only filter tips, RNA- and RNase-free tubes, and prepare all solutions RNase-free. 1.5 mL microcentrifuge tubes (autoclaved) (any vendor) 0.2 mL PCR tubes (any vendor) Sterile 15 mL polypropylene centrifuge tubes (any vendor) Sterile 50 mL polypropylene centrifuge tubes (any vendor) Sterile nuclease-free filter tips (10 µL, 200 µL, 1,000 µL) (any vendor) Sephadex G50 column (Cytiva, catalog number: 27534001) Cell culture 12-well plate (Greiner Bio-one, catalog number: 665180) Cell culture dishes, 150 cm2 (Greiner, catalog number: 639160) Serological pipette, 25 mL (any vendor) Serological pipette, 10 mL (any vendor) Serological pipette, 5 mL (any vendor) Petri dishes (Sarstedt, catalog number: 82.1473.001) Sterile surgical blades for the excision of DNA bands from gel (Swann-Morton, catalog number: 0208) QIAquick gel extraction kit protocol (Qiagen, catalog number: 28706) Plasmid Midi kit (Qiagen, catalog number: 12145) Hybond-N membrane (GE Healthcare, catalog number: RPN303B) Protran BA 85 nitrocellulose membrane, 0.45 µm pore size (VWR, catalog number: 10063-173) Filter paper (any vendor) Whatman paper 0.34 mm (Cytiva, catalog number: 3030-917) 0.22 µm filter (VWR, catalog number: 514-0061) Reagents (order of use) Sterile double distilled water (DDW), referred to as ultrapure or RNase-free water Tryptone (MP Biomedicals, catalog number: 091010817) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014) Yeast extract (MP Biomedicals, catalog number: 0210330391) Agar (Thermo Scientific, catalog number: 30391023) Sodium hydroxide (NaOH) EMSURE® (VWR, catalog number: 1.06498.1000) Agarose (Eurogentec, catalog number: EP-0010-05) DNA loading dye, 6× (Thermo Scientific, catalog number: R0611) Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Scientific, catalog number: 41966) Penicillin-streptomycin solution (Thermo Scientific, catalog number: 151401) Fetal calf serum (FCS) (Sigma-Aldrich, catalog number: F2442) Polyethyleneimine (PEI), branched (Sigma-Aldrich, catalog number: 108727) Opti-minimal essential medium (Opti-MEM) (Gibco, catalog number: 51985026) Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: 15496249) Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (VWR, catalog number: MERC1.06346) Sodium hydrogen carbonate (NaHCO3) EMSURE® (VWR, catalog number: 1.06329) Glucose (Invitrogen, catalog number: 15023021) Trypsin (Sigma-Aldrich, catalog number: T4799) Ethylenediaminetetraacetic acid (EDTA) (VWR, catalog number: 1.08418) Dulbecco’s phosphate buffered saline (PBS) (DPBS) (Life Technologies, catalog number: 14190) Tris (Sigma-Aldrich, catalog number: T1378) Hydrochloric acid (HCl), 37% (VWR, catalog number: 1.00317) Glycerol (Bio-Connect, catalog number: 4800688) NP-40 (IGEPAL®) (Sigma-Aldrich, catalog number: 56741) cOmplete protease inhibitor cocktail (Roche, catalog number: 1697498001) RNasin (Promega, catalog number: N2111) Streptavidin-conjugated SepharoseTM beads (GE Healthcare, catalog number: 17511301) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4390) β-Mercaptoethanol (β-ME) (Sigma-Aldrich, catalog number: M3148) Bromophenol blue (BFB) (Sigma-Aldrich, catalog number: B5525) Trizol reagent (Invitrogen, catalog number: 15596018) Chloroform, EMSURE® (Sigma-Aldrich, catalog number: 1.02445) Isopropanol, EMSURE® (Sigma-Aldrich, catalog number: 1.09634) GlycoBlue (Thermo Scientific, catalog number: AM9515) 25:24:1 phenol:chloroform:isoamyl alcohol (Invitrogen, catalog number: 15593049) Ethanol absolute, EMSURE® (Sigma-Aldrich, catalog number: 1.00986) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779) 5× transcription optimized buffer (Promega, catalog number: P1181) 100 mM NTPs (Affymetrix, catalog number: 77245) T7 RNA polymerase (Thermo Fisher Scientific, catalog number: EP0111) 32P-α-UTP (PerkinElmer, 3000 Ci/mmol, NEG502Z) Boric acid (Boom, catalog number: 61000501.1000) 40% (w/v) acrylamide:bisacrylamide (19:1) solution for RNA (Serva, catalog number: 10.679.02) Urea (Invitrogen, catalog number: 15505027) N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T7024) Ammonium persulfate (APS) (VWR, catalog number: 97064-594) Xylene cyanol FF (XCFF) (Sigma-Aldrich, catalog number: X4126) Monosodium phosphate (NaH2PO4) (Sigma-Aldrich, catalog number: L2887) Disodium hydrogen phosphate (Na2HPO4) (VWR, catalog number: 1.06580.1000) Sodium citrate (VWR, catalog number: 1.06448) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503) Ficoll® 400 (VWR, catalog number: 17-0300) Polyvinylpyrrolidone (Sigma-Aldrich, catalog number: PVP40) Herring sperm DNA (Sigma-Aldrich, catalog number: D6898) 30% (w/v) acrylamide:bisacrylamide (37.5:1) solution (Serva, catalog number: 10.688.02) Glycine (Merck, catalog number: 50046) Methanol (Thermo Scientific, catalog number: 11976961) Ponceau-S solution (Sigma-Aldrich, catalog number: P7170) Tween-20 (Merck, catalog number: 822184) Non-fat dry milk (any vendor) IRDye-conjugated secondary antibodies (LiCOR Biosciences) Luria-Bertani (LB) medium and agar (see Recipes) Complete DMEM medium (see Recipes) 10× tyrode solution (see Recipes) Trypsin-EDTA solution (see Recipes) Polyethyleneimine (PEI) (see Recipes) S1m lysis buffer (see Recipes) S1m incubation buffer (see Recipes) S1m wash buffer (see Recipes) 4× protein sample buffer (see Recipes) In vitro transcription (IVT) NTP mix (see Recipes) 1× TBE (see Recipes) RNA denaturing gel (see Recipes) 2× RNA sample buffer (see Recipes) Blotting buffer (see Recipes) 20× SSC (see Recipes) 100× Denhardt’s (see Recipes) 10 mg/mL sheared herring sperm DNA (see Recipes) Pre-hybridization buffer (see Recipes) SDS-PAGE running gel (see Recipes) SDS-PAGE stacking gel (see Recipes) SDS-PAGE running buffer (see Recipes) Western blotting buffer (see Recipes) Blot blocking solution (see Recipes) PBST (see Recipes) Optional: BioLux® Gaussia Luciferase assay kit (New England BioLabs, catalog number: E3300) Optional: Luminometer-compatible 96-well plate, opaque white or black (any vendor) Plasmids pGEM-3Zf(+)-RMRP (Pluk et al., 1999; available by request to the authors) pcDNA5/FRT/TO (Thermo Fisher, catalog number: V652020) Bacterial strains E. coli TOP10 competent cells (homemade) Equipment Benchtop centrifuge (Eppendorf, catalog number: 5415D) Refrigerated benchtop centrifuge (Eppendorf, catalog number: 5417R) Refrigerated centrifuge suitable for 50 mL volume (Heraeus Megafuge 1.0 R) Magnetic stirrer (IKA REO Basic C IKAMAG) Thermocycler (Bio-Rad T100) Shaker incubator (Infors HT Multitron 2) Spectrophotometer (DeNovix DS-11) Gel scanner (FLA-5100) Humidified 37 °C, 5% CO2 incubator (Heracell 150) Diagenode bioruptor Digital rotary mixer (Labinco, LD-76) Standard equipment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Odyssey imaging system (LiCOR Biosciences) Crosslinker, Stratalinker 1800 (Stratagene) with UV bulbs, λ = 254 nm (Ushio, catalog number: 3000016) Geiger counter (Thermo Scientific, Mini monitor 900) Phosphor imaging screens (Bio-Rad) Typhoon imager (GE Healthcare) Optional: Luminometer (Thermo Scientific, VL0000D0) Procedure S1m tagging Optimal site for S1m insertion/fusion The S1m aptamer sequence can be inserted in any region of the RNA of interest, but one has to make sure the tag will be available for purification. The streptavidin-binding aptamer must be properly folded, and the tertiary structure of the RNA and/or associated proteins must not interfere with the binding of streptavidin to the S1m aptamer. If the RNA structure and/or binding sites for RNA-binding proteins are known, place the S1m sequence in a non-conserved, exposed RNA region (Walker et al., 2008). In addition, the junction sites for the 5′- and 3′-ends of the S1m aptamer should be close in space to allow proper folding of the aptamer. We advise trying at least two distinct aptamer insertion sites and assessing which one leads to the most efficient purification. In case of RNase MRP, fusion to the 5′ or 3′ termini of the RNase MRP RNA appeared to be more efficient than insertion at an internal position. It should be noted that placing the affinity tag at the 5′ or 3′ position might decrease the RNA stability due to exonuclease degradation. Moreover, it is necessary to adjust the promoter to the gene of interest (pol I, II, or III) and to consider the required expression level. An inducible system such as a doxycycline-inducible RNA polymerase promoter might be desirable to avoid overexpression and unwanted effects. Tag gene of interest with S1m tag Insert the S1m aptamer sequence at the selected location(s) in the gene of interest, using a standard PCR-based strategy. The sequence of the S1m aptamer is AUGCGGCCGCCGACCAGAAUCAUGCAAGUGCGUAAGAUAGUCGCGGGUCGGCGGCCGCAU. This sequence can be commercially synthesized as an oligonucleotide and cloned by the use of restriction enzymes, as it was done for RMRP (for details, see Derksen et al., 2022). Another method is to add the aptamer sequence to the primers used for the amplification of the gene of interest and use PCR amplification and fusion-based cloning (such as In-Fusion®) to add the tag directly in the sequence of the gene of interest. This second method is faster, easier, and allows the introduction of the aptamer at any desired location. Confirm insertion of the aptamer by restriction digestion and gel electrophoresis. Note: We decided to use the S1m aptamer, which is a modified S1 aptamer containing an extended perfect strand complementarity in comparison with the original S1 basal stem, leading to a 2-fold higher affinity for streptavidin (Leppek and Stoecklin, 2014). Bacterial transformation and purification Once you obtain plasmids containing the insert, transform competent bacteria (e.g., E. coli TOP10) with the tagged gene following standard bacterial transformation protocols (Green and Rogers, 2013). Grow bacteria overnight on LB agar plates containing the appropriate antibiotics. The next day, pick single colonies and perform colony PCR to verify that they contain the correct plasmid. Inoculate bacteria from a single colony in 100 mL of selective medium and grow overnight at 37 °C in a shaking incubator at 200 rpm. Purify the plasmid from the bacterial culture using the Qiagen Plasmid Midi kit, following the manufacturer’s instructions. Confirm the sequence of the construct obtained by sequencing (e.g., Sanger sequencing). Check the concentration and purity of the plasmid by spectrometry and agarose gel electrophoresis.Note: Expected 260/280 and 260/230 ratios are >1.8 and >2.0, respectively. A lower 260/280 ratio may indicate protein or phenol contamination, and a lower 260/230 ratio may indicate contamination by organic compounds. Store purified plasmids at -20 °C or use directly for transfection (step B2). Cell culture and transfection Seed adherent HEK293T cells at 10% confluency in 150 cm2 dishes with 40 mL of complete DMEM medium. Around 24 h after seeding, when cells are approximately 20% confluent, transfect them with the S1m-containing construct generated in step A: Per 150 cm dish, mix in this order: 4 mL of Opti-MEM, 40 μg of DNA, and 120 μL of 1 mg/mL of PEI. Note: A PEI:DNA ratio of 3:1 (w/w) was optimal for transfection of RNase MRP constructs, but this has to be optimized for each construct to reach the optimal expression levels. Adapt the quantities for transfection in accordance with the surface area, considering 1 μg of DNA per milliliter of culture medium. As a mock control, transfect a dish with the same quantities of Opti-MEM and PEI without DNA. Incubate at room temperature for 30 min. Add the mixture dropwise to the cells and mix by swirling gently. Optional: If the transfected plasmid expresses Gaussia or Renilla luciferase, assess transfection efficiency by measuring luciferase expression level. Twenty-four to forty-eight hours after transfection, harvest 200 μL of culture medium and assess luciferase activity using the BioLux® Gaussia Luciferase assay kit following the manufacturer’s instructions. Forty-eight hours after transfection, harvest the cells by trypsinization. Remove the culture medium. Add 150 µL of 1× trypsin-EDTA per 10 cm2 dropwise to the cells at room temperature and incubate for 2–3 min at room temperature until more than 90% of the cells are detached. Add at least four volumes of pre-warmed complete DMEM medium to deactivate the trypsin and centrifuge at 500–1000 × g for 5 min. Wash the pellet once with at least three volumes of PBS. Note: The cell pellet can be stored at -20 °C. Purification of S1m-tagged complexes Streptavidin purification Lyse the harvested cells by resuspending the pellet in 1 mL of S1m lysis buffer and incubating on ice for 5 min. Sonicate 10 times in the bioruptor on high power for 30 s to shear genomic DNA. Centrifuge at 11,000 × g for 10 min at 4 °C and transfer the supernatant to a new tube to separate from insoluble material. Insoluble material can be discarded. Add 1 mL of S1m incubation buffer. Transfer 10% of the lysate to a new tube labeled as Input material for western and northern blotting analyses. Note: The amount of material to save at this stage depends on the expression level of the complex of interest, purification efficiency, and number of analyses. For RNase MRP, we used 0.6% of input material for western blotting analysis, and 1% for northern blotting analysis. Add the lysate and 3 μL of RNasin to 40 μL of packed pre-equilibrated streptavidin Sepharose beads. To pre-equilibrate the beads, wash them three times with 0.5 mL of S1m wash buffer and centrifuge at 800 × g for 2 min. Incubate overnight at 4 °C under agitation using a rotary mixer. Centrifuge at 800 × g for 2 min at 4 °C and transfer 10% of the supernatant to a new tube labeled as Non-bound material for western and northern blotting analyses. Note: Save the same amount as for the Input. Wash the beads three times for 5 min with 1 mL of S1m wash buffer by incubation at 4 °C under agitation and centrifugation at 800 × g for 2 min at 4 °C. The wash fractions can be discarded. During the last wash, split the resuspended beads into two tubes, to use one for protein extraction and the other for RNA extraction. Protein extraction from purified material Centrifuge one of the two tubes to pellet the beads and discard the supernatant. Add 20 µL of 4× protein sample buffer directly to the beads. Note: The amount of material to load on each gel depends on the purification efficiency. For RNase MRP, we used 10%–25% of bound material for western blotting analysis. Store the purified material at -20 °C or use directly for western blotting (step D2). RNA extraction from purified material Centrifuge the second tube to pellet the beads and discard the supernatant. Add 1 mL of TRIzol reagent directly to the beads and isolate the RNA following the manufacturer’s instructions. Note: We recommend performing an additional extraction step of the aqueous phase with one volume of chloroform and adding 10 µg of glycogen or GlycoBlue as a carrier during precipitation. Dissolve the RNA in 10–30 µL of RNase-free water. Store the purified RNA at -80 °C. Note: The amount of material to load on each gel depends on the purification efficiency. For RNase MRP, we used 15% of bound material for northern blotting analysis. Identification of the composition of purified material Northern blotting Verify the presence of your tagged RNA of interest by northern blot hybridization. The probe used to detect the RNA of interest should lead to the detection of both the endogenous RNA and the tagged RNA, in order to compare their expression levels. The analysis of a non-related RNA is recommended. This can serve as a loading control for the input fractions and to have a reference for the specificity of RNA purification. Here, we describe the procedure for northern blotting using an α-32P-labeled RNA probe (riboprobe), the most sensitive and specific type of radiolabeled probe. For RNAs expressed at a high level, 32P 5′-end-labeled DNA oligonucleotides or non-radioactive (e.g., fluorescently labeled) RNA or DNA probes can also be used. For detailed protocols describing these methods, see Tabor and Struhl (2001) and Franke et al. (2015), respectively. Probe cloning Insert (part of) the sequence of the RNA of interest in a plasmid, using a standard PCR-based strategy. The probe should be at least 100 nucleotides long. The vector should contain an in vitro transcription promoter (such as the promoter for T7 or SP6 RNA polymerase) upstream of the sense sequence of the RNA of interest, to generate a complementary probe after transcription. For RMRP, the pGEM-3Zf plasmid containing the complete sense sequence of RMRP downstream of the T7 promotor was used. Once you obtain the plasmid containing the probe sequence, follow steps A3a–g to amplify and purify the plasmid. In vitro transcription Linearize the plasmid by digestion with the appropriate restriction enzyme cleaving downstream of the probe sequence. Purify the linearized plasmid by 25:24:1 phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation [for details on the protocol, see Suchan (2020)]. Confirm linearization by agarose gel electrophoresis. Load both the circular and linear plasmid on the same gel and compare the size of the bands. The linear plasmid should migrate at a different position compared to the circular plasmid. Mix in the following order: RNase-free water (volume resulting in a total reaction volume of 20 µL), 2 µL of 100 mM DTT, 4 µL of 5× transcription optimized buffer, 2 µL of IVT NTP mix, 40 U of RNA polymerase, 2 µL of RNasin (80 units), 2 µL 32P-α-UTP (3000 Ci/mmol), and 1 µg of linearized plasmid. Incubate for 1 h at 37 °C. Purify the in vitro–transcribed probe using a Sephadex G50 column following the manufacturer’s instructions. Measure the amount of radioactivity in the purified probe and the column using a Geiger counter. Note: The radioactivity level should be higher in the purified RNA probe than in the column; the opposite indicates sub-optimal production of the labeled probe. Gel electrophoresis This protocol describes the procedure for the use of a polyacrylamide gel, but an agarose gel can also be used. Additional information can be found in more detailed protocols (Petrov et al., 2013). Prepare a polyacrylamide RNA denaturing gel. Pre-run the gel in 1× TBE for 30 min at 30 mA. Add one volume of RNA sample buffer to the Input, Non-bound, and purified RNA samples from transfected and non-transfected cells and incubate for 5 min at 95 °C to denature the RNA. Place samples back on ice immediately. After pre-running, clean the slots using a syringe and load the samples. Run the gel at 30 mA. Note: The running time depends on the size of the RNA molecules of interest. Use the colored dyes to determine when to stop running the gel (Petrov et al., 2013). For RNase MRP RNA, the gel should be run until the dark blue dye (bromophenol blue) reaches the bottom of the gel. RNA blotting and detection Transfer the RNA to a Hybond N+ membrane in northern blotting buffer for 1 h at 500 mA at room temperature. Allow the blot to dry and crosslink the RNA to the membrane by UV irradiation at 700 mJ/cm2. Note: Store the blot dry between filter paper at 4 °C if needed. Block the blot for 1 h at 65 °C with pre-hybridization buffer. Add the RNA probe to the pre-hybridization buffer and incubate at 65 °C overnight. Wash the blot twice with 1× SSC and 0.2% SDS, using enough buffer to cover the whole blot. Wash the blot once or twice with 0.1× SSC until no radioactivity is detected anymore at regions of the blot where no signal is expected. Wrap the blot in plastic foil and put it in a cassette with a Phosphor Screen. Develop with a phosphor imager to visualize the result (for an example see Figure 2). Figure 2. Northern blot analysis of S1m-tagged RNA. HEK293 cells were transfected with a S1m-RMRP construct to transiently express the tagged RNA and cultured for 24 or 48 h before extracting total RNA with TRIzol. As a control (- plasmid), cells subjected to the transfection procedure without the plasmid construct were used. Equal amounts of RNA were separated by denaturing polyacrylamide gel electrophoresis, and both wild-type and S1m-tagged RMRP were detected using a radiolabeled probe complementary to RMRP. Western blotting Protein gel electrophoresis Prepare an SDS-PAGE gel [see Recipes and detailed protocol in Sambrook and Russell (2006)]. After polymerization, remove the comb carefully and clean the slots using a syringe. Mix one volume of Input and Non-bound material with three volumes of 4× protein sample buffer. Load the Input, Non-bound, and purified protein samples after 2–5 min incubation at 95 °C and centrifugation to remove insoluble material. Note: Load up to 15 μL for a 0.75 mm thick gel and up to 30 μL for a 1.5 mm thick gel. Run the gel in SDS-PAGE running buffer at 90 V constant for approximately 15 min, until the dye reaches the bottom of the stacking gel. Then, run at 150 V constant for approximately 50–60 min, until the dye reaches the bottom of the running gel. Remove the gel from the running apparatus, carefully remove one of the glass plates and cut off the stacking gel. Protein blotting Assemble the transfer cassette: anode (-); foam mat; two sheets of Whatman paper; gel; nitrocellulose membrane; two sheets of Whatman paper, foam mat; cathode (+). Note: Soak all Whatman papers and the membrane in western blotting buffer before assembling the blotting sandwich. Avoid any air bubbles between the anode and cathode and gently remove any using a roller before closing the cassette to ensure efficient transfer on the whole surface of the blot. Insert the cassette into a blotting apparatus. Add an ice pack in the apparatus or keep the apparatus cold during the transfer. Transfer the proteins to a nitrocellulose membrane in western blotting buffer with magnetic stirring for at least 1 h at constant 100 V or overnight at constant 250 mA. Note: For thick gels (1.5 mm), transfer for at least 1.5 h. After transfer, remove the blot from the cassette and briefly incubate in Ponceau-S solution until clear bands appear for the Input and Non-bound fractions. Rinse the blot a few times with demineralized water and make an image of the stained blot. Immunodetection Block non-specific antibody binding by incubating the membrane in 10 mL of blot blocking solution at room temperature for at least 30 min with gentle shaking. Discard the blocking solution and add the primary antibody diluted to working concentration in 5–10 mL of blocking solution. Incubate for at least 1 h at room temperature or ideally overnight at 4 °C, with shaking or rotation. Wash the membrane three times for 5 min with 10–20 mL of PBST. Add the IRDye® 800CW- or 680RD-conjugated secondary antibody diluted to working concentration in 5–10 mL of blocking solution. Incubate for at least 1 h at room temperature, protected from the light. Note: Ensure that the secondary antibody used is targeted against immunoglobulins of the organism of origin of the primary antibody. Wash the membrane two times for 5 min with 10–20 mL of PBST. Briefly wash the membrane with 10 mL of PBS and keep it in PBS protected from the light until imaging. Note: We observe that after rinsing the membrane with PBS, less background fluorescence is observed when imaging. Detect the fluorescence using the Odyssey imaging system or Typhoon imager with the 700 nm or 800 nm channel, depending on the secondary antibody used. Data analysis Confirm by northern blotting that the band corresponding to the endogenous non-tagged RNA is visible in material from both transfected and non-transfected cells, in Input and Non-bound fractions. The RNA containing the aptamer should be visible in Input and purified material from transfected cells only. Normalize the expression level of the RNA of interest in different conditions to the control RNA. After western blotting, no or very weak bands are expected in the bound material, while the Ponceau-S staining is expected to be highly similar in Input and Non-bound fractions. Confirm the efficiency of the purification by immunodetection of known protein partners in the purified samples of transfected cells (for an example see Figure 3). Figure 3. Identification of proteins in the isolated complexes by western blotting. HEK293 cells were transfected with a S1m-RMRP construct to transiently express the tagged RNA and cultured for 48 hours. As a control (- plasmid), cells subjected to the transfection procedure without the plasmid construct were used. The cells were lysed, tagged ribonucleoprotein complexes were purified with streptavidin Sepharose beads, and proteins were eluted with protein sample buffer. Equal amounts of Input (I) and Flow-through (F) and three times more Bound (B) samples were separated by SDS-PAGE and analyzed by western blotting using specific antibodies. Rpp40 and Rpp25 are known components of the RNase MRP; the La protein, which does not interact with the complex, was used as a negative control. Recipes Luria-Bertani (LB) broth and agar 1% (w/v) tryptone 1% (w/v) NaCl 0.5% (w/v) yeast extract To prepare LB agar, add 1.5% (w/v) agar Adjust pH to 7.0 with NaOH Autoclave and store at room temperature To prepare LB agar plates, warm up to melt the agar and let it cool to <60 °C before adding antibiotics. Pour 10–20 mL per Petri dish in a sterile environment, let it cool, and store at 4 °C Complete DMEM medium DMEM 10% FCS 1% penicillin-streptomycin mix Store at 4 °C until use 10× tyrode solution 1.37 M NaCl 27 mM KCl 3.6 mM NaH2PO4·H2O 120 mM NaHCO3 111 mM glucose Mix all components except the glucose, adjust pH to 7.2 if necessary and autoclave. Then, add the glucose and keep sterile. Store at room temperature 10× Trypsin-EDTA solution 3.5% (w/v) trypsin 1% (w/v) EDTA Dissolve in 10× tyrode solution by shortly incubating at 37 °C Adjust pH to 7.4 with NaOH Filtrate through a sterile 0.22 µm filter Store at -20 °C or use directly to prepare 1× aliquots To prepare 1× solution for use in cell culture, dilute 10 times with sterile ultrapure water and store aliquots at -20 °C Polyethyleneimine (PEI) stock Add ultrapure water to PEI so that the final concentration is 1 mg/mL Incubate for 1 h at room temperature under agitation Incubate for 30 min at 50 °C Incubate for 30 min at room temperature under agitation Filtrate through a sterile 0.22 µm filter Store aliquots at -70 °C or -80 °C (up to three months) S1m lysis buffer 50 mM Tris-HCl, pH 7.6 10 mM MgCl2 100 mM KCl 10% glycerol 0.1% NP-40 Make fresh and add cOmplete protease inhibitor and 1 mM of DTT right before use S1m incubation buffer 50 mM Tris-HCl, pH 7.6 10 mM MgCl2 100 mM KCl 10% glycerol Make fresh and add cOmplete protease inhibitor and 1 mM of DTT right before use S1m wash buffer 50 mM Tris-HCl, pH 7.6 10 mM MgCl2 100 mM KCl 10% glycerol 0.05% NP-40 Make fresh and add cOmplete protease inhibitor and 1 mM of DTT right before use 4× protein sample buffer 200 mM Tris-HCl, pH 6.8 8% SDS 40% glycerol 5% β-mercaptoethanol 50 mM EDTA 0.05% bromophenol blue Store at 4 °C IVT NTP mix 1 mM ATP 1 mM GTP 1 mM CTP 0.1 mM UTP Store at -20 °C 1× TBE 100 mM Tris 100 mM boric acid 2 mM EDTA Store at room temperature RNA denaturing gel 1× TBE 8 M urea X% 19:1 acrylamide:bisacrylamide 0.1% TEMED 0.1% fresh APS (prepare 10% APS and use immediately or store single-use aliquots at -20 °C) Choose the percentage of acrylamide based on the size of the RNA of interest. Note: Prepare 500 mL of RNA denaturing gel mix without TEMED and APS and store at 4 °C if you plan to make multiple RNA gels. Before use, warm up at 37 °C to dissolve the urea if needed and add TEMED and APS. Beware: the gel will polymerize faster when the solution is warm. 2× RNA sample buffer 8 M urea 1× TBE 0.05% XCFF 0.05% BFB Store at room temperature Northern blotting buffer 18.4 mM NaH2PO4 6.5 mM Na2HPO4 Store at room temperature 20× SSC 3.0 M NaCl 0.3 M sodium citrate Adjust to pH 7.0 with HCl Store at room temperature 100× Denhardt’s 2% BSA 2% Ficoll® 400 2% polyvinylpyrrolidone Store at -20 °C 10 mg/mL sheared herring sperm DNA Dissolve herring sperm DNA at 10 mg/mL in ultrapure water Sonication is required for the proper preparation of the solution Store at -20 °C Pre-hybridization buffer 6× SSC 0.1 mg/mL sheared herring sperm DNA 0.2% SDS 10× Denhardt’s Store at room temperature SDS-PAGE running gel 0.4 M Tris-HCl, pH 8.8 X% 37:1 acrylamide:bisacrylamide 0.1% SDS 0.1% TEMED 0.1% fresh APS (prepare 10% APS and use immediately or store single-use aliquots at -20 °C) Choose the percentage of acrylamide based on the size of the proteins of interest. SDS-PAGE stacking gel 70 mM Tris-HCl, pH 6.8 4% 37:1 acrylamide:bisacrylamide 0.1% SDS 0.1% TEMED 0.1% fresh APS (Prepare 10% APS and use immediately or store single-use aliquots at -20 °C) SDS-PAGE running buffer Tris-glycine (25 mM Tris, 192 mM glycine) 0.1% SDS Store at room temperature Western blotting buffer Tris-glycine (25 mM Tris, 192 mM glycine) 20% methanol 0.1% SDS Store at room temperature Blot blocking solution PBS 0.1% Tween-20 5% non-fat dry milk Store single-use aliquots at -20 °C PBST PBS 0.1% Tween-20 Store at room temperature Acknowledgments This procedure was adapted from the original research article (Derksen et al., 2022), where we demonstrate the efficient isolation of RNase MRP and the possibility to use this method to study protein binding and substrate cleavage activity of wild-type and mutant RNase MRP RNA. Competing interests The authors declare no conflict of interest. References Bachler, M., Schroeder, R. and von Ahsen, U. (1999). StreptoTag: a novel method for the isolation of RNA-binding proteins. RNA 5(11): 1509-1516. Chang, D. D. and Clayton, D. A. (1987). A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J 6(2): 409-417. Coughlin, D. J., Pleiss, J. A., Walker, S. C., Whitworth, G. B. and Engelke, D. R. (2008). Genome-wide search for yeast RNase P substrates reveals role in maturation of intron-encoded box C/D small nucleolar RNAs. Proc Natl Acad Sci U S A 105(34): 12218-12223. Derksen, M., Mertens, V., Visser, E. A., Arts, J., Vree Egberts, W. and Pruijn, G. J. M. (2022). A novel experimental approach for the selective isolation and characterization of human RNase MRP. RNA Biol 19(1): 305-312. Franke, C., Grafe, D., Bartsch, H. and Bachmann, M. P. (2015). Use of Nonradioactive Detection Method for North- and South-Western Blot. Methods Mol Biol 1314: 63-71. Hermanns, P., Tran, A., Munivez, E., Carter, S., Zabel, B., Lee, B. and Leroy, J. G. (2006). RMRP mutations in cartilage-hair hypoplasia. Am J Med Genet A 140(19): 2121-2130. Gill, T., Cai, T., Aulds, J., Wierzbicki, S. and Schmitt, M. E. (2004). RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation. Mol Cell Biol 24(3): 945-953. Green, R. and Rogers, E. J. (2013). Transformation of chemically competent E. coli. Methods Enzymol 529: 329-336. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. and Altman, S. (1983). The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35(3 Pt 2): 849-857. Hou, S., Shi, L. and Lei, H. (2016). Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro. Methods Mol Biol 1421: 23-34. Lan, P., Zhou, B., Tan, M., Li, S., Cao, M., Wu, J. and Lei, M. (2020). Structural insight into precursor ribosomal RNA processing by ribonuclease MRP. Science 369(6504): 656-663. Leppek, K. and Stoecklin, G. (2014). An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins. Nucleic Acids Res 42(2): e13. Li, Y. and Altman, S. (2002). Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components. Nucleic Acids Res 30(17): 3706-3711. Lygerou, Z., Allmang, C., Tollervey, D. and Seraphin, B. (1996). Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro. Science 272(5259): 268-270. Mattijssen, S., Welting, T. J. and Pruijn, G. J. (2010). RNase MRP and disease. Wiley Interdiscip Rev RNA 1(1): 102-116. Mattijssen, S., Hinson, E. R., Onnekink, C., Hermanns, P., Zabel, B., Cresswell, P. and Pruijn, G. J. (2011). Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease. Cell Mol Life Sci 68(14): 2469-2480. Park, O. H., Ha, H., Lee, Y., Boo, S. H., Kwon, D. H., Song, H. K. and Kim, Y. K. (2019). Endoribonucleolytic Cleavage of m(6)A-Containing RNAs by RNase P/MRP Complex. Mol Cell 74(3): 494-507 e498. Petrov, A., Tsa, A. and Puglisi, J. D. (2013). Analysis of RNA by analytical polyacrylamide gel electrophoresis. Methods Enzymol 530: 301-313. Pluk, H., van Eenennaam, H., Rutjes, S. A., Pruijn, G. J. and van Venrooij, W. J. (1999). RNA-protein interactions in the human RNase MRP ribonucleoprotein complex. RNA 5(4): 512-524. Ramanathan, M., Porter, D. F. and Khavari, P. A. (2019). Methods to study RNA-protein interactions. Nat Methods 16(3): 225-234. Randau, L., Schroder, I. and Soll, D. (2008). Life without RNase P. Nature 453(7191): 120-123. Reiner, R., Ben-Asouli, Y., Krilovetzky, I. and Jarrous, N. (2006). A role for the catalytic ribonucleoprotein RNase P in RNA polymerase III transcription. Genes Dev 20(12): 1621-1635. Reiner, R., Krasnov-Yoeli, N., Dehtiar, Y. and Jarrous, N. (2008). Function and assembly of a chromatin-associated RNase P that is required for efficient transcription by RNA polymerase I. PLoS One 3(12): e4072. Robertson, H. D., Altman, S. and Smith, J. D. (1972). Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid precursor. J Biol Chem 247(16): 5243-5251. Sambrook, J. and Russell, D. W. (2006). SDS-Polyacrylamide Gel Electrophoresis of Proteins. CSH Protoc 2006(4). Srisawat, C. and Engelke, D. R. (2001). Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins. RNA 7(4): 632-641. Srisawat, C. and Engelke, D. R. (2002). RNA affinity tags for purification of RNAs and ribonucleoprotein complexes. Methods 26(2): 156-161. Suchan, T. (2020). Phenol-chloroform DNA purification. protocols.io: DOI: dx.doi.org/10.17504/protocols.io.re6d3he. Tabor, S. and Struhl, K. (2001). Enzymatic labeling of DNA. Curr Protoc Hum Genet Appendix 3: Appendix 3E. Walker, S. C., Scott, F. H., Srisawat, C. and Engelke, D. R. (2008). RNA affinity tags for the rapid purification and investigation of RNAs and RNA-protein complexes. Methods Mol Biol 488: 23-40. Welting, T. J., Kikkert, B. J., van Venrooij, W. J. and Pruijn, G. J. (2006). Differential association of protein subunits with the human RNase MRP and RNase P complexes. RNA 12(7): 1373-1382. Welting, T. J., Mattijssen, S., Peters, F. M., van Doorn, N. L., Dekkers, L., van Venrooij, W. J., Heus, H. A., Bonafe, L. and Pruijn, G. J. (2008). Cartilage-hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly. Biochim Biophys Acta 1783(3): 455-466. Yoon, J. H. and Gorospe, M. (2016). Identification of mRNA-Interacting Factors by MS2-TRAP (MS2-Tagged RNA Affinity Purification). Methods Mol Biol 1421: 15-22. Yoon, J. H., Srikantan, S. and Gorospe, M. (2012). MS2-TRAP (MS2-tagged RNA affinity purification): tagging RNA to identify associated miRNAs. Methods 58(2): 81-87. Article Information Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. How to cite Category Biochemistry > Protein > Isolation and purification Molecular Biology > RNA > RNA detection Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy Wenxin Zhang [...] Sharon A. Tooze Jan 5, 2024 650 Views Chromogranin B Purification for Condensate Formation and Client Partitioning Assays In Vitro Anup Parchure and Julia Von Blume Oct 20, 2024 286 Views Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization Jasmeet Kaur Shergill and Domenico Azarnia Tehran Jan 20, 2025 237 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,616	https://bio-protocol.org/en/bpdetail?id=4616&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis SK Shweta Kumar DN Dimple Nagesh VR Venketsubbu Ramasubbu AP Arathi Bangalore Prabhashankar NS Nagalingam Ravi Sundaresan Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4616 Views: 1742 Reviewed by: Giusy TornilloMohan BabuThirupugal Govindarajan Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Jan 2022 Abstract Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth. Keywords: Cardiac fibroblasts Fibrosis TGF-β1 Primary culture Cardiac fibrosis Background Cardiac fibroblasts or fibrocytes are one of several cells constituting a healthy heart. These are non-myocyte cells involved in developing and maintaining the cardiac architecture (Snider et al., 2009). In a physiological condition, they interact with other heart cells and maintain cardiac homeostasis (Kurose, 2021). Cardiac fibroblasts preserve the structural integrity of the heart by producing and depositing collagen I, III, V, and VI and fibronectin as part of the extracellular matrix (ECM) (Snider et al., 2009). Fibroblasts are the major players in modulating ECM by degrading its proteins through the secretion of matrix metalloproteinases (Eghbali and Weber, 1990; Chacar et al., 2017). In a healthy heart, one of the largest populations of cells is the fibroblasts (Camelliti et al., 2005). During cardiac stress or injury, fibroblasts divide and differentiate into myofibroblasts or activated fibroblasts with an increase in their activity (Rohr, 2011). To compensate for the loss of cardiomyocytes during a pathophysiological condition, activated fibroblasts deposit ECM proteins, such as collagen, to sustain the structural integrity of the heart (Kurose, 2021). However, if deposited in excess, the ECM expands the cardiac interstitium, a characteristic of cardiac fibrosis that leads to heart dysfunction (N. G. Frangogiannis, 2020) and premature death. Since cardiac fibroblasts hold great significance in the functioning of a heart, it is essential to characterize the various roles played by fibroblasts (Rohr, 2011). To investigate various cellular aspects of cardiac fibroblasts in detail, including changes in cellular morphology, regulatory molecular pathways, gene-expression levels, and changes in the expression level of proteins, cultured neonatal primary cardiac fibroblasts can prove to be a valuable model system. Cultured neonatal cardiac fibroblasts mimic various characteristics of fibroblasts in vitro. These cells have the propensity to phenotypically differentiate into myofibroblasts, thus recapitulating the conditions of a stressed or injured heart (Rohr, 2011). Obtaining pure primary cardiac fibroblasts and procuring homogeneity in the cells is indeed very advantageous. It allows for studies to be carried out on these specific cells without any interference from other heart cell types or other regulatory paracrine signaling pathways that can affect their activity. Cultured cardiac fibroblasts can also be subjected to treatments stimulating these cells to mimic cardiac pathologic conditions, such as cardiac fibrosis, in vitro. Transforming growth factor β (TGF-β) plays a significant role in fibrogenesis in the fibroblasts (N. Frangogiannis, 2020). There are three isoforms of TGF-β: TGF-β1, TGF-β2, and TGF-β3 (Schiller et al., 2004). All three have been shown to contribute to fibrogenesis. However, TGF-β1 has been reported to be the most important isoform involved in fibrosis (Yu et al., 2003). The signaling pathways involved in driving fibrosis are either Smad3-dependent or Smad3-independent pathways, involving various co-receptors and regulators (N. Frangogiannis, 2020). Primary cardiac fibroblasts can be used to study various signaling pathways involved in cardiac fibrosis, including the TGF- β/SMAD3 signaling pathway. Overall, primary cardiac fibroblasts act as a reliable in vitro model that can be used to induce and mimic fibrosis that takes place in vivo. Isolation and culture of primary cardiac fibroblasts from neonatal rat and mouse pups This protocol describes an easy procedure for isolating and culturing cardiac fibroblasts from neonatal rat and mouse pups. The described procedure involves the use of enzymes such as collagenase type II and trypsin for the digestion of the heart tissue (to digest the ECM and dissociate the cells). No additional growth supplements are required for the culture of primary cardiac fibroblasts. Tissue culture plates are coated with poly-L-lysine to enhance the attachment of cardiac fibroblasts. The procedure includes dissecting the rat/mouse pups and taking out the hearts. This is followed by making small chunks of the heart and digesting the heart tissue with the prepared digestion mixture (trypsin + collagenase). Eventually, collection of the digested cells is done in horse serum; the collected cells are plated in tissue culture plates and incubated in an incubator for further subculturing and experimentation. The whole procedure takes approximately 4–5 h (Figure 1). It is advised for the users to first consult the institutional guidelines on animal usage for experiments and to get the necessary permissions from the institute’s animal ethics committees. Gloves and lab coats should always be worn when handling animals, and there should be no direct contact of bare hands with animals and/or tissues. Carcasses and tissue wastes generated while harvesting the pups should be collected and sealed in double bags and disposed of in an incinerator, following institutional guidelines. Figure 1. Diagrammatic representation of the major steps involved in the isolation and culture of primary cardiac fibroblasts from murine pup hearts. The figure was designed using images adapted from Servier Medical Art by Servier. Original images are licensed under a Creative Commons Attribution 3.0 Unported License. Basic protocol: Isolation and culture of primary cardiac fibroblasts from neonatal murine pups Materials and Reagents Sterile cotton Sterile 10 cm Petri plates (Eppendorf, catalog number: EP0030702115) Sterile 1.5 mL microcentrifuge tubes (Eppendorf, catalog number: 0030121872) 50 mL conical centrifuge tubes (Eppendorf, catalog number: 0030122178) Rat pups or mouse pups (0–2 days old) Distilled water 70% ethanol (HIMEDIA, catalog number: MB228) (v/v) in double distilled water Horse serum (Gibco, catalog number: 16050122) Trypsin 0.25% solution 1× without phenol red (HIMEDIA, catalog number: TCL006) Complete media: Dulbecco’s Modified Eagle Medium (DMEM) (HIMEDIA, catalog number: AL007A), supplemented with 10% fetal bovine serum (FBS) (HIMEDIA, catalog number: RM-10432) and 1× antibiotic-antimycotic mix (Gibco, catalog number: 15640-055) (see Recipes) Phosphate-buffered saline (PBS), 1× (see Recipes) Phosphate-buffered saline buffer (1×) with D-glucose (PBSG) (see Recipes) Digestion mixture (DM) (see Recipes) Equipment Sterile surgical scissors (Harvard Apparatus, catalog number: ST2 72-8438) Forceps (Harvard Apparatus, catalog number: ST2 72-8949) Blades (Harvard Apparatus, catalog number: ST2 72-8366) Tissue culture laminar hood (vertical flow) (Thermo Scientific, model: 1338) Vortex mixer (SPINIX by Tarsons, 3020) Shaker incubator (Thermo, model: MAXQ 4450) CO2 incubator for cell culture (maintained at 37 °C and 5% CO2) Procedure Heart tissue harvesting Clean each mouse/rat pup with lukewarm distilled water followed by 70% ethanol, gently using a sterile absorbent cotton ball. Notes: This step is important to remove any filth or contaminants due to environmental exposure and is done to avoid any contamination in the later stages of cell culture. The number of pups required should be decided based on the planned experiments. Anesthetize pups and then decapitate them with the help of a pair of sterile scissors in a sterile dissection hood. Note: Pups should be 0–2 days old (P0–P2) as the yield of viable cells decreases with older pups. Culling of the pups should be done one by one. Make sure to wipe off the scissors with 70% ethanol before and after using them. Hold the animals in supine position, allowing access to the head, neck, and thoracic region of the animal. Make a horizontal incision in the neck region and then a vertical incision, cutting through thoracic ribs with the help of a pair of sterile scissors (the cut is shaped like a T). Note: Make the vertical cut inside the rib cage until it exposes the heart. Press a little with fingers from where the animal is held until the heart protrudes. Cut out the heart with the help of sterile scissors and quickly put the excised heart in ice-cold sterile PBSG. Note: Putting the heart in ice-cold PBSG is essential because it will decrease the rate of metabolism in the heart, which will lessen stress on the heart. After washing the hearts, transfer them to another dish containing fresh, ice-cold PBSG in a tissue culture laminar hood. With the help of sterile forceps, try to squeeze out any excess blood inside the heart and stop squeezing once the blood stops oozing out. Note: Get rid of parts of any other organs/tissues attached to the heart during its excision, like lungs, diaphragm, and any blood clots. Also, make sure to remove the top part of the heart that consists of the atria and large blood vessels. Digesting the heart tissue Cut a heart into 4–5 pieces (pieces should be around 1 mm3) with the help of a sterile surgical blade. Collect the pieces into a sterile 1.5 mL microcentrifuge tube containing ice-cold DM (approximately 130 μL per rat heart or two mice hearts). Notes: Try not to make very small pieces as it may be difficult to avoid taking them while collecting cells with a micropipette after rounds of digestion. One 1.5 mL microcentrifuge tube can accommodate up to three rat hearts and 5–6 mouse hearts. The volume of the DM should be adjusted according to the number of hearts in each tube. Crushing or grinding should not be included while cutting the heart pieces as it may lead to cell death. Start the digestion by placing the microcentrifuge tubes containing DM and the heart chunks into a shaker incubator, which should be maintained at 37 °C and set at 250 rpm for 7 min for each digestion cycle. After the first digestion cycle, vortex the tubes and then spin them for no more than 10 s at approximately 600 × g in a tabletop centrifuge to let the heart chunks settle at the bottom of the tube. Discard the supernatant. Note: Supernatant from the first digestion cycle is discarded as it contains parts of the ECM, RBCs, and other debris. Add fresh DM to these tubes in the tissue culture laminar hood and vortex briefly before placing them back in the shaker incubator, maintained at the same optimum conditions for 7 min. Note: Prolonged digestion or vortexing should be avoided, as excessive stress can cause damage and death of the cells. Once the digestion is complete, vortex and spin the tubes for less than 10 s at approximately 600 × g in a tabletop centrifuge. Inside the tissue culture laminar hood, collect the supernatant from the microcentrifuge tubes in a 50 mL conical centrifuge tube containing pre-warmed horse serum (at 37 °C). Keep this tube containing horse serum with cells in an incubator maintained at 37 °C and 5% CO2 saturation until the next digestion cycle is complete. Collect the supernatant from each digestion in the same tube. Note: To avoid contamination, a completely sterile environment should be maintained for the cells collected from this step onwards. Keep the screw cap of the 50 mL conical centrifuge tube partially open (not completely closed) to allow aeration. Continue the digestion process (add DM, keep in shaker incubator for 7 min, vortex and spin, collect the supernatant in horse serum in the 50 mL conical centrifuge tube, and place in the CO2 incubator) until the heart chunks are completely digested. It takes approximately 9–10 cycles to digest the mouse/rat heart tissue completely. Notes: The amount of horse serum in the 50 mL conical centrifuge tube used to collect the digested cells after each digestion cycle should be approximately double the total volume of supernatant collected upon all digestions. The enzymes present in the DM get neutralized by horse serum upon collection of the digested cells. This is important to prevent over-digestion, or the cells may be damaged. Sterile conditions should be maintained throughout the procedure. The number of digestions may be increased to get a higher cellular yield, but longer digestions may lead to damage to the cells, thus reducing the overall yield of viable fibroblasts. Plating of cells Mix the cells and horse serum to a near homogenous mixture before seeding the cells in poly-L-lysine–coated 10 cm culture dishes (Refer to the Support protocol for coating the cell culture dishes before seeding the fibroblasts). Incubate these dishes for 1 h in a CO2 incubator to allow the attachment of the fibroblasts. Notes: The number of dishes used will be based on the number of rat/mouse pups used according to the experiment. One 10 cm culture plate can accommodate fibroblasts from around 4–5 and 9–10 hearts from rat and mouse pups, respectively. The expected yield is approximately 2 × 105 cells per rat heart and approximately 1 × 105 cells per mouse heart. Fibroblasts attach to the plate because of the differential attachment properties of cardiac fibroblasts and cardiomyocytes. Cardiac fibroblasts get attached to the dishes before cardiomyocytes (within 1 h) in the presence of poly-L-lysine. Cardiomyocytes adhere poorly to the coated surface of the dishes within this short period and, therefore, can easily be washed and collected along with the supernatant. If the incubation time is increased, separating cardiac fibroblasts from cardiomyocytes may be challenging as the cardiomyocytes will also adhere to the coated surface of the 10 cm culture plates. Hence, the differential attachment properties of the two cell types have proved beneficial for the specific isolation of cardiac fibroblasts. After 1 h of incubation, remove the supernatant containing the unattached cardiomyocytes. The cardiomyocytes can either be discarded along with the serum or collected through centrifugation and can further be resuspended in fresh media to be used for experiments if needed. Wash the plates containing attached fibroblasts with fresh complete media by adding media to each plate (approximately 2 mL) and gently swirling it around before collecting it back. The collected media can be added to the collected serum supernatant from the last step before centrifugation. Note: Media wash is given to wash any remaining cardiomyocytes still there on the surface of the plate and obtain a pure culture of fibroblast cells. Add 10 mL of fresh complete media to each plate and keep in a CO2 incubator maintained at 37 °C and 5% CO2 saturation level. Propagation and seeding of fibroblasts Discard the media from the culture dishes in which the cells were seeded. Note: After removing used media using a serrated pipette and a pipette aid, keep the dishes in a slanting position to drain and collect the remaining media. Wash the cells (attached to the surface of the plate) with 1× PBS (4 mL per dish). Gently swirl the PBS and discard it in the same way as the used media in the previous step. Note: Proper washing of cells and removal of the used media is important because serum present in complete media may hamper the activity of trypsin. The serum contains protease inhibitors that inhibit trypsin activity. Add 1 mL of 0.25% trypsin-EDTA (1×) to each 10 cm culture dish containing primary cardiac fibroblasts and swirl the dish gently to cover all cells with the solution. Note: Keep the dishes with trypsin-EDTA in a CO2 incubator to activate trypsin’s activity for one to two minutes while constantly checking for the detachment of the cells (slight tapping can help the cells detach from the base). Add 4 mL of complete media to the trypsinized cells in the 10 cm culture dishes making the total volume as 5 mL (1 mL trypsin-EDTA + 4 mL complete media) in each dish. Note: Media is added as it contains serum that helps to prevent over-trypsinization of cells. If trypsin activity is not inhibited, prolonged trypsinization may lead to cell damage. Collect the cells + media + trypsin in a 15 mL conical centrifuge tube and spin at 100 × g for 5 min at room temperature in a centrifuge. Note: The media + trypsin solution in the plates can be used to flush the cells from the surface of the plates by pipetting them up and down several times in the culture dish. This step will help detach the maximum number of cells from the surface of the culture dish. After centrifugation, discard the supernatant and resuspend the cell pellet with fresh complete media. Note: Make sure to resuspend the cells to a near-homogenous mixture so that the seeding can be done appropriately for experiments. The resuspended cells can then be seeded in suitably labeled tissue culture dishes or other plates as per the requirements of the designed experiments (Refer ‘Support protocol’ for coating the cell culture dishes before seeding the fibroblasts). Note: Primary neonatal cardiac fibroblasts can be trypsinized and sub-cultured up to two times. Support protocol: Coating the tissue culture plates with poly-L-lysine for effective attachment of cardiac fibroblasts to the surface of the dishes Materials and Reagents 10 cm tissue culture plates (Eppendorf, catalog number: EP0030702115) 0.22 μm syringe filter (WhatmanTM Uniflo 25 syringe filter, Merck, catalog number: WHA9913-2502) 0.45 µm PVDF membrane (GE Healthcare, catalog number: 10600023) Poly-L-lysine (Sigma-Aldrich, catalog number: P8920) Trypsin 0.25% solution 1× without phenol red (HIMEDIA, catalog number: TCL006) Collagenase type II (Gibco Collagenase, Type II, powder, catalog number: 17101015) DMEM, high glucose (HIMEDIA, catalog number: AL007A) Fetal bovine serum (FBS) (Gibco, catalog number: RM-10432) 100× antibiotic-antimycotic solution (Gibco, catalog number: 15640-055) Formaldehyde solution (Sigma-Aldrich, catalog number: F8775) Triton X-100 (Sigma-Aldrich, catalog number: 9002-93-1) Bovine Serum Albumin (HIMEDIA, catalog number: MB-083) Tween-20 (Sigma-Aldrich, catalog number: P1379) Fluoromount (Sigma-Aldrich, catalog number: F4680) 2× Laemmli Sample Buffer (Bio-Rad, catalog number: 161-0737) β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148) Skimmed Milk Powder (HIMEDIA, catalog number: GRM-1254) Clarity Western ECL Substrate (Bio-Rad, catalog number: 1705061) RNAiso Plus Reagent (TAKARA, catalog number: 9108) PrimeScript 1st Strand cDNA Synthesis kit (TAKARA, catalog number: RR014B) SYBR-Green PCR master mix (Bio-Rad, catalog number: 1725124) TGF-β1 (Sigma, catalog number: T7039) KCl NaCl KH2PO4 Na2HPO4 Double-distilled water (DDW) HCl Glucose (Qualigens, catalog number: 15405) Antibodies (Table 1) Digestion mixture (see Recipes) DMEM with 10% FBS and antibiotic-antimycotic mix (complete media) (see Recipes) 0.01% (w/v) Poly-L-lysine (see Recipes) Phosphate-buffered saline (PBS), 1× (see Recipes) Phosphate-buffered saline-glucose (PBSG), 1× (see Recipes) Phosphate-buffered saline-Tween-20 (PBST), 1× (see Recipes) Tris-buffered saline-Tween-20 (TBST), 1× (see Recipes) Transfer Buffer (see Recipes) Table 1. List of antibodies used in the study S. No. Antibody Company Catalog no. Application 1 Anti-Actin, α-Smooth Muscle antibody, Mouse monoclonal Sigma A5228 Western Blotting (1:1,000), Immunofluorescence (1:500) 2 Fibronectin (EP5) mouse monoclonal Santa-Cruz sc-8422 Western Blotting (1:1,000), Immunofluorescence (1:250) 3 COL1A1 (C-18) goat polyclonal Santa-Cruz sc-8784 Western Blotting (1:1,000), Immunofluorescence (1:250) 4 COL3A1 (B-10) mouse monoclonal Santa-Cruz sc-271249 Western Blotting (1:1,000), Immunofluorescence (1:250) 5 Ms mAB to beta Actin Abcam ab8226 Western Blotting (1:2,000) Equipment -20 °C freezer Confocal Laser Scanning Microscope (LSM 880, Zeiss, USA) ChemiDoc Imaging System, Bio-Rad (Version 2.2.0.08) QuantStudio 6 Flex, Life Technologies Procedure Poly-L-lysine is an effective agent that promotes adherence of cells and ECM to coated solid surfaces. Polycationic poly-L-lysine molecules are strongly adsorbed onto the solid surfaces. The anionic sites on the cells recognize the exposed polycationic sites on these molecules, therefore enhancing the attachment of the cells to the surface of the coated tissue culture plates, forming a monolayer of cells (Mazia et al., 1975; Macieira-Coelho and Avrameas, 1972). For the seeding of fibroblasts, coating of tissue culture ware is done by covering the surface briefly with a 0.01% working solution of poly-L-lysine. Tissue culture plates coating Prepare sterile 0.01% working solution (in deionized water) from commercially available 0.1% stock solution of poly-L-lysine. Coat 10 cm tissue culture dishes by covering the entire surface with 0.01% poly-L-lysine. Note: 5 mL of working poly-L-lysine solution is sufficient for covering the surface of a 10 cm culture plate. Incubate the plates for half an hour in a CO2 incubator maintained at 37 °C and a 5% CO2 saturation level. Notes: Take the plates from the incubator and remove the excess poly-L-lysine solution. Collect any excess solution from the plates. Avoid putting any scratch on the coated surface with the pipette tips. Keep the plates open under UV for 30 min at room temperature inside a sterile tissue culture hood. Note: Avoid over-drying the coated plates, as it might compromise the attachment of cardiac fibroblasts because of coating disruption. These plates are now ready for seeding the cardiac fibroblasts after completing the digestion steps. Methodology Immunofluorescence protocol: Immunofluorescence experiments were performed with primary neonatal rat cardiac fibroblasts as described previously (Maity et al., 2020). 3.7% formaldehyde was used for the fixation of the cells for 10 min at room temperature, and permeabilization was done using 0.2% Triton X-100 for 5 min at room temperature. Three washes with 1× PBS for 3 min each were given to the cells before each step. Blocking the cells was done using 5% BSA in PBST buffer for 1 h at room temperature. Primary antibodies were prepared in 1% BSA in PBST and incubated with the cells overnight at 4 °C. After washing the cells thrice with 1× PBS for 3 min each, they were incubated with the secondary antibody prepared in 1% BSA in PBST for 1 h at room temperature. Nuclei were stained using Hoechst 33258 (1:2,000) for 10 min. Fluoromount aqueous mounting medium was used to mount the coverslips on clean glass slides, and imaging was done with a confocal microscope. The antibodies used have been listed in Table 1. Western Blotting: Western blotting was performed for primary neonatal rat cardiac fibroblasts as previously described (Maity et al., 2020). In short, total protein lysates were prepared using RIPA buffer supplemented with phosphatase inhibitors and protease inhibitors. Samples were prepared by mixing the lysates with 2× Laemmli sample buffer with 5% β-mercaptoethanol. The prepared samples were heated at 95 °C for 5 min and resolved on a 10% SDS-PAGE gel at 80 V. PVDF membrane (0.45 µm) was utilized for transferring the proteins through overnight wet transfer at 20 V, using transfer buffer. Blocking of the membrane after the transfer was done using 5% skimmed milk in TBST for 1 h at room temperature. Upon washing the blocked membrane with TBST, it was incubated with primary antibody (prepared in 5% BSA in TBST) overnight at 4 °C, followed by incubation with secondary antibody (prepared in 1% skimmed milk in TBST) for 1 h at room temperature. Antibodies bound to the PVDF membrane were visualized using a western ECL reagent in a chemiluminescence imager. The antibodies used have been listed in Table 1. Real-time PCR: qRT-PCR analysis was performed as described previously (Maity et al., 2020). RNAiso Plus reagent was used for the isolation of total RNA from the cells following the manufacturer’s protocol. Purity and integrity of the isolated RNA were confirmed, and cDNA synthesis was done using 1 μg of the RNA with the PrimeScript 1st Strand cDNA Synthesis kit. RT-PCR was performed with the SYBR-Green PCR master mix in a real-time PCR machine. The primers used in the study are listed in Table 2. Table 2. List of primers used in the study S. No. Name Sequence (5′-3′) 1 α-SMA Forward GGAGATGGCGTGACTCACAA 2 α-SMA Reverse CGCTCAGCAGTAGTCACGAA 3 FN1 Forward CCACCATCACTGGTCTGGAG 4 FN1 Reverse GGGTGTGGAAGGGTAACCAG 5 Col1a Forward CAATGGCACGGCTGTGTGCG 6 Col1a Reverse CACTCGCCCTCCCGTCTTTGG 7 Col3a Forward TGGCACAGCAGTCCAACGTA 8 Col3a Reverse AAGGACAGATCCTGAGTCACAGA 9 Actin Forward CACTGTCGAGTCGCGTCC 10 Actin Reverse TCATCCATGGCGAACTGGTG TGF-β1 treatment: Used media was removed from the cells and they were washed with PBS; 10 ng/mL TGF-β1 in serum-free DMEM was added to the cells, followed by incubation for 24 h at 37 °C and 5% CO2 saturation. Upon completion of the treatment, cells were either harvested for lysate preparation (western blot analysis) or RNA isolation (qRT-PCR analysis) according to the planned experiments. The treated cells could also be fixed using 4% formaldehyde for immunofluorescence experiments. Commentary: All major organs have resident fibroblasts, the most widely present active cells of the connective tissue (Kendall and Feghali-Bostwick, 2014). They play a crucial role in the proper functioning of the organs. Fibroblasts are essential for regulating gene expression affecting the site-specific differentiation of cells in different organs (Rinn et al., 2006). Interactions of fibroblasts with different cell types have been proven to play a significant role in the patterning of various organs, including the lung, skin, and gastrointestinal tract. Fibroblasts express specific genes, which help provide identity to cells in different organs, and, therefore, can be used to study organ-specific functions and the process of organogenesis (Rinn et al., 2006). Fibrosis, the excess production of ECM proteins, including collagen, to form scar tissue, is the underlying process for all fibrotic-related diseases. The production of scar tissue is important for sealing open wounds. However, if unregulated, it may act as the basis of numerous diseases such as idiopathic pulmonary fibrosis, systemic sclerosis, kidney fibrosis, liver cirrhosis, and cardiac fibrosis (Zhang and Zhang, 2020). Other than fibrosis, fibroblasts have also been shown to play a role in inflammation and cancer progression. In a published study, fibroblasts have been found to maintain inflammation, eventually leading to rheumatoid arthritis and supporting the growth of tumor mass (Mizoguchi et al., 2018; Winkler et al., 2020). In a healthy heart, fibroblasts can regenerate functional tissue. They are known to be involved in wound healing, inflammation, the proliferation of cells, deposition of ECM, and eventually remodeling (desJardins-Park et al., 2018). Cardiac fibroblasts play an important role in maintaining homeostasis and normal heart function, as well as in pathological conditions involving cardiac remodeling, such as hypertension and myocardial infarction. The functions that fibroblasts are involved in include the production and deposition of ECM proteins, the interaction and communication with other cell types including myocytes, and the intercellular signaling with endothelial cells and other fibroblasts. These interactions have been shown to regulate the electrophysiological properties of the cells, secretion of cytokines and growth factors, and angiogenesis. Several studies have been conducted to elucidate the role of fibroblasts in all these processes, except angiogenesis (Souders et al., 2009). Cardiac fibroblasts form a scaffold by secreting ECM, in which the cardiomyocytes are embedded to create the 3D structure of a healthy heart. This leads to dense packaging of cardiomyocytes and cardiac fibroblasts with each other, making it difficult to study each cell type separately. Hence, gaining information about these cells becomes difficult while they are in a three-dimensional arrangement in vitro. Cell culture in two-dimension has proven to be a very proficient approach for studying conceptual molecular biology. Though the cultured immortalized cell lines can be maintained for many passages, they do not accurately represent the in vitro conditions. Therefore, they are not the best model to study the physiological and pathophysiological aspects of the cell. Consequently, it is of utmost importance to be able to culture cardiac fibroblasts and cardiomyocytes separately in two-dimensional culture in vitro to study their functions individually. Most of what is already known about these cells, including their function and morphology in the heart and their behavior upon stimulation, has been possible by conducting experiments in two-dimensional primary tissue culture. In the present study, cultured primary cardiac fibroblasts proved to be of better biological relevance than immortalized cell lines. Upon stimulation, these cells in culture reflect various molecular changes in vitro (Landry et al., 2021). Therefore, these cells can be used to investigate multiple cellular responses under different conditions as these are the closest mimic of the processes that occur in vitro (Eghbali, 1992). However, upon passaging the cells 2–3 times, the cardiac fibroblasts tend to transdifferentiate into myofibroblasts, which are characterized by an increased α-SMA secretion (Rohr, 2011). Since fibroblasts are involved in the pathogenesis of various diseases, cultured fibroblasts can act as a suitable model for studying several diseases and pathological conditions. Moreover, cultured fibroblasts can be used to study drug toxicology and even drug screening. Fibroblasts cultured from human skin have been used in a published study to elucidate the cytotoxic effects and skin irritation of surfactants (Lee et al., 2000). In another study, fibroblasts isolated from lungs have been utilized to study the toxicity of antimicrobial drugs (Krempaska et al., 2020). Although cultured cardiac fibroblasts cannot completely mimic the in vitro phenotype, they prove to be an advantageous model in several aspects for both physiological and pathophysiological studies. For example, they can be selectively manipulated with numerous treatments without interference from cardiomyocytes as they are cultured as a distinct homogenous population (Ravi et al., 2021). These cells can be used for various genetic and/or drug screening purposes, which may be completed in comparatively less time than in vitro studies. Cultured cardiac fibroblasts are also a beneficial tool for studying the factors responsible for their transformation and regulating the production and degradation of the ECM. Moreover, they can be used to elucidate cardiac fibroblasts’ role in the endocrine activity (Grupp and Müller, 1999). Mouse and rats are used for culturing the primary cardiac fibroblasts due to several reasons. Rat and mice models are easily available without the requirement of high cost and skills. Additionally, these murine models are very well studied, and most of the studies on signaling pathways and metabolics that have been published have been carried out in these models (Zaragoza et al., 2011). Cultured primary fibroblasts act as a better model system in biological research when compared to immortal cancer cell lines. These cells lack mutations in the tumor repressor genes and the oncogenes. Therefore, they make for a preferred model system for studies on DNA repair, cell cycle regulation, and apoptosis (Alikhani et al., 2005; Schauble et al., 2012; Marthandan et al., 2016). Moreover, most existing protocols use sophisticated and specialized techniques like density gradient centrifugation. Our protocol, however, is a set of simple steps involving commonly available machines and chemicals that can be easily found in all laboratories. Although cultured primary fibroblasts are an excellent model for studying fibrosis, they have their limitations. The primary fibroblasts in culture may spontaneously transdifferentiate into myofibroblasts due to the contact with solid surfaces of the culture dishes. Additionally, since these cells are grown in isolation from other cell types present around them in a living organism, it is impossible to fully comprehend the interactions and signaling pathways regulated by those interactions between fibroblasts and the various other cell types in different organs. Results Using the described protocol, we successfully cultured primary cardiac fibroblasts from neonatal murine pups (P0-P2). Further, the cultured cells were treated with TGF-β1, a major regulator of fibrosis. Overall, our results demonstrate the expected outcome of the protocol as described. Moreover, we show that these cells can be transdifferentiated into activated myofibroblasts upon TGF-β1 treatment. Visualization and characterization of cultured cardiac fibroblasts Fibroblasts appear round during the initial stages of pre-plating and the beginning of attachment to the coated surface of the cell culture dishes. With time, the fibroblasts spread and attach firmly to the coated culture dishes. Eventually, they form a monolayer of plump, spindle-shaped cells (Figure 2A). Some cells remain rounded and do not attach even after a day; these cells can be washed off using PBS while passaging the cells or changing the medium. On keeping the fibroblasts in a confluent state, starving the cells of serum, or treating the cells with TGF-β1, the spindle-shaped fibroblasts transdifferentiate into myofibroblasts, which have a characteristic large surface area with various protrusions of the plasma membrane (Figure 2B). Treatment of cardiac fibroblasts with TGF-β1 TGF-β1 is one of the major regulators of fibrosis. TGF-β1 treatment is a well-established model for studying the induction of fibrosis in mice and cultured cells. Upon TGF-β1 treatment, the fibroblasts get activated and transdifferentiate into myofibroblasts. This can be characterized by enhanced production of α-smooth muscle actin (α-SMA), a myofibroblast-specific marker. α-SMA is a cytoskeletal protein responsible for activated myofibroblasts' mobility. Additionally, since TGF-β1 treatment results in the induction of fibrosis, it is accompanied by an increase in the production and deposition of the ECM proteins, including fibronectin 1 (FN1), collagen 1a (Col1a), and collagen 3a (Col3a). These act as fibrotic markers, and their levels can indicate whether fibrosis has set in upon the treatment. To show that the isolated cells are indeed cardiac fibroblasts and can be differentiated into myofibroblasts, we treated the cells with TGF-β1 (10 ng/mL) for 24 h to induce fibrosis. The cells with and without TGF-β1 treatment were stained with myofibroblast specific markers (α-SMA) and fibrotic markers (FN1, Col1a, and Col3a). We observed an increase in the protein levels of α-SMA and a difference in its arrangement, confirming the transdifferentiation of fibroblasts into myofibroblasts upon TGF-β1 treatment (Figure 2C). Moreover, we could note an increase in the protein levels of FN1, Col1a, and Col3a, all of which are fibrotic markers (Figure 2C). This implies that fibrosis is enhanced in the cells treated with TGF-β1 compared to the controls. To further solidify our claims, we performed a western blot analysis to check the protein levels of α-SMA, FN1, Col1a, and Col3a. The cells were treated with TGF-β1 (10 ng/mL) for 24 h in serum-free media, and the lysates were prepared and run in an SDS-PAGE gel. We found an increase in the levels of α-SMA, FN1, Col1a, and Col3a, thus confirming that there was an increased population of myofibroblasts than fibroblasts in the TGF-β1-treated samples as compared to control samples (Figure 3A). Further, we performed qRT-PCR analysis to check if there was any change in the mRNA levels of α-SMA, FN1, Col1a, and Col3a. Upon analysis of the qRT-PCR results, we found that the mRNA levels of Col1a were elevated in TGF-β1-treated samples compared to controls (Figure 3B). However, we could not see a significant difference in the mRNA levels of other genes. Our results prove that the cells isolated using the protocol are indeed cardiac fibroblasts and can be differentiated into myofibroblasts using TGF-β1 treatment. The cultured cardiac fibroblasts can be used for similar studies to evaluate the toxicity of various drugs and phenotypic and subcellular changes resulting from such treatments. Figure 2. Cultured primary cardiac fibroblasts treated with TGF-β1. (A) Bright-field image of attached neonatal rat cardiac fibroblasts on poly-L-lysine-coated dishes. Scale bar: 500 μm. (B) Bright-field image of primary cardiac fibroblasts with and without TGF-β1 treatment for 24 h. Scale bar: 100 μm. (C) Primary cardiac fibroblasts were treated with TGF-β1 for 24 h and stained with α-smooth muscle actin (α-SMA) as a marker for myofibroblasts, and fibronectin 1 (FN1), collagen 1a (Col1a), and Collagen 3a (Col3a) as fibrotic markers. Hoechst 33342 was used to stain the nuclei blue. Scale bar: 50 μm. Figure 3. Molecular analysis of cardiac fibroblasts treated with TGF-β1. (A) Western blot analysis of neonatal rat cardiac fibroblasts upon 24 h of TGF-β1 treatment. &Agr;-SMA was used as a myofibroblast marker, and FN1, Col1a, and Col3a were used as fibrotic markers. Actin was used as the loading control. (B) Relative mRNA levels of α-SMA, FN1, Col1a, and Col3a in neonatal rat primary cardiac fibroblasts treated with TGF-β1 for 24 h. Actin has been used for normalization (as an internal control). Student’s t-test used for statistical analysis (*p ≤ 0.05). Data presented as mean ± S.D. Time required The total amount of time required to carry out the primary cardiac fibroblast culture from neonatal rat pups according to the described protocol has been mentioned in Table 3. Table 3. Time requirement Procedure Time (h:min) Notes Basic protocol Washing and sterilization of pups with water and 70% ethanol, respectively 0:10 The indicated time periods are for 5–7 rat pups. Timings can be scaled up and down according to the number of animals being used Euthanasia of pups 0:15 Harvesting the hearts through dissection 0:15 Mincing the hearts into smaller pieces 0:10 Digestion of the cardiac tissue and collection of cells in horse serum 1:30 Seeding cells on culture dishes (Pre-plating) 1:30 Washing and resubstituting the cells with media (to remove unattached cardiomyocytes) 0:10 Total 4:00 The total time may vary depending upon the number of animals being used for the experiment and the expertise of the user. Support protocol Adding poly-L-lysine coating solution and incubating 0:30 Removing the solution and keeping for air-drying 0:30 The coating should be done either before or during the digestion of tissues. Notes Important parameters and troubleshooting The critical points to be noted while culturing primary cardiac fibroblasts using our protocol have been listed. Wherever possible, we have included tips along with the protocol to avoid potential issues for each step. We have also compiled a table listing all the possible problems that may be faced and their potential solutions in Table 4. Table 4. Troubleshooting for common problems Problem Possible reasons for problems Solution Low yield of cardiac fibroblasts ●Pieces of mouse/rat heart tissues are too big for proper digestion Taking large chunks would cause incomplete digestion of heart tissue. One heart should be cut into pieces of ~1 mm3 (4–5 pieces for rat pups and 2–3 pieces for mouse pups), for better yield. ●The digestion mixture used is insufficient For better digestion, sufficient DM should be added (130 μL per four mice or one rat heart). ●Optimum conditions such as speed (rpm) or temperature not maintained The shaker incubator should be set at 250 rpm and 37 °C for digestion. ●Improper digestion due to expired or substandard digestion reagents Ensure that the reagents used are fresh and new and stored appropriately at the recommended temperatures. ●Not enough cycles of digestion Digestion should be done for an adequate number of cycles (9–10 cycles). Less viability ●Too much digestion Digestion of the heart tissue should not exceed 10 cycles. Digestion time for one cycle should not be longer than 7 min. ●Excess mechanical stress Excessive mechanical stress should be avoided such as excessive mincing of the heart tissue and prolonged vortexing. The speed of the shaker incubator should not exceed 280 rpm. ●Optimum conditions not maintained during harvesting and digestion of heart The excised heart should be placed immediately in ice-cold PBSG buffer upon harvesting. After each cycle of digestion, cells should be collected in horse serum and the 50 mL tube should be kept inside a CO2 incubator maintained at 37 °C and a 5% CO2 saturation level. ●Improper coating of tissue culture plates Proper coating of tissue culture plates with appropriate adhesion reagent should be done. Contamination of cells by bacteria/fungi ●Use of unsterilized dissection tools and/or reagents All the steps should be performed under sterile conditions. All tools and reagents should be sterilized including scissors and forceps. Fresh blades and a new sterile 50 mL conical centrifuge should be used. Buffers should be made fresh and autoclaved. DM, horse serum, complete media, and the reagent used for coating the plates should be filter-sterilized. ●Contamination in CO2 incubator or tissue culture hood Heat-sterilize the incubator to decontaminate it. Fumigation or UV sterilization of the tissue culture hood should be practiced frequently. Before starting the digestion process, expose the tissue culture hood to UV for a minimum of 30 min. ●Use of expired or bad antibiotics Antibiotics should be added to the media right before usage. Repeated freeze-thaw cycles should be avoided for the media with antibiotics. Contamination by non-fibroblast cells ●Excessive pre-plating Pre-plating should not exceed 1 h as other cells (cardiomyocytes) may also start to get attached to the coated surface of the plate. ●Tissues from other organs not removed completely during harvesting of the hearts Non-cardiac tissues such as pieces of lungs, diaphragm, or any other connective tissue stuck to the heart should be completely removed before starting the digestion process. Recipes Digestion mixture (DM) DM with a composition of 0.4 mg per ml collagenase type II and 0.25% trypsin is prepared by mixing 8 mL trypsin 0.25% solution 1× without phenol red with 4 mg of collagenase type II and making up the volume to 10 ml by adding 1× sterile PBSG buffer. Sterilize this mixture by passing it through a 0.22 μm syringe filter. Prepared DM can be aliquoted into vials and stored in a -20 °C freezer for up to three months. DMEM with 10% FBS and antibiotic-antimycotic mix (complete media) 445 mL of DMEM, high glucose is supplemented with 50 mL of FBS (sterilized through filtration using a 0.22 μm syringe filter) and 5 mL of 100× antibiotic-antimycotic solution to prepare complete media. This media can be used for seeding the primary cardiac fibroblasts after the steps of digestion and for subculturing the cells. The media should be prepared fresh as the antibiotics and antimycotics may degrade upon prolonged storage in the fridge at 4 °C. 0.01% (w/v) Poly-L-lysine 0.1% (w/v) poly-L-lysine is diluted to a working concentration of 0.01% with deionized water before use. This diluted working solution can be stored in a refrigerator at 4 °C. The stored solution can be used for up to three months if no turbidity or bacterial growth is present (discard if contaminated). Phosphate-buffered saline (PBS) (1×) Prepare PBS buffer (1×) by dissolving 2.7 mM KCl, 137 mM NaCl, 1.8 mM KH2PO4, and 10 mM Na2HPO4 in DDW. The pH has to be set at 7.4 with the help of HCl. It should be autoclaved before use for cell culture and stored in the refrigerator at 4 °C (discard if contamination can be seen). Phosphate-buffered saline-glucose (PBSG) (1×) PBS is prepared as per the recipe and autoclaved. 0.01 M glucose in PBS is prepared by adding 1 mL of sterile 1 M D-glucose (in water) to 100 mL of autoclaved 1× PBS. PBSG cannot be autoclaved as glucose degrades upon autoclaving. The prepared buffer can be stored at 4 °C for up to one month. Phosphate-buffered saline with Tween-20 (PBST) (1×) PBS is prepared as per the recipe and autoclaved. 0.05% Tween-20 in PBS is prepared by adding 0.05 mL of Tween-20 to 100 mL of autoclaved 1× PBS. The prepared buffer can be stored at 4 °C for up to one month. Tris-buffered saline with Tween-20 (TBST) (1×) Tris-buffered saline with 0.05% Tween-20 is prepared by dissolving 20 M Tris, 160 mM NaCl and 0.05% Tween-20 in DDW. The pH has to be set at 7.4 with the help of HCl. The prepared buffer can be stored at 4 °C for up to one month. Transfer Buffer (1×) Transfer buffer is prepared by dissolving 12.5 mM Tris base, 230 mM glycine, 0.125% SDS, and 20% methanol in DDW. It can be stored at 4 °C and should be used up within a week. Acknowledgments The central animal facility, confocal facility (Department of Microbiology and Cell Biology), and Indian Institute of Science, Bengaluru, are acknowledged for their services and technical help. This work is supported by funding from the Department of Science and Technology, the Department of Biotechnology, the Indian Council of Medical Research, and the Department of Biotechnology-Indian Institute of Science partnership program for advanced research. N.R.S. is a recipient of the Innovative Young Biotechnologist Award (IYBA), the National Bioscience Award for Career Development, and the Ramalingaswami Re-entry Fellowship from the Department of Biotechnology, Government of India. The protocol has been modified and improved from the one used in a previous publication (Maity et al., 2020). Author Contributions Shweta Kumar: data curation, analysis, investigation, methodology, writing original draft, review, and editing, Dimple Nagesh: investigation, methodology, writing original draft, Venketsubbu Ramasubbu: data curation, investigation, analysis, methodology, Arathi Bangalore Prabhashankar: data curation, review, and editing, Ravi Sundaresan: funding acquisition, project administration, resources, supervision, review, and editing. Competing interests The authors declare no conflict of interest. Data Availability Statement The data supporting the study are available to the corresponding author upon request. References Alikhani, Z., Alikhani, M., Boyd, C. M., Nagao, K., Trackman, P. C. and Graves, D. T. (2005). Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. J Biol Chem 280(13): 12087-12095. Camelliti, P., Borg, T. K. and Kohl, P. (2005). Structural and functional characterisation of cardiac fibroblasts. Cardiovasc Res 65(1): 40-51. Chacar, S., Farès, N., Bois, P. and Faivre, J. F. (2017). Basic Signaling in Cardiac Fibroblasts. J Cell Physiol 232(4): 725-730. desJardins-Park, H. E., Foster, D. S. and Longaker, M. T. (2018). Fibroblasts and wound healing: an update. Regenerative Medicine 13(5): 491-495. Eghbali, M. (1992). Cardiac fibroblasts: function, regulation of gene expression, and phenotypic modulation. Basic Res Cardiol 87 Suppl 2: 183-189. Eghbali, M. and Weber, K. T. (1990). Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression. Mol Cell Biochem 96(1): 1-14. Frangogiannis, N. (2020). Transforming growth factor-β in tissue fibrosis. J Exp Med 217(3): e20190103. Frangogiannis, N. G. (2020). Cardiac fibrosis. Cardiovasc Res 117(6): 1450-1488. Grupp, C. and Müller, G. A. (1999). Renal fibroblast culture. Exp Nephrol 7(5-6): 377-385. Kendall, R. T. and Feghali-Bostwick, C. A. (2014). Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol 5: 123. Krempaska, K., Barnowski, S., Gavini, J., Hobi, N., Ebener, S., Simillion, C., Stokes, A., Schliep, R., Knudsen, L., Geiser, T. K., et al. (2020). Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls. (vol 21, 25, 2019). Respir Res 21(1). Kurose, H. (2021). Cardiac Fibrosis and Fibroblasts. Cells 10(7): 1716. Landry, N. M., Rattan, S. G. and Dixon, I. M. C. (2021). Soft Substrate Culture to Mechanically Control Cardiac Myofibroblast Activation. Methods Mol Biol 2299: 171-179. Lee, J. K., Kim, D. B., Kim, J. I. and Kim, P. Y. (2000). In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants. Toxicol In Vitro 14(4): 345-349. Macieira-Coelho, A. and Avrameas, S. (1972). Modulation of cell behavior in vitro by the substratum in fibroblastic and leukemic mouse cell lines. Proc Natl Acad Sci U S A 69(9): 2469-2473. Maity, S., Muhamed, J., Sarikhani, M., Kumar, S., Ahamed, F., Spurthi, K. M., Ravi, V., Jain, A., Khan, D., Arathi, B. P., et al. (2020). Sirtuin 6 deficiency transcriptionally up-regulates TGF-beta signaling and induces fibrosis in mice. J Biol Chem 295(2): 415-434. Marthandan, S., Menzel, U., Priebe, S., Groth, M., Guthke, R., Platzer, M., Hemmerich, P., Kaether, C. and Diekmann, S. (2016). Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence. Biol Res 49(1): 34. Mazia, D., Schatten, G. and Sale, W. (1975). Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. J Cell Biol 66(1): 198-200. Mizoguchi, F., Slowikowski, K., Wei, K., Marshall, J. L., Rao, D. A., Chang, S. K., Nguyen, H. N., Noss, E. H., Turner, J. D., Earp, B. E., et al. (2018). Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis. Nat Commun 9(1): 789. Ravi, V., Jain, A., Taneja, A., Chatterjee, K. and Sundaresan, N. R. (2021). Isolation and Culture of Neonatal Murine Primary Cardiomyocytes. Curr Protoc 1(7): e196. Rinn, J. L., Bondre, C., Gladstone, H. B., Brown, P. O. and Chang, H. Y. (2006). Anatomic demarcation by positional variation in fibroblast gene expression programs. PLoS Genet 2(7): e119. Rohr, S. (2011). Cardiac fibroblasts in cell culture systems: myofibroblasts all along? J Cardiovasc Pharmacol 57(4): 389-399. Schauble, S., Klement, K., Marthandan, S., Munch, S., Heiland, I., Schuster, S., Hemmerich, P. and Diekmann, S. (2012). Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts. PLoS One 7(8): e42150. Schiller, M., Javelaud, D. and Mauviel, A. (2004). TGF-beta-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing. J Dermatol Sci 35(2): 83-92. Snider, P., Standley, K. N., Wang, J., Azhar, M., Doetschman, T. and Conway, S. J. (2009). Origin of cardiac fibroblasts and the role of periostin. Circ Res 105(10): 934-947. Souders, C. A., Bowers, S. L. K. and Baudino, T. A. (2009). Cardiac Fibroblast: The Renaissance Cell. Circulation Research 105(12): 1164-1176. Winkler, J., Abisoye-Ogunniyan, A., Metcalf, K. J. and Werb, Z. (2020). Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat Commun 11(1): 5120. Yu, L., Border, W. A., Huang, Y. and Noble, N. A. (2003). TGF-β isoforms in renal fibrogenesis. Kidney Int 64(3): 844-856. Zaragoza, C., Gomez-Guerrero, C., Martin-Ventura, J. L., Blanco-Colio, L., Lavin, B., Mallavia, B., Tarin, C., Mas, S., Ortiz, A. and Egido, J. (2011). Animal models of cardiovascular diseases. J Biomed Biotechnol 2011: 497841. Zhang, M. and Zhang, S. (2020). T Cells in Fibrosis and Fibrotic Diseases. Front Immunol 11: 1142. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Cell Biology > Cell signaling > Intracellular Signaling Cell Biology > Cell isolation and culture > Cell isolation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems Ye Li [...] Annie C. Bowles-Welch Dec 20, 2023 1826 Views Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry Hui Jing Lim and Hamish E. G. McWilliam May 5, 2024 650 Views Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing Hongzhe Li [...] Stefan Scheding Jun 20, 2024 645 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,617	https://bio-protocol.org/en/bpdetail?id=4617&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Comprehensive Analyses of Muscle Function, Lean and Muscle Mass, and Myofiber Typing in Mice HD Hima Bindu Durumutla * CV Chiara Villa * MP Manoj Panta * MW Michelle Wintzinger AP Ashok Daniel Prabakaran KM Karen Miz MQ Mattia Quattrocelli (contributed equally to this work) Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4617 Views: 2559 Reviewed by: Vivien Jane Coulson-ThomasAllan-Hermann PoolNeha NandwaniAbhilash Padavannil Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Feb 2022 Abstract Skeletal muscle disorders commonly affect the function and integrity of muscles. Novel interventions bring new potential to rescue or alleviate the symptoms associated with these disorders. In vivo and in vitro testing in mouse models allows quantitative evaluation of the degree of muscle dysfunction, and therefore, the level of potential rescue/restoration by the target intervention. Several resources and methods are available to assess muscle function and lean and muscle mass, as well as myofiber typing as separate concepts; however, a technical resource unifying these methods is missing. Here, we provide detailed procedures for analyzing muscle function, lean and muscle mass, and myofiber typing in a comprehensive technical resource paper. Graphical abstract Keywords: Skeletal muscle Myofiber type Lean and muscle mass Muscle cross sectional area Muscle force Muscle function Background Skeletal muscle homeostasis and dysfunction depend on the adaptative changes in myofiber size, myofiber type, and force capacity. Equally important for both disease characterization and therapeutic evaluation are sensitive and precise methods for evaluating the parameters of muscle physiology in comprehensive assessments. This set of tools will enable scientists to assess not only the magnitude of the effect of a target intervention but also compare effects across different interventions. Multiple invasive and noninvasive methods have been developed to assess muscle strength in animal models, especially rodents (Takeshita et al., 2017). Invasive methods include in situ and in vitro measurement of muscle force, whereas noninvasive methods include in vivo assessment of strength and global performance. In this protocol, we present the methods for conducting both in situ invasive measurements of muscle force and in vivo non-invasive measurements of strength and aerobic exercise tolerance in response to a pro-ergogenic glucocorticoid treatment (Quattrocelli et al., 2022). Skeletal muscle is a metabolically active tissue made up of different types of muscle fibers, generally classified as type1, type 2A, type 2X, and type 2B according to the myosin isoform that is enriched in each of them (Talbot and Maves, 2016). Changes in muscle fiber typing are often correlated with changes in muscle strength, contractility, and/or endurance. Moreover, modifications in skeletal muscle fiber composition may be related to muscle myopathies such as muscular dystrophies, inherited myopathies preferentially affecting a specific myofiber type, and sarcopenia. Therefore, myofiber-type specification is important to provide insights for understanding the susceptibility and resistance of certain fiber types to muscle disease and to discover potential treatments based on muscle plasticity and metabolic shift. Histochemical staining for myosin ATPase or succinate dehydrogenase enzyme activity or quantification of metabolic enzymes are among the methods used to identify different myofiber types, but these cannot generally differentiate between myofibers subtypes (Reichmann and Pette, 1984). Therefore, here we report the immunofluorescence-based protocol that is routinely used in our lab for the differential staining of myosin heavy chain isoforms, which efficiently resolves the heterogeneity of muscle fibers. Apart from muscle function and myofiber typing, lean mass (Kalyani et al., 2014) and muscle mass (Lovering et al., 2005) are two important determinants associated with muscle disorders or muscle changes downstream of global conditions, such as aging or type-2 diabetes. Weight curves offer an immediate proxy estimate of overall growth and mass levels but evidently cannot provide information on fat vs. lean mass or actual muscle mass. Therefore, magnetic resonance imaging (MRI)-based systems to estimate lean and fat mass can provide a better estimation of body composition effects of interventions, such as circadian time–specific dosing of glucocorticoids (Quattrocelli et al., 2022). Using MRI-based systems, it is possible to simultaneously quantify changes in adiposity vs. lean mass (absolute or relative) over time. Overall, we provide a comprehensive method for the analysis of muscle function, performance, lean and muscle mass, and muscle fiber typing. These tests can be easily customized to evaluate overall muscle function, performance, and composition (especially in terms of muscle fiber types) in different situations, to study muscle physiopathology, or to assess the efficacy of therapeutic interventions. Materials and Reagents Microscope slides (Corning, catalog number: CLS294775X25) Microscope slide cover glass (Globe Scientific, catalog number: 1404-10) Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: 806552) Triton X-100 (Sigma-Aldrich, catalog number: T8787) 10% normal goat serum (Cell Signaling, catalog number: 5425) Ethanol, 200 proof (Thermo Scientific, catalog number: T038181000) Isoflurane (Covetrus, catalog number: 11695-6777-2) Ringer’s solution (Fisher Scientific, catalog number: S25512) Isopentane (Alfa Aesar, catalog number: 19387, CAS number: 78-78-4) Tissue-Tek OCT compound (Sakura Finetek, catalog number: 4583) PAP pen (Biotium, catalog number: 22006) ProLongTM Gold Antifade Mountant with DAPI (Invitrogen, catalog number: P36931) Primary antibodies (Table 1) Table 1. Primary antibodies for muscle fiber typing Antibody name Company and catalog number Dilution Rabbit anti(α)-Laminin Sigma, L9393 1:500 Myosin heavy chain, type IIA (SC-71) Developmental Studies Hybridoma Bank, SC-71 1:30 Myosin heavy chain, type IIB (BF-F3) Developmental Studies Hybridoma Bank, 10F5 1:10 Myosin heavy chain (slow) (BA-F8) Developmental Studies Hybridoma Bank, A4.840 1:10 Fluorescently tagged secondary antibodies (Table 2) Table 2. Secondary antibodies for muscle fiber typing Antibody Name Company and Cat No. Dilution Goat anti-rabbit IgG (H+L) Alexa FluorTM 488 Invitrogen, A-11034 1:1,000 Goat anti-mouse IgG2b Alexa FluorTM 647 Invitrogen, A-21242 1:1,000 Goat anti-mouse IgG1, Alexa FluorTM 488 Invitrogen, A21121 1:1,000 Goat anti-mouse IgM, Alexa FluorTM 594 Invitrogen, A-21044 1:1,000 Goat serum (Invitrogen, catalog number: 31872) Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: D1319) Prednisone powder (Sigma-Aldrich, catalog number: P6254) 1 mL insulin syringe with needle (BD, catalog number: 329424) Permeabilization reagent: 1% (v/v) Triton X-100 in PBS Blocking buffer: 10% (v/v) goat serum in PBS Prednisone stock (5 mg/mL): 5 mg prednisone powder in 1 mL DMSO Physiological solution: sterile 0.9% saline solution (Cytiva Z1379) Vehicle stock: 1 mL DMSO. Equipment Grip and treadmill assays Grip strength meter with single sensor for mice (max 1 kg capacity) with standard pull bars (Chatillon Instruments, Digital Force Gauge, catalog number: DFIS-2) Open treadmill with stimulus assembly, ~10% uphill incline, individual mouse lanes, drive motor to adjust speed (3–100 m/min range), and exercising belt with grip-prone texture (Columbus Instruments, catalog number: Exer3/6) Force-frequency analysis in tibialis anterior muscles Surgical scissors (Fine Science Tools, catalog number: 14060-10) Mouse handling forceps (Fine Science Tools, catalog number: 11036-20) Weighing scale (Ohaus, catalog number: 80000022) In-situ apparatus setup from 3-in-1 whole animal system for mice (Aurora Scientific, catalog number: 1300A) Ohaus Pioneer precision balance (Fisher Scientific, catalog number: 01-922-178) Digital caliper (Thermo Fisher, catalog number: 14-648-17) Isoflurane vaporizer (SurgiVet Classic T3, catalog number VCT302) and induction chamber (Patterson Scientific, catalog number: 07-8917760) Surgical suture (Ethicon, catalog number: K871) Myofiber typing Leica cryostat microtome (Leica Biosystems, catalog number: CM1510S) Humidified container (Thomas Scientific, catalog number: 1219D68) Nikon Eclipse Ti inverted microscope (Nikon Instruments, catalog number: Ti-E) MRI scans to determine lean mass ratios EchoMRI whole-body composition analyzer (EchoMRI, catalog number: 100H) Sample tubes dedicated to mice comprised between 20 and 40 g of body mass (EchoMRI, Houston, TX) Standard internal calibrator tube (canola oil) (EchoMRI, Houston, TX) Software Built-in software EchoMRI version 140320. Data were analyzed when hydration ratio was >85% ImageJ (Fiji, https://imagej.nih.gov/ji) ASI 611A Dynamic Muscle Analysis v5.300 ASI 610A Dynamic Muscle Control v5.500 Procedure The protocols and example data presented here are from wildtype (WT) male mice from the C57BL/6 background (JAX strain #000664). Number of animals used per cohort should be decided based on an appropriate power analysis tailored to the scope of the study. See notes at the end for considerations regarding age and sex of test mice. Quantitation of grip strength and treadmill performance Grip strength Record the body weight of each mouse prior to the grip assay. To record the force for a mouse, hold its tail, let the mouse grip to the bars of the grip strength meter with its forelimbs, and then pull the animal away from the metal grid. Ensure to pull with a constant, gentle movement, keeping the mouse parallel to the work surface (Figure 1). Repeat the grip force measurements three times per mouse. Let the mouse rest for a minute between the pulls and record the highest number. Normalize grip strength to body weight. Figure 1. In vivo assessment of muscle strength in mice using grip strength meter Notes: Examine the toes of the mice to make sure there are no visible wounds on them before the test. Make sure that the mouse is gripping nicely to the bars before pulling it; otherwise, this can result in a false low value. Ensure that the mouse is only holding bars with the forearms and the body is parallel to the worksurface. Treadmill assay Place each mouse in individual treadmill lanes. Each lane is equipped with a shock grid that delivers a foot shock. The shock current in the grid is usually set at the lowest level 1 (Figure 2). Start the conveyor belt at 3 m/min and gradually increase the speed by 1 m/min every minute (1 m/min2 acceleration). If analyses of weight-normalized work or power are required, regulate the treadmill belt on an upward incline (generally 10°). Monitor each mouse to ensure they are running (Figure 2). Mice that stop running are pushed to the shock grid through the moving treadmill belt. Signs of exhaustion (e.g., 30 s on the shock grid without successful efforts to start running again) are defined as treadmill assay endpoints. Record the distance (m) and the total time (s) until exhaustion. Analyze the treadmill assay performance as either time-to-exhaustion or distance-to-exhaustion. If an incline was used, weight-normalized work and/or power can be calculated using previously reported formulas (Castro and Kuang, 2017): Work (J) = body mass (kg) × gravity (9.81 m/s2) × vertical speed (m/s × angle expressed in radians) × time (s) Power (W) = work (J)/time (s) Calculations should be adjusted to take into account acceleration or varying speed, if used. Figure 2. In vivo evaluation of mouse muscle endurance and fatigue by treadmill Notes: This test is a form of forced exercise and requires aversive stimuli to keep the animal running. We use electric shock as an aversive stimulus and closely ensure that animals do not get excessive electric shocks, as it can affect the downstream assays. If assaying grip strength and treadmill on the same day, perform grip assay before the treadmill assay to avoid possible effects of fatigue. Allow mice to walk freely in the cage for 30 min between grip and treadmill assays. Also ensure that the experiments are conducted in a blinded fashion. Measure grip strength and treadmill performance at baseline (before injection) and post treatment (24 h after last prednisone dose). For both assays, keep operator-dependent variables (e.g., operator, instruments, room conditions, time of day) constant across cohorts and time points. Avoid collecting tissues immediately after a treadmill assay. It is recommended to collect tissues at least 24 h after the last treadmill assay. For the quantitation of muscle force Anesthetize mouse with 1.5% isoflurane inside the anesthesia induction chamber for approximately 2–5 min. The anesthesia system consists of an induction chamber, a transparent plastic box to confine one mouse at a time in a closed space connected to the oxygen flow, set to a maximum of 0.5 L per min, and to isoflurane gas, set to 1.5% (Figure 3). Figure 3. Anesthesia induction chamber Lay the mouse supine on the assay platform of the in-situ apparatus setup (Aurora). During the whole procedure, maintenance of anesthesia is supplied to animals through a nose cone delivering 1.5% isoflurane. Spray 70% ethanol to the specific hind limb (right or left) that you intend to perform the experiment on. Make a small incision near the ankle and remove the hindlimb skin to uncover the tibialis anterior (TA) muscle, ensuring tendons and muscles are not damaged. Detach the TA muscle from the tibia by sliding the forceps between the muscle and the bone. Using a surgical suture, secure the distal tendon to the force probe. Gently sever the tendon and separate the muscle from the tibia. The TA muscle should be linked to the force probe on the distal side and to the knee region on the proximal side. While keeping the mouse on the platform, block the knee with the dedicated clamp. Adjust the platform to ensure that the probe is in line with the knee. Place two electrical probes into the leg, one at the distal end of the TA and the other under the kneecap near the sciatic nerve (Figure 4). Figure 4. In vivo quantitation of muscle force Run test pulses to adjust muscle tension to the equilibrium point. The equilibrium point is the muscle tension at the initial resting point to which the muscle returns to after test pulses of electricity are administered. Record the muscle length in millimeters at equilibrium with a digital caliper (L0). Run the force-frequency test through tetanus stimulations at increasing frequency (i.e., from 25 to 200 Hz with intervals of 25 Hz). Pause 5 min between stimulation bouts. Record force (in N) for each isometric contraction (P0). Repeat procedure (B3–B12) and test on contralateral TA muscle. Proceed to euthanasia and collect and record each TA muscle with a precision scale. Calculate specific force (N/mm2) for each tetanus frequency as (P0 N)/[(muscle mass mg/1.06 mg/mm3)/Lf mm]. 1.06 mg/mm3 is the mammalian muscle density. Lf = L0 × 0.6, where 0.6 is the muscle to fiber length ratio in TA muscle (Burkholder et al., 1994). The specific force is often converted to and reported as N/cm2 units. Notes: Avoid delays while performing this test. Keep muscle hydrated by adding Ringer’s solution. Ensure that the electrodes are secured properly during the assessment. For the quantitation of muscle mass Tibialis anterior (TA) Immediately following the force experiment, perform the cervical dislocation on the mice. Position the mouse in supine position and pin all the limbs in the dissection pad. Remove the surgical suture from the distal tendon of the TA muscle that is used in the force experiment. Hold the distal tendon with the forceps. Measure the length (the muscle length will be used for the calculation of the force) and then resect the TA muscle at the knee region in the proximal side (Figure 5). Make sure to resect only the TA and not the extensor digitorum longus muscle, which is adjacent to the TA. Weigh the tibialis to the nearest 0.1 mg. Figure 5. Isolation of murine tibialis muscle Gastrocnemius (GA) and soleus Identify the GA and gently remove the fascia covering it. Cut the distal GA tendon and insert the forceps (Figure 6). Rub back and forth from the distal to proximal end with the forceps to open a gap. Once there is an opening, cut the tendon as close as possible to the foot. Lift the tendon with the forceps and cut the tendon as close as possible to the knee. Identify the soleus muscle (beneath the GA in darker pink color) and then use the fine forceps to hold one of its ends and cut it with the scissors. Hold the free end with forceps and then cut another end. Weigh the resected GA and soleus to the nearest 0.1 mg. Figure 6. Harvesting of GA muscle Quadriceps (QD) Identify the QD and remove the fascia. Cut closest to the knee. Hold the free end with forceps and cut towards the proximal end pointing the scissors down and forward (Figure 7). After reaching proximal end, resect the muscle. Weigh the QD to the nearest 0.1 mg. Figure 7. Isolation of murine QD muscle Calculation of muscle mass Calculate muscle mass as muscle weight of individual muscles over body weight and/or tibia length. For myofiber typing Sample preparation The myofiber typing can be performed in any muscle tissue. After weighing muscle for the muscle mass, it can be prepared for myofiber typing. Carefully, place the tissue on a syringe plunger and cover with OCT compound. Immediately snap-freeze the tissue by immersing in a metal cup filled with isopentane and cooled in liquid nitrogen for 30–40 s. Store the frozen tissue at -80 °C for future use. To section the frozen tissue, cut 10 μm transverse sections using the cryostat microtome. Use a brush to pick up the sections and place them on a microscope slide. Store the slides with appropriate sections at -80 °C for future use. Immunostaining Thaw the slides to room temperature for 30 min. Using a PAP pen, draw a perimeter around the tissue to create a hydrophobic barrier. Place the slides in a humidified chamber and add enough permeabilization reagent to cover the tissue (usually 200 μL per section). Incubate at 37 °C for 30 min followed by 10 min at room temperature. Gently wash the slides once with 1× PBS. Add freshly prepared blocking buffer to cover the section. Incubate for 1 h at room temperature. Wash the slides once in 1× PBS. Prepare the appropriate dilutions for the primary antibodies (see Table 1) in blocking buffer: BA-F8, SC-71, and BF-F3 and overlay onto the sections. SC-71, BF-F3, and BA-F8 antibodies are used for staining type IIA, type IIB, and type I myofibers, respectively. Incubate in the refrigerator (4 °C) overnight. Gently wash the sections once with 1× PBS to remove unbound primary antibody. Overlay the sections in secondary antibodies (see Table 2) diluted in blocking buffer: goat anti-mouse IgG2b Alexa FluorTM 647, goat anti-mouse IgG1, Alexa FluorTM 488, and goat anti-mouse IgM, Alexa FluorTM 594. Incubate for 1 h at room temperature in the dark. Gently wash the sections three times in 1× PBS. Mount slides with ProLongTM Gold Antifade Mountant with DAPI. If needed, store slides at 4 °C protected from light for future imaging. Slides can be saved for approximately one month for imaging. Quantitate myofiber types (Figure 8) by taking images with Nikon microscope in at least five serial sections and quantitate as the percentage of total counted myofibers. Conduct all analyses blinded to treatment. Figure 8. Representative image of immunohistochemical staining of TA section. Type 1 fibers stained in magenta, type 2A stained in green, type 2X showed no staining, and type 2B stained red. Nuclear staining in blue (original magnification, 20×). Myofiber cross-sectional area (CSA) The sample preparation for the myofiber CSA and myofiber typing is the same (see section D1 for sample preparation). Immunofluorescence Thaw the slides to room temperature for 30 min. Using a PAP pen, draw a perimeter around the tissue to create a hydrophobic barrier. Place the slides in a humidified chamber and add enough permeabilization reagent to cover the tissue (usually 200 μL per section). Incubate at 37 °C for 30 min followed by 10 min at room temperature. Gently wash the slides once with 1× PBS. Add freshly prepared blocking buffer to cover the section. Incubate for 1 h at room temperature. Wash the slides once in 1× PBS. Prepare anti(α)-Laminin antibody (see Table 1) in blocking buffer and overlay onto sections. Incubate in the refrigerator (4 °C) overnight. Gently wash the sections once with 1× PBS to remove unbound primary antibody. Dilute goat anti-rabbit IgG (H+L) Alexa FluorTM 488 (see Table 2) in blocking buffer and incubate for 1 h at room temperature in the dark. Gently wash the sections three times in 1× PBS. Mount slides with ProLongTM Gold Antifade Mountant with DAPI. If needed, store slides at 4 °C protected from light for future imaging. Slides can be saved for approximately one month for imaging. CSA quantitation Perform imaging with a Nikon Eclipse Ti inverted microscope using 10× and 20× (short-range) objectives (Figure 9). Conduct CSA quantitation on >400 myofibers per tissue per mouse using ImageJ software. For linear calibration, use the line selection tool to draw a line between the two points of known distance (such as the scale bar). Transform the line length from pixels into units of measure (μm) using command Analyze → Set scale. Manually outline the myofibers perimeter by means of an area selection tool (polygonal) (Figure 9). Myofibers at the edge of the image or with deformed/altered shapes () (Figure 9) are excluded from counting to remove the presence of artifacts. Display area measurements in a data window using the command Analyze → Measure. Figure 9. Representative immunofluorescence images of TA sections. Anti(α)-Laminin (green) staining (original magnification, 20×); () indicates representative myofibers with deformed shape. Note: The protocol for myofiber CSA quantitation can also be adopted for hematoxylin and eosin-stained sections. Quantitation of lean mass Open the EchoMRI program on the desktop. Create a folder to save the raw data automatically once the experiment is completed. Place the solid calibration holder (for mouse) into MRI and preform a system test by inserting the calibration holder. The process takes approximately 4 min to complete. The system is calibrated using the standard internal calibrator tube (canola oil). Remove the calibration holder from the machine. Holding the mice with its tail, place it in an appropriately sized holder and slide the adjustable barrier to minimize area of movement. Some degree of mobility is fine and will not compromise measurement (Figure 10). Figure 10. Body composition analysis using EchoMRI Record the reading and repeat the scan with an interval of 20 s. Remove the rodent from the tube holder and wipe out any urine/feces or gross debris with a brush/paper towels before proceeding to the next animal. When hydration ratio was >85%, analyze the data using the built-in software EchoMRI version 140320. Notes: If metal ear tags are used for mouse identification, use non-magnetic ones that will not affect the animal, machine, or measurements by EchoMRI. Once all measurements have been performed, wash tube holders and sanitize all parts. Sanitization washing must occur between different strains, species, and principal investigators’ animals to prevent potential cross contamination of rodent pathogens. Quantification of muscle function, lean and muscle mass, and myofiber typing in regimen-specific glucocorticoid treatment The data presented below (Figure 11) shows examples of implementation of our protocol on muscle function, lean and muscle mass, and myofiber typing. Here, we show the effects of dosage time of prednisone (a commonly used glucocorticoid); data presented are adapted from Quattrocelli et al. (2022). Glucocorticoid regimens Prior to injection, record the weight of each mouse. Prepare 50 μL (final volume) of physiological solution with the appropriate volume of prednisone stock to a final dose of 1 mg/kg [e.g., 4 μL of a 5 μg/μL stock (total of 20 μg) for a 20 g mouse]. For the vehicle, mix corresponding volumes of vehicle stock and sterile physiological solution. With a single-use insulin syringe per dose, load the appropriate solutions. Make sure no air is introduced. Expose the mice abdomen and perform an intraperitoneal injection. Frequency of intermittent dosing: once-weekly regimen consists of 1× prednisone dose (day 1) followed by 6× vehicle doses (day 2–6) per week. It is controlled by once-weekly vehicle injection. Time of dosing: Injections were conducted either at the beginning of the light phase (ZT0; lights-on) or at the beginning of the dark phase (ZT14; lights-off). Experiments were performed 24 h after the single pulse or the last injection in chronic treatment. Follow the regimen for a total of 12 weeks for chronic treatment. All in vivo, ex vivo, and postmortem analyses are conducted blinded to the treatment group. Figure 11. Effects of light-phase vs. dark-phase intermittent prednisone treatment on muscle bioenergetics. Results are shown after a 12-week-long treatment with intermittent once-weekly prednisone with dosing restricted to ZT0 (light phase) vs. ZT14 (dark phase). In wildtype (WT) mice, compared to isochronic vehicle controls, ZT0, but not ZT14, prednisone improved (A) treadmill performance, (B) grip test, (C) muscle weight, (D) lean mass, (E) muscle force, and (F) myofiber area. However, no changes were reported in (G) the distribution of myofiber types in tibialis anterior muscles. Lean mass, grip strength, myofiber cross-sectional area (CSA), and myofiber typing data were adapted from Quattrocelli et al. (2022). = p < 0.05, = p < 0.01, * = p < 0.001; 1-way ANOVA + Sidak (A-D, F-G), 2-way ANOVA (curves in E). Notes Here, we used 1 mg/kg prednisone dose to successfully discriminate the effects of diverging the once-weekly day dosing from the once-weekly night dosing in WT mice lines. Other glucocorticoids, dosages, and intermittence intervals should be tested to evaluate differences/similarities with analyses and trends discussed here. A 12-week-long treatment enables for stabilization of the divergent regimen–specific changes, with the additional consideration of ages at start and endpoint. Here, we report methods and data related to glucocorticoid treatments in 4-months-old mice at start. This age-at-start is convenient for 12-week-long regimens. Indeed, treated mice will be 4 months old at start and approximately 7 months old at endpoint. Analyses at different ages will yield different results and therefore age-at-analysis should be considered when performing these tests. Regarding sex as biological variable, biological sex impacts almost every virtual parameter of muscle physiology and its response to drug treatments, including glucocorticoid intermittence (Salamone et al., 2022). Therefore, analyses in sufficiently powered cohorts of both male and female mice are recommended, as well as data reporting as aggregated and disaggregated by sex. Acknowledgments This protocol has been adapted from our previous work published in Quattrocelli et al. (2022). This work was supported by AG078174, HL158531, DK121875 and DK130908 (NIH), Start-up funds (CCHMC), Trustee Award (CCHMC), Heart Institute Translational Funds (CCHMC) and CuSTOM pilot grant (CCHMC). Competing interests MQ is listed as co-inventor on a patent application related to intermittent glucocorticoid use filed by Northwestern University (PCT/US2019/068618). All other authors declare they have no competing interests. Ethics Mice were housed in a pathogen-free facility in accordance with the American Veterinary Medical Association and under protocols fully approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Medical Center (#2020-0008). References Burkholder, T. J., Fingado, B., Baron, S. and Lieber, R. L. (1994). Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb. J Morphol 221(2): 177-190. Castro, B. and Kuang, S. (2017). Evaluation of Muscle Performance in Mice by Treadmill Exhaustion Test and Whole-limb Grip Strength Assay. Bio Protoc 7(8): e2237. Kalyani, R. R., Corriere, M. and Ferrucci, L. (2014). Age-related and disease-related muscle loss: the effect of diabetes, obesity, and other diseases. Lancet Diabetes Endocrinol 2(10): 819-829. Lovering, R. M., Porter, N. C. and Bloch, R. J. (2005). The muscular dystrophies: from genes to therapies. Phys Ther 85(12): 1372-1388. Quattrocelli, M., Wintzinger, M., Miz, K., Levine, D. C., Peek, C. B., Bass, J. and McNally, E. M. (2022). Muscle mitochondrial remodeling by intermittent glucocorticoid drugs requires an intact circadian clock and muscle PGC1alpha. Sci Adv 8(7): eabm1189. Reichmann, H. and Pette, D. (1984). Glycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles. Histochemistry 80(5): 429-433. Salamone, I. M., Quattrocelli, M., Barefield, D. Y., Page, P. G., Tahtah, I., Hadhazy, M., Tomar, G. and McNally, E. M. (2022). Intermittent glucocorticoid treatment enhances skeletal muscle performance through sexually dimorphic mechanisms. J Clin Invest 132(6). Takeshita, H., Yamamoto, K., Nozato, S., Inagaki, T., Tsuchimochi, H., Shirai, M., Yamamoto, R., Imaizumi, Y., Hongyo, K., Yokoyama, S., et al. (2017). Modified forelimb grip strength test detects aging-associated physiological decline in skeletal muscle function in male mice. Sci Rep 7: 42323. Talbot, J. and Maves, L. (2016). Skeletal muscle fiber type: using insights from muscle developmental biology to dissect targets for susceptibility and resistance to muscle disease. Wiley Interdiscip Rev Dev Biol 5(4): 518-534. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Biological Sciences > Biological techniques Cell Biology > Cell imaging > Fixed-cell imaging Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections Bernhard A. Kramer [...] Gabriele Gut Jul 5, 2023 1576 Views Studying Cellular Focal Adhesion Parameters with Imaging and MATLAB Analysis Ling-Yea Yu [...] Feng-Chiao Tsai Nov 5, 2023 524 Views Automated Layer Analysis (ALAn): An Image Analysis Tool for the Unbiased Characterization of Mammalian Epithelial Architecture in Culture Christian Cammarota [...] Tara M. Finegan Apr 20, 2024 2866 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,618	https://bio-protocol.org/en/bpdetail?id=4618&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Detection of Zebrafish Retinal Proteins by Infrared Western Blotting JZ Jingjing Zang SN Stephan Neuhauss Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4618 Views: 709 Reviewed by: Prashanth N SuravajhalaAnsul LokdarshiMasfique Mehedi Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in eLIFE Sep 2021 Abstract The zebrafish retina is a canonical vertebrate retina. Since the past few years, with the continually growing genetic toolbox and imaging techniques, zebrafish plays a crucial role in retinal research. This protocol describes a method to quantitatively evaluate the expression of Arrestin3a (Arr3a) and G-protein receptor kinase7a (Grk7a) in the adult zebrafish retina at protein levels by infrared fluorescence western blot. Our protocol can be easily adapted to measure protein levels in additional zebrafish tissues. Keywords: Infrared fluorescence western blot Visual transduction Circadian rhythms Zebrafish Retina Background Circadian rhythms are the inner clocks that drive the daily fluctuations of physiology and behavior (Vatine et al., 2011; Brown et al., 2019; Frøland Steindal and Whitmore, 2019). The molecular mechanisms of circadian rhythms are based on a transcription-translation feedback loop, in which core clock genes’ mRNA and protein levels fluctuate in an approximate 24-h period. Certain core clock genes regulate the transcription of downstream clock-controlled genes, which in turn generate the circadian output. Strikingly, nearly 20% of the genes in zebrafish genome are thought to be regulated by the circadian clock (Li et al., 2013). Circadian fluctuations are commonly studied at the transcript level, using qRT-PCR or RNA-Seq. Circadian rhythms at the protein level are less accessible to study, partially because the daily changes are not very large. However, in numerous instances, circadian fluctuations in mRNA expression are not reflected in corresponding changes of protein levels. It is easy to conceptualize that proteins with a slow turnover rate will have stable overall protein levels even in the face of rhythmic mRNA expression. Hence, it is important to measure changes in protein concentration of any process, before interpreting their involvement in any observed rhythmic behavior. In a recent study, we linked circadian expression of mRNA and proteins of the cone visual transduction cascade to visual sensitivity assayed by electrophysiological and behavioral means (Zang et al., 2021). The transcription of regulators of the visual transduction cascade, such as Recoverins, Arrestins, Opsin kinases, and Regulator of G-protein signaling displayed a robust circadian rhythm. Importantly, this rhythm was also observed at the protein level, with a relative delay likely due to translation. These changes in protein concentration can be directly correlated with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. This rhythm can be reversed by an inverted light cycle, persists in constant darkness, and is disturbed by constant light, as is expected for a bona fide circadian rhythm. Interestingly, the cyclic expression of orthologous genes in the nocturnal mouse retina are anti-phasic to the ones of the diurnal zebrafish. While general chemiluminescence western blotting can detect the analyzed proteins, infrared fluorescence western blot can simultaneously stain the protein of interest as well as the loading control. Additionally, the signal detection for infrared fluorescence western blot is straightforward. Here, we describe the method of infrared fluorescence western blot to quantify the relative protein level change in adult zebrafish retina throughout the day, based on the standard polyacrylamide gel electrophoresis and protein transfer protocols provided by Bio-Rad and detection protocol provided by LI-COR Biosciences with modifications. The current protocol can be easily adapted to work with variable zebrafish tissues at different development stages or used to study protein level changes under other conditions, for example in the mutants or fish under drug treatment. Materials and Reagents Nitrocellulose membrane, 0.45 µm (Bio-Rad, catalog number: 1620116) Sterile filter, 0.45 µm (SARSTEDT AG&Co. KG, catalog number: 83.1826) 4%–15% Mini-protean® TGXTM gels, 15-well comb, 15 µL (Bio-Rad, catalog number: 4561086) Adult zebrafish (WIK wild-type strain) Rabbit anti-ARR3a antibody (Renninger et al., 2011) Rabbit antt-GRK7a antibody (Rinner et al., 2005) Mouse anti-β-Actin (Sigma-Aldrich, catalog number: A1978) Bovine serum albumin (BSA) (Roche, catalog number: 10735094001) Tween® 20 (Sigma-Aldrich, catalog number: P1379) Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71380) TRIS (Biosolve, catalog number: 200923) Triton® X-100 (Fluka, catalog number: 93418) Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750) Sodium dodecyl sulfate (SDS) (Roche, catalog number: 11667289001) Protease inhibitor cocktail (PIC) tablets (Roche, catalog number: 04906845001) Glycerol (Sigma-Aldrich, catalog number: G7757) Bromophenol blue indicator (Fluka, catalog number: 32712) β-mercaptoethanol (Sigma-Aldrich, catalog number: 444203) Glycine (Sigma-Aldrich, catalog number: 50046) Precision Plus protein kaleidoscope marker (Bio-Rad, catalog number: 1610375) Phosphate buffer saline (PBS) (VWR, catalog number: 75801-006) IRDye® 800CW goat anti-rabbit IgG (LI-COR, catalog number: 925-32211) IRDye® 680RD goat anti-mouse IgG (LI-COR, catalog number: 925-68070) PierceTM BCA Protein Assay kit (Thermo Scientific, catalog number: 23227) Safe-Lock micro test tubes, 1.5 mL (Eppendorf, catalog number: EP0030120167) Methylalkohol (MeOH) (Sigma-Aldrich, catalog number :1424109) Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: 1099730001) Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881) Radioimmunoprecipitation assay (RIPA) buffer (see Recipes) PIC (see Recipes) 5× Laemmli sample buffer (see Recipes) 10× running buffer (see Recipes) 10× transfer buffer (see Recipes) Phosphate buffered saline with Tween 20 (PBST) (see Recipes) Blocking buffer (see Recipes) Equipment Eyeball scoop (Chiru-Instrumente) Micro scissors Shaker Sonicator (Bandelin Sonopuls) PowerPac basic power supply (Bio-Rad, catalog number: 164-5050) Mini-PROTEAN® Tetra vertical electrophoresis cell (Bio-Rad, catalog number: 165-8004) Mini Trans-Blot® module (Bio-Rad, catalog number: 170-3935) Odyssey® DLx imaging system (LI-COR) NanoDropTM One (Thermo Scientific, catalog number: ND-ONE-W) pH meter (SCHOTT Instruments, model: Lab 850) Software Image Studio software (LI-COR, version: 5.2, https://www.licor.com/bio/image-studio-lite/) Microsoft 365 Excel Procedure Pictorial methodology is illustrated in Figure 1. Figure 1. Western blotting workflow Eye dissection Place the fish in ice water at 1–2 °C at the desired time of the day. Leave the fish in the ice water for 2–5 min and decapitate the fish. Remove the eyeballs with a scoop (Figure 2B) and cut the white optic nerve using micro scissors (Video 1). Figure 2. Items used during western blotting. (A) BSA standards (1–7) and protein sample (*). (B) Eye scoop. (C) Incubator. (D) Transfer cassette. (E) Transfer module. Video 1. Zebrafish eye dissection Shock-freeze the eyes (3–4 eyes per sample) in liquid nitrogen. Notes: In the original paper, western blot was used to evaluate protein level changes in a 24-h period. The eyes were collected every 3 h at eight different time points. Therefore, it was easier to store the eyes and perform the protein extraction for all the samples at once. Depending on the purpose of the experiment, the eyeballs can be collected directly in RIPA buffer and processed for the protein extraction right after the dissection. The dissection should be carried out under dim red light if light adaptive changes are to be avoided. Protein extraction Perform all steps on ice. Collect eyes and put them in 150 µL of cold RIPA buffer with 1× PIC, which is added right before the extraction. Homogenize eyes with sonicator three times for 3 s at 70% maximum power. Incubate on shaker at 4 °C for 2 h, centrifuge at 5,590 × g for 30 min at 4 °C, and transfer the supernatant to a fresh 1.5 mL tube. Perform BCA assay to determine the protein concentration: Use 1 ampule (1 mL, 2.0 mg/mL) of albumin standard from the PierceTM BCA Protein Assay kit to prepare BSA dilutions in RIPA buffer according to Table 1. Note: Duplicates are normally prepared. Prepare the BCA working reagents (WR) and calculate the total amount of WR according to sample to WR ratio (1:20). Prepare WR by mixing reagent A from the kit with reagent B (50:1). Mix well 15 µL of each standard with 300 µL of WR in separate 1.5 mL tubes. Mix well protein lysate with 300 µL of WR in another 1.5 mL tube. Incubate the tubes in a heating block at 37 °C for 30 min. Cool them down to room temperature (Figure 2A). Use a photospectrometer to determine concentration (we used a NanoDrop device). i. Input the number of replicates and concentrations of BSA standards according to Table 1. ii. Measure the protein concentration for each standard and replicate according to NanoDrop instructions. iii. Measure the sample concentration according to the instructions. The result will be shown on the screen and can be exported. Continue with the following steps or store the lysate at -80 °C until use. Table 1. Dilutions for preparing BSA standards Vial Volume of RIPA Buffer(µL) Volume of Source of BSA (µL) Final BSA Concentration (µg/ml) 1 0 40 of Stock 2000 2 10 30 of Stock 1500 3 20 20 of Stock 1000 4 20 20 from vial 2 dilution 750 5 20 20 from vial 3 dilution 500 6 30 10 from vial 5 dilution 125 7 40 0 0 Electrophoresis Mix lysates with 1× loading buffer containing 5% β-mercaptoethanol freshly added and place the samples on ice. Insert gel(s) into the electrophoresis cell, fill with 1× running buffer completely covering the gel, remove combs, and check/remove bubbles in each well. Load 10 µL of Precision Plus protein kaleidoscope marker and 20 µg of each sample. Note: An asymmetric loading scheme will make it easier to recognize the orientation of the membrane later. Run electrophoresis at around 100 V until the blue front band reaches the bottom of the separating gel. Note: The higher the voltage is applied, the faster the proteins travel. However, the bands sometimes do not run at the same height throughout the gel (“smile effect”). Remove the gel from electrophoresis cell. Blotting Work under the fume hood with good ventilation. Prepare the electroblotting system: For each gel, cut one piece of nitrocellulose membrane and two pieces of filter paper to the size of the gel. Equilibrate the membrane, filter paper, and two filter pads in 1× transfer buffer containing 20% MeOH in an incubator (Figure 2C). Mount the transfer cassette according to the scheme and remove air bubbles by gently pressing on the gel or membrane (Figure 2D). Cathode pole (black) Filter pad Filter paper Gel Membrane Filter paper Filter pad Anode pole (white) Insert the transfer cassette into the Trans-Blot® module (Figure 2E), insert an ice block and a magnetic stir bar, and fill with 1× transfer buffer containing 20% MeOH completely covering the transfer module. Place the whole transfer module on a magnetic stirrer plate, which allows continuously stirring the buffer inside the module to keep the temperature low. Blot at 100 V/350 mA for around 40 min for protein Arrestin3a (Arr3a) and around 1 h for G-protein receptor kinase7a (Grk7a). Note: Blotting time varies according to protein size. In this protocol, we focused on Arr3a (around 41 kDa), Grk7a (around 63 kDa), and loading control β-Actin (around 42 kDa). In general, for proteins smaller than 50 kDa, blotting takes around 40 min. For proteins between 50 and 150 kDa, blotting takes around 1 h. For proteins larger than 150 kDa, blotting takes around 1 h in 1× transfer buffer without MeOH. Transfer the membrane into a dish with PBS and mark membrane on one of the corners to remember which side is the top side. Antibody staining Place the membrane in a container (Figure 2C) and rinse twice for 5 min with 1× PBST. Block for 1 h in blocking solution on the shaker and shake gently. Prepare the first antibodies (rabbit anti-Arr3a: 1:4,000 and mouse anti-β-Actin: 1:6,000, or rabbit anti-Grk7a: 1:3,000 and mouse anti-β-Actin: 1:6,000) in blocking solution at 4 °C and incubate the membrane overnight at 4 °C on the shaker. Note: The first antibody solution can be kept at 4 °C and reused later. Depending on the antibody, this solution may be reused five times. Wash for 5 min in blocking solution at room temperature. Wash three times for 10 min in 1× PBST. Prepare the secondary antibodies (IRDye® 800CW goat anti-rabbit IgG and IRDye® 680RD goat anti-mouse IgG, both at 1:20,000) in blocking solution and incubate the membrane for 1 h on the shaker. Protect from light by covering the container completely with foil or placing it in a black box from now on. Wash three times for 5 min in 1× PBST. Signal detection Clean the scanning area of the imager with water and place the membrane. Define the area of interest in the Image Studio software and scan. Acquire images at 700 nm to visualize the signal of Arr3a or Grk7a, and at 800 nm to visualize the signal of β-Actin. Note: A membrane image example is shown in Figure 3 (Zang et al., 2021). Data analysis Perform the initial analysis with Image Studio. Draw a rectangle around each band. The signals at 700 and/or 800 nm will be calculated automatically by the software. Normalize the Arr3a or Grk7a signal to β-Actin signal in the appropriate software (e.g., Excel). At least three independent experiments should be performed. Note: The daily rhythms in the protein level of Arr3a and Grk7a were evaluated in the original paper. Statistical analysis was performed by “RAIN” as previously described (Thaben and Westermark, 2014). Figure 3. Arr3a protein levels show daily changes in adult zebrafish eyes. This figure was modified according to Figure 3 (Zang et al., 2021; https://elifesciences.org/articles/68903). One sample image of infrared fluorescence western blot was shown. Protein of interest was Arr3a. β-Actin was used as loading control. Eyes were collected every 3 h throughout a 24-h period. The highest protein level was normalized to 1. ZT = Zeitgeber time. Recipes RIPA buffer NaCl, 150 mM Triton X-100, 1% v/v Sodium deoxycholate, 0.5% w/v Tris, 50 mM, pH 8 adjusted with HCl and/or NaOH EDTA, 1 mM SDS, 0.1% w/v Filter-sterilize PIC Dissolve one PIC tablet in 1.5 mL of double-distilled H2O to obtain 7× stock solution Add 1× PIC to RIPA buffer before preparing lysates 5× Laemmli sample buffer Tris, 250 mM, pH 6.8 adjusted with HCl and/or NaOH Glycerol, 50% v/v SDS, 10% w/v Bromophenol blue, 1% w/v β-mercaptoethanol, 5% v/v (add right before use) Filter-sterilize 10× running buffer Tris, 250 mM Glycine, 1.9 M SDS, 0.50% w/v Autoclave 10× transfer buffer Tris, 200 mM Glycine, 1.5 M Autoclave PBST 1× PBS pH 7 Tween 20, 0.1% v/v Blocking buffer 1× PBST BSA, 1% w/v Acknowledgments Swiss National Science Foundation (SNF) 310030_204648. We would like to thank Dr. Matthias Gesemann for critical comments on the manuscript. This protocol is derived from the original research paper (Zang et al., 2021). Competing interests The authors have no conflict of interest. Ethics All animal experiments were carried out in line with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Veterinary Authorities of Kanton Zurich, Switzerland (TV4206). References Brown, A. J., Pendergast, J. S. and Yamazaki, S. (2019). Peripheral Circadian Oscillators. Yale J Biol Med 92(2): 327-335. Frøland Steindal, I. A. and Whitmore, D. (2019). Circadian Clocks in Fish-What Have We Learned so far? Biology (Basel) 8(1): 17. Li, Y., Li, G., Wang, H., Du, J. and Yan, J. (2013). Analysis of a gene regulatory cascade mediating circadian rhythm in zebrafish. PLoS Comput Biol 9(2): e1002940. Renninger, S. L., Gesemann, M. and Neuhauss, S. C. (2011). Cone arrestin confers cone vision of high temporal resolution in zebrafish larvae. Eur J Neurosci 33(4): 658-667. Rinner, O., Makhankov, Y. V., Biehlmaier, O. and Neuhauss, S. C. (2005). Knockdown of cone-specific kinase GRK7 in larval zebrafish leads to impaired cone response recovery and delayed dark adaptation. Neuron 47(2): 231-242. Thaben, P. F. and Westermark, P. O. (2014). Detecting rhythms in time series with RAIN. J Biol Rhythms 29(6): 391-400. Vatine, G., Vallone, D., Gothilf, Y. and Foulkes, N. S. (2011). It's time to swim! Zebrafish and the circadian clock. FEBS Lett 585(10): 1485-1494. Zang, J., Gesemann, M., Keim, J., Samardzija, M., Grimm, C. and Neuhauss, S. C. (2021). Circadian regulation of vertebrate cone photoreceptor function. Elife 10: e68903. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). How to cite Category Neuroscience > Basic technology > Optogenetics Biochemistry > Protein > Quantification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Quantitative Determination of Ca2+-binding to Ca2+-sensor Proteins by Isothermal Titration Calorimetry Seher Abbas and Karl-Wilhelm Koch Apr 5, 2020 4872 Views Compartment-Resolved Proteomics with Deep Extracellular Matrix Coverage Maxwell C. McCabe [...] Kirk C. Hansen Dec 5, 2024 336 Views An Assay System for Plate-based Detection of Endogenous Peptide:N-glycanase/NGLY1 Activity Using A Fluorescence-based Probe Hiroto Hirayama and Tadashi Suzuki Jan 5, 2025 201 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,619	https://bio-protocol.org/en/bpdetail?id=4619&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Far-western Blotting Detection of the Binding of Insulin Receptor Substrate to the Insulin Receptor JP Jinghua Peng BR Balamurugan Ramatchandirin AP Alexia Pearah LH Ling He Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4619 Views: 1083 Reviewed by: Suresh KumarRitu Gupta Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in The Journal of Biological Chemistry Mar 2022 Abstract Far-western blotting, derived from the western blot, has been used to detect interactions between proteins in vitro, such as receptor–ligand interactions. The insulin signaling pathway plays a critical role in the regulation of both metabolism and cell growth. The binding of the insulin receptor substrate (IRS) to the insulin receptor is essential for the propagation of downstream signaling after the activation of the insulin receptor by insulin. Here, we describe a step-by-step far-western blotting protocol for determining the binding of IRS to the insulin receptor. Keywords: Signal transduction Insulin receptor Insulin receptor substrate Protein–protein binding Post-translational modification Background Insulin, secreted by the pancreatic β-cells, is the most powerful anabolic hormone known to regulate the metabolism of glucose, lipids, and amino acid metabolism through the activation of the insulin signaling pathway. Insulin binding to the insulin receptor (IR), a tetrameric complex consisting of two extracellular α-subunits and two transmembrane β-subunits, leads to a conformational change, the activation of tyrosine kinase activity in the β-subunits, and the transphosphorylation of β-subunits at Y972 (Sweet et al., 1987; Cheatham and Kahn, 1995; Yip and Ottensmeyer, 2003). The phosphorylation of the β-subunit at Y972 generates a NPXpY motif, which the downstream mediator insulin receptor substrate (IRS) subsequently recognizes and binds to in the insulin receptor b (IRβ), resulting in the activation of PI3K-AKT signaling (Machado-Neto et al., 2018; White et al., 1988). Therefore, the binding of the IRS to the IRβ subunit plays a critical role in the activation of insulin signaling (Peng and He, 2018). Far-western blotting is an effective technique to assess protein–protein interactions, including receptor–ligand interactions (Kaido et al., 2007; Wu et al., 2007), in in vitro assays. In a far-western blotting analysis, one protein is first separated in an SDS-PAGE gel and transferred to a membrane, followed by the binding of a non-antibody secondary protein. Then, a specific antibody against the secondary protein will be employed to determine its binding to the first protein that is being transferred onto the membrane (Figure 1). This method can be used in a regular laboratory, without the need of expensive equipment, to determine the interaction of proteins in other methods, such as the surface plasmon resonance system. We introduce a far-western blotting analysis that has been successfully used to determine the interaction of IRβ to its downstream mediator, the IRS, and to assess the importance of post-translational acetylation of IRS in affecting its binding to IRβ, as well as the activation of the insulin signaling pathway in our studies (Cao et al., 2017; Peng et al., 2022). Figure 1. Procedure of far-western blotting to examine the binding of IRS to IRβ. A. Sequential steps of the procedure. B. Diagram depicts the detection of the binding with antibody. C. Purified IRS1 and IRS2 proteins were subjected to SDS-PAGE and stained with colloidal blue. D. Similar quantities of IRS2 and acetylated IRS2 by acetyltransferase P300 protein were employed in an SDS-PAGE and transferred onto a membrane; after renaturation, membranes were incubated with IRβ, followed by incubation with anti-IRβ antibody. Materials and Reagents 1.5 mL Eppendorf tubes (Eppendorf, catalog number: 022363204) Immuno-Blot PVDF membrane (Bio-Rad, catalog number: 1620177) Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: T5941) Sodium dodecyl sulfate (Sigma-Aldrich, catalog number: L3771) Glycerol (Sigma-Aldrich, catalog number: G5516) β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148) EDTA (Sigma-Aldrich, catalog number: E9884) Bromophenol blue (Sigma-Aldrich, catalog number: B8026) NuPAGETM LDS sample buffer (4×) (Thermo Fisher Scientific, catalog number: NP0007) NuPAGETM 3%–8%, NovexTM tris-acetate 1.5 mm mini protein gel, 10 well (Thermo Fisher Scientific, catalog number: EA0378BOX) NuPAGETM tris-acetate SDS running buffer (20×) (Thermo Fisher Scientific, catalog number: LA0041) IRS proteins were prepared as described previously (Cao et al., 2017; Peng et al., 2022). To determine the effect of IRS acetylation on its binding to IRb, IRS1 and IRS2 proteins were acetylated by acetyltransferase P300 protein. In the acetylation assay, 2 μg of IRS1 or IRS2 were added to the reaction containing 50 mM Tris-HCl (pH 8.0), 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM dithiothreitol (DTT), 1 mM PMSF, 10 mM sodium butyrate, 0.2 μg of acetyl-CoA, and 0.2 μg of P300 (Active Motif). Samples were incubated at 30 °C for 1 h. In another acetylation assay set, acetyl-CoA was not added but it served as a positive control. Recombinant human IRβ protein (Creative Biomart, catalog number: INSR-5093H) Anti-IRβ (Cell Signaling Technology, catalog number: 3020); dilution: 1:500 Protein ladder (Thermo Fisher Scientific, catalog number: 26634) Tris (Sigma-Aldrich, catalog number: T1503) Glycine (Sigma-Aldrich, catalog number: G8898) Methanol (Sigma-Aldrich, catalog number: 34860) NaCl (Sigma-Aldrich, catalog number: 9888) Tween-20 (Sigma-Aldrich, catalog number: P1379) Skim milk powder (Bio-Rad, catalog number: 1706404XTU) DTT (Sigma-Aldrich, catalog number: D0632) KH2PO4 (Sigma-Aldrich, catalog number: P5655) Na2HPO4 (Sigma-Aldrich, catalog number: S9763) Guanidine-HCl (Sigma-Aldrich, catalog number: G3272) ECL kit (PierceTM ECL western blotting substrate) (Thermo Fisher Scientific, catalog number: 32106) Loading buffer (see Recipes) Wet transfer buffer (see Recipes) Denaturing and renaturing buffers (see Table 1) (see Recipes) Protein-binding buffer (see Recipes) PBST buffer (see Recipes) Equipment Mini gel tank (Thermo Fisher Scientific, catalog number: A25977) PowerPacTM basic power supply (Bio-Rad, catalog number: 1645050) ChemiDoc XRS+ gel imaging system (Bio-Rad) Mini trans-blot electrophoretic transfer cell (Bio-Rad, catalog number: 1703930) Procedure Denaturing the protein samples Prepare 4× loading buffer. Mix the loading buffer (4×) and NuPAGETM LDS sample buffer (4×) in 1:1 volume. Apply the mix to the purified protein of mouse IRS1 (0.2–0.4 μg/μL) and IRS2 (0.2–0.5 μg/μL) (final 1×) in 1.5 mL Eppendorf tubes, at 95 °C for 10 min, and put on ice right after. Notes: The mixture of loading buffer and NuPAGETM LDS sample buffer (final 1×) can be used as a negative control. During the heating process, pay close attention to prevent the sample from volatilizing due to the tubes’ caps opening. Before loading the sample, collect the water vapor on the cap by completing a short spin. Separating protein samples by electrophoresis Load the denatured IRS1 and IRS2 protein samples onto the NovexTM mini protein gel along with the protein ladder. Run at 80 V in the stacking gel and then turn to 120–150 V in the resolving gel, until your target protein migrates to the middle of the resolving gel. Notes: Sample volume should not be >50 μL for a 1.5 mm 10-well gel. In our experiment, we load 5–10 μg of IRS1 (180 kD) protein and 5–12 μg of IRS2 (185 kD) protein. When the protein samples run into the resolving gel, transfer the electrophoresis tank into the refrigerator set at 4 °C. Based on the migration of the protein ladder, the position of the target protein in the gel can be estimated. Transferring the protein to the membrane Membrane preparation: pre wet the PVDF membrane first in methanol for 20 s, then place in ultrapure water for one quick rinse, followed by sitting in ultrapure water for 5 min, and finally in the wet transfer buffer (for at least 15 min). After electrophoresis is complete, wash the electrophoresis plates with pure water, place the gel over the blotting paper, carefully place the pre wet PVDF membrane onto the gel, place a second blotting paper onto the PVDF membrane, and place a sponge support pad. In this assay, we use wet transfer [100 V/gel, 2 h (for 1.5 mm gel), room temperature (RT)] or 10 mA/gel, overnight at 4 °C. Caution: To open and remove gel(s) from the pre-cast gel cassettes, please refer to the “NuPAGE Technical Guide” at https://www.thermofisher.cn/order/catalog/product/EA0375PK2. Make sure that there are no air bubbles present in the transfer sandwich. Denaturing/renaturing of proteins on the membrane After the transfer is complete, take the membrane out of the sandwich and rinse with ultrapure water twice. Incubate the membrane in the denaturing and renaturing buffers (freshly prepared) from high to low concentrations of guanidine-HCl as shown in Table 1. Finally, incubate the membrane in the denaturing and renaturing buffer without guanidine-HCl overnight at 4 °C. Table 1. Denaturing and renaturing buffers with varying concentrations of guanidine-HCl Guanidine-HCl concentration 6 M 3 M 1 M 0.1 M 0 M 80% glycerol (mL) 3.125 3.125 3.125 3.125 3.125 5 M NaCl (mL) 0.5 0.5 0.5 0.5 0.5 1 M Tris pH 7.5 (mL) 0.5 0.5 0.5 0.5 0.5 0.5 M EDTA (mL) 0.05 0.05 0.05 0.05 0.05 10% tween-20 (mL) 0.25 0.25 0.25 0.25 0.25 8 M guanidine-HCl (mL) 18.75 9.3 3.13 0.31 0 Milk powder (g) 0.5 0.5 0.5 0.5 0.5 1M DTT (μL) 25 25 25 25 25 ddH2O (mL) 1.825 12.195 17.445 20.265 20.575 Total volume (mL) 25 25 25 25 25 Time/temperature 30 min/RT 30 min/RT 30 min/RT 30 min/4 °C Overnight/4 °C Caution: Completely renaturing proteins on the membrane is critical for subsequent protein–protein binding assays. Blocking the membrane with 5% milk in PBST buffer for 1 h, at RT Incubating the membrane with recombinant human IRβ protein After blocking, wash the membrane with PBST buffer three times for 5 min. Apply 3–10 μg of IRβ in PBST buffer containing 3% milk and incubate with the membrane at 4 °C overnight. Detecting the IRβ bound to IRS1/2 on the membrane Wash off un-bound IRβ with PBST buffer three times for 10 min. Incubate with anti-IRβ (1:250 dilution) in 3% milk PBST, at 4 °C overnight. Wash the membrane with PBST buffer, three times for 10 min. Incubate the membrane with the secondary antibody (1:5,000 dilution) for 1 h at RT. Wash the membrane with PBST buffer, three times for 10 min. Rinse the membrane with PBS for 5 min. Developing the membrane with the chemiluminescent ECL kit according to the instructions Briefly, prepare the chemiluminescent substrate reagent by mixing the detection reagent 1 and 2 at 1:1 volume. Incubate the blot with the chemiluminescent substrate for 2 min. Remove the blot from the substrate and place it in the ChemiDoc XRS+ gel imaging system to get image. Caution: Use a sufficient volume to ensure that the blot is completely wet with the chemiluminescent substrate and does not become dry (0.1 mL/cm2). Recipes Loading buffer 50 mM Tris-HCl (pH 6.8) 2% sodium dodecyl sulfate 10% glycerol 1% β-mercaptoethanol 12.5 mM EDTA 0.02% bromophenol blue This loading dye can be stored at RT (-20 °C) for up to six months. Wet transfer buffer 25 mM Tris 192 mM glycine 20% methanol The buffer should be freshly prepared. Denaturing and renaturing buffers 100 mM NaCl 20 mM Tris (pH 7.6) 10% glycerol 0.1% Tween-20 2% skim milk powder 1 mM DTT This should be freshly prepared. Protein-binding buffer 100 mM NaCl 20 mM Tris (pH 7.6) 0.5 mM EDTA 10% glycerol 0.1% Tween-20 2% skim milk powder 1 mM DTT The solution should be freshly prepared. PBST buffer 4 mM KH2PO4 16 mM Na2HPO4 115 mM NaCl (pH 7.4) 0.05% Tween-20 Prepare 10× PBST and store at RT for up to three months. Dilute to 1× before use. Acknowledgments This work was supported in part by grants from the NIH: R01DK107641 and R01DK120309. This protocol was modified from a previous publication (Wu et al., 2007). Competing interests We declare no competing interest. References Cao, J., Peng, J., An, H., He, Q., Boronina, T., Guo, S., White, M. F., Cole, P. A. and He, L. (2017). Endotoxemia-mediated activation of acetyltransferase P300 impairs insulin signaling in obesity. Nat Commun 8(1): 131. Cheatham, B. and Kahn, C. R. (1995). Insulin action and the insulin signaling network. Endocr Rev 16(2): 117-142. Kaido, M., Inoue, Y., Takeda, Y., Sugiyama, K., Takeda, A., Mori, M., Tamai, A., Meshi, T., Okuno, T. and Mise, K. (2007). Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana. Mol Plant Microbe Interact 20(6): 671-681. Machado-Neto, J. A., Fenerich, B. A., Rodrigues Alves, A. P. N., Fernandes, J. C., Scopim-Ribeiro, R., Coelho-Silva, J. L. and Traina, F. (2018). Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis. Clinics 73(suppl 1): e566s. Peng, J. and He, L. (2018). IRS posttranslational modifications in regulating insulin signaling. J Mol Endocrinol 60(1): R1-R8. Peng, J., Ramatchandirin, B., Wang, Y., Pearah, A., Namachivayam, K., Wolf, R. M., Steele, K., MohanKumar, K., Yu, L., Guo, S., White, M. F., Maheshwari, A. and He, L. (2022). The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor. J Biol Chem 298(3): 101621. Sweet, L. J., Morrison, B. D. and Pessin, J. E. (1987). Isolation of functional alpha beta heterodimers from the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex. A structural basis for insulin binding heterogeneity. J Biol Chem 262(15): 6939-6942. White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A. and Kahn, C. R. (1988). Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell 54(5): 641-649. Wu, Y., Li, Q. and Chen, X. Z. (2007). Detecting protein-protein interactions by Far western blotting. Nat Protoc 2(12): 3278-3284. Yip, C. C. and Ottensmeyer, P. (2003). Three-dimensional structural interactions of insulin and its receptor. J Biol Chem 278(30): 27329-27332. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Biochemistry > Protein > Immunodetection Cell Biology > Cell signaling > Intracellular Signaling Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons Katarina Trajkovic [...] Dimitri Krainc Jan 5, 2018 7066 Views Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor Rubén Martínez-Barricarte [...] Jean-Laurent Casanova Jun 5, 2019 5089 Views Capillary Nano-immunoassay for Quantification of Proteins from CD138-purified Myeloma Cells Irena Misiewicz-Krzeminska [...] Norma C. Gutiérrez Jun 20, 2019 5716 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,620	https://bio-protocol.org/en/bpdetail?id=4620&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Automated Sleep Deprivation Setup Using a Shaking Platform in Mice WB Wen-Jie Bian LL Luis de Lecea Published: Vol 13, Iss 4, Feb 20, 2023 DOI: 10.21769/BioProtoc.4620 Views: 781 Reviewed by: Christine Muheim Shaorong Ma Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Neuroscience May 2022 Abstract The functions of sleep remain largely unclear, and even less is known about its role in development. A general strategy to tackle these questions is to disrupt sleep and measure the outcomes. However, some existing sleep deprivation methods may not be suitable for studying the effects of chronic sleep disruption, due to their lack of effectiveness and/or robustness, substantial stress caused by the deprivation method, or consuming a large quantity of time and manpower. More problems may be encountered when applying these existing protocols to young, developing animals, because of their likely heightened vulnerability to stressors, and difficulties in precisely monitoring sleep at young ages. Here, we report a protocol of automated sleep disruption in mice using a commercially available, shaking platform–based deprivation system. We show that this protocol effectively and robustly deprives both non-rapid-eye-movement (NREM) sleep and rapid-eye-movement (REM) sleep without causing a significant stress response, and does not require human supervision. This protocol uses adolescent mice, but the method also works with adult mice. Graphical abstract Automated sleep deprivation system. The platform of the deprivation chamber was programmed to shake in a given frequency and intensity to keep the animal awake while its brain and muscle activities were continuously monitored by electroencephalography and electromyography. Keywords: Sleep deprivation Mice Automated system Adolescence Shaking platform Background The functions of sleep and its long-term impact on behavior and physiology have been largely understudied. Studies focusing on the acute effect of sleep deprivation (SD) in juvenile mice, rats, and cats revealed that sleep affects synaptic structure and plasticity during adolescent development (Frank et al., 2001; Dumoulin Bridi et al., 2015; Shaffery et al., 2006; Shaffery et al., 2002; Maret et al., 2011; Yang et al., 2014; W. Li et al., 2017; de Vivo et al., 2017). However, whether these changes result in long-lasting behavioral changes remains unknown. To evaluate the impact of long-term sleep perturbation, especially during development, chronic sleep disruption is required, and so is longitudinal sleep monitoring using electroencephalogram (EEG) along the manipulation process. Automated SD in rodents often uses physical stimulations or setups that keep the animal in constant motion, such as using continuously moving treadmills or rotating wheels (Colavito et al., 2013). In an alternating platform setup, the animal was forced to move between two small platforms that continuously and alternatively move above and below water (Pierard et al., 2007). In a setup of spinning disk above a water tank, the disk rotated whenever sleep onset was detected, and the animal would need to stay awake and mobile in order not to fall into the water (they woke up in the case they did fall) (Rechtschaffen et al., 1983). Another method is the so-called “grid over water,” where placing the animal on a grid floor suspended over the water surface significantly reduced NREM and REM sleep and increased sleep latency, but the reduction of sleep was only in half (Shinomiya et al., 2003). REM-selective deprivation can be achieved by placing the animal on a small platform surrounded by and slightly above the water surface (Morden et al., 1967). The animal was allowed to have NREM sleep by crouching on the platform; however, upon transition to a REM episode, the loss of muscle tone caused the animal to touch or fall into the water and thus aroused the animal (Morden et al., 1967). These automated SD paradigms all produce stress, anxiety, and/or excessive physical stimuli, making them unsuitable for adolescent mice, which likely have heightened vulnerability to stressors (Romeo, 2013). In comparison, more mild methods such as the “gentle touch” protocol requires constant monitoring of the mouse behavior or EEG by an experimenter, as whenever the mouse exhibits signs of sleep (e.g., staying still for longer than 5 s, or low-frequency EEG), the experimenter directly intervenes by touching the animal using a soft tool (e.g., a brush), keeping the animal awake (Eban-Rothschild et al., 2016; W. Li et al., 2017; Yang et al., 2014). However, this method is labor-intensive, and may not be easily replicated between experimenters; thus, it is not ideal if high throughput or chronic intervention is desired. Another “gentle” protocol requires exposing the animal to novel objects, a different environment, and/or nesting materials, which leads to exploratory or nest-building behaviors, respectively, and helps maintain wakefulness. This method does not cause significant stress (Kopp et al., 2006), but the animal tends to become habituated quickly, and the forced wakefulness usually does not last longer than 2 h. In addition, pre-exposure to the enrichment materials may be a confounder, if a behavior task such as the novel object recognition test is performed subsequently. Here, we report an alternative protocol for automated SD in mice, which is effective for 4 h or longer and robust across many days, does not cause significant stress even in adolescent animals, and can be performed in high throughput (up to four mice at a time for each chamber) without human intervention. The protocol is ideal for chronic sleep disruptions over days, in both developing and adult mice. In combination with a closed loop algorithm, it is also possible to achieve REM-selective automated deprivation. Materials and Reagents Paper towels Male or female mice with a C57BL/6J background produced by breeding pairs purchased from Jackson Laboratory, or with a 129S2/SvPasCrl background produced by breeding pairs purchased from Charles River. They were born and housed at constant temperature (22 ± 1 °C) and humidity (40%–60%), under a 12/12 h light:dark cycle [lights-on: 7:00–19:00, zeitgeber time (ZT) 0–12; lights-off: 19:00–7:00, ZT 12–24], with access to food and water ad libitum. Animals were single-housed in customized home-cages after implantation of the electrodes for EEG and electromyography (EMG) (see Equipment). (Optional) An ultra-light extension cord (~50 cm in length, Cooner wires, catalog number: CW8183) was used for reducing the cable weight on adolescent mice during EEG/EMG recording; however, this is not required for adult mice. Corticosterone ELISA kit (Enzo, catalog number: ADI-900-097, storage temperature: 4 °C) Microvette® CB 300 Lithium heparin (Sarstedt Inc., catalog number: 16.443.100) Hydrogel (ClearH2O, catalog number: 70-01-5022) 70% ethanol Equipment Sleep Deprivation System (ViewPoint Life Sciences, Inc., Lyon, France), composed of a deprivation chamber (PVC cylinder, height: 46 cm, width: 30 cm, weight: 5 Kg) with a shaking platform at the bottom, a control box, and a computer with the controlling program Shaker Driver (Figure 1A, B). A video from the vendor illustrating the setup and its use can be found in https://youtu.be/LNTc7c5yFZM. A customized home-cage (Figure 1C) was made by taping two standard mouse cages together (without the lid, dimensions, 28 cm × 17.5 cm × 12.5 cm). A big opening (approximately 22 × 11 cm) was made on the top for the extension cord/recording cable to pass through, and a small hole was drilled on the side of bottom part, for water access. Regular mouse food pellets were placed inside the cage together with the bedding. Eppendorf centrifuge 5424 (Eppendorf, Hamburg, Germany). Figure 1. Automated sleep deprivation system and the customized home-cage. A. Schematics of the automated sleep deprivation system with simultaneous EEG/EMG recording. B. Sleep deprivation chamber. C. Customized home-cage. Software Shaker Driver v1.9.4 (ViewPoint Life Sciences) (Figure 2) Standard data processing and statistics software: Excel, Prism (GraphPad; version 8 or higher), etc. Procedure The procedures of electrode implantation surgery, EEG/EMG recoding, and data processing have been described in detail in our previous studies (Eban-Rothschild et al., 2016; S. B. Li et al., 2018; S. B. Li et al., 2020; Bian et al., 2022; S. B. Li et al., 2022), and therefore will not be a focus of this protocol. The SD system can accommodate up to four mice with EEG/EMG recording at the same time, using a custom-made divider (cardboard, Makrolon plastic, or other materials) which prevents the tangling of recording cables (Figure 1B). Wean the mouse pups on postnatal day (P) 21 and assign them randomly to Sleep Deprivation (SD) or Control (Ctrl) groups. Implant the electrodes for EEG/EMG recording at P28–P30 (Bian et al., 2022). After the surgery, individually house the animals in the customized home-cage (Figure 1C). At least one day before EEG/EMG recording, habituate the animal to the ultra-light extension cord for at least 24 h, by connecting the cord to the mouse headset. Fix the other end of the extension cord above the home-cage. Allow an ample length of the extension cord, for the free moving of the animal, but avoid leaving too much of the cord in the cage to prevent the animal from biting it. Mice will be kept connected for the entire experimental period. Baseline recording of spontaneous sleep/wake cycle in the home-cage (P36, Day 0). Connect the extension cord to the main EEG/EMG cable and the recording setup. Start recording at 7:00 (ZT 0) and stop recording at 7:00 the next morning (ZT 24). Set up the SD and Ctrl protocols in the Shaker Driver (Figure 2 and Video 1). In Stimulation Designer, set High Level Duration (ms) to 15, Total Duration (ms) to 500, and Delay (ms) to 0. Test the communication amongst the computer, the control box, and the deprivation chamber by clicking the Test button in Shaker Driver. It will generate a pulse as you programmed in Stimulation Designer, and the platform should shake accordingly. Set up the delivery sequence of pulses for the SD and Ctrl protocols. For the SD protocol, set Protocol duration (hours) to 4 in Sequence Designer, check “Use a randomized number of stimulations,” and set Number of Stimulations per sequence from 2 to 8. Check “Use a randomized Time between 2 sequences (min)” and set Time from 0.2 to 0.8. This will generate a train of randomized 2–8 pulses (of 15 ms in duration) delivered at 2 Hz every randomized 0.2–0.8 minutes, for a total duration of 4 h. For the Ctrl protocol, set Protocol duration (hours) to 4 in Sequence Designer, uncheck “Use a randomized number of stimulations,” and set Number of Stimulations per sequence to 5. Uncheck “Use a randomized Time between 2 sequences (min)” and set Time to 0.5. This will generate a train of five pulses (of 15 ms in duration) delivered at 2 Hz every 0.5 min, for a total duration of 4 h. Figure 2. Software interface of Shaker Driver Video 1. Setting up Shaker Driver for the SD and Ctrl protocols Perform the Ctrl/SD protocol (P37–P41, Days 1–5) Place bedding materials on the platform. Other materials/objects (e.g., shredded paper, nestlets, or paper tubes) may also be added as environmental enrichment, as they help to prevent distress in animals. Place a few food pellets and hydrogels (see Notes) on the platform, so the animals have access to food and water ad libitum. Transfer the SD animals to the deprivation chamber at 9:00 (ZT 2). No prior acclimation or habituation to the chamber is needed. The novel environment of the chamber also helps to maintain the animals awake. Place each animal in each compartment separated by the cardboard divider and allow the extension cord to pass through from the top of the apparatus (Figure 1A, B and Video 2). Start the SD procedure in Shaker Driver. After 4 h (ZT 2–6), the procedure will stop, or it can be manually terminated by clicking the Stop button. Video 2. Programmed platform shaking during an SD session For Ctrl animals, transfer them to the deprivation chamber and start the Ctrl protocol at 19:00 (ZT 12) and stop at 23:00 (ZT 16). After either an SD or Ctrl session, immediately return the animals to their home-cages. Discard the bedding, food pellets, and hydrogels. Clean the apparatus with 70% ethanol. Repeat steps a–e for the remaining days of the Ctrl/SD protocol. EEG/EMG recording is continuously performed on Day 3 from 7:00 (ZT 0) to 7:00 the next morning (ZT 24). It can also be performed in other days during the five days of Ctrl/SD (see Notes). One day after Ctrl/SD (P42, Day 6), record the 24-h EEG/EMG signals in the home-cage. To examine the stress response potentially caused by the SD or Ctrl protocols, measure the plasma corticosterone level by enzyme-linked immunosorbent assay (ELISA): Use a different cohort of animals without EEG/EMG recording and subject them to steps 6 and 7. On Days 1 and 5, immediately after the Ctrl/SD session, anesthetize the animal using isoflurane and use a capillary to collect a small quantity of blood (~100 μL) from the retro-orbital sinus into heparin-treated microvette tubes (Sarstedt Inc., 16.443.100). The animal should recover from at least one blood collection. Collect no-shake control samples from naïve animals without any manipulation at the same ZT. Separate the plasma from the whole blood sample by centrifugation (Eppendorf, Centrifuge 5424) at 1,000 × g and room temperature for 15 min. Transfer the supernatant (plasma) to a clean tube, avoiding contamination with the blood cells in the precipitate. Measure the corticosterone level in the plasma samples using a corticosterone ELISA kit (Enzo, ADI-900-097), according to the manufacturer’s instructions. Data analysis The processing and quantification of EEG/EMG data was described in our previous studies (Eban-Rothschild et al., 2016; S. B. Li et al., 2018; S. B. Li et al., 2020; Bian et al., 2022; S. B. Li et al., 2022), and is subject to change in different EEG/EMG systems—thus, it is not characterized here. States of Wake, NREM, and REM were defined based on the following criteria: Wake: desynchronized small-amplitude EEG and extensive EMG activity; NREM: large-amplitude, delta (0.25–4 Hz)-dominant oscillations in EEG, and reduced EMG activity compared to wakefulness; REM: high frequency (4–9 Hz-dominant) and relatively small and uniform amplitude of EEG, often associated with a flat EMG (muscle atonia). Any epoch that lasts longer than 4 s in SleepSign (Kissei Comtec Co.) (Eban-Rothschild et al., 2016) or 5 s in a custom MATLAB script (S. B. Li et al., 2018; S. B. Li et al., 2020; Bian et al., 2022; S. B. Li et al., 2022) was counted as a state. Calculate the percentage of time in Wake, NREM, and REM states in each hour, and plot it against the ZT to evaluate the effect of SD. Perform repeated-measure two-way analysis of variance (RM 2-way ANOVA) followed by Bonferroni's multiple comparisons to examine the difference between the groups. Representative data can be found in Extended Data Figure 1d–f of our previous study (Bian et al., 2022). Calculate the parameters of sleep architecture, such as the total time of each state during the entire 4-h SD session, total number of bouts of each state, average bout length, sleep rebound in the following 6 h (Bian et al., 2022), and reduction of NREM and REM sleep caused by the SD protocol compared to the Ctrl protocol (Figure 3A). Other parameters can be calculated form EEG as well, such as the power spectrum of each state, before or after SD. Representative data can be found in Extended Data Figure 1g–m of our previous study (Bian et al., 2022). The sleep recording one day after SD (P42, Day 6) or on additional following days can be used to evaluate any long-lasting alterations in sleep architecture, by comparing it to baseline recording, or to the Ctrl animals. The plasma corticosterone measurements from mice receiving no shaking, or immediately after the SD session on Days 1 or 5 showed no significant difference, confirming that the SD protocol does not cause significant acute stress. These results can be found in Extended Data Figure 1n of our previous study (Bian et al., 2022). The corticosterone levels on Day 5 were also not significantly different between Ctrl and SD groups (Figure 3B). Figure 3. Representative data. A. Reduction of NREM or REM sleep amount (s) during 4 h Ctrl/SD relative to their respective baseline sleep during the same ZT period. The Ctrl protocol began immediately at dark phase onset and lasted for 4 h (ZT 12–16), and the SD protocol was performed in early light phase onset (ZT 2–6) in adolescent mice (P35–P42). N = 4 mice in Ctrl; 9 mice in SD. Welch’s t-test, ΔNREM, t = 10.63, df = 5.13, P = 0.0001; ΔREM, t = 8.74, df = 10.99, P = 0.000003. B. Five days of SD did not induce significant stress compared to the Ctrl protocol. N = 7 mice in Ctrl; 10 mice in SD. Welch’s t-test, t = 0.3331, df = 14.14, P = 0.74. Data are shown as means ± S.E.M. * P < 0.05; P < 0.01; * P < 0.001; n.s., not significant. SD values were extracted from the data published in Bian et al. (2022). Notes The system robustly deprives the animals from sleep and can accommodate up to four mice at a time with EEG recording by using a divider. The randomness in the SD protocol helps to reduce habituation to the shaking, and thus prevents sleep, while the non-random design in the Ctrl protocol does the opposite, i.e., minimizing the sleep disturbance as much as possible. Indeed, during Ctrl sessions we found that some mice can retain a small amount of sleep, which is not significantly different from the spontaneous sleep during the early dark hours (ZT 12–16). We recommend avoiding using the same animals for corticosterone measurement and EEG/behavioral tests, due to potential confounding effects and poor physical conditions after repeated blood collections. The corticosterone measurements reported in this protocol do not examine the acute/transient stress during the treatment. If that is a concern for the study, additional stress controls or more timepoints for stress measurements should be added. Due to the short age window for manipulations in adolescent mice, we used a minimum of 24 h for habituating the animal to the recording cable. However, longer habituation time is strongly encouraged when doing less age-sensitive experiments. We have compared the EEG recordings in adolescent mice after 24 h (1 d) and 5 days (5 d) of habituation, respectively, and found that the overall sleep/wake pattern and the total time of each state were not significantly different between 1 d and 5 d (Supplementary Figure 1A and 1B). However, the average bout length of Wake and NREM episodes did appear to be increased as the habituation time increased, although only the difference in the dark-phase NREM category was statistically significant (Supplementary Figure 1C). These results suggest that 1 d habituation is sufficient for restoring the normal sleep/wake cycle, although the animal might go through slight sleep fragmentation, which is likely improved with extended habituation. The deprivation chamber comes with a water bottle held on the cylinder. However, the water bottle has serious leaking issues during shaking, so we did not use it; instead, we just placed hydrogels on the chamber floor. ViewPoint also offers a SleepScore software that, according to the vendor, enables real-time detection of vigilance states and specific deprivation of NREM and REM sleep (https://www.viewpoint.fr/en/p/software/sleepscore). However, we have not yet tested the performance of this software. Acknowledgments This work was supported by Human Frontier Science Program fellowship LT000338/2017-L (W.-J.B.), Brain & Behavior Research Foundation NARSAD Young Investigator grant 29952 (W.-J.B.) and National Institutes of Health grants R01 MH102638 (L.d.L.), R01 MH087592 (L.d.L.), R01 MH116470 (L.d.L.). Data derived from the use of the protocols described here have been published recently (Bian et al., 2022). Competing interests The authors declare no competing interests. Ethics All experimental protocols were approved by the Stanford University Animal Care and Use Committee and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. References Bian, W. J., Brewer, C. L., Kauer, J. A. and de Lecea, L. (2022). Adolescent sleep shapes social novelty preference in mice. Nat Neurosci 25(7): 912-923. Colavito, V., Fabene, P. F., Grassi-Zucconi, G., Pifferi, F., Lamberty, Y., Bentivoglio, M. and Bertini, G. (2013). Experimental sleep deprivation as a tool to test memory deficits in rodents. Front Syst Neurosci 7: 106. de Vivo, L., Bellesi, M., Marshall, W., Bushong, E. A., Ellisman, M. H., Tononi, G. and Cirelli, C. (2017). Ultrastructural evidence for synaptic scaling across the wake/sleep cycle. Science 355(6324): 507-510. Dumoulin Bridi, M. C., Aton, S. J., Seibt, J., Renouard, L., Coleman, T. and Frank, M. G. (2015). Rapid eye movement sleep promotes cortical plasticity in the developing brain. Sci Adv 1(6): e1500105. Eban-Rothschild, A., Rothschild, G., Giardino, W. J., Jones, J. R. and de Lecea, L. (2016). VTA dopaminergic neurons regulate ethologically relevant sleep-wake behaviors. Nat Neurosci 19(10): 1356-1366. Frank, M. G., Issa, N. P. and Stryker, M. P. (2001). Sleep enhances plasticity in the developing visual cortex. Neuron 30(1): 275-287. Kopp, C., Longordo, F., Nicholson, J. R. and Luthi, A. (2006). Insufficient sleep reversibly alters bidirectional synaptic plasticity and NMDA receptor function. J Neurosci 26(48): 12456-12465. Li, S. B., Borniger, J. C., Yamaguchi, H., Hedou, J., Gaudilliere, B. and de Lecea, L. (2020). Hypothalamic circuitry underlying stress-induced insomnia and peripheral immunosuppression. Sci Adv 6(37): eabc2590. Li, S. B., Damonte, V. M., Chen, C., Wang, G. X., Kebschull, J. M., Yamaguchi, H., Bian, W. J., Purmann, C., Pattni, R., Urban, A. E., et al. (2022). Hyperexcitable arousal circuits drive sleep instability during aging. Science 375(6583): eabh3021. Li, S. B., Nevarez, N., Giardino, W. J. and de Lecea, L. (2018). Optical probing of orexin/hypocretin receptor antagonists. Sleep 41(10): zsy141. Li, W., Ma, L., Yang, G. and Gan, W. B. (2017). REM sleep selectively prunes and maintains new synapses in development and learning. Nat Neurosci 20(3): 427-437. Maret, S., Faraguna, U., Nelson, A. B., Cirelli, C. and Tononi, G. (2011). Sleep and waking modulate spine turnover in the adolescent mouse cortex. Nat Neurosci 14(11): 1418-1420. Morden, B., Mitchell, G. and Dement, W. (1967). Selective REM sleep deprivation and compensation phenomena in the rat. Brain Res 5(3): 339-349. Pierard, C., Liscia, P., Philippin, J. N., Mons, N., Lafon, T., Chauveau, F., Van Beers, P., Drouet, I., Serra, A., Jouanin, J. C. and Beracochea, D. (2007). Modafinil restores memory performance and neural activity impaired by sleep deprivation in mice. Pharmacol Biochem Behav 88(1): 55-63. Rechtschaffen, A., Gilliland, M. A., Bergmann, B. M. and Winter, J. B. (1983). Physiological correlates of prolonged sleep deprivation in rats. Science 221(4606): 182-184. Romeo, R. D. (2013). The Teenage Brain: The Stress Response and the Adolescent Brain. Curr Dir Psychol Sci 22(2): 140-145. Shaffery, J. P., Lopez, J., Bissette, G. and Roffwarg, H. P. (2006). Rapid eye movement sleep deprivation revives a form of developmentally regulated synaptic plasticity in the visual cortex of post-critical period rats. Neurosci Lett 391(3): 96-101. Shaffery, J. P., Sinton, C. M., Bissette, G., Roffwarg, H. P. and Marks, G. A. (2002). Rapid eye movement sleep deprivation modifies expression of long-term potentiation in visual cortex of immature rats. Neuroscience 110(3): 431-443. Shinomiya, K., Shigemoto, Y., Okuma, C., Mio, M. and Kamei, C. (2003). Effects of short-acting hypnotics on sleep latency in rats placed on grid suspended over water. Eur J Pharmacol 460(2-3): 139-144. Yang, G., Lai, C. S., Cichon, J., Ma, L., Li, W. and Gan, W. B. (2014). Sleep promotes branch-specific formation of dendritic spines after learning. Science 344(6188): 1173-1178. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Neuroscience > Behavioral neuroscience > Sleep and arousal Neuroscience > Development Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Simultaneous Microendoscopic Calcium Imaging and EEG Recording of Mouse Brain during Sleep Sasa Teng and Yueqing Peng May 5, 2023 911 Views Using Fiber Photometry in Mice to Estimate Fluorescent Biosensor Levels During Sleep Mie Andersen [...] Celia Kjaerby Aug 5, 2023 796 Views Measuring Sleep and Activity Patterns in Adult Zebrafish Fusun Doldur-Balli [...] Allan I. Pack Jun 20, 2024 728 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,621	https://bio-protocol.org/en/bpdetail?id=4621&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Molecular Surveillance of Malaria Using the PF AmpliSeq Custom Assay for Plasmodium falciparum Parasites from Dried Blood Spot DNA Isolates from Peru JK Johanna Helena Kattenberg ND Norbert J. van Dijk CF Carlos A. Fernández-Miñope PG Pieter Guetens MM Mathijs Mutsaers DG Dionicia Gamboa AR Anna Rosanas-Urgell Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4621 Views: 698 Reviewed by: Alka MehraMariana Barnes Rita Marie Celine Meganck Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Microbiology Spectrum Feb 2023 Abstract Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes. Keywords: Plasmodium falciparum Malaria Molecular surveillance Drug resistance AmpliSeq custom assay Sequencing Background Surveillance of Plasmodium parasites has been identified by the World Health Organization (WHO) as one of the essential pillars to move towards malaria elimination in its endemic areas (WHO, 2019). Molecular parasite genotyping tools can strengthen malaria surveillance systems by monitoring the emergence and spread of drug resistance, histidine rich protein 2 and 3 (hrp2 and hrp3) deletions, quantification of malaria importation risk, and characterization of changing transmission intensity. As molecular surveillance platforms transition from proof-of-concept research studies to operational incorporation into national malaria control programs (NMCPs), simple standardized laboratory protocols feasible on benchtop sequencers and automated analysis pipelines are necessary to generate reproducible results and decrease the time from sample collection to results, thereby ensuring rapid turnover of up-to-date reports for decision making and policy (WHO, 2016; MPA Committee, 2019). Various methods have been developed to investigate drug resistance markers, usually based on the amplification of loci of interest using PCR-based techniques, with amplicons detected in real time assays by amplicon sequencing (Taylor et al., 2013; Menard et al., 2016; Nsanzabana et al., 2018), or analyzed using restriction fragment length polymorphism (Eldin de Pecoulas et al., 1995; Duraisingh et al., 1998). In the past, population surveillance widely utilized genotyping tools targeting surface antigens by PCR to distinguish parasite clones (Falk et al., 2006; Koepfli et al., 2009), followed by panels of microsatellites (MS) that are not under evolutionary selection pressure and are, therefore, more suitable to inform population genetic changes (Anderson et al., 2000; Imwong et al., 2007; Karunaweera et al., 2008). More recently, genome-wide single nucleotide polymorphism (SNP) panels, capable of defining a molecular barcode to capture the diversity of parasite populations, have been developed and can be investigated with methods such as microarrays, real-time PCR, and deep sequencing (Daniels et al., 2008, 2013 and 2015; Neafsey et al., 2008; Baniecki et al., 2015; Koepfli and Mueller, 2017; Fola et al., 2020; Tessema et al., 2022). Surveys to detect these gene deletions rely on molecular methods based on PCR assays that classify deletions based on failure to amplify targets in the hrp2 and hrp3 exon region (and sometimes flanking genes). Currently, there is no multifunctional tool that includes a combination of more than two types of markers (i.e., SNP-barcodes, drug resistance, etc.) to serve several use cases. In addition, the characterization of hrp2 and hrp3-deletions relies on PCR assays that classify deletions based on failure to amplify targets. The few existing tools that combine population markers with drug resistance target short regions around validated drug resistance SNPs, missing the potential to detect novel resistance-associated mutations. Furthermore, many SNP-panels were designed from genomes across the world and lack the resolution to study subtle patterns on a smaller geographical scale. Therefore, we have developed a targeted amplicon next-generation sequencing (NGS) assay for molecular surveillance of Plasmodium falciparum parasites that combines a specifically designed barcode for the target country, 13 full-length resistance-associated genes (Table 1), hrp2 and hrp3, and an apical membrane antigen 1 (ama1) microhaplotype region. The novelty of the assay is its high number of targets multiplexed in one easy workflow, using AmpliSeq deep sequencing technology (Illumina, 2018), and combining phenotypic markers with a 28-SNP barcode with in-country resolution to investigate parasite gene flow in Peru. The assay uses overlapping amplicons to cover large genes. The 28 SNP-barcode was designed with in-country resolution to monitor parasite strains circulating in Peru over space and time (Kattenberg et al., 2023). These SNPs were selected from a South American P. falciparum genome dataset selecting SNPs with a frequency of minor alleles below 0.35 and those that were not under selective pressure, prioritizing SNPs that differentiated the Peruvian samples from other parasite populations in the dataset. Noteworthy, the seq-based tool can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes using different SNP-barcodes. The technology applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure, and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. Table 1. Targeted genes of interest for drug resistance in the PF AmpliSeq assay Gene ID Chromosome Gene with resistance Drug associated with resistance* PF3D7_1218300 12 ap2-mu ART; QN PF3D7_1251200 12 coronin DHA PF3D7_0709000 7 crt CQ; PPQ; AMQ mal_mito_3 mitochondrial cytochrome B ATQ PF3D7_0417200 4 dhfr PYR; PG PF3D7_0810800 8 dhps SULF PF3D7_1362500 13 exonuclease PPQ PF3D7_1343700 13 k13 ART PF3D7_0523000 5 mdr1 CQ; PPQ; MQ; QN; HF; AMQ; LF PF3D7_0112200 1 mrp1 ART, MQ, LF PF3D7_1408000 14 plasmepsin II PPQ PF3D7_API04900 apicoplast 23s rRNA CM PF3D7_0104300 1 ubp-1 ART AMQ: amodiaquine; ART: artesunate; ATQ: atovaquone; CM: clindamycin; CQ: chloroquine; DHA: dihydroartemisinin; HF: halofantrine; LF: lumefantrine; MQ: mefloquine; PG: proguanil; PPQ: piperaquine; PYR: pyrimethamine; SULF: sulfadoxine; QN: quinine. The list of drugs to which resistance has been observed is non-exhaustive. Validated mutations are listed in the WHO report on antimalarial drug efficacy, resistance, and response: 10 years of surveillance (2010–2019). Materials and Reagents AmpliSeq Library PLUS kit for Illumina (96 reactions; Illumina, catalog number: 20019102) Contains: 1× Lib Amp mix 10× Library Amp primers DNA ligase 5× AmpliSeq HiFi mix FuPa Reagent Low TE (Tris-EDTA buffer) Switch solutions AmpliSeq Custom DNA panel for Illumina (Illumina, catalog number: 20020495, custom design IAD179763_241; see Supplementary manifest file and APPENDIX 1 for oligo sequences) Contains: Primer pool 1 (red cap) Primer pool 2 (blue cap) AmpliSeq CD Indexes Set A for Illumina (96 indexes, 96 samples; Illumina, catalog number: 20019105) Note: Optionally, you can also use Index Set B, C, or D. Absolute ethanol (EtOH) (Sigma-Aldrich, Merck, catalog number: 1.00983.1000) Agencourt AMPure XP beads (Beckman Coulter, catalog number: A63881) 96-well PCR plate, 0.2 mL (Greiner Bio-One, catalog number: 652201) 8-well PCR strips (Greiner Bio-One, catalog number: 673210) Strip caps for PCR pates (Greiner Bio-One, catalog number: 373250) Adhesive seals for PCR plates (Westburg Life Science, catalog number: WB2-3800) 1.5 mL DNA LoBind® Tubes (Eppendorf, catalog number: 022431021) Nuclease-free water (Lonza, catalog number: BE51200) KAPA Library Quantification kit (KAPA Biosystems, Roche, catalog number: 07960298001) DNA standards 1–6 (80 µL each) Primer mix (1 mL) KAPA SYBR® FAST qPCR Master mix (5 mL) Qubit® dsDNA HS Assay kits (Invitrogen, Thermo Fischer Scientific, catalog number: Q32851) QubitTM dsDNA HS reagent (250 µL) QubitTM dsDNA HS buffer (50 mL) QubitTM dsDNA HS standard #1 (1 mL) QubitTM dsDNA HS standard #2 (1 mL) Thin-wall, clear, 0.5 mL PCR tubes (e.g., Qubit® assay tubes, catalog number: Q32856) Pipette filter tips, 10 µL (e.g., Greiner Bio-One, catalog number: 771353; Biofil, catalog number: PPT150010) Combitips 0.2 and 0.5 mL (Eppendorf, catalog numbers: 0030089774 and 0030089421) Miseq Reagent kit v3 (600 cycle) (Illumina, catalog number: MS-102-3003) Reagent cartridge HT1 (hybridization buffer, 5 mL) PR2 (incorporation buffer, 500 mL) Flow cell 1 N sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 22146-5) Tris-hydrochloride (HCl), pH 8.0(Sigma-Aldrich, catalog number: T3038-1L) , adjusted to pH 7.0 Tween® 20 (Sigma-Aldrich, catalog number: 022431021) PhiX Control v3 (Illumina, catalog number: FC-110-3001) Optional to include as controls and take along in the library preparation: DNA extracted from laboratory strain(s) of P. falciparum (e.g., 3D7 or Dd2) as positive control Note: Laboratory strains can be obtained from BEI Resources, NIAID, NIH (https://www.beiresources.org/): P. falciparum, strain 3D7, MRA-102, contributed by Daniel J. Carucci; P. falciparum, strain Dd2, MRA-150, contributed by David Walliker; P. falciparum, strain Dd2_R539T, MRA-1255 and P. falciparum, strain CamWT_C580Y, MRA-1251, contributed by David A. Fidock; and P. falciparum, strain IPC 4912, MRA-1241, contributed by Didier Ménard. Genomic DNA of these strains is also available from BEI Resources. DNA extracted from uninfected human blood as negative control Equipment Multichannel pipettes 10 µL, 0.1 mL and 0.3 mL Electronic multi-dispenser pipette (Eppendorf, Multipette © E3x, catalog number: 4987000029) Centrifuge (Sigma Laborzentrifugen GmbH, Sigma 4-16KS, catalog number: 150507) Vortex (Scientific Industries, Vortex Genie 2, catalog number: SI-0236) 96-well magnet stand (Invitrogen, ThermoFisher Scientific, DynamagTM-96 Side Skirted, #12027; RNA Life Technologies, Ambion® Magnetic Stand-96, catalog number: 1307065) Qubit 2.0 (or higher) fluorometer (Invitrogen, Thermo Fischer Scientific, catalog number: Q32866) Conventional cycler (Biometra, Westburg, Professional Thermocycler Basic 96 × 0.2 mL, catalog number: 846-070-701) LightCycler® 480 real-time PCR system (Roche, catalog number: 04640268001) MiSeqTM system (Illumina, catalog number: SY-410-1003) Software Illumina experiment Manager (Illumina) Microsoft Excel (Microsoft) In-house Linux-based pipeline Ubuntu (Canonical, ubuntu.com) Trimmomatic (Bolger et al., 2014, https://doi.org/10.1093/bioinformatics/btu170) Burrows Wheeler Aligner (Li et al., 2009, https://doi.org/10.1093/bioinformatics/btp324) Picard tools (Broad Institute, https://broadinstitute.github.io/picard/) Genome Analysis Toolkit (GATK) (Broad Institute, https://gatk.broadinstitute.org/) SnpEff (Cingolani et al., 2012) On-Off instrument alignment Local Run Manager (Illumina) DNA Amplicon Module (Illumina) Procedure In this protocol, target regions of parasite DNA are amplified in two reactions, followed by a library preparation where indexes are added and subsequently libraries are amplified and cleaned, quantified, pooled, and sequenced on a MiSeq system (Figure 1). Figure 1. Schematic flowchart of library preparation procedures P. falciparum–infected human blood sample preparation This section quantifies and prepares the DNA extracted from dried blood spots (DBS) for library preparation. Isolate DNA from DBS according to standardized protocols (e.g., DNA extractions using the Qiagen 96 DNA blood kit or E.Z.N.A. DNA mini kit). Note: DNA extracted from whole blood or white blood cell–depleted blood can also be used as input material. However, take note of the DNA concentrations as described in step A3. Quantify 3–10 µL of DNA of (a subset of) samples using the Qubit® dsDNA HS Assay kits, according to manufacturer’s protocol. Set up the required number of 0.5 mL tubes for standards and samples. The Qubit® dsDNA HS Assay requires two standards. Prepare the Qubit® working solution by diluting the Qubit® dsDNA HS reagent 1:200 in Qubit® dsDNA HS buffer. Use a clean plastic tube each time you prepare Qubit® working solution. For 96 samples, prepare 20 mL working solution by mixing: i) 19.9 mL Qubit® dsDNA HS buffer. ii) 100 µL Qubit® dsDNA HS reagent. Prepare the standard tubes: i) Add 190 μL of Qubit working solution to each of the two tubes used for standards. ii) Add 10 μL of each Qubit® standard to the appropriate tube, then mix by vortexing 2–3 s. Be careful not to create bubbles. Prepare the sample tubes: i) Add 200 μL minus the sample volume (see next step) of Qubit working solution to each of the tubes used for the libraries. ii) Add 3–10 μL of each DNA sample to the appropriate tube, then mix by vortexing 2–3 s. Be careful not to create bubbles. Note: If you suspect the sample concentration is too low, you can use a higher volume (10 μL) of sample in the Qubit reaction. Allow all tubes to incubate at room temperature (RT) for 2 min. On the home screen of the Qubit® 2.0 fluorometer, press DNA, then select dsDNA High Sensitivity as the assay type. The Standards screen is displayed. On the Standards screen, press Yes to read the standards. Insert the tube containing standard #1 into the sample chamber, close the lid, then press Read. When the reading is complete (~3 s), remove standard #1. Insert the tube containing standard #2 into the sample chamber, close the lid, then press Read. When the reading is complete, remove standard #2. When the calibration is complete, the instrument displays the Sample screen. Insert a sample tube into the sample chamber, close the lid, then press Read. When the reading is complete (~3 s), remove the sample tube. The instrument displays the results on the Sample screen. The value displayed corresponds to the concentration after your sample was diluted into the assay tube. To find the concentration of your original sample, you can record this value and perform the calculation later in the Excel template. To calculate the concentration of your sample, use the following equation: Concentration = measured value (concentration of diluted sample) × (200/V), where V is the volume of sample that you started the reaction in step A2 d.ii. Repeat steps j-k until all samples have been read. Recommended input concentration from the library preparation kit is 10 ng of high-quality DNA per reaction with one of the two primer pools. (The kit supports 1–100 ng DNA input. Please note, however, that due to the nature of our samples, we have a mixture of human and parasite DNA.) Dilute samples with too high DNA concentration (>100 ng) in low TE (supplied with AmpliSeq Library PLUS kit) to an input concentration of ~1–10 ng/μL. Note: In our procedures, mean DNA concentration after DNA extraction from DBS was 6.1 ± 0.3 ng/μL and we used 7.5 μL of undiluted sample (~23 ng total input per reaction) in the library preparation reactions. It is important not to exceed the 100 ng DNA input as this will negatively affect the quality of the sequencing result. For DBS samples, in our experience, the DNA concentration was always below the upper limit. After testing the DNA concentration of a few samples with varying parasite densities at the start of a study, we do not routinely check the concentration every time we do a plate to save time and costs. It is important to consider that for P. falciparum DBS samples, we have a mixture of human and parasite DNA. To ensure you have sufficient high-quality parasite DNA, it is important to quantify your parasite DNA in advance with a qPCR. From our validation procedure (see notes at the end of the document) we recommend including samples with P. falciparum densities ≥60 parasites/µl as determined by Mangold PCR (Mangold et al., 2005). This limit depends also on the amount of blood spotted on filter paper and the amount of filter paper (e.g., two pieces of ~0.5 cm2 as in our case), which can impact the amount of template DNA in your sample. Prepare a 96-well layout with the sample IDs (see Excel template “Template_AmpliSeq.xlsx”). Note: We usually do the library preparation for 96 samples at once, so we use one 96 sample AmpliSeq Library PLUS kit per time. You could increase or decrease the number of samples and kits used for one run on a MiSeq, but this will impact the depth per sample. Add 7.5 µL of (diluted) DNA for each sample in the corresponding well of your layout. SAFE STOPPING POINT: Store the 96-well plate with prepared samples at 4 °C overnight or at -20 °C for longer periods if not immediately commencing with the library preparation procedures. Amplify DNA targets This section uses PCR to amplify the (overlapping) target regions of the DNA samples in two reactions with two different primer pools from the AmpliSeq Custom DNA panel. As a first step, the DNA is mixed with the AmpliSeq HiFi Mix (including a buffering solution and enzyme for amplification) and subsequently split over two new PCR plates. To the wells in each of the plates, one of the two pre-mixed pools of primers is added for amplification on a thermal cycler. There are two pools of primers (and not just one amplifying all targets), to allow for the overlap between amplicons spanning large genes (see Figure 2). Figure 2. Overlapping amplicon design and primer pools. In order to allow for amplification of large genes, the custom DNA panel includes short overlapping amplicons. By using two pools of primers alternating the overlapping amplicons, we prevent amplification of the wrong primer combination in the overlapping region. After the initial two PCR reactions with the two primer pools, you add both products together and continue with a single library preparation in the next step. Thaw the following reagents: 96-well plate with 7.5 µL of DNA prepared in previous section: thaw on ice if frozen. 5× AmpliSeq HiFi Mix (red cap; AmpliSeq Library PLUS kit): thaw on ice and invert to mix. One aliquot of 2× AmpliSeq Custom DNA panel pool 1 (red cap, AmpliSeq Custom DNA panel): thaw at RT, vortex to mix. One aliquot of 2× AmpliSeq Custom DNA panel pool 2 (blue cap, AmpliSeq Custom DNA panel): thaw at RT, vortex to mix. Briefly centrifuge the 96-well plate and prepared tubes to collect all liquid at the bottom of the wells. Remove the lids/seal from the 96-well plate with DNA. To each well with sample [volume (V) = 7.5 µL], add 5 µL of 5× AmpliSeq HiFi Mix using a multi-dispenser pipette and 0.5 mL of Combitip. Centrifuge the plate briefly to collect all liquid at the bottom of the well. Mix the DNA–HiFi mixture with a multichannel pipette and transfer 5 µL to the corresponding column of a new PCR plate (labelled pool 1) and 5 µL to a second new PCR plate (labelled pool 2). To the first new plate (pool 1), add 5 µL of 2× AmpliSeq Custom DNA pool 1 (red cap) to each well using a multi-dispenser pipette and 0.5 mL Combitip. To the second new plate (pool 2), add 5 µL of 2× AmpliSeq Custom DNA pool 2 (blue cap) to each well using a multi-dispenser pipette and 0.5 mL Combitip. Seal both plates with an adhesive seal or lids and briefly centrifuge to collect all liquid at the bottom of the well. Check for air bubbles, remove by tapping/flicking the side of the well with your fingers, and centrifuge again. Place the 96-well plate in the thermocycler, set the volume to 10 µL (if applicable), and run the AMP_DNA program as in Table 2 (with heated lid on at 105 °C): Table 2. PCR cycling conditions (AMP_DNA) Cycles Temperature Time 1× 99 °C 2 min 21× 99 °C 60 °C 15 s 8 min Hold 10 °C up to 24 h SAFE STOPPING POINT: If you are stopping, leave the plate on the thermal cycler at 10 °C for up to 24 h. For longer durations, store at -25 °C to -15 °C. Partially digest amplicons This section uses the FuPa Reagent to digest primer dimers and partially digest amplicons. From this section forward, work in a clean hood that you can radiate with ultraviolet or clean to denature DNA. Thaw the following reagents: 96-well plate with amplified pool 1 prepared in previous section: thaw on ice and vortex. 96-well plate with amplified pool 2 prepared in previous section: thaw on ice and vortex. FuPa Reagent (brown cap; AmpliSeq Library PLUS kit): thaw on ice. If continuing with the next section (D) immediately after this section: Switch solution (yellow cap; AmpliSeq Library PLUS kit): thaw at RT. Briefly centrifuge the 96-well plates with amplified pool 1 and pool 2 and prepared tubes to collect all liquid at the bottom of the wells. Remove the seals from the 96-well plates with amplified pool 1 and pool 2. For each sample, use a multichannel pipette to combine the 10 µL reaction from amplified pool 2 in the corresponding well of amplified pool 1. This will result in a total volume per sample of 20 µL. Aliquot 30 µL of FuPa reagent in each well of a clean 8-well PCR strip. From here, using a multichannel pipette, transfer 2 µL of FuPa reagent to each well of the combined pool 1 + 2 product and mix. Discard the tips before continuing with the next column. Note: The reagent is very viscous and due to the low volume, it is very difficult to do this with a multi-dispenser pipette and Combitip. So, use a multichannel pipette with 10 µL tips for more accurate pipetting. Seal the plate with an adhesive seal, vortex briefly, and briefly centrifuge to collect all liquid at the bottom of the well. Place the 96-well plate in the thermocycler, set the volume to 22 µL (if applicable), and run the FUPA program as in Table 3 (with heated lid on at 105 °C): Table 3. PCR cycling conditions (FUPA) Cycles Temperature Time 1 50 °C 10 min 1 55 °C 10 min 1 62 °C 20 min Hold 10 °C Up to 1 h SAFE STOPPING POINT: If you are stopping, leave the plate on the thermal cycler at 10 °C for up to 1 h. For longer durations, store at -25 °C to -15 °C. Ligate indexes This section ligates Index 1 (i7) and Index 2 (i5) adapters to the fragments of each sample. The indexes are premixed in a single-use plate to ensure unique combinations. Each library must have a unique index combination for dual-index sequencing. When more than 96 samples are being included in the same sequencing run, make sure to use different index sets (four sets are available from Illumina with different combinations: Set A, Set B, set C, and/or Set D, allowing multiplexing of maximum 384 libraries). For more information see the Illumina Index Adapter Pooling Guide (https://support-docs.illumina.com/SHARE/IndexAdapterPooling/Content/SHARE/IndexAdapterPooling/AmpliSeq/Pooling_fAS.htm). To avoid library prep failure, do not combine the reagents for this section together outside the wells with the digested amplicons. Thaw the following reagents: Switch solution (yellow cap; AmpliSeq Library PLUS kit): thaw at RT, vortex to mix. AmpliSeq CD Index set: thaw at RT, vortex to mix. DNA ligase (blue cap; AmpliSeq Library PLUS kit): thaw on ice. If continuing with the next section E immediately after this section: Equilibrate AMPure XP beads to RT (at least 30 min). Vortex vigorously to resuspend. Briefly centrifuge the 96-well plate with partially digested amplicons, index plate, and the prepared tubes of reagents to collect all liquid at the bottom of the wells. Remove the seals from the 96-well plate with partially digested amplicons and from the index plate. Add (in the listed order) to each well of the 96-well plate with partially digested amplicons: 4 µL of switch solution (yellow cap) using a multi-dispenser pipette and 0.2 mL Combitip (multi-dispenser pipette set to 48 steps of 4 µL; so, you need to aspirate and dispense twice). Note: Alternatively, use an 8-well strip with 50 µL of switch solution aliquots and multichannel to pipette 4 µL of switch solution into each well of the 96-well plate. 2 µL of the AmpliSeq CD Index to the corresponding well of the 96-well plate with partially digested amplicons + switch solution using a multichannel pipette and 10 µL tips. Aliquot 30 µL of DNA ligase reagent (blue cap) in each well of a clean 8-well PCR strip. From here, using a multichannel pipette, transfer 2 µL of DNA ligase reagent to each well of the 96-well plate with partially digested amplicons + switch solution + index. Seal the plate with an adhesive seal, vortex briefly, and briefly centrifuge to collect all liquid at the bottom of the well. Place the 96-well plate in the thermocycler, set the volume to 30 µL (if applicable), and run the LIGATE program as in Table 4 (with heated lid on at 105 °C): Table 4. PCR cycling conditions (LIGATE) Cycles Temperature Time 1 22 °C 30 min 1 68 °C 5 min 1 72 °C 5 min Hold 10 °C up to 24 h If the index plate contains unused indexes, seal the plate and return to storage (-25 °C to -15 °C). SAFE STOPPING POINT: If you are stopping, leave the plate on the thermal cycler at 10 °C for up to 24 h. For longer durations, store at -25 °C to -15 °C. Clean up library This section uses Agencourt AMPure XP beads to clean up the library. The library will be bound to the beads, which are carried over to the next section. Prepare the following reagents: Equilibrate AMPure XP beads to RT (at least 30 min). Vortex vigorously to resuspend. Freshly prepare 50 mL of 70% ethanol (EtOH) solution (mix 35 mL of 100% EtOH and 15 mL of Nuclease-free water). For the next section (F) that should immediately be continued after this section, prepare: Four tubes of 1× Lib Amp mix (black cap; AmpliSeq Library PLUS kit): thaw on ice and invert to mix. Two tubes of 10× Library Amp Primers (pink cap; AmpliSeq Library PLUS kit): thaw at RT and vortex to mix. Briefly centrifuge the 96-well library plate (with amplicons and index) and the prepared tubes of reagents to collect all liquid at the bottom of the wells. Remove the seals from the 96-well library plate. Add 30 µL of AMPure XP beads (vortex thoroughly before pipetting) to each well with library in the 96-well plate using a multi-dispenser pipette and 2.5 or 5 mL Combitips. Seal the plate with an adhesive seal and vortex briefly. Inspect each well to ensure the mixture is homogenous (see Figure 3); then, centrifuge briefly (low speed ~500–1,000 rpm). Figure 3. Homogenized library and beads mix Incubate the plate at RT for 5 min. Place the plate on a magnetic stand, remove the seal, and wait until the mixture is clear (at least 2 min) (see Figure 4). Figure 4. Bead mixture on magnetic stand. Left: mixture is not yet entirely clear and still a bit yellowish. Right: Mixture is clear, and the blue color of indexes added in the previous step can be seen. While on the magnetic stand, the library is bound to the beads: Use a multichannel pipette (200 or 300 µL tips) to remove and discard the entire supernatant from each well. Wash twice: i) Add 150 µL of freshly prepared 70% EtOH to each well using a multi-dispenser pipette and 0.5 mL Combitips. ii) Incubate at RT until the solution is clear (> 30 s). iii) Without disturbing the beads, remove and discard supernatant. Seal the plate with an adhesive seal and centrifuge briefly (low speed ~500–1,000 rpm). Place the plate on a magnetic stand (make sure to place in the same orientation as in steps E8 and E9 to keep the beads on the same side of the well), remove the seal, and wait until the mixture is clear (~30 s). Remove any residual EtOH as follows: Use a 10 µL multichannel pipette to remove as much residual EtOH from each well as you can. Air-dry the 96-well plate on the magnetic stand without seal for at least 10 min. Inspect the wells to make sure the residual EtOH has evaporated. If it remains in some wells, try to remove with 10 µL pipette and continue to air-dry until the EtOH is no longer visible (Figure 4). Note: Overdried or cracked beads do not negatively affect the performance of the assay. However, residual EtOH causes library preparation failing in the next section by inhibiting amplification. Amplify library This section uses PCR to amplify the libraries to ensure sufficient quantity for sequencing on Illumina systems. The amplification reactions contain the beads, which are carried over from the previous section. The libraries are amplified using universal primers. In the previous section you should have already prepared the following reagents: Four tubes of 1× Lib Amp mix (black cap; AmpliSeq Library PLUS kit): thaw on ice and invert to mix. Two tubes of 10× Library Amp primers (pink cap; AmpliSeq Library PLUS kit): thaw at RT and vortex to mix. If continuing with the next section (G) immediately after this section: Equilibrate AMPure XP beads to RT (at least 30 min). Vortex vigorously to resuspend. Prepare the amplification master mix in a 15 mL falcon tube (for 96 reactions) by combining the reagents as in Table 5. Table 5. Amplification master mix recipe Reagent Volume (µL) for one library Volume (µL) for 96 libraries (make for 100×) Volume (µL) for X libraries 1× Lib Amp mix (black cap) 45 4,500 10× Library Amp primers (pink cap) 5 500 Total Volume (µL) 50 5,000 Vortex the amplification master mix briefly and centrifuge. Remove the 96-well library plate (from the previous section) from the magnet. Add 50 µL of amplification master mix to each well using the multi-dispenser pipette and 5 mL Combitip. Seal the plate with an adhesive seal, vortex briefly, and briefly centrifuge to collect all liquid at the bottom of the well. Place the 96-well plate in the thermocycler, set the volume to 50 µL (if applicable), and run the AMP_7 program as in Table 6 (with heated lid on at 105 °C): Table 6. PCR cycling conditions (AMP_7) Cycles Temperature Time 1 98 °C 2 min 7 98 °C 64 °C 15 s 1 min Hold 10 °C up to 24 h SAFE STOPPING POINT: If you are stopping, leave the plate on the thermal cycler at 10 °C for up to 24 h. For longer durations, store at -25 °C to -15 °C. Second cleanup This section performs the second cleanup with AMPure XP beads for two rounds of purification. First round: High molecular weight DNA is captured by the beads and discarded. The library and primers are retained in the supernatant and transferred to a fresh plate for the second round of purification. Second round: Libraries in the saved supernatant are captured by the beads while primers remain in the supernatant. The bead pellet is saved, and libraries are subsequently eluted from the beads. Prepare the following reagents: Equilibrate AMPure XP beads to RT (at least 30 min). Vortex vigorously to resuspend. Low TE (bottle; AmpliSeq Library PLUS kit): thaw at RT for 45 min, vortex to mix. Can be stored at RT. Freshly prepare 50 mL of 70% EtOH solution (mix 35 mL of 100% EtOH and 15 mL of nuclease-free water). Briefly centrifuge the 96-well library plate to collect all liquid at the bottom of the wells. First round: Add 25 µL of AMPure XP beads (vortex thoroughly before pipetting) to each well with ~50 µL library in the 96-well plate using a multi-dispenser pipette and 2.5 mL Combitips. Note: This step adds beads to the beads already in the reaction. Seal the plate with an adhesive seal and vortex briefly, then centrifuge briefly (low speed ~500–1,000 rpm). The beads do not need to be fully resuspended. Incubate at RT for 5 min. Place the plate on a magnetic stand, remove the seal, and wait until the mixture is clear (at least 5 min). Transfer the entire supernatant (~75 µL) to a new plate. Small amounts of bead carryover do not affect performance. THE SUPERNATANT CONTAINS THE DESIRED AMPLICON LIBRARY! Second round: Add 60 µL of AMPure XP beads to each well with the transferred supernatant in the new 96-well plate using a multi-dispenser pipette and 5 mL Combitips. Seal the plate with an adhesive seal and vortex briefly; then, centrifuge briefly (low speed ~500–1,000 rpm). Incubate at RT for 5 min. Place the plate on a magnetic stand, remove the seal, and wait until the mixture is clear (at least 5 min). While on the magnetic stand, perform the following steps. This time, the library is bound to the beads. Use a multichannel pipette (200 or 300 µL tips) to remove and discard the entire supernatant from each well. Wash twice: i) Add 150 µL of freshly prepared 70% EtOH to each well using a multi-dispenser pipette and 0.5 mL Combitips. ii) Incubate at RT until the solution is clear (> 30 s). iii) Without disturbing the beads, remove and discard supernatant. Use a 10 µL multichannel pipette to remove as much residual EtOH from each well as you can. Air-dry the 96-well plate on the magnetic stand without seal for at least 5 min. Remove the plate from the magnet. Add 30 µL of low TE to each well. Seal the plate with an adhesive seal and vortex briefly. Then, centrifuge briefly (low speed ~500–1,000 rpm). To ensure the beads are well resuspended, if necessary, you can mix by pipetting. Place the plate on a magnetic stand, remove the seal, and wait until the mixture is clear (at least 5 min). Transfer 27 µL of the supernatant (containing the libraries) to a new 96-well plate. SAFE STOPPING POINT: If you are stopping, store at -25 °C to -15 °C (up to 30 days). Quantify libraries Libraries should be quantified to pool the libraries of the samples at equimolar ratios for balanced sequencing output for every sample. There are two options for quantifying your libraries: using a KAPA qPCR kit or, alternatively, using the Qubit high-sensitivity reagents and fluorometer, depending on the availability of equipment in your laboratory. OPTION 1: KAPA library quantification Follow the manufacturer’s procedures of the Library Quantification kit for Illumina Platforms (KAPA Biosystems), which is a qPCR-based method for quantification of Illumina libraries flanked by the P5 and P7 flowcell oligo sequences. Dilute the libraries to be tested 1:1,000,000 in DNA dilution buffer [10 mM Tris-HCl, pH 8.0–8.5 (25 °C) + 0.05% Tween® 20]. For accurate dilution and for keeping the volume manageable, serially dilute three times 100×. So, start with mixing 3 µL of DNA + 297 µL of dilution buffer and briefly vortex, for a first 100× dilution. Then, mix 10 µL of 100× diluted DNA + 900 µL of dilution buffer, for a second dilution to 10,000× dilution, and briefly vortex. Then, mix 10 µL of 10,000× diluted DNA + 900 µL of dilution buffer for a final dilution to 1,000,000× dilution, and briefly vortex. Ensure that all components of the KAPA Library Quantification kit are completely thawed and thoroughly mixed. If the kit is used for the first time, add the primer premix (10×) (1 mL) to the bottle of KAPA SYBR® FAST qPCR Master mix (2×) (5 mL). Mix thoroughly using a vortex mixer. Prepare the required internal control dilutions (e.g., PhiX library dilution) in the same way as the samples. Each library, including the controls, is measured in triplicate (so, for 96 libraries, multiple plates with KAPA Library quantification need to be performed). Prepare PCR plate layouts and include six standards and internal control (DNA standard 0 of the standards), which are all provided by the KAPA kit, as per the instructions of the manufacturer, and non-template controls (NTC, e.g., nuclease-free water or buffer). Dispense 6 µL of the master mix [KAPA SYBR FAST qPCR Master mix (2×) + primer premix (10×)] into each well of a 96-well qPCR plate. Add 4 μL of nuclease-free water to all NTC wells. Dispense 4 μL of each DNA standard into the appropriate well/tube(s), working from the most dilute (standard 6) to the most concentrated (standard 1). Dispense 4 μL of each dilution of libraries and internal controls to be assayed. Seal the PCR plate and transfer to the qPCR instrument (Roche LightCycler® 480). Perform qPCR with the cycling protocol of Table 7, selecting the Absolute Quantification option in the instrument software. Adjust run parameters (e.g., reporters, reference dyes, gain settings, etc.) as required. Table 7. qPCR cycling conditions (KAPA quantification) Cycles Temperature Time 1 95 °C 5 min 35 95 °C 60 °C 30 s 45 s Melt curve 65–95 °C OPTION 2: Qubit DNA concentration measurement Use the Qubit® dsDNA HS Assay kits to determine the concentration of double-stranded DNA in the libraries, following the manufacturers’ procedures. Set up the required number of 0.5 mL tubes for standards and samples. The Qubit® dsDNA HS Assay requires two standards. Prepare the Qubit® working solution by diluting the Qubit® dsDNA HS reagent 1:200 in Qubit® dsDNA HS buffer. Use a clean plastic tube each time you prepare Qubit® working solution. For 96 samples, prepare 20 mL working solution by mixing: 19.9 mL Qubit® dsDNA HS buffer. 100 µL Qubit® dsDNA HS reagent. Add 190 μL of Qubit working solution to each of the two tubes used for standards. Add 10 μL of each Qubit® standard to the appropriate tube, then mix by vortexing 2–3 s. Be careful not to create bubbles. Add 197 μL of Qubit working solution to each of the tubes used for the libraries. Add 3 μL of each sample library to the appropriate tube, then mix by vortexing 2–3 s. Be careful not to create bubbles. Note: If you suspect the library concentration is too low, you can use a higher volume (5–20 μL) of library in the Qubit reaction. If this is the case, also adjust the volume of Qubit working solution in step H5 to make sure the final volume after adding the library is 200 μL. Take care not to use too much library as you will need sufficient volume (up to 6 µL) to make the pool in the next section. If the library concentration is too high (i.e., beyond the linear range of the Qubit kit) after the first measurement, dilute the library (e.g., 1:2 or 1:4) and measure again to get an accurate concentration. Allow all tubes to incubate at RT for 2 minutes. Note: You can add the libraries to the working solution in batches of 16–24, then incubate and measure. In the meantime, keep the unused tubes in the dark, for example by covering with aluminum foil. On the Home screen of the Qubit® 2.0 fluorometer, press DNA, then select dsDNA High Sensitivity as the assay type. The Standards screen is displayed. On the Standards screen, press Yes to read the standards. Insert the tube containing standard #1 into the sample chamber, close the lid, then press Read. When the reading is complete (~3 s), remove standard #1. Insert the tube containing standard #2 into the sample chamber, close the lid, then press Read. When the reading is complete, remove standard #2. When the calibration is complete, the instrument displays the Sample screen. Insert a sample tube into the sample chamber, close the lid, then press Read. When the reading is complete (~3 s), remove the sample tube. The instrument displays the results on the Sample screen. The value displayed corresponds to the concentration after your sample was diluted into the assay tube. To find the concentration of your original sample, you can record this value and perform the calculation later in the Excel template. To calculate the concentration of your sample, use the following equation: Concentration = measured value (concentration of diluted sample) × (200/V), where V is the volume of library that you added, in this case 3 µL. Repeat steps 12-13 until all samples have been read. Dilute and pool libraries Libraries of each sample and control will be diluted with low TE to the same concentration (2 nM) individually and then pooled at equimolar ratios for a balanced sequencing output for all samples. Library quantity will vary depending on template input amount (e.g., differing parasite densities). Thaw frozen low TE buffer (AmpliSeq library prep kit) at RT. Using the library concentrations determined with the KAPA or Qubit kit (previous section), determine the molarity of the library. For measurements with the KAPA kit, use the template provided by the company and standard curve included in the PCR plate to determine the size-adjusted molarity. Note: With KAPA kit, a library prep from a dried blood spot sample with parasite density ≥5 p/µL usually has a size-adjusted molarity within the range of 10–1000 nM. For Qubit measurements, we use the following formula: (c 106) / (660 * library size), where c is the concentration (ng/µL) as measured with the Qubit. Note: You can determine the mean size of your library with, for example, the Tapestation (Agilent) or alternatively using 350 bp as the default for AmpliSeq. (With Qubit, a library preparation from a DBS sample with parasite density ≥5 p/µL usually has a size-adjusted molarity within the range of 1–100 nM.) Calculate the dilution you need to make to reach a concentration of 2 nM for each library. Dilute each library to 2 nM using low TE in a 96-well plate as indicated in the template. If the concentration (molarity) of the library (undiluted) is below 2 nM, then add 6 µL of the undiluted library to the final 2 nM plate. Note: Take care not to start with a too low volume of library (at least 3 µL) for the dilution, as this will increase the inaccuracy of the final concentration. Also, take care to keep the final volume of your dilution below 200 µL; otherwise, the well of your dilution plate will overflow. If needed, dilute in a separate Eppendorf tube for larger volumes, then transfer 50 µL of the diluted library to the appropriate well in the dilution plate. Make a pool with equal volumes of all 2 nM libraries: Use a multichannel pipette to combine 5 µL from each well from the rows of the 96-well plate into one 8-well strip (i.e., make a pool from each row). Mix by pipetting 10 times. Combine the entire volume of the row pools from the 8-well strip into one 1.5 mL LoBind® tube. Note: This example for the pooling is for 96 samples. When sequencing more than 96 samples in one run, pool all 2 nM libraries of all samples to be included in the run into one final pool at equal volumes. When running more than 96 samples, take care to combine libraries prepared with different index sets; otherwise, you will not be able to demultiplex the sequences from the different plates. Preferably, proceed to sequencing the diluted library pool soon after the dilution. If not possible, store diluted library at -20 °C up to one week. Preparing files for sequencing on MiSeq This section describes the steps and settings needed to prepare the sample sheet that is required to set up the sequencing reaction on the MiSeq. Using the Illumina Experiment Manager software, create a sample plate: From the main screen, select Create Sample Plate. Select AmpliSeq CD Indexes plate A (or B, C, or D, depending on the index kit that you used during the library prep), and then select Next. In the Unique Plate Name field, enter a unique name for the sample plate. (Do not use special characters and spaces in sample IDs and plate names.) Select 2 (Dual) for the Index Reads, which should be the default setting. Select Next. Select the Table or Plate tab, depending on your preferred view. Enter a unique Sample ID for each well (for example by copy pasting from an Excel layout). Setting the indexes (in the Table tab). When using the default layout from the 96-well index plates, you can auto populate the indexes: select the Apply Default Index Layout button at the bottom left. When you are using a different layout than the standard index plate layout: i) Select a well in the Index Well field. ii) In the Index1 and Index2 fields, select the index adapter being used for each Index Read. Check your layout; valid entries for all samples will have turned white instead of brown/grey. If everything is valid, select Finish, and then save the sample plate file in a desired location. Using the Illumina Experiment Manager software, create a sample sheet: From the main screen, select Create Sample Sheet. Select MiSeq and then Next. Select the appropriate application (Other -> FASTQ Only, to only generate fastq files for subsequent analysis) and then select Next. (Alternatively, variants can be analyzed directly using the DNA Amplicon application and the manifest file in the basespace cloud system or local run manager.) In the Reagent Kit Barcode field, enter the reagent kit ID from the label of box 1 or box 2 of the SBS kit that starts with RGT, followed by eight digits located underneath the barcode. (If unknown at this stage, you can correct this later.) Select the appropriate Library Prep Workflow (AmpliSeq Library PLUS for Illumina). Select the appropriate Index Adapter (AmpliSeq CD Indexes plate A, B, C, or D, or 384 when combining more than 96 samples). Index Reads should be 2 (Dual) by default. Enter the Experiment Name, Investigator Name, Description, and Date. Enter the expected date of sequencing. Select the Paired End Read Type. In the Cycles Read fields, enter one more than the number of cycles (301 for the MiSeq v3 600 cycle reagent kit) for both read 1 and for read 2. In the workflow specific settings, select Use Adapter Trimming (default). Select Next to continue to Select Samples for a MiSeq Sample Sheet. Select samples by selecting Select Plate and then navigate to the sample plate prepared in step J1. Choose wells to include in the sequencing run (Select all). Select Add Selected Samples. Note: Make sure that all the libraries included in the pool are in the sample sheet with corresponding indexes. So, if you are running more than 96 samples in a run, add all plates to the one sample sheet. Select Finish, and then save the sample sheet file (*.csv) in the desired location. Review the sample sheet in Excel (there should be no spaces or special characters in the sample IDs). Final preparation for sequencing In this section, the pooled library is denatured and diluted before loading on the MiSeq cartridge for sequencing. A control library (PhiX) is added to the pool at the same concentration and will act as a control for the sequencing reaction on the MiSeq. In addition, by adding PhiX, you will increase the nucleotide diversity during the sequencing run, which will increase the sequencing quality, as the P. falciparum genome, and hence also the targeted regions in this assay, are high in A and T nucleotides. Prepare a fresh dilution of 0.2 N NaOH: Combine the reagents in an Eppendorf tube: i) 800 µL nuclease-free water. ii) 200 µL 1.0 N NaOH. Mix the tube by inverting several times. Use within 12 h. Prepare HT1: Thaw the HT1 buffer (Miseq reagent kit v3, Illumina) at RT. Store the HT1 buffer at 2–8 °C until needed, if not used immediately when thawed. Denature libraries: Combine 5 µL of library pool with 5 µL of 0.2 N NaOH. Vortex briefly, then centrifuge briefly. Incubate at RT for 5 min. Add 5 µL of 200 mM Tris-HCl, pH 7.0. Add 985 µL of pre-chilled HT1 buffer to the tube of denatured pool. The result is a 10 pM denatured library. Vortex and centrifuge briefly. Place the 10 pM libraries on ice until you are ready to proceed to final dilution. Dilute library pool to final loading concentration. Dilute with pre-chilled HT1 buffer until the final loading concentration at a final volume of 600 μL: 18 pM for KAPA quantified libraries 7 pM for Qubit quantified libraries If you are stopping, seal the tube and store at 25°C to 15°C. Spike library pool with 1%–5% PhiX spike-in to include quality controls (5% is recommended for PF AmpliSeq libraries as GC content, and therefore library diversity, is lower). Dilute stock of PhiX spike-in to a concentration similar to the final library (7 or 18 pM). i) Take 570 µL of 7 pM library ii) Add 30 µL of PhiX You obtained a 7 pM library pool with 5% PhiX spike-in for loading onto the reagent cartridge and the MiSeq according to the directions of the manufacturer and Reagent kit. Keep on ice until loading. Preparation of the MiSeq and loading the library pool Perform a pre-run wash of the MiSeq as per the instructions on MiSeq control software after selecting Wash. Thaw the reagent cartridge from the MiSeq V3 Reagent kit in a water bath (or sink filled with a layer of water) at RT. Do not submerge the entire cartridge, only the base. There is a maximum water level indicator line on the cartridge that you should not surpass. It will take 1–2 h before the entire cartridge is thawed. Invert the reagent cartridge 10 times to mix the reagents. Inspect that all reagents are thawed, fully mixed, and free of precipitates. Gently tap the cartridge on the bench to reduce air bubbles in the reagents. Set the reagent cartridge aside if not using immediately, store on ice or at 2–8 °C for up to six hours. For best results, proceed directly to loading the sample and setting up the run. Locate the seal that is covering the reservoir labelled Load Samples and clean the foil with a tissue/Kimwipe and pierce it with a clean 1 mL pipette tip. Add 600 µL of prepared denatured 7 pM library pool with 5% PhiX spike-in into the reservoir marked Load Samples; avoid touching the seal. Set up the run on the MiSeq System: Select Sequence using the MiSeq control software. Select Set up a run with the Local Run Manager and follow the steps until the Local Run Manager is opened. Select Create Run and the appropriate analysis module: i) Select the module GenerateFASTQ to sequence and generate fastq files that can be analyzed with the Linux pipeline described in the data analysis option. ii) Select the module DNA Amplicon to use the manifest file to perform the variant calling on the MiSeq system. Note: You will need to have this module installed on your MiSeq system in order to use it. You will also need to copy the manifest file to the MiSeq system, as well as the reference genome following the instructions of the Local Run Manager Amplicon Analysis Module Workflow Guide (https://support.illumina.com/content/dam/illumina-support/documents/documentation/software_documentation/local-run-manager/local-run-manager-dna-amplicon-workflow-guide-1000000048047-03.pdf). Select Import sample sheet to import the sample sheet prepared in section J. Check the settings, samples, and indexes and add a name for the run and a description (optional). Select Save Run. Your run is now ready for sequencing. With the local run manager still open, select the run you just created; then, select Next at the bottom right. Remove the previously loaded flow cell from the MiSeq machine by releasing the clamp with the button. Carefully clean the new flow cell with a Kimwipe or ethanol wipe until the surface is clean. Place it in the MiSeq system, close the clamp, and close the lid of the flow cell compartment. The machine will check the ID and correct placement of the flow cell. Select Next. Invert the PR2 bottle to mix and open it. Open the reagent compartment door, raise the handle until it locks, and replace the wash bottle with the PR2 bottle. Empty the waste reservoir. Close the handle and the reagent compartment door. The MiSeq will check if all materials are placed correctly and read the ID of the PR2 bottle. Click Next when the check is completed. Open the reagent compartment door and the reagent chiller door and remove the wash cartridge. Insert the reagent cartridge with your loaded library into the reagent chiller, sliding it to the back. Close the reagent chiller door and the reagent compartment door. The MiSeq will check if all materials are placed correctly and read the ID of the reagent cartridge. Click Next when the check is completed. Check the parameters of the run [e.g., Read type (Paired end), read length (2 × 300 + index reads)] and click Next when correct or Edit to make changes. The system will perform a complete pre-run system check. When complete and if everything is in order, select Start Run to start the sequencing. The run with a 600-cycle MiSeq Reagent kit V3 will take ~56 h to complete. When the run is complete, the fastq files will be ready to copy to a USB and then your computer for analysis (or, if you selected to share the run on Basespace, you can access and download it in your basespace account). Data analysis The Miseq sequencer generates fastq files with the raw sequencing reads that are automatically demultiplexed (i.e., using the indexes in the sample sheet generated in step J, the reads are separated for each sample and individual fastq files are generated for each individual sample). We processed these fastq files with an in-house analysis pipeline on a Unix operating system desktop computer, which is described in Kattenberg et al. (2022) and described below. Alternatively, for fast automated variant calling after sequencing, the fastq files of your run can be analyzed with the local run manager (Illumina) on the MiSeq and the DNA-Amplicon approach using the manifest file (Additional Material), according to the local run manager instructions explained in the previous section (Figure 5). Figure 5. Flowchart of variant calling procedures Variant calling In our in-house pipeline (https://github.com/Ekattenberg/Plasmodium-AmpliSeq-Pipeline), the resulting sequences are processed to generate a variant file (vcf), containing only the positions where the samples had a sequence that was different from the reference sequence. In the series of scripts, the following steps are performed: Quality control reports are generated using FastQC. FastQ Screen was used to determine sources of contamination (e.g., human sequences). Fastq files are trimmed using Trimmomatic (settings: ILLUMINACLIP: 2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36) to remove adapter sequences and poor-quality reads. Trimmed reads are subsequently aligned to the 3D7 reference genome (version plasmoDB-44) using Burrows-Wheeler aligner (v0.7.17) ( Li et al., 2009). Alignment statistics are generated using Picard’s CollectAlignmentSummaryMetrics. Subsequently, variants in amplicons including overlapping regions were jointly called using HaplotypeCaller (GATK, v4.1.2) and GenotypeGVCFs (GATK, v4.1.2). Variants are hard filtered [QUAL > 30, overall depth (DP) > 100, RMSMappingQuality (MQ) > 50, QualByDepth (QD) > 1.0, ReadPosRankSumTest (ReadPosRankSum) > -10, StrandOddsRatio (SOR) < 4, genotype field depth (DP) > 5]. Finalize by annotation with SnpEff (v4.3T), resulting in 2,146 high-quality genotypes for statistical and population genetic analysis. Microsatellite (MS) variant-calling MS alleles were called using a different approach because of the short tandem repeat length. The raw fastq files were aligned to reference sequences containing only the four MS amplicon regions (poly-alpha, TA81, ARAII, and PfPK2) using Burrows-Wheeler aligner (v0.7.17) (Li et al., 2009). Subsequently, reads were realigned on repeats using Genotan v0.1.5 (Tae et al., 2014). Short tandem repeat length was determined using HipSTR (Willems et al., 2017). Note: As HipSTR is made for diploid genomes, only the two predominant MS genotypes present in the sequencing reads are called. While this does not allow us to give exact estimates of complexity of infection (COI, i.e., number of co-infected clones), we can distinguish between single clone (COI =1) vs. multiple clone infections (COI ≥ 2, if two MS alleles are found for ≥1 MS locus). Calculation of performance measures To measure the sequencing depth for each sample and amplicon in each sample, we calculated the median sequencing depth of all loci in the variant file for each sample or for each amplicon. To calculate the mean, we used the depth of coverage after filtering at each position in the vcf (format field DP in the vcf file). Aligned coverage is calculated as the number of bases that passed filter (from the AlignmentSummaryMetrics report) divided by the number of bases (57445 bp) targeted in the PF AmpliSeq assay. Inclusion criteria for analysis For the final analysis we include only samples with good quality data (<50% of genotype calls missing and mean aligned coverage >15) and retaining only one library in case of replicates, with the lowest proportion of genotype missingness and highest aligned coverage. Statistical analysis The presence or absence of the hrp2 and hrp3 genes was determined for each sample using the mean read depth of respective amplicons compared to the mean depth of all amplicons, resulting in a depth ratio. Log-transformed mean depth ratios of previously typed samples were used to define thresholds for classification for each amplicon (Kattenberg et al., 2022). A final classification of presence/absence of hrp2 and hrp3 was based on the proportion of amplicons with a deletion. Due to the repetitive nature and homologies of the hrp2 and hrp3 genes, misalignment between reads of hrp3 with hrp2 occurred; therefore, we used a conservative cut-off value, which sometimes resulted in a grey zone where deletion/presence was left inconclusive when most amplicons were not in accordance, as is explained in detail in Kattenberg et al. (2022). One amplicon for hrp2 (AMPL3593062) was not used for the classification, as it offered no discriminatory power. A final variable for rapid diagnostic test (RDT) failure (classified as both hrp2 and hrp3 absent) vs. RDT detectable (hrp2 and/or hrp3 was present) was created, allowing also the classification of samples that were inconclusive in one of the two genes, in case the other gene was present. Allele frequencies (AF) at barcode loci were calculated from allele depths in the vcf file to reflect true population allele frequencies in complex infections using an in-house R script. First, AF was calculated for each position in each sample by calculating the ratio of the allele depths in the vcf. Next, we summed the AF from the previous step for all samples, resulting in SUM-AF. Then, SUM-AF calculated in the previous step was divided by the sum of within-sample allele frequencies (first step) for all alleles at that locus. Notes The assay has been validated with a low error rate, high accuracy, high depth of coverage for an optimal performance to analyze blood samples collected on filter papers with P. falciparum parasite densities ≥60 p/µL, as determined by Mangold PCR (Mangold et al., 2005). In P. falciparum samples with parasite densities < 60 p/µL, selective whole-genome amplification (sWGA) prior to the PF AmpliSeq assay increases the number of reads and genotype calls, but also the error rate. The mitochondrial targets are sequenced at a higher depth than nuclear targets, due to the higher abundance of the mitochondrial genomes in the cell. In later versions of the assay, we have removed the mitochondrial targets, which for our study purpose where less interesting. sWGA prior to library preparation balances the coverage of nuclear vs. mitochondrial amplicons. Library quantification, in our experience, is quicker and more robust (better equalized libraries) with the Qubit kit rather than the PCR-based quantification with the KAPA kit. Acknowledgments The development and validation of the PF AmpliSeq assay was funded by the Belgium Development Cooperation (DGD) under the Framework Agreement Program between DGD and ITM (FA4 Peru, 2017-2021). This protocol was derived from the original research paper titled Malaria molecular surveillance in the Peruvian Amazon with a novel highly multiplexed Plasmodium falciparum AmpliSeq assay by J.H. Kattenberg et al., currently in press in Microbiology Spectrum (DOI: https://doi.org/10.1128/spectrum.00960-22). Competing interests The authors declare that they have no competing interests. Ethics Samples that were used for validation of the PF AmpliSeq assay were collected in Peru by Universidad Peruana Cayetano Heredia (UPCH) and the U.S. Naval Medical Research Unit 6 (NAMRU-6). Individuals were included in this study only if signed informed consent included a future-use clause, and secondary-use was approved through the Institutional Review Board of the Institute of Tropical Medicine Antwerp (reference 1417/20). References Anderson, T. J., Haubold, B., Williams, J. T., Estrada-Franco, J. G., Richardson, L., Mollinedo, R., Bockarie, M., Mokili, J., Mharakurwa, S., French, N., et al. (2000). Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol 17(10): 1467-1482. Baniecki, M. L., Faust, A. L., Schaffner, S. F., Park, D. J., Galinsky, K., Daniels, R. F., Hamilton, E., Ferreira, M. U., Karunaweera, N. D., Serre, D., et al. (2015). Development of a single nucleotide polymorphism barcode to genotype Plasmodium vivax infections. PLoS Negl Trop Dis 9(3): e0003539. Bolger, A. M., Lohse, M. and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15): 2114-2120. Cingolani, P., Platts, A., Wang le, L., Coon, M., Nguyen, T., Wang, L., Land, S. J., Lu, X. and Ruden, D. M. (2012). A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6(2): 80-92. Daniels, R., Volkman, S. K., Milner, D. A., Mahesh, N., Neafsey, D. E., Park, D. J., Rosen, D., Angelino, E., Sabeti, P. C., Wirth, D. F., et al. (2008). A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking. Malar J 7: 223. Daniels, R., Chang, H. H., Séne, P. D., Park, D. C., Neafsey, D. E., Schaffner, S. F., Hamilton, E. J., Lukens, A. K., Van Tyne, D., Mboup, S., et al. (2013). Genetic surveillance detects both clonal and epidemic transmission of malaria following enhanced intervention in Senegal. PLoS One 8(4): e60780. Daniels, R. F., Schaffner, S. F., Wenger, E. A., Proctor, J. L., Chang, H. H., Wong, W., Baro, N., Ndiaye, D., Fall, F. B., Ndiop, M., et al. (2015). Modeling malaria genomics reveals transmission decline and rebound in Senegal. Proc Natl Acad Sci U S A 112(22): 7067-7072. Duraisingh, M. T., Curtis, J. and Warhurst, D. C. (1998). Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol 89(1): 1-8. Eldin de Pecoulas, P., Basco, L. K., Abdallah, B., Dje, M. K., Le Bras, J. and Mazabraud, A. (1995). Plasmodium falciparum: detection of antifolate resistance by mutation-specific restriction enzyme digestion. Exp Parasitol 80(3): 483-487. Falk, N., Maire, N., Sama, W., Owusu-Agyei, S., Smith, T., Beck, H. P. and Felger, I. (2006). Comparison of PCR-RFLP and Genescan-based genotyping for analyzing infection dynamics of Plasmodium falciparum. Am J Trop Med Hyg 74(6): 944-950. Fola, A. A., Kattenberg, E., Razook, Z., Lautu-Gumal, D., Lee, S., Mehra, S., Bahlo, M., Kazura, J., Robinson, L. J., Laman, M., et al. (2020). SNP barcodes provide higher resolution than microsatellite markers to measure Plasmodium vivax population genetics. Malar J 19(1): 375. Illumina. (2018). AmpliSeq for Illumina Custom and Community Panels: Reference Guide. San Diego, California, USA. Imwong, M., Nair, S., Pukrittayakamee, S., Sudimack, D., Williams, J. T., Mayxay, M., Newton, P. N., Kim, J. R., Nandy, A., Osorio, L., et al. (2007). Contrasting genetic structure in Plasmodium vivax populations from Asia and South America. Int J Parasitol 37(8-9): 1013-1022. Kattenberg, J. H., Fernandez-Miñope, C. A., van Dijk, N. J., Llacsahuanga Allca, L., Guetens, P., Valdivia, H. O., et al. (2023). Malaria molecular surveillance in the Peruvian Amazon with a novel highly multiplexed Plasmodium falciparum Ampliseq assay. Microbiol Spectr https://doi.org/10.1128/spectrum.00960-22. Karunaweera, N. D., Ferreira, M. U., Munasinghe, A., Barnwell, J. W., Collins, W. E., King, C. L., Kawamoto, F., Hartl, D. L. and Wirth, D. F. (2008). Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax. Gene 410(1): 105-112. Koepfli, C., Mueller, I., Marfurt, J., Goroti, M., Sie, A., Oa, O., Genton, B., Beck, H. P. and Felger, I. (2009). Evaluation of Plasmodium vivax genotyping markers for molecular monitoring in clinical trials. J Infect Dis 199(7): 1074-1080. Koepfli, C., and Mueller, I. (2017). Malaria Epidemiology at the Clone Level. Trends Parasitol 33(12): 974-85. Li, H. and Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14): 1754-1760. Mangold, K. A., Manson, R. U., Koay, E. S., Stephens, L., Regner, M., Thomson, R. B., Jr., Peterson, L. R. and Kaul, K. L. (2005). Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol 43(5): 2435-2440. Menard, D., Khim, N., Beghain, J., Adegnika, A. A., Shafiul-Alam, M., Amodu, O., Rahim-Awab, G., Barnadas, C., Berry, A., Boum, Y., et al. (2016). A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms. N Engl J Med 374(25): 2453-2464. MPA Committee. (2019). Technical consultation on the role of parasite and anopheline genetics in malaria surveillance. Geneva: World Health Organization. Neafsey, D. E., Schaffner, S. F., Volkman, S. K., Park, D., Montgomery, P., Milner, D. A., Jr., Lukens, A., Rosen, D., Daniels, R., Houde, N., et al. (2008). Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence. Genome Biol 9(12): R171. Nsanzabana, C., Djalle, D., Guerin, P. J., Menard, D. and Gonzalez, I. J. (2018). Tools for surveillance of anti-malarial drug resistance: an assessment of the current landscape. Malar J 17(1): 75. Tae, H., Kim, D., McCormick, J., Settlage, R.E., Garner, H.R. (2014) Discretized Gaussian mixture for genotyping of microsatellite loci containing homopolymer runs. Bioinformatics 30(5): 652-659 Taylor, S. M., Parobek, C. M., Aragam, N., Ngasala, B. E., Martensson, A., Meshnick, S. R. and Juliano, J. J. (2013). Pooled deep sequencing of Plasmodium falciparum isolates: an efficient and scalable tool to quantify prevailing malaria drug-resistance genotypes. J Infect Dis 208(12): 1998-2006. Tessema, S. K., Hathaway, N. J., Teyssier, N. B., Murphy, M., Chen, A., Aydemir, O., Duarte, E. M., Simone, W., Colborn, J., Saute, F., et al. (2022). Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome. J Infect Dis 225(7): 1227-1237. WHO. (2019). World Malaria Report. Geneva: World Health Organization. WHO. (2016). Minutes of the Evidence Review Group meeting on the emergence and spread of multidrug‐resistant Plasmodium falciparum lineages in the Greater Mekong subregion. Geneva: World Health Organization. Willems, T., Zielinski, D., Yuan, J., Gordon, A., Gymrek, M., Erlich, Y. (2017) Genome-wide profiling of heritable and de novo STR variations. Nature Methods 14(6): 590-592. Wingett, S. W. and Andrews, S. (2018). FastQ Screen: A tool for multi-genome mapping and quality control. F1000Res 7: 1338. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). How to cite Category Molecular Biology > DNA > DNA sequencing Microbiology > Microbial genetics > DNA Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Identifying Antigenic Switching by Clonal Cell Barcoding and Nanopore Sequencing in Trypanosoma brucei Abdoulie O. Touray [...] Igor Cestari Dec 20, 2023 889 Views Identification of Mycobacterium tuberculosis and its Drug Resistance by Targeted Nanopore Sequencing Technology Chen Tang [...] Guangxin Xiang Feb 5, 2025 47 Views Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking Mengya Jia [...] Haixing Li Feb 5, 2025 63 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,622	https://bio-protocol.org/en/bpdetail?id=4622&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed A Miniature Sucrose Gradient for Polysome Profiling Ansul Lokdarshi AA Albrecht G. von Arnim Published: Vol 13, Iss 6, Mar 20, 2023 DOI: 10.21769/BioProtoc.4622 Views: 2936 Reviewed by: Wenrong HeYao Xiao Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Plant Physiology Feb 2020 Abstract Polysome profiling by sucrose density gradient centrifugation is commonly used to study the overall degree of translation (messenger RNA to protein synthesis). Traditionally, the method begins with synthesis of a 5–10 mL sucrose gradient onto which 0.5–1 mL of cell extract is layered and centrifuged at high speed for 3–4 h in a floor-model ultracentrifuge. After centrifugation, the gradient solution is passed through an absorbance recorder to generate a polysome profile. Ten to twelve fractions (0.8–1 mL each) are collected for isolating different RNA and protein populations. The overall method is tedious and lengthy (6–9 h), requires access to a suitable ultracentrifuge rotor and centrifuge, and requires a substantial amount of tissue material, which can be a limiting factor. Moreover, there is often a dilemma over the quality of RNA and protein populations in the individual fractions due to the extended experiment times. To overcome these challenges, here we describe a miniature sucrose gradient for polysome profiling using Arabidopsis thaliana seedlings that takes ~1 h centrifugation time in a tabletop ultracentrifuge, reduced gradient synthesis time, and also less tissue material. The protocol described here can be easily adapted to a wide variety of organisms and polysome profiling of organelles, such as chloroplasts and mitochondria. Key Features • Mini sucrose gradient for polysome profiling that requires less than half the processing time vs. traditional methods. • Reduced starting tissue material and sample volume for sucrose gradients. • Feasibility of RNA and protein isolation from polysome fractions. • Protocol can be easily modified to a wide variety of organisms (and even polysome profiling of organelles, such as chloroplast and mitochondria). Graphical Overview Figure 1. Graphical overview of polysome profiling using mini sucrose gradient. A. One milliliter each of 15% (w/v) and 50% (w/v) sucrose gradient solution is added to the individual chambers of the gradient maker. While mixing with a small magnetic stirrer in the 50% solution chamber, base station knob is turned to open position, allowing sucrose gradient solution to slowly flow through the outlet into a 2.2 mL gradient tube. After centrifugation at 50,000 rpm (213,626.2 × g) in a swinging bucket rotor for 70 min at 4 °C, the gradient tube is stored at 4 °C for the next steps. B. Cell extract from 12-day-old vertically grown Arabidopsis thaliana seedlings is centrifuged twice and 100 µL of supernatant is gently layered on the pre-made sucrose gradient from step A. After centrifugation as described in step A, polysome profile is obtained by feeding the gradient solution through an absorbance recorder (A254 nm). Eight (200 µL) fractions are collected for RNA and protein isolation. Keywords: Mini sucrose density gradient Polysome profiling Ultracentrifugation Translation Erb binding protein 1 (EBP1) Target of rapamycin (TOR) Ribosome fractionation GCN2 Salt stress Arabidopsis meristem Background Gene expression control is studied at multiple levels and, among these, translational control provides unparalleled insights about the functional readout of the genome in a spatiotemporal manner (Urquidi Camacho et al., 2020). For example, translatome studies provide highly valuable information that explain differences between the transcriptome (total mRNA pool) and the proteome (changes in total protein levels). Translation is governed by the dynamic distribution of cellular mRNAs into the non-translating and actively translating pool. The actively translating mRNAs loaded with multiple 80S ribosomes (polysome) are larger in size with higher density than the non-translating mRNAs, which are either free or associated with just the 40S ribosomal subunit. Polysome profiling by sucrose gradient centrifugation is a powerful technique for studying changes related to the distribution of mRNAs, as well as genome-wide effects on the translatome (Zuccotti and Modelska, 2016). Traditionally, for translational control studies in plants (Mustroph et al., 2009; Layat et al., 2014; Missra et al., 2015; Yamasaki et al., 2015; Bai et al., 2017; Enganti et al., 2017; Lokdarshi et al., 2020a and 2020b), the method begins with the synthesis of a continuous sucrose gradient with variable density, for example, 15%–50% (w/v) sucrose gradient solution in a 5–10 mL centrifuge tube. Around 0.5–1 g of plant tissue material is ground and 0.5–1 mL of cellular lysate in polysome extraction buffer is gently layered onto the top of the gradient. The centrifuge tubes are spun at high speed for 3–4 h at 4 °C. RNA and protein components that are smaller in size and less dense (e.g., ribosome-free mRNAs, 40S ribosome subunit) travel less far into the gradient and therefore distribute at the top of the gradient, while larger species (e.g., 60S and 80S ribosome subunit, polysomes) travel farther into the gradient. After centrifugation, the gradient tube is assembled into a fractionator and 10–12 fractions (each with 1–1.2 mL) from the top (smaller, slower traveling) to bottom (bigger, faster traveling) are collected for RNA and protein extraction. Polysome profiles are constructed by measuring the optical density of the fractions while they are fed through an absorbance recorder (254 nm). Even though the traditional procedure continues to be a gold standard for studying translational control, this protocol requires ample starting material and suffers from being too lengthy, which further raises doubts over the quality of both RNA and the protein components. To overcome some of these drawbacks, we designed and successfully showed the use of a mini sucrose gradient for polysome profiling with Arabidopsis thaliana seedlings that has tremendous advantages, such as greatly reduced gradient synthesis and centrifuge times and low sample tissue requirement (Lokdarshi et al., 2020b and 2020c). The protocol described here can be easily adapted to a wide variety of organisms (e.g., bacteria, yeast, fungus, and plant and animal tissue) and even for the polysome profiling of organelles such as chloroplasts and mitochondria. Materials and Reagents Biological materials Twelve-day-old Arabidopsis thaliana ecotype Columbia (Col_0) seedlings Solutions DEPC-treated water Milli-Q water treated with 0.2% (v/v) DEPC for 24 h at room temperature and autoclaved for 20 min in a glass bottle. Cool down the bottled water at room temperature for 24 h before use. (Can be stored indefinitely if the bottle is unopened) All reagent stocks (except sucrose solution, cycloheximide, RNase inhibitor and DTT) should be made in a glass beaker and stored in a 100 mL glass bottle in the dark at room temperature. (Replace with new stock after three months) Cycloheximide (CHX, 50 mg/mL) stock is made by resuspending 50 mg of cycloheximide in 1 mL of ethanol in a 1.5 mL tube. Store stock solution at -20 °C. (Replace with new stock after three months) Chloramphenicol (CHL, 50 mg/mL) stock is made by resuspending 50 mg of chloramphenicol in 1 mL of ethanol in a 1.5 mL tube. Store stock solution at -20 °C. (Replace with new stock after three months) Dithiothreitol (DTT, 1 M) stock is made by resuspending 0.154 g of DTT in 1 mL of water in a 1.5 mL tube. Store stock solution at -20 °C. (Replace with new stock after two months) Sucrose solutions made in a glass beaker need to be filtered through a 0.2 µm PVDF filter bottle and stored in the dark at 4 °C. (Replace with new stock after one month. Before each use, always shake the bottle to check for any cloudy appearance. Discard and make new solutions as necessary) Punch solution should be made in a glass bottle and autoclaved for 20 min on liquid cycle. (Can be stored indefinitely at room temperature in the dark) 100 mL of 1 M Tris-HCl (pH 8.4) is made by dissolving 12.14 g of Tris in 80 mL of deionized water and adjusting the pH to 8.4 using 0.1 N HCl. After attaining the desired pH, the volume of 1 M Tris-HCl solution is adjusted to final 100 mL using deionized water. (Solution is stored at room temperature and it is recommended to replace with new stock after three months) Lysis buffer (1,000 µL in a 2 mL tube) (see Recipes) 15% (or 50%) sucrose solution (100 mL) (see Recipes) Punch solution (100 mL) (see Recipes) Recipes Lysis buffer (1,000 µL in a 2 mL tube) Make fresh from stock solutions for each experiment. Reagent Final concentration Amount Tris-HCl (1 M, pH 8.4) KCl (0.5 M) MgCl2 (0.5 M) Deoxycholic acid (10%) Polyoxyethylene 10 tridecyl ether [20% (w/v)] Cycloheximide (50 mg/mL) RNase inhibitor (40 U/µL) H2O 200 mM 50 mM 25 mM 1% (v/v) 2% (v/v) 50 µg/mL 40 U/mL n/a 200 µL 50 µL 25 µL 100 µL 100 µL 1 µL 1 µL 523 µL Total n/a 1,000 µL 15% (or 50%) sucrose solution (100 mL) Reagent Final concentration Amount Tris-HCl (1 M, pH 8.4) KCl (0.5 M) MgCl2 (0.5 M) Sucrose H2O 200 mM 50 mM 25 mM 15% or 50% (w/v) n/a 20 mL 10 mL 5 mL 15 or 50 g 40 mL Total n/a Make up final volume to 100 mL Punch solution (100 mL) Note: Water should be added in increments of 10 mL. Allow the solution to mix completely before adding any more water. Sucrose will take a substantial volume when fully dissolved. Reagent Final concentration Amount Sucrose Bromophenol blue (0.1% (w/v)) Tris-HCl (1 M, pH 8.4) H2O 60% (w/v) 0.02% (v/v) 1 mM n/a 60 g 20 mL 100 µL see note Total n/a 100 mL Laboratory Supplies 1.5 and 2 mL tubes (Thermo Fisher, catalog numbers: AM12450 and AM12475) Pipettes (200–1,000, 20–200, 2–20, and 0.2–2 µL) DNase/RNase free tips (1,000, 200, 10 µL) 1.5 mL tube rack 7/16 × 13/8 in. (11 × 34 mm) 2.2 mL polypropylene tube (Beckman Coulter, catalog number: 347357) 19 Gauge needle for gradient tube piercing (Fisher Scientific, catalog number: 14-826-52) Parafilm (Fisher Scientific, catalog number: S37440) Disposable bottle top filters (Thermo Fisher, catalog number: 597-4520) Disposable bottle filter units (Thermo Fisher, catalog number: 568-0020) Permanent ink markers Clear tubing (6 inch in length) for gradient mixer outlet (1/16 in diameter) Wipes (Fisher Scientific, catalog number: 06-666C) Flat-tip forceps (Fisher Scientific, catalog number: 16-100-116) Porcelain pestle and mortar (Fisher Scientific, catalog number: S27075) Ice bucket (Styrofoam box can be used as an alternative) Basic binder paper clip (medium) Gloves Stainless steel spatula with tapered end Tris (Molecular Grade) (Promega, Fisher Scientific catalog number: PR-H5133) KCl (Fisher Scientific, catalog number: AC418205000) MgCl2 (Fisher Scientific, catalog number: M33-500) Deoxycholic acid (Fisher Scientific, catalog number: BP349-100) Polyoxyethylene 10 tridecyl ether (Millipore Sigma, catalog number: P2393-100G) Cycloheximide (Millipore Sigma, catalog number:01810-G) Chloramphenicol (Millipore Sigma, catalog number: 220551-25GM) 40 U/mL RNase inhibitor (Promega, catalog number: N2515) Sucrose, molecular biology grade (Millipore Sigma, catalog number: 573113) Bromophenol blue (Fisher Scientific, catalog number: AAA1846909) Dithiothreitol (DTT) (Thermo Fisher Scientific, catalog number: R0861) Murashige and Skoog (MS) plant basal salt (MP Biomedicals, Fisher Scientific catalog number: ICN2623022) RNaseZapTM RNase decontamination solution (ThermoFisher, catalog number: AM9780) Liquid nitrogen Aluminum foil Equipment Gradient mixer (Buchler Instruments). Catalog number for this specific product is not available as no vendor supports this product anymore. For custom manufacturing of the gradient mixer, see Figure 2. Tabletop ultracentrifuge (Beckman Coulter, catalog number: A95761 or equivalent) TLS-55 swinging-bucket rotor with four buckets for up to four gradients (Beckman Coulter, catalog number: 346936) Gradient station or any equivalent gradient fractionators (Biocomp, catalog number: 153-002) ISCO UA 5 100 absorbance/fluorescence monitor. Catalog number for this specific product is not available as the vendor is out of market. Alternative product information: BR-188 Density Gradient Fractionation System with Peak Chart Data Acquisition System (Brandel) Tabletop centrifuge with rotor to hold 1.5 mL tube. If refrigerated centrifuge is not available, then a non-refrigerated centrifuge can be placed in a 4 °C cold room as an alternative Magnetic plate stirrer Magnetic stirrer bar (Fisher Scientific, catalog number: 14-513-93). Cut the magnetic stirrer bar into half with the plier and store the halves in a 1.5 mL tube (micro-stirrer bar). Note: Cleaning of this micro stirrer bar should be done with warm water and stored carefully. Weighing balance with 0.001 g accuracy Vortex Burette stand Software and Datasets The BR-188 Density Gradient Fractionation System comes with the Peak Chart Data Acquisition System Software. See note in section E to develop polysome profile without a Peak Chart Data Acquisition System Software. Procedure Growing Arabidopsis seedling and tissue storage This step is a standard procedure in plant biology and various recourses are available that outline the method for germinating Arabidopsis seeds on 1/2× MS plant media. After 12 days of growth, seedlings are harvested into a 1.5 mL tube, flash frozen in liquid nitrogen, and then stored at -80 °C. Note: It is recommended to use this sample within 1–2 months for best reproducibility of the experiment. Readiness of gradient maker and general setup (see Figure 1A and Figure 2A for details) Note: Application of RNaseZapTM RNase decontamination solution on gloves is recommended before beginning the following procedure. One day prior to the polysome profiling experiment, rinse the gradient maker with diluted dish washing soap in warm tap water for 1–2 min and then wash with warm tap water for few minutes. Micro-stirrer bar needs to be cleaned with just warm water and dried with wipes. Rinse the gradient maker with DEPC-treated water twice and air dry on a few layers of wipes. Arrange the following items on a stable bench: pipettes, tips, burette stand, magnetic stirrer plate, magnetic stirrer bar, 2.2 mL polypropylene centrifuge tubes, 1.5 mL tube stand, trash cans for liquid and solid waste, and stock solutions of 15% and 50% sucrose, CHX, CHL, and DTT. Attach the gradient maker support rod to the burette stand holder and adjust the height of the holder such that the base of the gradient maker is ~0.5–1 inch above the magnetic stirrer plate. Add the micro-magnetic stirrer bar into the chamber connected to the outlet tubing (Well-II) and turn the magnetic stirrer plate ON to confirm rotation of the micro-stirrer bar. Turn the push-pull valve to face towards self, closing the gradient conduit between the mixing chambers (Well-II and Well-I). Clamp a binder clip to the outlet tubing (1 inch away from the gradient maker outlet valve). Attach a 10 µL pipette tip at the end of the outlet tubing using the broad side for the outlet tubing and the tip side for elution. CHL-CHX-DTT mix: Add 15 µL each of CHX and CHL stock and 1.5 µL of DTT stock into the same 1.5 mL tube and mix by vortexing at room temperature for 10–20 s. Store on ice. Figure 2. Schematics of the gradient mixer. A. Original view of the gradient mixer. The push-pull valve at the bottom of the apparatus is facing towards self, indicating closed connection between the two gradient chambers. B–D. Outline of the gradient mixer in different view angles showing specific measurements. Abbreviations: in = inches, i.d. = inner diameter Preparation of 2 mL sucrose gradients for pre-run before polysome profiling Add 2.1 µL of CHL-CHX-DTT mix along the side wall of each Well-I and II. Add 1 mL of 15% sucrose solution into Well-I and 1 mL of 50% sucrose solution in Well-II (magnetic stirrer should be rotating in Well-II while 50% sucrose solution is added to Well-II) (see Figure 1A). Remove the binder clamp gently to allow the 50% sucrose solution to flow temporarily approximately 1 cm into the outlet tubing. Quickly clamp back the tubing. Note: For a precise control over the flow of the 50% sucrose solution, the tip of the outlet tubing can be carefully inserted into the tip of a 200 µL tip attached to a 20–200 µL pipette. Once the clamp is removed, the pipette is adjusted to allow 50% sucrose solution exactly 1 cm into the outlet tubing. On completion of this step, clamp the tubing again and remove the 200 µL tip, and proceed to the next step. Turn the push-pull valve to face horizontal position, opening the gradient conduit between the mixing chambers (Well-I and Well-II). Air bubbles trapped in the passage between the two wells need to be removed by sealing the top of the Well-I with a gloved finger and then slightly pressing down to increase the pressure. Note: This will release air bubbles trapped in the valve passage into the Well-II. While making sure that the outlet is at the same height with the bottom of Well-II, place the tip of the outlet tubing into the bottom of a 2.2 mL ultracentrifuge tube and gently open the clamp. Lower the centrifuge tube slowly and let the solution flow along the walls of the centrifuge tube by gravity. Make sure that the solutions drain evenly from both Well-I and Well-II. When the solution in Well-II is almost over (~200 µL remaining), tilt the gradient maker clockwise until the base of the gradient maker touches the magnetic stirrer plate, while making sure the tip at the end of the outlet tube touches the top of the finished gradient. Note: This step will ensure all liquid flows out. Place the finished gradient in a 1.5 mL tube rack and cover it with aluminum foil. Repeat the steps B6–9 and C1–6 for making more gradients. When all the gradients are done, adjust the weight of each gradient tube to 2.800 g by gently adding 15% sucrose solution on top of the finished gradient. For pre-run, place the gradient tube using forceps into a TLS-55 rotor bucket (pre-chilled at 4 °C) and make sure to match the weight with other tubes using 15% sucrose solution. Note: It is important to spin all the four buckets of the rotor with equal weights. Spin at 50,000 rpm (213,626.2 × g) for 70 min at 4 °C (deceleration = 0, meaning no brakes). After the run, take the gradient tubes out using forceps and place in a 1.5 mL tube rack. Cover all the gradients with aluminum foil and store at 4 °C on a stable base without any shaking. Note: Pre-run gradients are good for up to 24 h. Cell lysis and sucrose density gradient centrifugation Note: Two days prior to polysome profiling, wash mortars and pestles with dish soap and rinse with tap water. Use a new set for each sample to prevent cross contamination. Air dry and wrap one mortar with pestle as a pair with aluminum foil and autoclave for 30 min using dry setting. One day before the experiment (day of steps A and B), place the mortar-pestle set at -20 °C (or preferably at -80 °C). (This practice is done to reduce time spent for chilling the mortar-pestle set and save on liquid nitrogen.) *The term pre-chilled in the following section refers to the use of liquid nitrogen to achieve the coldest temperature. Add liquid nitrogen to the cold mortar-pestle until no boiling of liquid nitrogen is observed. Collect your tissue samples from the -80 °C freezer and keep them floating in a dewar with liquid nitrogen until it is their turn. Add the frozen tissue from the storage tube into the mortar and grind in liquid nitrogen to a fine powder using the pestle. (This step is crucial and usually takes 4–5 min. After every 1 min, add some liquid nitrogen gently into the mortar to keep the ground material cold.) It also helps to have the mortar sit inside a Styrofoam box that is kept cold with liquid nitrogen. Using a pre-chilled spatula, weigh 150 mg of pulverized tissue powder in a pre-chilled 1.5 mL tube. Note: During waiting periods, 1.5 mL tube with open cap should be kept in a rack that is placed inside a Styrofoam container with some liquid nitrogen. The cap is left open to allow liquid nitrogen to evaporate. Cover the container to avoid condensation buildup on the top of the 1.5 mL tube. Add 100 µL of freshly prepared lysis buffer (see Recipes) into the 1.5 mL tube with tissue powder and place the tube in a new rack kept at room temperature. After 40–50 s, close the cap of the tube and vortex at highest setting for 1 min at room temperature. Repeat this step twice to achieve complete homogeneity. Spin the tube at 21, 000 × g for 5 min at 4 °C. Transfer 125 µL of the supernatant into a new 1.5 mL tube and repeat centrifugation at 21,000 × g for 5 min at 4 °C. Carefully layer 100 µL of the supernatant from step 7 onto the pre-made 15–50% sucrose gradient (see section C). Notes: Tubes can be numbered with a marker at this stage. For a blank gradient, layer 100 µL of lysis buffer on a pre-made 15%–50% sucrose gradient. Place the gradient tube using forceps into a TLS-55 rotor bucket (chilled at 4 °C) and make sure to match the weight with other tubes. 15% sucrose solution can be used for this purpose. Note: It is very important to use all the four rotor buckets with equal weights. Spin at 50,000 rpm (213,626.2 × g) for 70 min at 4 °C (deceleration = 0, meaning no brakes). Take the gradient tubes out using forceps and place the tubes in a 1.5 mL tube rack (Figure 3A). Cover all the gradients with aluminum foil and place on ice. Polysome profiling and gradient fractionation Note: To begin Section E, the gradient should be clamped down in the gradient fractionator and sealed against the top assembly with the outlet tube. (Refer to manufacturer’s manual for details.) The general idea is that the gradient tube gets pierced at the bottom and the heavy push solution is pumped into the bottom of the gradient tube. This way, the gradient is displaced up through the outlet tube at the top into the absorbance recorder and finally out for fractionation. After each gradient run, the fractionator tubing should be washed with 50 mL of warm water by siphoning it from one end. Dry both the inlet and outlet with wipes and apply vacuum to get rid of any residual water in the fractionator tubing. The baseline absorbance (A254nm) should return to zero. Run the punch solution through the fractionator tubing at lowest speed (for example, 375 μL/min in the ISCO Teledyne gradient fractionator). Continue pumping the punch solution until it comes out of the piercing needle. Carefully wrap a small piece of parafilm (2 cm wide) around the circumference of a 2.2 mL tube with just water and load the tube into the holder of the gradient fractionator. Note: Parafilm is installed for better grip of the tube with the adapter for gradient fractionator. Rotate the collector piston to pierce the gradient tube and start the pump as described in step E1 (Figure 3B). As the water starts to come out of the fractionator tubing, adjust the absorbance (A254nm) to zero for recording baseline at sensitivity of 0.5 in the ISCO Teledyne gradient fractionator. On completion of the water tube run, take the tube out (see step E6) and load the blank gradient tube (see step D8b) for recording blank reading (Figure 3C). After each gradient run, the fractionator tubing should be washed with 50 mL of warm water by siphoning it from one end. Dry both the inlet and outlet with wipes and apply vacuum to get rid of any residual water in the fractionator tubing. The baseline absorbance (A254nm) should return to zero. After blank gradient recording is complete, take the tube out and load the first sample gradient tube (Figure 3A). Collect eight 200 µL fractions in individual 1.5 mL tubes. Place tubes on ice immediately after each fraction collection. Figure 3. Miniature gradient tube setup and polysome profile. A. Gradient tube with sample (Arabidopsis cell extract) after ultracentrifugation. B. Setup of the gradient tube in the ISCO Teledyne gradient fractionator with punch solution inside the tube. C. Example of 15%–50% miniature sucrose gradient. The position of the 40S, 60S, monosome (80S), and the polysome is indicated on the sample gradient profile. Notes If a gradient fractionator is not available, individual fractions can be collected by carefully aliquoting 200 µL of the finished gradient from the top using a 20–200 µL pipette. For polysome profile, RNA extracted from these fractions can be quantified using a spectrophotometer and plotted with x-axis as fraction # and y-axis representing absorbance. Acknowledgments This project was developed during the postdoctoral tenure of Dr. Ansul Lokdarshi (corresponding author) in the lab of Dr. Albrecht von Arnim (Professor, Dept. of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville). This work was supported by grants from the National Science Foundation (IOS-1456988 and MCB-1546402) and the National Institutes of Health NIH R15 GM129672 to Dr. Albrecht von Arnim. We also thank Ricardo Urquidi Camacho (Graduate Research Assistant, University of Tennessee, Knoxville) for helpful discussions during the optimization of this procedure. The protocol discussed in this manuscript has been successfully used for polysome profiling (Lokdarshi et al., 2020b and 2020c) and furthermore for RNA and protein analysis from the gradient fractions (Lokdarshi et al., 2020c). Please see “Figure 8: Ribosome-RNA profile of wild-type and gcn2 mutant under salt stress.”(Lokdarshi et al., 2020b). Please see “Figure 6. EBP1 associates with cytosolic ribosomes and supports ribosome biogenesis.” (Lokdarshi et al., 2020c). Competing interests Authors declare no competing interests. References Bai, B., Peviani, A., van der Horst, S., Gamm, M., Snel, B., Bentsink, L. and Hanson, J. (2017). Extensive translational regulation during seed germination revealed by polysomal profiling. New Phytol 214(1): 233-244. Enganti, R., Cho, S. K., Toperzer, J. D., Urquidi-Camacho, R. A., Cakir, O. S., Ray, A. P., Abraham, P. E., Hettich, R. L. and von Arnim, A. G. (2017). Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals. Front Plant Sci 8: 2210. Layat, E., Leymarie, J., El-Maarouf-Bouteau, H., Caius, J., Langlade, N. and Bailly, C. (2014). Translatome profiling in dormant and nondormant sunflower (Helianthus annuus) seeds highlights post-transcriptional regulation of germination. New Phytol 204(4): 864-872. Lokdarshi, A., Guan, J., Urquidi Camacho, R. A., Cho, S. K., Morgan, P. W., Leonard, M., Shimono, M., Day, B. and von Arnim, A. G. (2020a). Light Activates the Translational Regulatory Kinase GCN2 via Reactive Oxygen Species Emanating from the Chloroplast. Plant Cell 32(4): 1161-1178. Lokdarshi, A., Morgan, P. W., Franks, M., Emert, Z., Emanuel, C. and von Arnim, A. G. (2020b). Light-Dependent Activation of the GCN2 Kinase Under Cold and Salt Stress Is Mediated by the Photosynthetic Status of the Chloroplast. Front Plant Sci 11(431). doi: 10.3389/fpls.2020.00431. Lokdarshi, A., Papdi, C., Pettko-Szandtner, A., Dorokhov, S., Scheres, B., Magyar, Z., von Arnim, A. G., Bogre, L. and Horváth, B. (2020c). ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem. Plant Physiol 182(2): 919-932. Missra, A., Ernest, B., Lohoff, T., Jia, Q., Satterlee, J., Ke, K. and von Arnim, A. G. (2015). The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading. Plant Cell 27(9): 2582-2599. Mustroph, A., Zanetti, M. E., Jang, C. J., Holtan, H. E., Repetti, P. P., Galbraith, D. W., Girke, T. and Bailey-Serres, J. (2009). Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 106(44): 18843-18848. Urquidi Camacho, R. A., Lokdarshi, A. and von Arnim, A. G. (2020). Translational gene regulation in plants: A green new deal. Wiley Interdiscip Rev RNA: e1597. Yamasaki, S., Matsuura, H., Demura, T. and Kato, K. (2015). Changes in Polysome Association of mRNA Throughout Growth and Development in Arabidopsis thaliana. Plant Cell Physiol 56(11): 2169-2180. Zuccotti, P. and Modelska, A. (2016). Studying the Translatome with Polysome Profiling. Methods Mol Biol 1358: 59-69. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). How to cite Category Plant Science > Plant molecular biology > Genetic analysis Molecular Biology > RNA > mRNA translation Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Phylogenomics of Plant NLR Immune Receptors to Identify Functionally Conserved Sequence Motifs Toshiyuki Sakai [...] Hiroaki Adachi Jul 5, 2024 1109 Views A Step-by-step Protocol for Crossing and Marker-Assisted Breeding of Asian and African Rice Varieties Yugander Arra [...] Wolf B. Frommer Sep 20, 2024 428 Views An Effective and Safe Maize Seed Chipping Protocol Using Clipping Pliers With Applications in Small-Scale Genotyping and Marker-Assisted Breeding Brian Zebosi [...] Erik Vollbrecht Feb 5, 2025 62 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,623	https://bio-protocol.org/en/bpdetail?id=4623&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Laser Capture Microdissection (LCM) of Human Skin Sample for Spatial Proteomics Research QZ Qiyu Zhang * HG Huizi Gong * JM Jie Ma JL Jun Li LL Ling Leng (*contributed equally to this work) Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4623 Views: 931 Reviewed by: Zinan ZhouDarrell Cockburn Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Nature Communications Jul 2022 Abstract In mammals, the skin comprises several distinct cell populations that are organized into the following layers: epidermis (stratum corneum, stratum granulosum, stratum spinosum, and basal layer), basement membrane, dermis, and hypodermal (subcutaneous fat) layers. It is vital to identify the exact location and function of proteins in different skin layers. Laser capture microdissection (LCM) is an effective technique for obtaining pure cell populations from complex tissue sections for disease-specific genomic and proteomic analysis. In this study, we used LCM to isolate different skin layers, constructed a stratified developmental lineage proteome map of human skin that incorporates spatial protein distribution, and obtained new insights into the role of extracellular matrix (ECM) on stem cell regulation. Keywords: Skin Laser capture microdissection (LCM) Spatial proteomics Stem cell Extracellular matrix (ECM) Background Skin, the largest barrier organ in the body, has a tough and pliable structure to adapt to external conditions by quickly repairing mechanical, chemical, and biological injuries. In mammals, the skin consists of several distinct layers: the epidermal, dermal, and subcutaneous fat layers. The epidermis is the outermost layer, with stratified cell layers maintained by keratinocytes including stem cells and an abundance of mature cells (Gonzales and Fuchs, 2017). The basal layers of the epidermis, which express the keratins KRT5 and KRT14, are the location of undifferentiated proliferative epidermal stem cells (Blanpain and Fuchs, 2006). The progenitor cells replenish basal layers and differentiate into mature keratinocytes in the granulosum and spinosum layers, which express KRT1, KRT10, and involucrin. Stratum corneum, the outermost layer, consisting of terminally differentiated and dead cells, acts as a scaffold for the lipid bilayers that comprise the epidermal barrier on the skin surface (Fuchs, 2007; Koster and Roop, 2007). Keratins, which constitute the cytoskeleton of epithelial cells, are highly expressed in the epidermis, especially in the stratum corneum (Li et al., 2022). In the epidermis, the transition of keratinocytes from the proliferative basal to the supra-basal cell layer during terminal differentiation and keratinization is characterized by keratin expression transition from basal cell keratins (KRT5, KRT14, and KRT15) to supra-basal cell keratins (KRT1 and KRT10) (Moll et al., 2008).The dermis provides nourishment and support for the skin and contains an abundance of extracellular matrix (ECM) proteins including collagens, glycosaminoglycans, hyaluronic acid, fibronectin, elastin, and laminin (Nyström and Bruckner-Tuderman, 2019). Basement membrane, a specialized layer of ECM proteins connecting the dermis and epidermis, serves as an important microenvironment for basal stem cells; understanding its composition is crucial to the study of basal stem cell fate and function. However, there is a lack of detailed information about the molecular composition and regulatory function of specialized proteins localized in different skin layers that are difficult to separate, especially the basement membrane zone, during the dynamic processes of skin development, homeostasis, and regeneration for wound healing (Dengjel et al., 2020; Dyring-Andersen et al., 2020). As described above, the skin is a complex tissue composed of heterogeneous cell types with different spatial distributions. Studying the unique physiological and pathological functions of different spatially distributed cell types is essential for understanding the molecular characteristics of human skin. Laser capture microdissection (LCM) is a powerful technique for separating target cell populations with extremely high microscopic precision, thus providing a perfect solution to the problem of skin tissue heterogeneity. Here, we describe a protocol for the isolation of different skin layers by LCM for spatial proteomics research (Li et al., 2022). Using a combination of previously developed tissue engineering decellularization methods, dedicated to the removal of epidermis and separation of basement membrane (Leng et al., 2020, Liu et al., 2020), LCM, and mass spectrometry (MS), we isolated proteins from six skin layers (stratum corneum, stratum granular-spinous, basal layer, basement membrane, superficial dermis, and deep dermis) and constructed a stratified developmental lineage proteome map of human skin. By obtaining different skin samples, the protocol enables the analysis of spatial protein expression in normal and disease-specific skin tissues, which is important for understanding the pathogenesis of different skin diseases and discovering potential therapeutic targets. Materials and Reagents MMI MembraneSlidesTM (MMI GmbH, catalog number: 50102) MMI IsolationCaps transparent, 0.5 mL (MMI GmbH, catalog number: 50204) Phospholipase A2 (Sigma-Aldrich, catalog number: P6534) Sodium deoxycholate (Sigma-Aldrich, catalog number: V900388) PBS (TBD Science, catalog number: PB2004Y) EDTA (Sigma-Aldrich, catalog number: E9884) NaCl (Sigma-Aldrich, catalog number: S9888) DNase (Sigma-Aldrich, catalog number: 11284932001) RNase (Sigma-Aldrich, catalog number: 10109134001) OCT compound (SAKURA, catalog number: 4583) 4% paraformaldehyde (BOSTER Biological Technology, catalog number: AR1068) Sucrose (Sigma-Aldrich, catalog number: V900116) Delipidation solution (see Recipes) DNase-RNase solution (see Recipes) 3.4 M NaCl solution (see Recipes) 30% sucrose solution (see Recipes) Equipment Laser microdissection system (MMI, cellcut plus) Freezing microtome (Leica, model: CM1950) Shaker (tcsysb, THZ-C) Scalpel (BELEVOR MEDICAL, catalog number: 03.0030.01) Procedure Obtain native frozen skin tissue sections Rinse skin tissue three times with cold PBS. Fix tissue with 4% paraformaldehyde at room temperature for 24 h. Perform dehydration with 10 mL of 30% sucrose solution at 4 °C until the tissue sinks to the bottom. Freeze at -80 °C and then embed the skin tissues. Apply a layer of OCT compound in the groove of the embedding mold and place the tissue section down into the groove. Fill with embedding agent (OCT compound) and put the sample on the freezer until the embedding agent solidifies. Cut 20-μm-thick sections of native skin tissues. Place the MembraneSlidesTM (with the MMI logo face up) close to the slice, which will stick to it. Reverse the MembraneSlidesTM, gently press them at the corresponding position below the slice with your fingers, and the sample will completely adhere to the MembraneSlidesTM. Mount sections on MMI MembraneSlidesTM and store at -20 °C. Obtain decellularized frozen skin tissue sections Rinse skin tissue three times with cold PBS containing 0.1% EDTA. Perform delipidation by treating tissue with 25 mL of delipidation solution (see Recipes) for 4 h in a shaker at 37 °C until the tissue segments become oyster white. Gently scrap the surface of the skin with the back of the scalpel to remove the epidermis. Rinse the decellularized dermal scaffolds with 3.4 M NaCl (see Recipes) at 37 °C for 1 h. Wash the samples with DNase-RNase solution (see Recipes) at 37 °C for 1 h. Rinse skin tissue three times with cold PBS. Fix tissue with 4% paraformaldehyde at room temperature for 24 h. Perform dehydration with 10 mL of 30% sucrose solution in a 15 mL centrifuge tube at 4 °C until the tissue sinks to the bottom. Freeze and embed the skin tissues as in step A2. Cut 20-μm-thick sections of decellularized skin tissues as in step A3. Mount sections on MMI MembraneSlidesTM and store slides at -20 °C. Laser capture microdissection (LCM) of frozen skin sections Open LCM system. Install MembraneSlides and IsolationCaps transparent. Switch to 4× objective and start slide scan and navigation. Switch to 10× objective for subsequent dissection. Set laser position indicator. Click the button to fire the laser and check whether the laser position matches the green cross. If not, click “CellCut” – “Laser” – “Set Laser Position” and click at the location where the laser is emitted. Calibrate the parameters for the laser. Select the freehand tool, draw a long line on the empty area without tissues on the slides, and cut. Adjust “Cut velocity,” “Laser focus,” and “Laser power” to make the cutting line clear and precise. Move to the target position, draw cut lines, and start cutting. Increase the cut velocity, cut power, and number of cuts appropriately to get the best cutting efficiency. Pick up the sample under manual mode. Click the collecting button to attach the cutout to the EP tube cover and check if the tissue was successfully collected. If not, repeat collection or try to adjust the position of the EP tube and even recut. Repeat steps C6 and C7 to isolate stratum corneum, granulosum-spinosum, basal layer, superficial dermis, and deep dermis on native sections and basement membrane on decellularized sections successively. Check the entire slice to ensure that the target area is cut and collected. Label the samples and store them at -20 °C for subsequent MS analysis. Notes To obtain a clearer view of tissue structure when cutting, hematoxylin-eosin staining can be performed on the sections. The LCM systems allow simultaneous cutting of the same structure for up to 3–4 slices and collection of multiple structures with multiple tubes by using the group function. When the sample is difficult to be dissected or collected by the EP tube cover, first check if the objective magnification is correct. Then, optimize laser parameters, such as increasing laser power or slowing down the cutting speed, and repeat the cuts. Finally, move the position of the EP tube cover to an area with less adherent samples or replace the collection tube with a new one. The cutting circle should not be too small, or the tissue will be easily repelled away by the laser. Also, the cutting line should not be too long, or the tissue will break during collection. For laser parameters calibration, first decrease “Cut velocity” and “Laser power” to a low value (as 5 μm/s and 5%) and cut. Then, change “Laser focus” until the cutting line becomes clear and spacious. Finally, decrease the “Laser power” to get a fine line and change the “Laser focus” again to find the finest line. After calibration, increase the “Cut velocity” to a high value (as 70 μm/s) and increase the “Laser power” accordingly to get a fast and precise cutting line. For best cutting efficiency, change “Cut velocity” and “Laser power” according to different tissue locations and thickness. Videos 1–5 show the continuous process of cutting the stratum corneum, stratum granulosum-spinosum, basal layer, superficial dermis, and deep dermis of the native skin tissue sections from outside to inside. Video 6 shows the process of cutting the basement membrane of the decellularized skin tissue sections. Video 1. Isolation of stratum corneum Video 2. Isolation of stratum granulosum-spinosum Video 3. Isolation of basal layer Video 4. Isolation of superficial dermis Video 5. Isolation of deep dermis Video 6. Isolation of basement membrane Recipes Delipidation solution Dissolve 10 g of sodium deoxycholate powder in 1 L of deionized water. Add phospholipase A2 to above solution before delipidation treatment to make a final concentration of 2,000 U/L. DNase-RNase solution Dissolve 1 mg of DNase powder and 0.5 mg of RNase powder in 100 mL of PBS buffer before use. 3.4 M NaCl solution Dissolve 198.9 g of NaCl powder with 800 mL of deionized water. After dissolving, fill with deionized water to 1 L. 30% sucrose solution Dissolve 300 g of NaCl powder with 800 mL of deionized water. After dissolving, fill with deionized water to 1 L. Acknowledgments This work was supported by Beijing Municipal Science and Technology Commission (Z191100006619011), Capital’s Funds for Health Improvement and Research (2020-2-4016), CAMS Innovation Fund for Medical Sciences (CIFMS 2020-I2M-C&T-B-048), and National Science and Technology Major Project (2021YFA1301603). This protocol was adapted from Li et al. (2022). Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics The authors declare no conflict with respect to ethical grounds. References Blanpain, C. and Fuchs, E. (2006). Epidermal stem cells of the skin. Annu Rev Cell Dev Biol 22: 339-373. Dengjel, J., Bruckner-Tuderman, L. and Nystrom, A. (2020). Skin proteomics - analysis of the extracellular matrix in health and disease. Expert Rev Proteomics 17(5): 377-391. Dyring-Andersen, B., Lovendorf, M. B., Coscia, F., Santos, A., Moller, L. B. P., Colaco, A. R., Niu, L., Bzorek, M., Doll, S., Andersen, J. L., et al. (2020). Spatially and cell-type resolved quantitative proteomic atlas of healthy human skin. Nat Commun 11(1): 5587. Fuchs, E. (2007). Scratching the surface of skin development. Nature 445(7130): 834-842. Li, J., Ma, J., Zhang, Q., Gong, H., Gao, D., Wang, Y., Li, B., Li, X., Zheng, H., Wu, Z., et al. (2022). Spatially resolved proteomic map shows that extracellular matrix regulates epidermal growth. Nat Commun 13(1): 4012. Moll, R., Divo, M. and Langbein, L. (2008). The human keratins: biology and pathology. Histochem Cell Biol 129(6): 705-733. Gonzales, K. A. U. and Fuchs, E. (2017). Skin and Its Regenerative Powers: An Alliance between Stem Cells and Their Niche. Dev Cell 43(4): 387-401. Koster, M. I. and Roop, D. R. (2007). Mechanisms regulating epithelial stratification. Annu Rev Cell Dev Biol 23: 93-113. Leng, L., Ma, J., Sun, X., Guo, B., Li, F., Zhang, W., Chang, M., Diao, J., Wang, Y., Wang, W., Wang, S., Zhu, Y., He, F., Reid, L. M. and Wang, Y. (2020). Comprehensive proteomic atlas of skin biomatrix scaffolds reveals a supportive microenvironment for epidermal development. J Tissue Eng 11: 2041731420972310. Liu, B., Zhang, S., Wang, W., Yun, Z., Lv, L., Chai, M., Wu, Z., Zhu, Y., Ma, J. and Leng, L. (2020). Matrisome Provides a Supportive Microenvironment for Skin Functions of Diverse Species. ACS Biomaterials Science & Engineering 6(10): 5720-5733. Nyström, A. and Bruckner-Tuderman, L. (2019). Matrix molecules and skin biology. Semin Cell Dev Biol 89: 136-146. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Cell Biology > Tissue analysis > Tissue isolation Biochemistry > Protein > Quantification Stem Cell > Adult stem cell > Epithelial stem cell Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Protein Level Quantification Across Fluorescence-based Platforms Hector Romero [...] M. Cristina Cardoso Oct 5, 2023 854 Views Compartment-Resolved Proteomics with Deep Extracellular Matrix Coverage Maxwell C. McCabe [...] Kirk C. Hansen Dec 5, 2024 336 Views Cochlear Organ Dissection, Immunostaining, and Confocal Imaging in Mice Chenyu Chen [...] Dongdong Ren Jan 20, 2025 1602 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,624	https://bio-protocol.org/en/bpdetail?id=4624&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed 3D Compartmentalised Human Pluripotent Stem Cell–derived Neuromuscular Co-cultures PH Peter Harley AP Amaia Paredes-Redondo GG Gianluca Grenci VV Virgile Viasnoff YL Yung-Yao Lin * IL Ivo Lieberam * (contributed equally to this work) Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4624 Views: 1077 Reviewed by: Sébastien Gillotin Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Advances Sep 2021 Abstract Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)–derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)–like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study disease-specific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo–like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study. Graphical abstract Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ). Keywords: Tissue engineering Neuromuscular co-culture Compartmentalised microdevice Human pluripotent stem cells Optogenetics Motor neuron Myofiber DMD ALS Background Neuromuscular diseases are a diverse group of disorders with considerable unmet clinical need. For instance, many muscular dystrophies, such as Duchenne muscular dystrophy (DMD) that lead to progressive muscle wasting, currently lack effective treatments (Duan et al., 2021). Similarly, neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), caused by degeneration of motor neurons (MNs) that innervate and control movement of skeletal muscle, also lack effective treatments (Hardiman et al., 2017). Early peripheral phenotypes like axonal and neuromuscular junction (NMJ) dysfunction have been implicated in a wide range of divergent subsets of neuromuscular disorders, including both ALS and DMD (Fischer et al., 2004; van der Pijl et al., 2016). Emerging evidence suggests that therapeutically targeting neuromuscular synapse dysfunction is a viable option for extending lifespan and reducing disease severity in these disorders (Bruneteau et al., 2013; Cantor et al., 2018; Paredes-Redondo et al., 2021). However, a major challenge has been the generation of human-relevant disease models that accurately recapitulate in vivo human neuromuscular physiology and distinct neuromuscular disease phenotypes. While mouse models have been widely used for studying neuromuscular physiology, growing evidence shows that there are substantial differences between human and mouse neuromuscular synapses, including: contrasting synaptic morphologies, divergent transcriptomes and proteomes, and altered homeostasis and maintenance (Jones et al., 2017). With the development of differentiation protocols to derive enriched populations of motor neurons and myofibers (MFs) from human pluripotent stem cells (PSCs) (Du et al., 2015; Chal et al., 2016; Fernandopulle et al., 2018; Rao et al., 2018; Cheesbrough et al., 2022; Harley et al., 2022), a number of neuromuscular co-culture platforms have been established. A major challenge for generating human PSC-derived neuromuscular co-cultures has been stabilising contractile myofibers in order to prevent detachment upon contraction and allow long-term culture and maturation (Abd Al Samid et al., 2018). A notable example of an approach that overcame this issue was the development of a microphysiological neuromuscular disease model of ALS by Kamm and colleagues, in which bundles of myofibers were supported between micropillar cantilevers capable of deflecting upon contraction (Uzel et al., 2016; Osaki et al., 2018). Building on this work, we have developed a model of human neuromuscular physiology with a number of innovative features. These include a compartmentalised design comprising three chambers connected by microchannels, which mimics the spatial organization of tissues in vivo. This design enabled us to compare two genetically different motor neuron populations—one sensitive to optogenetic stimulation and the other insensitive—in their ability to innervate the same myofiber targets in response to optogenetic entrainment (Machado et al., 2019). Likewise, it allowed us to study phenotypes of the same motor neurons innervating an isogenic pair of DMD and CRISPR-corrected myofibers (Paredes-Redondo et al., 2021). Furthermore, incorporation of ALS-related TDP-43G298S motor neurons and CRISPR-corrected controls enabled us to recapitulate key ALS-related neuromuscular phenotypes (Harley et al., 2022). In all these experiments, motor neurons were embedded in a scaffold of mouse embryonic stem cell (ESC)–derived astrocytes to promote maturation and provide neurotrophic support. In future studies, it will allow different reconfigurations of the device to support different co-cultures. Such combinations of tissues may include a full corticomotor tract (cortical neurons, motor neurons, muscle) or the incorporation of sensory neurons into a sensory feedback compartment. Secondly, the proximity of the individual compartments to the optical polymer allows easy and rapid high-power imaging without the need for additional tissue processing and microdissection. Combined with the microchannels for axonal outgrowth, this would easily allow for high-resolution live axonal transport imaging and live synaptic imaging assays to be developed in future studies. Thirdly, myofibers are stabilized by a thin layer of UV-cured resin applied to the surface of the optical polymer, similar to the anchor points in the original microdevices (Machado et al., 2019). Finally, we provide evidence that formation of functional in vitro human neuromuscular circuits is activity dependent. We found that optogenetic entrainment of the motor neurons over the course of the culture dramatically enhanced synapse formation and myofiber contractility. In future studies, the microdevices described here may be used to understand how different stimulation paradigms and competitive innervation of common synaptic targets facilitates the wiring of human neuromuscular circuits. Materials and Reagents 15 mL Falcon tubes (Corning, catalog number: 430791 or equivalent) 50 mL Falcon tubes (Corning, catalog number: 352070 or equivalent) U-bottom 96-well plates (Corning, catalog number: 353227) Silicon master mould (CAD design available in supplementary materials) Channelrhodopsin-2 (CHR2)-YFP+ human PSC-motor neurons (+recommended primary or PSC-astrocytes) (Paredes-Redondo et al., 2021; Harley et al., 2022). Request of relevant cells from the Lieberam and Lin groups are subject to approval of Material Transfer Agreements with the institutions at which they were generated. Human PSC-myogenic progenitors (Paredes-Redondo et al., 2021; Harley et al., 2022). Request of relevant cells from the Lieberam and Lin groups are subject to approval of Material Transfer Agreements with the institutions at which they were generated. Polydimethylsiloxane (PDMS) (Dow Corning, Sylgard-184, catalog number: 4019862); store in the dark at room temperature (RT) NOA-73 (Norland products, catalog number: NOA73); store in the dark at RT Triton X-100 (Thermo Fisher, catalog number: 85111) Growth factor reduced (GFR) matrigel (Corning, catalog number: 356238); store at -20 °C Lipidure (Amsbio, catalog number: CM5206); store 0.5% solution in 100% ethanol at RT in the dark TrypLE (Gibco, catalog number: 12605010); store at 4 °C Accutase (Gibco, catalog number: A1110501); store at 4 °C DNase-I (Roche, catalog number: 10104159001); store at 4 °C Fibrinogen from bovine plasma (Sigma, catalog number: F8630); stock solution 24 mg/mL 0.9% NaCl; store at -80 °C Thrombin from bovine plasma (Sigma, catalog number: 9002-04-4); store at -80 °C Bovine serum albumin (BSA) fraction V (Roche, catalog number: 10735078001), dissolved in D-PBS (Gibco, catalog number: 14190250) to get 5% stock solution, and then sterile filtered. Store at 4 °C 1 M CaCl2 solution (Sigma, catalog number: 10043-52-4); store at RT 1,000× antioxidant supplement (Sigma, catalog number: A1345); store at 4 °C DMEM/F-12 (Gibco, catalog number: 11320033) Advanced DMEM/F-12 (Gibco, catalog number: 12634028) Neurobrew-21 (Miltenyi Biotec, catalog number: 130-093-566) Skeletal muscle cell growth medium (M2 in Figure 4) (PromoCell, catalog number: C-23060) N2 supplement (Gibco, catalog number: 17502001) L-glutamine (Gibco, catalog number: 25030149) Penicillin/streptomycin (Gibco, catalog number: 15140122) β-mercaptoethanol (Gibco, catalog number: 21985023) Insulin-transferrin-selenium (Gibco, catalog number: 41400045) GDNF (stock 100 µg/mL; PeproTech, catalog number: 450-10) BDNF (stock 100 µg/mL; PeproTech, catalog number: 450-02) Mouse IgM anti Titin (DSHB, clone: 9D10); store single aliquots at -80 °C Mouse IgG2a anti TUBB3 (R&D Systems, catalog number: MAB1195, clone: Tuj1); store single aliquots at -80 °C Rat IgG anti AChR (DSHB, clone: MAB35); store single aliquots at -80 °C Mouse IgG1 anti-SV2A (DSHB, clone: SV2); store single aliquots at -80 °C Goat anti mouse IgM 405 AlexaFluor (Abcam, catalog number: ab175662); store single aliquots at -80 °C Goat anti mouse IgG2a 488 AlexaFluor (Thermo Fisher, catalog number: A-21131); store single aliquots at -80 °C Goat anti rat IgG 555 (Thermo Fisher, catalog number: A-21434); store single aliquots at -80 °C Goat anti mouse IgG1 647 AlexaFluor (Thermo Fisher, catalog number: A-21240); store single aliquots at -80 °C Vectashield antifade mounting medium without DAPI (Vector Laboratories, catalog number: H-1900-10) ADFNB medium (M1) (see Recipes) Skeletal muscle secondary differentiation medium (M3) (see Recipes) Motor neuron medium (M4); optional (see Recipes) 0.1% PBT (see Recipes) Equipment Blunt forceps (Millipore, catalog number: XX6200006P) Vacuum chamber (Thermo Scientific/Nalgene, catalog number: 5305-0609 or equivalent) Vacuum pump (Welch, model: 2522Z-02 or equivalent) Oven for PDMS curing (Binder, model: VD23 or equivalent) UV cross-linker (Vilber-Lourmat, model: BLX-254 or equivalent) Tissue culture hood (Thermo Scientific, model: HeraSafe KS or equivalent) Stereo microscope (Olympus, model: SZX10 or equivalent) Hemocytometer (Neubauer, catalog number: 10360141 or equivalent) Centrifuge (Eppendorf, model: 5810R) with rotor A-4-81 and multi-well plate buckets 35 mm plastic bottom dishes (Ibidi, catalog number: 81156) 15 cm tissue culture dish (Thermo Scientific, catalog number: 353025 or equivalent) 10 cm dish (Corning, catalog number: 430591 or equivalent) Multichannel pipette (Thermo Fisher, catalog number: 4661050N or equivalent) Tissue culture incubator (Thermo Scientific, model: HeraCell 150i or equivalent) Custom LED stimulator for entrainment (Wefelmeyer et al., 2015) 470 nm LED light source (Thorlabs, model: M470F3) LED driver (Thorlabs, model: DC2200) Cell scraper (Falcon/VWR, catalog number: 734-0385 or equivalent) Software CellSens (Olympus) Matlab R2021A (Mathworks) PIVLab 2.53 (Thielicke, 2014) IMARIS 9.1.2 (Oxford Instruments) Procedure Preparation of compartmentalised microdevices (Figure 1, Video 1) Figure 1. Manufacture of compartmentalised polydimethylsiloxane (PDMS) microdevice arrays in 35 mm tissue culture dishes. A. Schematic showing the steps for generating microdevice arrays by curing PDMS on a silicon master mould and adhering the arrays onto 35 mm tissue culture dishes using the UV-curable polymer NOA-73. B. Pictures showing the final microdevice array in the 35 mm tissue culture dishes. Video 1. Preparation of compartmentalized microdevices Mix 2 g of PDMS base 10:1 with curing agent in a 50 mL Falcon tube by pipetting for 1 min and apply uniformly across the silicon master mould. Avoid introducing air bubbles during mixing. Degas in a 4.7 L vacuum chamber at 800 psi for 20 min. Cure at 80 °C in the oven for 2 h. Carefully peel the PDMS wafer using blunt forceps and place into a sterile 15 cm2 tissue culture dish microchannel side down to prevent debris from accumulating inside them. Use a scalpel to cut wafer into desired arrays (2 × 3 or 3 × 3 device arrays work well for a 35 mm dish). Apply 0.5 μL of the UV-curable polymer NOA-73 to a plastic bottom 35 mm dish and spread evenly using a cell scraper. Mark on the side of the dish the orientation of the final spread since myofibers align better along the same direction as the ridges in the polymer. Partially UV-cure at 55 J/cm2 for 10 s in a UV crosslinker. Caution: Partial curing is important to increase the viscosity of the resin while still retaining adhesiveness. If the UV exposure is too short, the resin will fill the microgrooves. If it is too long, the PDMS will not adhere to the dish. Transfer a 2 × 3 or 3 × 3 array onto the 35 mm dish ensuring that the long axis of the compartment follows the direction of the NOA-73 spread. Fully UV-cure the device at 55 J/cm2 for 1 min on each side. UV sterilize under UV lamp for 20 min. Apply 1 mL of 1:50 GFR matrigel in DMEM/F12 to the dish. Under a stereomicroscope placed in a tissue culture hood, use a fine pipette to remove air bubbles from each large compartment. Degas the microchannels in a vacuum chamber at 800 psi for 2 h. Use immediately or seal culture dish with parafilm and store at 4 °C for up to two weeks. Generating neural spheroids This protocol is designed to work with multiple human PSC differentiation protocols. The human PSC-motor neuron differentiation protocol used for this study is from Zhang and colleagues (Du et al., 2015) with additional magnetic-activated cell sorting purification steps outlined in (Machado et al., 2019). Astrocytes (ACs) were derived from mouse ESCs as described in (Machado et al., 2019). Other notable protocols for generating human PSC-motor neurons and astrocytes include (Julia et al., 2017; Canals et al., 2018; Fernandopulle et al., 2018). For the protocol used for these experiments, follow those outlined in (Harley et al., 2022). Consult the original protocol to determine the best point to dissociate and replate the motor neurons/astrocytes. To generate non-adherent 96-well plates neural spheroids, add 100 μL of 0.5% lipidure dissolved in 100% ethanol absolute to each well of a U-bottom 96-well plate. Leave plates near the air vents of the tissue culture hood with the lids ajar to dry overnight. The next day, dissociate motor neurons and astrocytes into single cells using TrypLE or accutase, respectively. For motor neuron dissociation choose a stage in the protocol that allows live cell sorting (Harley et al., 2022) and replating into different culture formats; if motor neurons are too mature with large axonal projections, then dissociation will result in cell death. Astrocyte dissociation may require longer incubations (5–20 min) and addition of DNase-I to prevent cell clumping. Count the cells using a hemocytometer and plate 15,000 motor neurons and 5,000 astrocytes in each well of the lipidure plate. Calculate the quantity needed for a full plate and use a multichannel pipette. Plate into ADFNB medium. [Optional]: Combine ADFNB medium 50:50 with motor neuron medium. We have successfully used both medium conditions for human PSC-motor neuron spheroid formation. Briefly spin the plates at 1,200 rpm (290 × g) for 2 min. After 48 h, neural spheroids of uniform size and cell type composition should have formed. Check that the edges are well defined and the overall morphology is round (Figure 2). Figure 2. Examples of poor (A) and good quality (B) motor neuron (MN)/astrocyte (AC) spheroids. Scale bar = 50 μm. Plating neural spheroids into the compartmentalised microdevices (Figure 3) Thaw GFR matrigel, fibrinogen, and thrombin on ice. Place metal tube rack, metal block, and 35 mm dish on ice to cool. Make hydrogel mix containing 160 μL of fibrinogen, 40 μL of GFR matrigel, and 0.5 μL of CaCl2 (1 M stock) and leave on ice. Use a multichannel pipette to transfer neural spheroids from the U-bottom 96-well lipidure plates to a 10 cm dish containing 5 mL of ADFNB medium. Pipette up and down several times to dislodge the spheroids but do not be too vigorous as it risks breaking up the spheroids. Swirl the plate and transfer all the spheroids in 1 mL of medium to a 15 mL Falcon tube. Remove 950 μL of medium and add 50 μL of hydrogel mix so all the neural spheroids are in 100 μL of 50:50 medium to hydrogel. Transfer these spheroids to the pre-cooled 35 mm dish and leave on the cool metal block to prevent hydrogel polymerization. Remove GFR matrigel from the compartmentalised microdevices in the 35 mm dishes (from step A). Directly apply the aspirator to the PDMS array to remove medium from the chambers. Add 500 μL of ADFNB medium to the edge of the 35 mm dish to prevent drying. Ensure that this does not infiltrate the compartmentalised microdevice array. Using a fine pipette tip to transfer three neural spheroids to each outer chamber of the microdevice array under a sterile hood microscope. After completing the whole array, leave to rest for 5 min at RT. Add extra hydrogel mix to each chamber to ensure there is a meniscus of hydrogel above the PDMS array (this ensures that addition of thrombin does not disturb the neural spheroids). Now, add ~30 μL of thrombin across the compartmentalised PDMS array to convert fibrinogen to fibrin and initiate polymerisation. Do not add directly to the chambers; just apply at the corners. Do this relatively fast as the thrombin acts quickly to polymerise the hydrogel. Leave to rest at RT for 5 min. Now, carefully add 2 mL of ADFNB medium to the dish and place in the incubator. Plating myogenic progenitors into the compartmentalised microdevices (Figure 3) Figure 3. Schematic outlining the different steps for plating human pluripotent stem cell (PSC)–neural spheroids and human PSC-myogenic progenitors into the compartmentalised microdevices. MN: motor neuron; AC: astrocyte; MPCs: myogenic progenitor cells; NMJ: neuromuscular junction. This protocol is designed to work with multiple human PSC-myogenic differentiation protocols. The one used for this study is from Pourquie and colleagues (Chal et al., 2016), although similar results were also obtained using forward programming of human PSCs by forced expression of PAX7, as described in Rao et al. (2018) and Cheesbrough et al. (2022). Cells should be replated when they are at an equivalent progenitor/myoblast stage, before myotube fusion has occurred. After plating the neural spheroids, wait for 24 h before plating the myogenic progenitors. During this time, motor axons will grow through the microchannels into the central compartment. Thaw GFR matrigel, fibrinogen, and thrombin on ice. Place metal tube rack and metal block on ice to cool. Make hydrogel mix containing 160 μL of fibrinogen, 40 μL of GFR matrigel, and 0.5 μL of CaCl2 (1M stock) and leave on ice. Dissociate myogenic progenitors into single cells using TrypLE or accutase. Count cells and resuspend at a concentration of 20,000 cells/μL. Mix myogenic progenitor suspension with hydrogel mix 50:50, so that cells are now at a concentration of 10,000 cells/μL, and keep on ice. Remove medium from the edge of the 35 mm dish containing the neural spheroids plated in the outer compartments of the microdevice array. Add 500 μL of skeletal muscle growth medium to the edge of the 35 mm dish to prevent the neural spheroids in the compartments from drying. Ensure that the medium does not spill onto the microdevice array in the centre of the dish. Under the sterile hood microscope, use a fine pipette tip to remove residual medium from the empty central chambers of each microdevice. Now transfer ~1 μL of myogenic progenitor/hydrogel suspension to each central chamber. Leave to rest for 5 min. Top up with additional hydrogel mix to create a meniscus above the chamber and leave to rest for another 5 min. Carefully add ~30 μL of thrombin across the compartmentalised PDMS array. Do not add directly to the chambers; just apply at the corners. Do this relatively fast as the thrombin acts quickly to polymerise the hydrogel. Wait a further 5 min. Now, carefully add 2 mL of skeletal muscle cell growth medium to the dish and place in the incubator. Wait 24 h after plating the myogenic progenitors, then change the skeletal muscle cell growth medium to skeletal muscle secondary differentiation medium to induce the formation of a 3D sheet of myofibers in the central chamber. Maintenance and optogenetic entrainment (optional) of neuromuscular co-cultures (Figure 4) Figure 4. Optogenetic entrainment enhances neuromuscular junction (NMJ) formation and myofiber contractility in human pluripotent stem cell (PSC)–derived neuromuscular circuits. A. Schematic showing optogenetic entrainment of wildtype human PSC neuromuscular co-cultures. B. Particle image velocimetry analysis of optogenetically evoked myofiber contractions in non-entrained and entrained co-cultures (Scale bar = 200 μm). C. Quantification of peak optogenetically evoked myofiber contraction velocity in non-entrained and entrained co-cultures, n = 6. D. Immunofluorescence staining for TUBB3, SV2, and AChR alongside SV2/AChR (NMJ) colocalization channels in non-entrained and entrained co-cultures (Scale bar = 50 μm). Quantification of axon outgrowth and NMJ number in non-entrained and entrained conditions, n = 6. Error bars represent the SEM, unpaired non-parametric t-tests used to determine statistical significance. p < 0.01, *p < 0.001. The optogenetic entrainment section of the protocol is designed for neuromuscular co-cultures containing motor neurons harbouring an optogenetic actuator. In our studies, we used CHR2-YFP stably integrated into the human PSC lines with a PiggyBac transposition system (Harley et al., 2022). Furthermore, this section relies on the use of LED stimulator placed in a tissue culture incubator. In our work, we used a custom built stimulator (Machado et al., 2019), although commercial versions are also widely available. While optogenetic entrainment was not essential to generate functional neuromuscular circuits, it did significantly improve circuit formation and contractility and provides an interesting paradigm to investigate the activity dependence of synapse formation in future studies. Refresh skeletal muscle secondary differentiation medium every 2–3 days. [Optional]: Combine skeletal muscle secondary differentiation medium with motor neuron medium 50:50 at this stage onwards. Four days after plating the myogenic progenitors, motor axons should have begun to grow through the microchannels into the central muscle compartment. Before optogenetic entrainment, add 1:1,000 antioxidant supplement to the 35 mm dish. Place on top of the LED stimulator in the incubator and stimulate at 20 Hz for 1 h at 40% LED intensity. After stimulation, completely change the medium to remove toxic free radicals induced by photo-stimulation. Repeat this for five days. Note: The length of entrainment depends on the experimental conditions. We noticed that when treated with drugs that promote myofiber contractility, five days of entrainment would lead to too much detachment, so shorter entrainment periods were used (Paredes-Redondo et al., 2021). Recording and particle image velocimetry (PIV) analysis of optogenetic motor neuron–evoked myofiber contractions (Figures 4A–4D, 5, 6) Figure 5. Particle image velocimetry (PIV) analysis of optogenetically evoked myofiber contractions. A. PIV analysis steps using the PIVLab Matlab plug-in: 1. Load images as time resolved sequence; 2. Set PIV setting; 3. Calibrate image to reference and time step; 4. Apply vector validation; 5. Extract velocity measurements. B. PIV analysis of optogenetically evoked myofiber contractions with and without treatment with the AChR blocker d-Tubocurarine (DTC). Figure 6. Neuromuscular co-cultures can be used to model amyotrophic lateral sclerosis (ALS) and Duchenne muscular dystrophy (DMD)–related neuromuscular disease phenotypes. A. Optogenetically evoked myofiber contractions in neuromuscular co-cultures containing myofibers from DMD patient–derived DMD-R3381X PSCs compared to CRISPR-corrected controls (CORR-R3381X). B. Optogenetically evoked myofiber contractions in neuromuscular co-cultures containing motor neurons from ALS patient–derived TDP-43M337V PSCs compared to wildtype controls. To briefly test if neuromuscular circuits were functional during the course of the culture, an Olympus X73 microscope with a fluorescent lamp was used to optogenetically stimulate the motor neurons, simply by opening and closing the fluorescent shutter, and to observe associated myofiber contractions. However, for a more controlled analysis, a Thorlabs LED stimulator was used to provide precise optogenetic stimulation via an optical fibre light guide, measured to be 0.2 mW/mm2 of light intensity (Paredes-Redondo et al., 2021), which is sufficient to trigger action potentials in CHR2-expressing neurons. Place the optical fibre coupled with a 470 nm LED light guide >1 cm beneath the 35 mm dish, just out of frame of the microscope’s internal light beam. Set the LED intensity on the LED driver to 100% and the stimulation interval to 500 ms. Record 5 s videos at 20 frames per second (FPS) using the CellSens software; manually trigger the LED stimulator during the recording. Export video recordings as TIFF image sequences. Download and install the PIVLab Matlab plug-in. Load images as a time resolved sequence (A + B), (B + C), and (C + D). In PIV settings, select FTT in the PIV algorithm, Interrogation area = 64 pixels and Step = 32 in Pass 1, and Interrogation area of 32 px for Pass 2, 3, and 4. Calibrate the image to a reference image with a pattern of known dimensions (for example an image of a hemocytometer chamber taken with the same microscope and settings) and set the time calibration to the interval between pixels, which is 50 ms for a 20 FPS recording. Click: Analysis > ANALYZE! For post-processing, apply 8 to Standard deviation filter and Apply to all frames. Export results as text (ASCII) chart for all frames as area mean value using the velocity magnitude (m/s) parameter and draw the area of interest for extraction (the central myofiber compartment in this case). Finally, multiply the velocity magnitude values by 106 to derive the velocity magnitude in μm/s. Multiple recordings of the same conditions are combined by aligning the contractile peaks. Immunohistochemistry and analysis of axon outgrowth and NMJ number/morphology (Figures 4E, 7) Figure 7. Immunocytochemistry analysis of axonal outgrowth and neuromuscular junction (NMJ) number and morphology. A. Steps used in IMARIS software to analyse colocalization of SV2 and AChR objects to infer NMJ number and morphology. B. Representative immunofluorescence images for Titin, TUBB3, SV2, and AChR alongside composite images and the colocalization channel for SV2 and AchR (Scale bar = 50 μm). Following contraction analysis, wash the neuromuscular co-cultures in PBS and fix for 20 min in 4% PFA, 15% sucrose solution. Wash three times in PBS; then, block in 3% BSA, 0.1% Triton-X-100, and 10% DMSO for 1 h at RT. Add primary antibodies (Titin 1:20, TUBB3 1:500, SV2 1:200, and AChR 1:100) in blocking solution and leave overnight at 4 °C. Wash three times in 0.1% PBT and incubate secondary antibodies (1:1,000 dilution) in 0.1% PBT, 10% DMSO overnight at 4 °C. Wash three times in PBS; then, mount using an 18 mm coverslip and vectashield antifade mounting medium. To mount the samples, add 3–4 drops of mounting medium to the edge of the array, tilt the dish so that the medium flows over the compartments, and then slowly drop the coverslip onto the array, starting with one side. Image using a confocal microscope over a 40 μm Z-stack at 2 μm intervals. Using IMARIS image analysis software, create a surface for each channel. Use the colocalization (Coloc) plug-in to generate a colocalization channel for the SV2 and AChR channels by using the automatic threshold calculation tool. Create a surface for this colocalization channel. Now, export object data for each channel, as well as morphology related data, such as area and volume. Use object number and mean area/volume data from the colocalization channel to infer NMJ number and morphology. Use sum area/volume data from the TUBB3 channel to infer axonal outgrowth. Notes Owing to the complex and multi-lineage composition of these co-cultures, there is normally a reasonable degree of interexperimental variability in contractility, axonal outgrowth, and NMJ formation. As such, care should be taken to include all appropriate control groups each time co-cultures are generated. Normalisation of data between experiments should also be considered to compare multiple datasets. We recommend that at least four technical replicates are performed for each condition in each experiment, and that the experiments are repeated at least three times to ensure reproducibility. Recommended statistical tests: PIV measurements of myofiber contractions, two-way ANOVA with Sidak’s multiple comparisons test; NMJ number and morphology, one-way ANOVA with Tukey’s or Dunnet’s multiple comparison test. Recipes ADFNB medium (M1) Reagent Final concentration Amount (for 50 mL) Advanced DMEM/F-12 1 part 23.25 mL Neurobasal 1 part 23.25 mL Neurobrew-21 1× 1 mL N2 supplement 1× 0.5 mL L-glutamine 2 mM 0.5 mL Penicillin/streptomycin 1× 0.5 mL β-mercaptoethanol 1× 50 μL BSA (5% stock) 0.1% 1 mL Skeletal muscle secondary differentiation medium (M3) Reagent Final concentration Amount (for 50 mL) DMEM/F-12 1 part 48.4 mL N2 supplement 1× 0.5 mL Insulin-transferrin-selenium 1× 0.5 mL Penicillin/streptomycin 1× 0.1 mL L-glutamine 2 mM 0.5 mL Motor neuron medium (M4) (Optional) Reagent Final concentration Amount (for 50 mL) DMEM/F-12 1 part 23.75 mL Neurobasal 1 part 23.75 mL Neurobrew-21 1× 1 mL N2 supplement 1× 0.5 mL L-glutamine 2 mM 0.5 mL Penicillin/streptomycin 1× 0.5 mL GDNF 10 ng/mL 5 μL BDNF 10 ng/mL 5 μL 0.1% PBT Reagent Final concentration Amount (for 500 mL) PBS 1 part 500 mL Triton X-100 0.1% 5 mL 10% stock diluted in PBS Acknowledgments This research was funded in part by a Wellcome Trust “Cell Therapies & Regenerative Medicine” PhD studentship 108874/Z/15/Z to P.H; a QMUL-Blizard PhD studentship and QMUL-Life Sciences Initiative grant to A.P.-R.; Medical Research Council grant MR/N025865/1 to I.L.; and Royal Society grant RG130417, Newlife Charity grant SG/14-15/14, Action Duchenne grant AD1801Y, Duchenne Parent Project grant 19.017, and Barts Charity grant MGU0426 to Y.-Y.L. The authors acknowledge Cancer Research UK Centre of Excellence Award to Barts Cancer Centre grant C16420/A18066 and Medical Research Council Centre grant MR/N026063/1. For the purpose of open access, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission. This protocol is derived from the original research paper (Paredes-Redondo et al., 2021). Competing interests Y.-Y.L. is the principal investigator of a research grant received from Pfizer. Ethics * For DMD patient-derived cells, informed consent was obtained under appropriate ethics approved by Hammersmith and Queen Charlotte’s and Chelsea Hospital (REC reference 06/Q0406/33) and by National Research Ethics Service Committee London-Stanmore (REC reference 13/LO/1826; IRAS project ID: 141100). * For cells derived from patients with ALS, informed consent was obtained under appropriate ethics approved by King’s College NHS Foundation Trust (Prof Chris Shaw - Ethics number: 10/S1103/10; Trust R&D registration number: KCH12-047). * I.L. has approval from the UK Stem Cell Bank steering committee (no. SCS11-06) to import human H9 ESCs. References Abd Al Samid, M., McPhee, J. S., Saini, J., McKay, T. R., Fitzpatrick, L. M., Mamchaoui, K., Bigot, A., Mouly, V., Butler-Browne, G. and Al-Shanti, N. (2018). A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts. Stem Cells and Cloning:Advances and Applications 11: 85-93. Bruneteau, G., Simonet, T., Bauche, S., Mandjee, N., Malfatti, E., Girard, E., Tanguy, M. L., Behin, A., Khiami, F., Sariali, E., et al. (2013). Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression. Brain 136: 2359-2368. Canals, I., Ginisty, A., Quist, E., Timmerman, R., Fritze, J., Miskinyte, G., Monni, E., Hansen, M. G., Hidalgo, I., Bryder, D., et al. (2018). Rapid and efficient induction of functional astrocytes from human pluripotent stem cells. Nature Methods 15(9): 693-696. Cantor, S., Zhang, W., Delestree, N., Remedio, L., Mentis, G. Z. and Burden, S. J. (2018). Preserving neuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonist antibody. Elife 7: e34375. Chal, J., Al Tanoury, Z., Hestin, M., Gobert, B., Aivio, S., Hick, A., Cherrier, T., Nesmith, A. P., Parker, K. K. and Pourquie, O. (2016). Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nat Protoc 11(10): 1833-1850. Cheesbrough, A., Sciscione, F., Riccio, F., Harley, P., R'Bibo, L., Ziakas, G., Darbyshire, A., Lieberam, I. and Song, W. H. (2022). Biobased Elastomer Nanofibers Guide Light-Controlled Human-iPSC-Derived Skeletal Myofibers. Adv Mater 34(18): e2110441 Du, Z. W., Chen, H., Liu, H. S., Lu, J. F., Qian, K., Huang, C. L., Zhong, X. F., Fan, F. and Zhang, S. C. (2015). Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun 6: 6626. Duan, D. S., Goemans, N., Takeda, S., Mercuri, E. and Aartsma-Rus, A. (2021). Duchenne muscular dystrophy. Nature Reviews Disease Primers 7(1). https://doi.org/10.1038/s41572-021-00248-3. Fernandopulle, M. S., Prestil, R., Grunseich, C., Wang, C., Gan, L. and Ward, M. E. (2018). Transcription Factor–Mediated Differentiation of Human iPSCs into Neurons. Curr Protoc Cell Biol 79(1): e51. Fischer, L. R., Culver, D. G., Tennant, P., Davis, A. A., Wang, M. S., Castellano-Sanchez, A., Khan, J., Polak, M. A. and Glass, J. D. (2004). Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol 185(2): 232-240. Hardiman, O., Al-Chalabi, A., Chio, A., Corr, E. M., Logroscino, G., Robberecht, W., Shaw, P. J., Simmons, Z. and van den Berg, L. H. (2017). Amyotrophic lateral sclerosis. Nature Reviews Disease Primers 3. Harley, P., Neves, G., Riccio, F., Barcellos Machado, C., Cheesbrough, A., R'Bibo, L., Burrone, J. and Lieberam, I. (2022). Pathogenic TDP-43 Disrupts Axon Initial Segment Structure and Neuronal Excitability in a Human iPSC Model of ALS. bioRxiv: 2022.2005.2016.492186. Jones, R. A., Harrison, C., Eaton, S. L., Hurtado, M. L., Graham, L. C., Alkhammash, L., Oladiran, O. A., Gale, A., Lamont, D. J., Simpson, H., et al. (2017). Cellular and Molecular Anatomy of the Human Neuromuscular Junction. Cell Rep 21(9): 2348-2356. Julia, T. C. W., Wang, M. H., Pimenova, A. A., Bowles, K. R., Hartley, B. J., Lacin, E., Machlovi, S. I., Abdelaal, R., Karch, C. M., Phatnani, H., et al. (2017). An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells. Stem Cell Rep 9(2): 600-614. Machado, C. B., Pluchon, P., Harley, P., Rigby, M., Sabater, V. G., Stevenson, D. C., Hynes, S., Lowe, A., Burrone, J., Viasnoff, V., et al. (2019). In Vitro Modeling of Nerve-Muscle Connectivity in a Compartmentalized Tissue Culture Device. Adv Biosyst 3(7): 1800307. Osaki, T., Uzel, S. G. M. and Kamm, R. D. (2018). Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons. Sci Advs 4(10): eaat5847. Paredes-Redondo, A., Harley, P., Maniati, E., Ryan, D., Louzada, S., Meng, J. H., Kowala, A., Fu, B. Y., Yang, F. T., Liu, P. T., et al. (2021). Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections. Sci Adv 7(37): eabi8787. Rao, L. J., Qian, Y., Khodabukus, A., Ribar, T. and Bursac, N. (2018). Engineering human pluripotent stem cells into a functional skeletal muscle tissue. Nat Commun 9. Thielicke, W. (2014). PIVlab – Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB. In: Stamhuis, E. J. (Ed.). Journal of Open Research Software, p.e30. Uzel, S. G. M., Platt, R. J., Subramanian, V., Pearl, T. M., Rowlands, C. J., Chan, V., Boyer, L. A., So, P. T. C. and Kamm, R. D. (2016). Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units. Sci Adv 2(8): e1501429. van der Pijl, E. M., van Putten, M., Niks, E. H., Verschuuren, J., Aartsma-Rus, A. and Plomp, J. J. (2016). Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models. Eur J Neurosci 43(12): 1623-1635. Wefelmeyer, W., Cattaert, D. and Burrone, J. (2015). Activity-dependent mismatch between axo-axonic synapses and the axon initial segment controls neuronal output. Proc Natl Acad Sci U S A 112(31): 9757-9762. Supplementary information Masks used to manufacture silicon master mould: Harley_etal_MBI 223_DF.gds Harley_etal_MBI 218_no frame (BF).gds Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). How to cite Category Biological Engineering > Biomedical engineering Cell Biology > Cell isolation and culture > Co-culture Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Medullary Thymic Epithelial Cell Antigen-presentation Assays Alexia Borelli [...] Magali Irla Nov 5, 2023 605 Views Protocol for Immune Cell Isolation, Organoid Generation, and Co-culture Establishment from Cryopreserved Whole Human Intestine Enrique Gamero-Estevez [...] Martin Resnik-Docampo Jan 5, 2025 729 Views An Optimized Protocol for Simultaneous Propagation of Patient-derived Organoids and Matching CAFs Jenny M. Högström and Taru Muranen Jan 20, 2025 1768 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,625	https://bio-protocol.org/en/bpdetail?id=4625&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Isolation of Intact Vacuoles from Arabidopsis Root Protoplasts and Elemental Analysis CJ Chuanfeng Ju DF Dali Fu Cun Wang ZZ Zhenqian Zhang Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4625 Views: 721 Reviewed by: Wenrong HeYao XiaoYe Xu Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Molecular Plant May 2021 Abstract The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins. Graphical abstract Keywords: Isolation of intact vacuoles Arabidopsis protoplasts Elemental analysis Mn transporter MTP8 Background The central vacuole is the largest compartment of a mature plant cell and participates in plenty of physiological processes. The determination of its elemental concentrations is essential for researching the function and regulation of plant tonoplast transporters. The transport rates of different anions and the factors affecting the uptake of chloride ions across the tonoplast were detected in isolated barley vacuoles (Martinoia et al., 1986). Transport of phosphate across the tonoplast was also detected in intact vacuoles, which were isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don (Massonneau et al., 2000). Shimaoka et al. (2004) modified the method for extracting intact vacuoles, which is summarized in this article, and detected the tonoplast proteins combined with proteomic analysis. The function and regulatory mechanisms of the tonoplast manganese (Mn) transporter MTP8 were resolved via this method (Eroglu et al., 2016; Zhang et al., 2021; Ju et al., 2022). In conclusion, this protocol is feasible and also applied for the research to investigate tonoplast transporters in the future. Materials and Reagents 40 μm cell strainer (Corning, catalog number: 431750) 50 mL tubes (Sangon Biotech, catalog number: F602788) Cellulase R10 (Yakult, catalog number: L0012) Macerozyme R10 (Yakult, catalog number: L0021) D-mannitol (Sigma-Aldrich, catalog number: M9647) MES hydrate (Sigma-Aldrich, catalog number: M2933) Tris-base (Thermo Fisher Scientific, catalog number: BP152-5) Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670) Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Percoll (GE Healthcare, catalog number: 17-0891-09) HEPES (Sigma-Aldrich, catalog number: H3375) EGTA (Sigma-Aldrich, catalog number: H3889) Sucrose (Sigma-VETEC, catalog number: V900116) K-gluconate (Sigma-Aldrich, catalog number: G4500) D-sorbitol (Sigma-Aldrich, catalog number: S1876) MnSO4 (Sigma-Aldrich, catalog number: M7634) HNO3 (GHTECH, catalog number: 1.14003.018) H2O2 (Sinopharm Chemical Reagent, catalog number: 10011208) Murashige and Skoog (MS) base salts with vitamins (Phytotech Labs, catalog number: M519) Agar (Sigma-Aldrich, catalog number: A1296) KNO3 (Sinopharm Chemical Reagent, catalog number: 10017218) Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: SC278601) NH4H2PO4 (Sinopharm Chemical Reagent, catalog number: 10002808) MgSO4·7H2O (Sinopharm Chemical Reagent, catalog number: 10013018) H3BO3 (Sinopharm Chemical Reagent, catalog number: 10004808) MnCl2·4H2O (Sigma-Aldrich, catalog number: SM500501) (NH4)6Mo7O24·4H2O (Sinopharm Chemical Reagent, catalog number: 10002318) ZnSO4·7H2O (Sinopharm Chemical Reagent, catalog number: 10024018) CuSO4·5H2O (Sinopharm Chemical Reagent, catalog number: 10008218) EDTA-Fe(III)Na (Biotopped, catalog number: Q0028-100g) BSA (Sigma-Aldrich, catalog number: V900933) β-mercaptoethanol (14.3 M) (Millipore, catalog number: 444203) 1/2 MS medium (see Recipes) 1/5 Hoagland solution (see Recipes) Protoplasting solution (see Recipes) Medium B (see Recipes) Medium I (see Recipes) Medium II (see Recipes) Medium III (see Recipes) Medium IV (see Recipes) Medium V (see Recipes) Medium VI (see Recipes) Medium VII (see Recipes) Medium VIII (see Recipes) Equipment Artificial illumination incubator (PERCIVAL, model: LT-36VL) Horizontal centrifuge (Eppendorf, model: Centrifuge 5810R) Dissolver (LabTech, model: DigiBlock ED54) ICP-MS (Thermo Fisher Scientific, model: ICAP Qc) Perfluoroalkoxy alkane (PFA) vessels (LabTech, model: GC-36-L) Fuchs-Rosenthal hemacytometer (Marienfeld, model: Dark-Line 0650010) Procedure Protoplast preparation Sterilize approximately 50 Arabidopsis seeds, plant on 1/2 MS medium, and then grow in an artificial illumination incubator under normal light conditions (100 µmol m-2 s-1) with a long-day cycle (16:8 h light/dark) at 22 °C. After seven days, clamp the seedlings with a small sponge and place them on a reticulated floating board; immerse the roots in 1/5 Hoagland solution and then grow in an artificial illumination incubator under normal light conditions (100 µmol m-2 s-1) with a short-day cycle (8:16 h light/dark) at 22 °C. Change the solution every three days. After seven weeks, transfer the seedlings to the same solution containing 240 μM MnSO4 for another seven days. Take 10–15 seedling roots and cut them into small pieces with a blade, depositing them into a 100 mL flask containing the protoplasting solution (see Recipes). Generally, 20 mL of protoplasting solution is used per 15 seedling roots. Shake the flasks gently (75 rpm) at room temperature for 1 h. A longer incubation time may increase the protoplast yield. Filter the protoplast solution with a 40 μm cell strainer and carefully pour into a 50 mL tube. Vacuole extraction In order to reduce the disruption of protoplasts, first add 5 mL of medium I to the bottom of the tube and then gently mix the filtered protoplasts. After centrifugation (200 × g, 10 min), discard the supernatant with a pipette to obtain sedimented protoplasts. Slowly add 5 mL of medium I into the tube and gently mix with the sedimented protoplasts. Using a pipette, add 5 mL of medium II to the tube against the wall, and then 5 mL of medium III. Each layer of liquid should be added slowly to ensure that the layers do not break apart. After adding medium III, the gradient is formed and can be clearly seen in the tube. After centrifugation (800 × g, 10 min), purified colorless protoplasts are obtained in the interfaces between medium I and II or between medium II and III. Discard the latter protoplasts (between medium II and III) with the pipette to avoid contamination of the vacuolar fractions with protoplasts. Gently mix approximately 2 mL of the remaining purified protoplasts with 2 mL of medium B and incubate on ice for 5 min. Gently mix the mixture of protoplasts, vacuoles, and lysate (from step B4) with 5 mL of medium IV first. Then, a gradient is formed by consecutive overlaying with 5 mL each of medium V, medium VI, medium VII, and medium VIII. After centrifugation (800 × g, 10 min), discard medium VIII with a pipette. Carefully aspirate approximately 1 mL of liquid between medium VII and medium VIII as the purified vacuoles. Elemental analysis Determine the number of vacuoles using a Fuchs-Rosenthal hemacytometer. (Approximately 10 vacuoles per square millimeter of the Fuchs-Rosenthal hemacytometer, whose height is 0.1 mm through microscope observation; so, there are 100 vacuoles in each cubic millimeter of solution.) The total number of vacuoles in the solution is obtained by multiplying the number of vacuoles per cubic millimeter by the volume of the solution. Digest the vacuoles in PFA vessels with 1.5 mL of HNO3 (65%) and 0.6 mL of H2O2 (30%) for 15 min in the dissolver at 170 °C. After cooling down to room temperature, transfer the digestion solution to a plastic volumetric flask and dilute to 5 mL with ultrapure water (18.25 MΩ cm−1). Determine Mn in the digestion solution using an inductively coupled plasma mass spectrometer (ICP-MS). Data analysis The vacuolar elemental concentration of each plant material requires three biological replicates to be measured. The mean value of three biological replicates was calculated and statistically analyzed (Student’s t-test). Detailed data and statistical methods could be found in the previously published articles (Figure 5B in Zhang et al., 2021 and Figure 4B in Ju et al., 2022). Recipes 1/2 MS medium (adjust the pH to 5.7 using Tris) Reagent Final concentration Amount MS base salts with vitamins 0.222% 2.22 g Sucrose 1% 10 g Agar ddH2O Total 1% n/a n/a 10 g Up to 1 L 1 L 1/5 Hoagland Solution Reagent Final concentration Amount KNO3 1 μM 0.51 g Ca(NO3)2·4H2O 1 μM 1.18 g NH4H2PO4 MgSO4·7H2O H3BO3 MnCl2·4H2O (NH4)6Mo7O24·4H2O ZnSO4·7H2O CuSO4·5H2O EDTA-Fe(III)Na ddH2O Total 0.2 μM 0.4 μM 3 nM 0.5 nM 1 nM 0.4 nM 0.2 nM 20 nM n/a n/a 0.12 g 0.49 g 1.85 μg 0.99 μg 12.36 μg 1.15 μg 0.05 μg 8.42 μg Up to 1 L 1 L MES-Tris buffer Reagent Final concentration Amount MES hydrate 0.5 M 9.76 g Tris-base Adjust pH to 5.7 ddH2O n/a Up to 100 mL Total n/a 100 mL Protoplasting solution Reagent Final concentration Amount Cellulase R10 1.25% 1.25 g Macerozyme R10 0.3% 0.3 g D-mannitol (0.8 M) 0.4 M 50 mL MES-Tris buffer (0.5 M, pH 5.7) 20 mM 4 mL KCl (1 M) 20 mM 2 mL Heat the solution to 55 °C for 10 minutes and let it cool down to room temperature BSA 0.1% 0.1 g CaCl2 (1 M) 10 mM 1 mL β-mercaptoethanol (14.3 M) 5 mM 35 μL ddH2O n/a Up to 100 mL Total n/a 100 mL Medium B Reagent Final concentration Amount HEPES-Tris (1 M, pH 7.2) 30 mM 3 mL K-gluconate 30 mM 0.7 g MgCI2 (1 M) 2 mM 0.2 mL EGTA (0.5 M) 2 mM 0.4 mL ddH2O n/a Up to 100 mL Total n/a 100 mL Medium I Reagent Final concentration Amount Sucrose 400 mM 13.69 g Percoll 50% 50 mL Medium B n/a Up to 100 mL Total n/a 100 mL Medium II Reagent Final concentration Amount Sucrose 400 mM 13.69 g Percoll 7.5% 7.5 mL Medium B n/a Up to 100 mL Total n/a 100 mL Medium III Reagent Final concentration Amount D-sorbitol 400 mM 7.27 g Medium B n/a Up to 100 mL Total n/a 100 mL Medium IV Reagent Final concentration Amount D-sorbitol 200 mM 3.64 g Percoll 25% 25 mL Medium B n/a Up to 100 mL Total n/a 100 mL Medium V Reagent Final concentration Amount Sucrose 200 mM 6.85 g Percoll 7.5% 7.5 mL Medium B n/a Up to 100 mL Total n/a 100 mL Medium VI Reagent Final concentration Amount Sucrose 200 mM 6.85 g Percoll 5% 5 mL Medium B n/a Up to 100 mL Total n/a 100 mL Medium VII Reagent Final concentration Amount Sucrose 200 mM 6.85 g Percoll 2.5% 2.5 mL Medium B n/a Up to 100 mL Total n/a 100 mL Medium VIII Reagent Final concentration Amount Sucrose 200 mM 6.85 g Medium B n/a Up to 100 mL Total n/a 100 mL Acknowledgments This research was funded by a grant from the National Natural Science Foundation of China (31770289 to C.W.), Northwest A&F University (Z111021604 to C.W.), and the National Natural Science Foundation of China (31900236 to Z.Z.). The initial publication on preparation of the plant material is published in Eroglu et al. (2016); the initial publication on protoplast preparation of the plant material is published on Bargmann et al. (2010); the initial publication on vacuole extraction is published on Shimaoka et al. (2004). Competing interests Non-financial competing interests on behalf of all authors. References Bargmann, B. O. and Birnbaum, K. D. (2010). Fluorescence activated cell sorting of plant protoplasts. J vis exp (36). Eroglu, S., Meier, B., von Wirén, N. and Peiter, E. (2016). The vacuolar manganese transporter MTP8 determines tolerance to iron deficiency-induced chlorosis in Arabidopsis. Plant Physiol 170(2): 1030-1045. Ju, C., Zhang, Z., Deng, J., Miao, C., Wang, Z., Wallrad, L., Javed, L., Fu, D., Zhang, T., Kudla, J., Gong, Z. and Wang, C. (2022). Ca2+-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis. Mol Plant 15(3): 419-437. Martinoia, E., Schramm, M. J., Kaiser, G., Kaiser, W. M. and Heber, U. (1986). Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system. Plant Physiol 80(4): 895-901. Massonneau, A., Martinoia, E., Dietz, K. J. and Mimura, T. (2000). Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don. Planta 211(3): 390-395. Shimaoka, T., Ohnishi, M., Sazuka, T., Mitsuhashi, N., Hara-Nishimura, I., Shimazaki, K., Maeshima, M., Yokota, A., Tomizawa, K. and Mimura, T. (2004). Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. Plant Cell Physiol 45(6): 672-683. Zhang, Z., Fu, D., Sun, Z., Ju, C., Miao, C., Wang, Z., Xie, D., Ma, L., Gong, Z. and Wang, C. (2021). Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis. Mol Plant 14(5): 805-819. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Plant Science > Plant cell biology > Organelle isolation Plant Science > Plant biochemistry > Other compound Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols A Quick Method to Quantify Iron in Arabidopsis Seedlings Chandan Kumar Gautam [...] Wolfgang Schmidt Mar 5, 2022 2603 Views Sorghum bicolor Extracellular Vesicle Isolation, Labeling, and Correlative Light and Electron Microscopy Deji Adekanye [...] Jeffrey L. Caplan Oct 5, 2024 275 Views Optimized Isolation of Lysosome-Related Organelles from Stationary Phase and Iron-Overloaded Chlamydomonas reinhardtii Cells Jiling Li and Huan Long Nov 20, 2024 178 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,626	https://bio-protocol.org/en/bpdetail?id=4626&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Assessment of Oxidative Stress Biomarkers in Rat Blood YS Yuri K. Sinzato TR Tiago Rodrigues LC Larissa L. Cruz VB Vinícius S. Barco MS Maysa R. Souza GV Gustavo T. Volpato DD Débora C. Damasceno Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4626 Views: 1024 Reviewed by: Vivien Jane Coulson-ThomasSudhir VermaXiao Lin Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Frontiers in Cell and Developmental Biology May 2022 Abstract Redox status assessments are time-consuming, require a large volume of samples and great reagent amounts, and are not adequately described for methodological reproducibility. Here, the objective was to standardize redox balance determination, based on previously described spectrophotometric tests in pregnant rats, to improve precision, time dispensed, and the volume of samples and reagents, while maintaining accuracy and adequate cost benefits. This protocol summarizes oxidative stress markers, which focus on spectrophotometric tests for the assessment of thiobarbituric acid–reactive substances, reduced thiol groups, and hydrogen peroxide, as well as the antioxidant activity of superoxide dismutase, glutathione peroxidase, and catalase in washed erythrocyte and serum samples from full-term pregnant rats. For non-pregnant rats and other species, it is necessary to standardize these determinations, especially the sample volume. All measurements were normalized by the estimated protein concentrations in each sample. To establish optimum conditions for the reproducibility of the proposed methods, we describe all changes made in each assay’s steps based on the reference method reassessed for the new standardizations. Furthermore, the calculations of the concentrations or activities of each marker are presented. Thus, we demonstrate that the analysis of serum samples is easier and faster, but it is impossible to detect catalase activity. Furthermore, the proposed methods can be applied for redox balance determination, especially using smaller reagent amounts and lower sample volumes in lesser time without losing accuracy, as is required in obtaining samples during rat pregnancy. Keywords: Oxidative stress Lipid peroxidation Biomarker Redox status Pregnancy Background Reactive oxygen species (ROS) are free radicals; their non-radical intermediates, produced in a limited amount in response to physiological stimuli, mediate inter- and intracellular signaling (Fujii et al., 2005). Hydroxyl radicals are free radicals that are capable of causing lipid peroxidation in the plasma membrane or in organelles that contain large quantities of polyunsaturated fatty acid side chains (Burton and Jauniaux, 2011). Lipid peroxidation is mostly assessed by measuring thiobarbituric acid–reactive substance (TBARS) concentration (De Souza et al., 2010). Furthermore, amino acids are also a target for oxidative damage. The extraction of hydrogen ions from the thiol group of cysteine can form disulfide bonds and abnormal protein folding, which can lead to functional impairment and protein aggregation (Burton and Jauniaux, 2011). To assess reduced thiol concentrations, the determination of the content of reduced sulfhydryl groups (-SH) provides useful and early indications of antioxidant capacity and structural and functional alterations in the cell membrane (Kumar and Maurya, 2013). Another oxygen-free radical is the superoxide anion (O2-), which is mainly generated by mitochondria and the endoplasmic reticulum (Burton and Jauniaux, 2011). The superoxide anion is detoxified by superoxide dismutase (SOD) enzymes, which convert O2- to hydrogen peroxide (H2O2). Hydrogen peroxide is not a free radical, making it less reactive than the superoxide anion. However, it comes under the term ROS as it is intimately involved in the generation and detoxification of free radicals. H2O2is, in turn, detoxified to water by the enzymes catalase (CAT) and glutathione peroxidase (GSH-Px) (Burton and Jauniaux, 2011). An imbalance between ROS production and antioxidant defenses causes oxidative stress, which might damage macromolecular classes (lipids, protein, and DNA), leading to loss of function and even cell death (Burton and Jauniaux, 2011; Birben et al., 2012). The redox status is an important measurement to evaluate pathophysiological mechanisms involved in several disorders in pregnancy (Agarwal et al., 2005 and 2012). Several methods to assess redox status in reproductive processes have been reported, but the type of sample and techniques employed for oxidative stress measurement have yielded conflicting results that hinder comparisons. Most of these studies have measured total antioxidant capacity (TAC) (Pisoschi and Negulescu, 2012). However, the measurement of single components or TAC alone may not be a reliable indicator of oxidative stress under physiological and pathological conditions. Investigating the scavenging activity of antioxidant enzymes such as SOD, GSH-Px, and CAT might be very useful, as the target oxygen radicals of each of these enzymes would be known. Techniques such as spectrophotometry, fluorimetry, and high-performance liquid chromatography have been used for assessing oxidative stress and require a large sample volume and great reagent amounts, along with being very time-consuming (Wang and Joseph, 1999; Del Rio et al., 2005; Ćebović et al., 2013). Spectrophotometry, however, seems to be the most easily available and cost-effective technique. Other studies must be performed to develop more accurate and low-cost techniques that are relevant for oxidative stress evaluation. Furthermore, investigators need blood samples for the determination of different biomarkers during pregnancy in rats. However, there are many methodologies that use a large volume for each marker, making it difficult to determine the different biomarkers in the same animal. Therefore, our objective was to standardize the redox balance determination based on previously described spectrophotometric tests in pregnant Wistar/Sprague-Dawley rats, to improve precision, time dispensed, and the volume of samples and reagents concentrations, while maintaining accuracy and adequate cost-benefits. Additionally, sample collection and processing steps are described in detail to provide reproducibility. Materials and Reagents Paper towel (Garden®, catalog number: 6610) 50 × 50 cm filter paper (Qualy®, catalog number: 1282) Laboratory film (Parafilm®, catalog number: PM-996) 1.5 mL microtube (Eppendorf®, catalog number: 0030120086) 2.0 mL microtube (Eppendorf®, catalog number: 0030120094) 0.1–10 μL tip (Eppendorf®, catalog number: 0030000838) 2–200 μL tip (Eppendorf®, catalog number: 0030000889) 50–1,000 μL tip (Eppendorf®, catalog number: 0030000927) 96-well microplate (Corning®, catalog number: 3595) Quartz cuvettes (Daigger Scientific®, catalog number: EF22153L/FX22153L) 10 mL volumetric flask (ISO: 3819, Laborglas®, catalog number: 9110608) 50 mL volumetric flask (ISO: 3819, Laborglas®, catalog number: 9110617) 100 mL volumetric flask (ISO: 3819, Laborglas®, catalog number: 9110624) 250 mL volumetric flask (ISO: 3819, Laborglas®, catalog number: 9110636) 600 mL volumetric flask (ISO: 3819, Laborglas®, catalog number: 9110648) 1,000 mL volumetric flask (ISO: 3819, Laborglas®, catalog number: 910654) 10 mL beaker (ISO: 4788, Laborglas®, catalog number: 9139608) 100 mL beaker (ISO: 4788, Laborglas®, catalog number: 9139624) 250 mL beaker (ISO: 4788, Laborglas®, catalog number: 9139636) 500 mL beaker (ISO: 4788, Laborglas®, catalog number: 9139744) 1,000 mL beaker (ISO: 4788, Laborglas®, catalog number: 9139754) Glass sticker (Laborglas®, catalog number: 9108805) 1,000 mL Amber bottle (Laborglas®, catalog number: 91806545) Purified water 2-mercaptoethanol (purity ≥99%) (Sigma-Aldrich®, catalog number: 8057400250) 2-thiobarbituric acid (TBA, purity ≥98%) (Sigma-Aldrich®, catalog number: T5500) 5,5-dithiobis (2-nitro-benzoic acid) (DTNB, purity ≥98%) (Sigma-Aldrich®, catalog number: D8130) 5-sulfosalicylic acid hydrate (purity ≥95%) (Sigma-Aldrich®, catalog number: 390275) Bovine serum albumin (BSA) (Sigma-Aldrich®, catalog number: A-4503) Calcium chloride (CaCl2, A.C.S.) (Sigma-Aldrich®, catalog number: C1016) Ethylenediaminetetraacetic acid (EDTA, purity ≥99%) (Sigma-Aldrich®, catalog number: S26-36) Glutathione reductase (GSH-Rd, 100–300 units/mg protein) (Sigma-Aldrich®, catalog number: G3664) Horseradish peroxidase (HRP) (25,000 units) (Sigma-Aldrich®, catalog number: P8250) L-glutathione reduced (GSH, purity 98%) (Sigma-Aldrich®, catalog number: G4251) Magnesium chloride (MgCl2, A.C.S.) (Sigma-Aldrich®, catalog number: 208337) Phenol red (Sigma-Aldrich®, catalog number: 114529) Phosphate buffered saline (PBS, 0.138 M NaCl, 0.0027 M KCl, pH 7.4) (Sigma-Aldrich®, catalog number: P3813) Potassium cyanide (purity ≥97%) (Sigma-Aldrich®, catalog number: 31252) Potassium ferricyanide (purity ≥99%) (Sigma-Aldrich®, catalog number: 702587) Sodium hydroxide (NaOH, purity ≥97%) (Sigma-Aldrich®, catalog number: 221465) Sodium phosphate monobasic monohydrate (NaH2PO4·H2O, A.C.S.) (Sigma-Aldrich®, catalog number: S9638) T -butyl hydroperoxide 70% aqueous solution (Sigma-Aldrich®, catalog number: B2633) β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH, purity ≥93%) (Sigma-Aldrich®, catalog number: N1630) Protein assay dye reagent concentrate (Bio-Rad®, catalog number: 500-0006) TRIS-UltrapureTM (purity ≥99.9%) (Invitrogen®, catalog number: 15504-020) Hydrochloric acid (HCl, A.C.S., purity 36.5%–38%) (Synth®, catalog number: A1028.01.BJ) Pyrogallic acid (purity ≥99.9%) (Synth®, catalog number: A1052.01.AE) Di-potassium hydrogen phosphate trihydrate (K2HPO4 , purity ≥99%) (Merck®, catalog number: AO150099.010) Ethyl alcohol (A.C.S., purity 99.5%) (Merck®, catalog number: 108543) Sodium bicarbonate (NaHCO3 , purity ≥99.5%) (Merck®, catalog number: 6329) Potassium chloride (KCl, A.C.S.) (Vetec®, catalog number: 104) Sodium chloride (NaCl, A.C.S.) (Vetec®, catalog number: V003132) Hydrogen peroxide (H2O2, A.C.S., purity 35%) (Dinâmica®, catalog number: 1857) Potassium dihydrogen phosphate (KH2PO4, A.C.S.) (Dinâmica®, catalog number: P.10.0513.009.00) Sodium phosphate dibasic (Na2HPO4, A.C.S.) (Dinâmica®, catalog number: P.10.0513.012.00) Stabilizing solution (2.7 mM EDTA and 0.7 mM 2-mercaptoethanol) (see Recipes) Drabkin’s solution (see Recipes) 1 μg/μL stock solution (see Recipes) 3% (w/v) 5-sulfosalicylic acid hydrate (for 100 samples) (see Recipes) Thiobarbituric acid solution (TBA) 0.67% (for 200 samples) (see Recipes) 1 M HCl (see Recipes) 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 (for 250 duplicate sample analysis) (see Recipes) 10 mM DTNB (for 250 duplicate washed erythrocytes samples and 100 duplicate serum samples) (see Recipes) Solution 1: 1.71 M NaCl, 34 mM KCl, 14 mM K2HPO4, 0.1 M Na2HPO4 (see Recipes) Solution 2: 90 mM CaCl2 (see Recipes) Solution 3: 0.11 M MgCl2 (see Recipes) Solution 4: 0.1% phenol red (see Recipes) Phosphate buffer solution A (see Recipes) Peroxidase solution (HRP) (see Recipes) Buffer solution b (see Recipes) 1 M NaOH (see Recipes) 1 M TRIS/HCl 5 mM EDTA pH 8.0 (for 150 duplicate sample analysis) (see Recipes) 10 mM pyrogallol solution (for 250 duplicate sample analysis) (see Recipes) 50 mM TRIS/HCl 5 mM EDTA (for 500 duplicate sample analysis) (see Recipes) 1% NaHCO3 and 1 mM EDTA (see Recipes) 10 mM HCl solution (see Recipes) 0.1 M glutathione reduced (GSH) (for 60 duplicate sample analysis) (see Recipes) 10 U/mL glutathione reductase (GSH-Rd) (for 60 duplicate sample analysis) (see Recipes) 7 mM t -butyl hydroxiperoxide (for 125 duplicate sample analysis) (see Recipes) 4 mM NADPH (for 60 duplicate sample analysis) (see Recipes) 0.1 M NaH2PO4·H2O (for 200 samples analysis) (see Recipes) 1 M hydrogen peroxide (H2O2) (see Recipes) Equipment 0.1–2.5 μL micropipette (Eppendorf®, catalog number: 4924000010) 0.5–10 μL micropipette (Eppendorf®, catalog number: 4924000029) 2–20 μL micropipette (Eppendorf®, catalog number: 4924000045) 10–100 μL micropipette (Eppendorf®, catalog number: 4924000053) 20–200 μL micropipette (Eppendorf®, catalog number: 4924000061) 100–1,000 μL micropipette (Eppendorf®, catalog number: 4924000088) Centrifuge (Eppendorf®, model: 5804R) Water purifier (GEHAKA®, Master System) Microplate reader (Biotek®, Power Wave XS) Spectrophotometer (Shimadzu®, model: UV1800) Digital microplate mixer (IKA®, model: MS3) Water bath (Quimis®, model: Q334M-24) Analytical balance (Denver Instrument®, model: APX200) -80 °C ultra-freezer (ColdLab®, model: CL200-86V) Refrigerator (Electrolux®, model: RDE 32) Vortex agitator mixer (Quimis®, model: G220) Magnetic agitator (Quimis®, model: Q2461-22) pH meter (Tecnal®, model: Tec05) Autoclave (Ecel®, model: EC 21D) Fume hood (Permution®, model: NBR 7094) Software Gen5 (Bio Tek®) UVProbe (Shimadzu®) Statistica (StatSoft®) Microsoft Office 365 (Microsoft®) Procedure Sample collection Collect whole blood samples from pregnant rats at term through a cut at the end of the tail in both dry/free of anticoagulant (approximately 5.0–9.0 mL of blood) and heparinized (approximately 1.5 mL of blood in 0.2 mL of heparin) tubes. These volumes of blood are sufficient to process samples for oxidant and antioxidant biomarker measurements. Store under refrigeration at 4–8 °C for 30 min after collection. Note: The samples cannot be stored on wet ice. Use the heparinized tubes to obtain washed erythrocyte samples (Figure 1): Figure 1. Procedures to obtain the washed erythrocytes. Created with BioRender.com. Centrifuge the blood samples in heparinized tubes at 185 × g for 10 min at 4 °C. Discard the plasma (light yellow supernatant) and the buffy coat (leukocytes and platelets, translucid supernatant). Notes: 1) If the phases do not separate appropriately, repeat centrifuging. If the phases do not separate again, discard the sample. 2) If there is high lipid concentration in the samples, the plasma color will be white. Add 2 mL of PBS 1× (pH 7.4) to the erythrocyte samples for washing. Centrifuge at 1,575 × g for 1 min at 4 °C. Discard the supernatant (translucid supernatant). Repeat procedures A3c–e three times. Identify three microtubes for Hb, TBARS, and SH level measurements (Figure 2): i) Pipette 50 μL of washed erythrocyte samples (pellet obtained from previous steps) directly in each microtube (three in total) for later measurements of Hb, TBARS, and SH levels. ii) Add 950 μL of purified water in each microtube containing 50 μL of the washed erythrocyte. iii) Gently homogenize the samples by inversion. iv) Store aliquots at -80 °C until assays (stable for up to six months). Figure 2. Aliquots for Hb, TBARS, and SH level measurements in the washed erythrocytes using purified water. Created with BioRender.com. Identify five microtubes for Hb, SOD, H2O2, GSH-Px, and CAT level measurements (Figure 3): i) Pipette 50 μL of the washed erythrocyte samples (pellet obtained from previous steps) directly in each microtube (five in total) for activity measurements of Hb, SOD, H2O2 GSH-Px, and CAT. ii) Add 950 μL of the stabilizing solution (see Recipe 1) in each microtube. iii) Gently homogenize the samples by inversion. iv) Store the aliquots at -80 °C until assays (stable for up to six months). Figure 3. Aliquots for Hb, SOD, H2O2, GSH-Px, and CAT level measurements in the washed erythrocytes using a stabilizing solution. Created with BioRender.com. Serum sample collection in dry/free anticoagulant tubes (mentioned in step 1,Figure 4): Centrifuge the blood samples in dry tubes at 1,575 × g for 10 min at 4 °C. Collet the serum using a micropipette and put it in another tube. Next, discard the pellet. Pipette the serum aliquots directly into six microtubes for protein quantification (20 μL), TBARS (550 μL), SH (10 μL), H2O2 (20 μL), SOD (5 μL), and GSH-Px (5 μL) using specific identification in each microtube. Store at -80 °C until analysis (stable for up to six months). Figure 4. Procedures to obtain the serum. Created with BioRender.com. Hemoglobin (Hb) quantification in the washed erythrocytes Take identified microtubes with Hb [diluted with purified water (step A3g) and stabilizing solution (step A3h)]. Note: Hemoglobin concentration measurements are similar in purified water and/or stabilizing solution because they are necessary for the determination of the normalization of oxidant and antioxidant concentrations. Thus, two microplates will be used for Hb determination (one for the purified water and another for the stabilizing solution). Homogenize the samples in the vortex agitator mixer. Pipette 200 μL of Drabkin’s solution (see Recipe 2) in two wells of the microplate as a blank. Pipette 197.5 μL of Drabkin’s solution and 2.5 μL of samples (washed erythrocytes) in each well of the microplate (in duplicates). Cover the microplate and mix using a microplate mixer for 1 min at 750 rpm for homogenization. Rest the microplate for 10 min at 25 °C. Lightly tap the sides of the microplate several times to gently mix. Measure the absorbance at a wavelength of 546 nm using Gen5 software. Calculate Hb concentrations (see Data analysis). Quantification of protein concentrations in serum samples by Bradford’s method (Bradford, 1976) Dilute one part of the serum samples in 100 parts of purified water in a microtube. Note: Other dilutions can be tested according to the experiment. Identify six microtubes for the BSA solutions: 0, 0.1, 0.2, 0.4, 0.8, and 1.0 μg/μL. Prepare the BSA solutions for a standard curve setting from the 1 μg/μL stock solution (see Recipe 3), pipetting the following volumes of BSA and purified water: Concentration (µg/µL) Stock solution (µL) Purified water (µL) 0 0 50 0.1 5 45 0.2 10 40 0.4 20 30 0.8 40 10 1.0 50 0 Dilute the protein assay dye reagent concentrate in a ratio of one part of the reagent to four parts of purified water to prepare Bradford’s reagent. For example: dilute 10 mL of the protein assay dye reagent concentrate in 40 mL of purified water. Note: Calculate the quantity for Bradford’s reagent according to the number of wells that will be used. Pipette 190 μL of Bradford’s reagent into each well of the microplate (standard curve and sample wells). Pipette 10 μL of each BSA solution into the standard curve wells (in duplicates). Homogenize with the tip. Pipette 10 μL of the serum samples into other wells (in duplicates). Homogenize with the tip. Measure the absorbance at a wavelength of 595 nm using Gen5 software. Calculate total protein concentrations (see Data analysis). Thiobarbituric acid reactive substances (TBARS) Prepare 3% (w/v) 5-sulfosalicylic acid hydrate (see Recipe 4) and TBA 0.67% (see Recipe 5) solutions. Add 500 μL of serum or washed erythrocytes to a test microtube containing 500 μL of 3% 5-sulfosalicylic acid hydrate. Vortex the microtube for 10 s. Centrifuge the microtube at 18,000 × g for 3 min at 4 °C. Rest the microtube for 15 min at 25 °C. Pipette the supernatant and store it in new microtubes. Note: Avoid pipetting the brown granules. Add 500 μL of purified water in a tube as a blank. Add 500 μL of the supernatant to 500 μL of the TBA solution in a tube for each sample. Vortex the tubes for 10 s. Heat the mixtures at 95 °C for 30 min in a water bath with the tubes partially sealed. Cool on ice for 10 min to stop further reactions. Do not exceed 30 min for the next steps (a–e): Equilibrate the blanks and samples at 25 °C. Pipette 300 μL of purified water in two wells as a blank. Pipette 300 μL of the samples into each well of the microplate (in duplicates). Lightly tap the sides of the microplate several times to gently mix. Measure the absorbance at a wavelength of 535 nm using Gen5 software. Calculate TBARS concentrations (see Data analysis). Reduced thiol groups (-SH) Prepare 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 solution (see Recipe 7). Prepare the 10 mM DTNB solution (see Recipe 8). For the -SH quantification in the washed erythrocytes: Pipette 200 μL of 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 in two wells as a blank. Pipette 195 μL of 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 solution into other wells (in duplicates). Pipette 1.0 μL of the washed erythrocyte (in duplicates except in blank wells). Pipette 4.0 μL of the 10 mM DTNB solution into the sample wells (in duplicates). Cover the microplate and mix using a microplate mixer for 10 min at 750 rpm for homogenization. Measure the absorbance at a wavelength of 412 nm. For the -SH serum quantification: Pipette 200 μL of 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 in two wells as a blank. Pipette 175 μL of 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 solution into other wells (in duplicates). Pipette 5.0 μL of serum (in duplicates except in blank wells). Pipette 20 μL of the 10 mM DTNB solution into the sample wells (in duplicates). Mix the microplate using a microplate mixer at 750 rpm for 10 min. Measure the absorbance at a wavelength of 412 nm using Gen5 software. Calculate -SH concentrations (see Data analysis). Hydrogen peroxide (H2O2) Prepare the solutions below: Solution 1 (see Recipe 9). Solution 2 (see Recipe 10). Solution 3 (see Recipe 11). Solution 4 (see Recipe 12). Phosphate buffer solution A (see Recipe 13). Peroxidase solution (HRP) (see Recipe 14). Prepare buffer solution B (see Recipe 15). Pipette 110 μL of the buffer solution B into two wells of the microplate as a blank. For the H2O2 quantification in the washed erythrocytes: Pipette 5 μL of the washed erythrocyte sample into other wells (in duplicates). Pipette 105 μL of the buffer solution B into the sample wells. Mix the microplate using a microplate mixer at 750 rpm for 10 s. Incubate the microplate at 37 °C for 10 min. Pipette 10 μL of 1 M NaOH (see Recipe 16) to stop the reaction. Measure the absorbance at a wavelength of 620 nm. For the H2O2 quantification in the serum samples: Pipette 10 μL of the serum sample into other wells (in duplicates). Pipette 100 μL of the buffer solution B into the sample wells. Mix the microplate using a microplate mixer at 750 rpm for 10 s. Incubate the microplate at 37 °C for 10 min. Pipette 10 μL of 1 M NaOH to stop the reaction. Measure the absorbance at a wavelength of 620 nm using Gen5 software. Calculate H2O2 concentrations (see Data analysis). Superoxide dismutase (SOD) Prepare 1 M TRIS/HCl 5 mM EDTA pH 8.0 solution (see Recipe 17). Prepare 10 mM pyrogallol solution (see Recipe 18). Pipette 147 μL of 1 M TRIS/HCl 5 mM EDTA pH 8.0 solution into two wells of the microplate as a blank. Pipette 144 μL of 1 M TRIS/HCl 5 mM EDTA pH 8.0 into other wells (in duplicates). Pipette 3.0 μL of the washed erythrocyte/serum (in duplicates except in the blank wells). Lightly tap the sides of the microplate several times to gently mix. Add 3.0 μL of 10 mM pyrogallol solution into all wells including the blank (30 s for pipetting the solution in all microplates). Lightly tap the sides of the microplate several times to gently mix. Measure the absorbance during the time intervals of 0 (T0) and 10 (T10) min at 25 °C at a wavelength of 420 nm. Calculate SOD concentrations using Gen5 software (see Data analysis). Glutathione peroxidase (GSH-Px or GPx) Prepare the solutions below: Note: All solutions (except 50 mM TRIS/HCl 5 mM EDTA pH 7.6 and 7 mM t-butyl hydroperoxide) and reagents must be kept on ice until the moment of the assay. 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 solution (see Recipe 7). 50 mM TRIS/HCl 5 mM EDTA pH 7.6 solution (see Recipe 19). 1% (w/v) NaHCO3 and 1 mM EDTA solution (see Recipe 20). 10 mM HCl solution (see Recipe 21). 0.1 M glutathione reduced (GSH) solution (see Recipe 22). 10 U/mL glutathione reductase (GSH-Rd) solution (see Recipe 23). 7 mM t-butyl hydroperoxide solution (see Recipe 24). 4 mM NADPH solution (see Recipe 25). Prepare a MIX solution containing: Note: The MIX solution must be prepared immediately before the assay. It is recommended to trigger the reaction in up to 16 duplicate samples. If you prepare more than 16 duplicate samples, the results will be different because of the long time between the reactions. 240 μL of the 0.1 M GSH solution. 1,200 μL of the 10 U/mL GSH-Rd solution. 1,200 μL of the 4 mM NADPH. Keep this MIX solution on ice. Pipette 100 μL of the 50 mM TRIS/HCl 5 mM EDTA pH 7.6 solution in all wells of the microplate. Pipette 1.0 μL of the serum or 0.5 μL of the washed erythrocytes into all wells (in duplicates). Lightly tap the sides of the microplate several times to gently mix. Pipette 50 μL of the MIX solution into all wells. Lightly tap the sides of the microplate several times to gently mix. Pipette 20 μL of the 7 mM t-butyl hydroperoxide solution into all wells (to initiate a reaction). Note: It takes 15 s to pipette the 7 mM t-butyl hydroperoxide solution into the sample wells and trigger the reaction in up to 16 duplicate samples. Quickly homogenize the microplate. Measure the absorbance every one-minute interval for 5 min at 25 °C in a microplate at a wavelength of 340 nm using Gen5 software. Calculate GSH-Px concentrations (see Data analysis). Catalase (CAT) Prepare 0.1 M NaH2PO4·H2O solution (see Recipe 26). Prepare 1 M H2O2 solution (see Recipe 27). Verify hydrogen peroxide molarity: Pipette 1,000 μL of the 0.1 M NaH2PO4·H2O solution into a quartz cuvette (cuvette 1) as a blank in the reference position. Pipette 990 μL of the 0.1 M NaH2PO4·H2O solution into another quartz cuvette (cuvette 2) in the reading position. Select AUTO ZERO in a UV1800 spectrophotometer for calibration. Wait until the absorbance reaches zero. Keep the cuvette 1 in the reference position. Add 10 μL of 1 M H2O2 into cuvette 2. Quickly homogenize. Return cuvette 2 to the reading position. Measure the absorbance at a wavelength of 240 nm. Calculate the hydrogen peroxide real molarity (see Data Analysis and Notes). Wash both cuvettes. Pipette 999 μL of the 0.1M NaH2PO4·H2O solution with 1.0 μL of washed erythrocytes into a quartz cuvette (cuvette 1) as a blank (in the reference position). Pipette 978 μL of the 0.1M NaH2PO4·H2O solution with 1.0 μL of the washed erythrocytes into another quartz cuvette (cuvette 2) in the reading position. Select AUTO ZERO in a UV1800 spectrophotometer. Wait until the absorbance reaches zero. Add 21 μL of 1 M H2O2 into the quartz cuvette 2 for a quick homogenization. Perform the kinetics measurements for 2 min at 25 °C in a UV1800 spectrophotometer at 240 nm using UVProbe software. Calculate CAT concentrations (see Data analysis). Data analysis To ensure sample homogeneity, all measurements were adjusted according to the protein concentration estimated in each sample (hemoglobin and total protein for erythrocytes and serum, respectively). Assays To determine the minimum sample volume and reagent concentrations required to reproduce each reference method in pregnant rats at term, decreasing amounts of reagents were tested until the results obtained were equal to the reference values of other articles established in the literature. All samples were analyzed in duplicates at 25 °C. Hemoglobin Absorbance, expressed in grams (g) of hemoglobin per deciliter (dL), was measured at 546 nm (Tentori and Salvati, 1981) and calculated using the extinction coefficient of the complex cyanmethemoglobin (hemoglobin-cyanide) monomer at 546 nm (11.0 mM -1 cm -1) (Assendelft and Zijlstra, 1975) and the pathlength (0.6 cm) of the solution in the well, using the Gen5 software. Hb = hemoglobin concentration in g/dL A = mean sample absorbance – blank absorbance 80 = dilution factor (Drabkin’s solution volume/sample volume) 16114.5 = hemoglobin monomer molar mass (complex cyanmethemoglobin–hemoglobin–cyanide monomer at 546 nm) 10 = conversion from L to dL 1000 = conversion from mg to g Total protein concentration Total protein absorbance values were determined by Bradford’s method (1976) for the normalization of oxidative stress biomarkers using Gen5 software. The total protein concentration is determined by the linear equation obtained from the standard curve. Standard curve is an increasing or decreasing succession of points obtained from the relationship between the concentration of the standard species and its signal intensity from the detection system. The most important part of the graph is the quadratic equation, from which the concentration can be calculated. The graph also provides the value of R2 , which is the value of the angular coefficient. The closer to 1 the value of R is, the better is the line described by the linear regression of the points aiming to obtain lines ≥0.99. Quantification requires knowing the dependence between the measured response and the concentration of the analyte. Linearity is obtained by internal or external standardization and formulated as a mathematical expression used to calculate the concentration of the analyte to be determined in the real sample. The equation of the line that relates the two variables is: y=ax+b y = measured response (absorbance) x = concentration a = slope of the analytical curve = sensitivity b = intersection with the y axis when x = 0 To calculate the linear equation from the standard curve in Microsoft Office Excel®, follow the steps depicted in Figures 5–8: Figure 5. Steps 1, 2, and 3 to calculate the line equation from the standard curve Figure 6. Step 4 to calculate the line equation from the standard curve Figure 7. Step 5 to calculate the line equation from the standard curve Figure 8. Steps 6 and 7 to calculate the line equation from the standard curve y = measured response (absorbance) x = total protein concentration in μg/μL 457.94 = slope of the analytical curve = sensitivity 127.53 = intersection with the y axis when x = 0 0.9978 = angular coefficient Note: If R2 value < 0.98, repeat the standard curve measurements. To calculate total protein concentration in serum samples, follow the example below: Considering an absorbance of 500 of the serum sample, the calculation is: y=457.94x+127.53 500=457.94x+127.53 x=(500-127.53)/457.94 x = 0.81 μg/μL of total protein in the sample. To convert μg/μL to g/dL, divide the concentration value by 10. Note: If the absorbance value of the serum sample is higher than the absorbance of the 1.0 BSA solution concentration (standard curve), a dilution of 1:2 with purified water must be performed in the serum sample and the absorbance must be measured again. Then, the total protein concentration value (g/dL) must be multiplied by the dilution factor (in this case, multiplied by 2). Thiobarbituric acid reactive substances (TBARS) Absorbance was measured at 535 nm using Gen5 software. Results were expressed as nM of TBARS per milligram of Hb or total protein (nM/mg Hb or total protein) in washed erythrocyte or serum samples, respectively. The MDA-TBA complex extinction coefficient of 1.56 × 105 M-1 cm-1 at 25 ºC (Buege and Aust, 1978) and 0.9 cm pathlength were used for the calculations. TBARS = Thiobarbituric acid reactive substance concentration in nM/mg Hb or total protein A = mean sample absorbance – blank absorbance 2 = sample dilution factor 109 = conversion from M to nM Hb = concentration obtained by Drabkin’s solution expressed as g/dL Total protein (PTN) = as PTN concentration obtained by Bradford’s method is expressed in μg/μL, it is necessary to divide this by 10 to convert to g/dL Follow the steps below to obtain Hb or PTN mass (mg): To convert g/dL (Hb or PTN concentrations) to mg/mL, it is necessary to multiply by 10 (g to mg and dL to mL). In addition, the used volume (0.5 mL) must be inserted by multiplication after the last conversion. Finally, the total conversion is: Hb or PTN concentration (g/dL × 10) = Hb or PTN concentration (mg/mL × 0.5 mL) = Hb or PTN mass (mg) Reduced thiol groups (-SH) Absorbance was measured at 412 nm using Gen5 software. The results were expressed as mM of SH per mg Hb or total protein (mM/mg of Hb or total protein). The 5-thio-2-nitrobenzoic acid (TNB) extinction coefficient (14150 M-1 cm-1 at 25 °C) Riddles et al., 1983) and a 0.6 cm pathlength were used in the calculations. SH = concentration in mM/mg Hb or total protein A = mean sample absorbance – blank absorbance 103 = conversion from M to mM Hb = concentration obtained by Drabkin’s solution expressed as g/dL PTN = as PTN concentration obtained by Bradford’s method is expressed as μg/μL, it is necessary to divide this by 10 to convert to g/dL Follow the steps below to obtain Hb or PTN mass (mg): To convert g/dL (Hb or PTN concentrations) to mg/mL, it is necessary to multiply by 10 (g to mg and dL to mL). In addition, the sample volume used in the assay (mL) must be inserted by multiplication after the last conversion Finally, the total conversion is: Hb or PTN concentration (g/dL × 10) = Hb or PTN concentration (mg/mL × sample volume used in the assay) = Hb or PTN mass (mg) Hydrogen peroxide (H2O2) Absorbance was measured at a wavelength of 620 nm using Gen5 software. The results were expressed as µM of H2O2 per mg of hemoglobin or total protein (µM/mg Hb or total protein). The extinction coefficient of phenol red oxidation by H2O2 was calculated from an adjusted standard curve in our laboratory at 37 °C (0.0296 µM-1 cm-1 ) and 0.33 cm pathlength. H2O2 = concentration in µM/mg Hb or total protein A = mean sample absorbance – blank absorbance Hb = concentration obtained by Drabkin’s solution expressed as g/dL PTN = as PTN concentration obtained by Bradford’s method is expressed as μg/μL, it is necessary to divide this by 10 to convert to g/dL Follow the steps below to obtain Hb or PTN mass (mg): To convert g/dL (Hb or PTN concentrations) to mg/mL, it is necessary to multiply by 10 (g to mg and dL to mL). In addition, the sample volume used in the assay (mL) must be inserted by multiplication after the last conversion. Finally, the total conversion is: Hb or PTN concentration (g/dL × 10) = Hb or PTN concentration (mg/mL × sample volume used in the assay) = Hb or PTN mass (mg) Superoxide dismutase (SOD) Absorbance was immediately measured during the time intervals of 0 (T0) and 10 (T10) min at 25 °C in a microplate at 420 nm using Gen5 software. For the calculations, the absorbances of the blanks and samples at times T0 and T10 were used. The activity of SOD is calculated indirectly by the percentage of inhibition of pyrogallol by the sample. Therefore, the calculations were made following these steps: Calculation of the percentage of inhibition of the pyrogallol autoxidation by the SOD samples: x% = percentage of pyrogallol autoxidation by SOD sample Thus, the inhibition of pyrogallol autoxidation is equal to 100 – x%. Calculation of SOD activity considering that 1 IU is the volume capable of inhibiting 50% of the pyrogallol oxidation (Marklund and Marklund, 1974): y = sample volume (mL) that inhibits 50% of pyrogallol autoxidation 50% = percentage inhibition of pyrogallol that equals 1 U of SOD 0.003 = volume (mL) of the sample in the assay 100-x% = percentage of pyrogallol inhibition by the sample SOD enzymatic activity: [SOD] = enzymatic activity in IU/mg Hb Y = sample volume (mL) that inhibits 50% of pyrogallol autoxidation 100 = conversion factor from dL to mL referring to the value of Hb (1 dL = 100 mL) Hb = concentration obtained by Drabkin’s solution expressed as g/dL PTN = as PTN concentration obtained by Bradford’s method is expressed as μg/μL, it is necessary to divide this by 10 to convert to g/dL Follow the steps below to obtain Hb or PTN mass (mg): To convert g/dL (Hb or PTN concentrations) to mg/mL, it is necessary to multiply by 10 (g to mg and dL to mL). In addition, the sample volume used in the assay (mL) must be inserted by multiplication after the last conversion. Finally, the total conversion is: Hb or PTN concentration (g/dL × 10) = Hb or PTN concentration (mg/mL × sample volume used in the assay) = Hb or PTN mass (mg) Glutathione peroxidase (GSH-Px) Absorbance change (ΔA340) per min was calculated using Gen5 software and the results were expressed as mM of GSH-Px/min/mg Hb or total protein. The NADPH extinction coefficient (6.220 M -1 cm-1 at 25 °C) (Fruscione et al., 2008) and 0.5 cm pathlength were used for calculations. GSH-Px = activity in mM/min/mg Hb or total protein ΔA340/min = mean delta absorbance 103 = conversion from M to mM Hb = concentration obtained by Drabkin’s solution expressed as g/dL PTN = as PTN concentration obtained by Bradford’s method is expressed as μg/μL, it is necessary to divide this by 10 to convert to g/dL Follow the steps below to obtain Hb or PTN mass (mg): To convert g/dL (Hb or PTN concentrations) to mg/mL, it is necessary to multiply by 10 (g to mg and dL to mL). In addition, the sample volume used in the assay (mL) must be inserted by multiplication after the last conversion. Finally, the total conversion is: Hb or PTN concentration (g/dL × 10) = Hb or PTN concentration (mg/mL × sample volume used in the assay) = Hb or PTN mass (mg) Catalase (CAT) Hydrogen peroxide molarity: After verifying the H2O2 absorbance, perform the equation to know the solution molarity. For example, the H2O2 absorbance was 0.416 and the molar absorptivity of H2O2 at 25 °C is 43.6. Therefore: C=A/ε → C=0.416/43.6 → C = 0.0095 M = 9.5 mM C = concentration (mMol/L) A = Absorbance E = Molar absorptivity (M-1) Thus, although the solution concentration in the cuvette should be 10 mM (theoretical value), it is instead 9.5 mM. To correct the solution concentration, perform the following equation: C1 × V1 = C2 × V2 C 1 = Real concentration V1 = Volume to be pipetted C2 = Concentration of the prepared H2O2 solution V2 = Volume to be pipetted in the cuvette 2 9.5 mM × V1 = 10 mM × 10 µL V1 = 10.5 µL – volume that must be pipetted in the cuvette to obtain a final solution at 10 mM. In the assay, the final concentration of H2O2 must be 20 mM. Therefore, the volume of the solution that will be pipetted in the cuvette will be 21 µL ( V1 × 2). CAT activity Absorbance change per min was calculated using UVProbe software from UV1800 spectrophotometer and the results expressed as mM of CAT/min/mg Hb (mM/min/mg Hb). The H2O2 extinction coefficient (43.6 M-1 cm-1 at 25 °C) (Noble and Gibson, 1970) and a 1.0 cm pathlength were used for the calculations. CAT = activity in mM/min/mg of Hb = ∆A240/min mean delta absorbance 103 = conversion from mol to mmol Hb = concentration obtained by Drabkin’s solution expressed as g/dL Follow the steps below to obtain Hb mass (mg): To convert g/dL (Hb concentration) to mg/mL, it is necessary to multiply by 10 (g to mg and dL to mL). In addition, the sample volume used in the assay (mL) must be inserted by multiplication after the last conversion. Finally, the total conversion is: Hb concentration (g/dL × 10) = Hb concentration (mg/mL × sample volume used in the assay) = Hb mass (mg) All data was obtained and analyzed using the software Statistica (StatSoft). A p-value ≤ 0.05 was considered statistically significant. Results The mean total protein and hemoglobin concentrations are presented inFigure 9. These measurements were used to normalize the activity of antioxidant enzymes and redox status markers concentrations in serum and washed erythrocytes, respectively. Hemoglobin concentrations in samples diluted into a stabilizing solution were higher compared to hemoglobin concentrations diluted into purified water (p = 0.002, t -test) (Figure 9). Figure 9. Protein analyses. (A) Hemoglobin concentration from samples diluted in stabilizing solution and purified water. (B) Total protein concentrations from serum samples. Raw data are presented for each marker. TBARS and SH measurements were performed in washed erythrocytes samples diluted into purified water solution because the stabilizing solution interfered with the results of the assays (Figure 10). The intra-assay coefficient of variation in all assays was considered low (up to 10%). CAT activity was not detected in the serum samples. Although sample volumes from 1 to 50 µL were tested for this assay, no activity was detected in healthy animals. Moreover, although concentrations around 0.044 mM of -SH per milligram of total protein in serum were detected, these concentrations were approximately 60-fold lower than those found in washed erythrocytes (2.74 mM/mg Hb) (Figure 11). Figure 10. Biomarkers of oxidative stress status in the washed erythrocyte samples. Antioxidant enzyme activities and the redox status markers in washed erythrocyte samples from pregnant rats at term. (A) Superoxide dismutase (SOD); (B) Glutathione peroxidase (GSH-Px); (C) Catalase (CAT); (D) Thiobarbituric acid reactive substances (TBARS); (E) Reduced thiol groups (-SH); (F) Hydrogen peroxide (H2O2). Raw data are presented for each marker. Figure 11. Biomarkers of oxidative stress status in serum samples. Activity of antioxidant enzymes and the redox status markers in serum samples from pregnant rats at term. (A) Superoxide dismutase (SOD); (B) Glutathione peroxidase (GSH-Px); (C) Thiobarbituric acid reactive substances (TBARS); (D) Reduced thiol groups (-SH); (E) Hydrogen peroxide (H2O2). Raw data are presented for each marker. Notes Hemoglobin (Hb) concentration was determined and used to normalize the oxidative stress biomarkers (SOD, GSH-Px, CAT, -SH, TBARS, and H2O2) in washed erythrocytes according to the diluent used (purified water or stabilizing solution). Thus, hemoglobin concentration must be twice determined: in purified water and in stabilizing solution. It is relevant to emphasize that the Hb and total protein concentrations should be transformed in mass (mg) in all formulas of oxidative stress biomarkers. In TBARS assay, all the samples must be thawed at 25 °C. After TBA solution preparation, the pH should be below 2.0. If the pH is above 2.0, it is possible that the purified water conductivity is impaired. In reduced thiol groups (-SH), hydrogen peroxide (H2O2), superoxide dismutase (SOD), GSH-Px, and CAT assays, all the samples must defrost on ice and be kept cooled to not allow degradation to occur. Hydrogen peroxide (H2O2) is a volatile substance; thus, it is necessary to perform the equation for H2O2 real molarity when executing the assay. To reduce the sample volume and reagents to obtain the same results between microplates and cuvettes, the researchers decided to use these final equations of each biomarker following the light pathlength of cuvette methodologies (2 mL), but the sample and reagent volumes were adjusted to the microplate volume (well total volume 360 μL). Recipes Stabilizing solution (2.7 mM EDTA and 0.7 mM 2-mercaptoethanol) EDTA 10 mL 2-mercaptoethanol 0.5 μL Note: Solution stable for up to six months at 2–8 °C. Drabkin’s solution Potassium ferricyanide 200 mg Potassium cyanide 50 mg NaHCO3 1 g Purified water 1,000 mL Note: Drabkin’s solution is light sensible. Store at 4 °C in an Amber bottle to protect from light. This solution is stable for up to six months at 2–8 °C. 1 μg/μL stock solution Dilute 1 g of BSA in 1 mL of purified water. Note: Solution stable for up to six months at 2–8 °C. 3% (w/v) 5-sulfosalicylic acid hydrate (for 100 samples) Sulfosalicylic acid 3 g Ultrapure water 100 mL Dilute sulfosalicylic acid in a volumetric flask adding purified water. Seal it with laboratory film. Keep at 25 °C. Note: Solution must be freshly prepared. After use, discard remaining solution. Thiobarbituric acid solution (TBA) 0.67% (for 200 samples) Dilute 0.67 g of TBA in 95 mL of purified water in an Amber bottle. Keep the bottle partially sealed and heat the solution to 80 °C for 30 min in a water bath. Wait until the solution reaches 25 °C. Adjust pH to 2.0 by adding 1 M NaOH (see Recipe 16). Add the solution to a beaker and complete to 100 mL using purified water. Note: Solution must be freshly prepared at the assay day. This solution is light sensible. Store in Amber bottle. After use, discard remaining solution. 1 M HCl Dilute 8.3 mL of HCl in 100 mL of purified water. Note: Solution stable for undetermined time at 2–8 °C. 0.1 M TRIS/HCl 0.5 mM EDTA pH 8.0 (for 250 duplicate samples) Dilute 1.2114 g of TRIS-UltrapureTM and 0.0146 g of EDTA in 90 mL of purified water. Adjust pH to 8.0 by adding 1 M HCl (see Recipe 6). Transfer the solution to a beaker and complete to 100 mL using purified water. Note: Solution stable for undetermined time at 2–8 °C. 10 mM DTNB (for 250 duplicate washed erythrocytes samples and 100 duplicate serum samples) Dilute 0.0079 g of DTNB in 2 mL of ethyl alcohol. Aliquot the total volume in two microtubes (1 mL each) Seal and store at -20 °C. Note: Solution must be freshly prepared and stored in a sealed tube at -20°C. After use, discard remaining solution. Solution 1: 1.71 M NaCl, 34 mM KCl, 14 mM K2HPO4 , 0.1 M Na2HPO4 Mix 8 g of NaCl, 0.2 g of KCl, 0.2 g of K2HPO4 , and 1.15 g of Na2HPO4 in a volumetric flask. Add 80 mL of purified water and seal with laboratory film. Keep at 25 °C until buffer solution B preparation (Recipe 15). Store at 2–8 °C after buffer solution B preparation. Note: Solution stable for undetermined time. Solution 2: 90 mM CaCl2 Dilute 0.3 g of CaCl2 in 30 mL of purified water. Homogenize and seal the solution with laboratory film. Keep at 25 °C until buffer solution B preparation (Recipe 15). Store at 2–8 °C after buffer solution B preparation. Note: Solution stable for undetermined time. Solution 3: 0.11 M MgCl2 Dilute 0.3 g of MgCl2 in 30 mL of purified water. Homogenize and seal the solution with laboratory film. Keep at 25 °C until buffer solution B preparation (Recipe 15). Store at 2–8 °C after buffer solution B preparation. Note: Solution stable for undetermined time. Solution 4: 0.1% phenol red Dilute 0.3 g of phenol red in 30 mL of purified water in a tube. Homogenize the solution at 54 °C in water bath until complete dissolution. Remove the solution from the water bath. Seal the tube with laboratory film. Keep at 25 °C until buffer solution B preparation (Recipe 15). Store at 25 °C after buffer solution B preparation. Note: Solution stable for undetermined time. Phosphate buffer solution A Add 2.61 g of K2HPO4 to 150 mL of purified water. Homogenize and seal the solution with laboratory film. Keep the solution at 25 °C. Add 2.06 g of KH2PO4 to 150 mL of purified water. Homogenize and seal the solution with laboratory film. Keep the solution at 25 °C. Add 100 mL of K2HPO4 solution in a volumetric flask and add KH2PO4 solution until reaching pH 7.0. Store at 2–8 °C. Note: Solution stable for undetermined time. Peroxidase solution (HRP) Dissolve 5 mg of HRP in 1 mL of phosphate buffer solution A. Note: Solution must be freshly prepared. After use, discard remaining solution. Buffer solution B 10.56 mL of purified water 0.960 mL of Solution 1 0.120 mL of Solution 2 0.120 mL of Solution 3 0.120 mL of Solution 4 0.120 mL of peroxidase solution (HRP) Note: Solution must be freshly prepared. After use, discard remaining solution. 1 M NaOH Dilute 0.040 g of NaOH in 0.10 mL of purified water. Note: Solution stable for undetermined time at 2–8 °C. 1 M TRIS/HCl 5 mM EDTA pH 8.0 (for 150 duplicate samples) Dilute 6.0570 g of TRIS-UltrapureTM and 0.0731 g of EDTA in 45 mL of purified water. Adjust pH to 8.0 by adding 1 M HCl (see Recipe 6). Transfer the solution to a beaker and complete to 50 mL using purified water. Store in a volumetric flask. Note: Solution stable for undetermined time at 2–8 °C. 10 mM pyrogallol solution (for 250 duplicate sample analysis) Dilute 0.0063 g of pyrogallic acid in 5 mL of 10 mM HCl solution (see Recipe 21). Note: The solution must be freshly prepared. Minimum volume of 5 mL due to the pyrogallol quantity to be weighted. After use, discard remaining solution. 50 mM TRIS/HCl 5 mM EDTA pH 7.6 (for 500 duplicate sample analysis) Dilute 0.6057 g of TRIS-UltrapureTM and 0.1461 g of EDTA in 90 mL of purified water. Adjust pH to 7.6 by adding 1 M HCl (see Recipe 6). Transfer the solution to a beaker and complete the solution to 100 mL using purified water. Store in a volumetric flask. Note: Solution stable for undetermined time at 2–8 °C. 1% NaHCO3 and 1 mM EDTA Dilute 1 g of NaHCO3 and 0.0292 g of EDTA to 100 mL of purified water in a volumetric flask. Note: Solution stable for undetermined time at 2–8 °C. 10 mM HCl solution Dilute 50 μL of HCl in 60 mL of purified water. Note: Solution stable for undetermined time at 2–8 °C. 0.1 M glutathione reduced (GSH) (for 60 duplicate sample analysis) Dilute 0.0184 g of GSH in 600 μL of 10 mM HCl solution (see Recipe 21). Note: The solution must be freshly prepared and kept on ice. After use, discard remaining solution. 10 U/mL glutathione reductase (GSH-Rd) (for 60 duplicate sample analysis) Add 60 μL of GSH-Rd to 2.940 mL of 0.1M TRIS/HCl 0.5 mM EDTA pH 8.0 (see Recipe 7). Note: The solution must be freshly prepared and kept on ice. After use, discard remaining solution. 7 mM t-butyl hydroxiperoxide (for 125 duplicate sample analysis) Dilute 5 μL of t -butyl hydroperoxide in 5 mL of purified water. Note: The solution must be freshly prepared. After use, discard remaining solution. 4 mM NADPH (for 60 duplicate sample analysis) Dilute 0.010 g of NADPH in 3 mL of 1% NaHCO3 1 mM EDTA. Note: Solution must be freshly prepared and kept on ice during the assay. Solution stable for up to 4 h. 0.1 M NaH2PO4·H2O (for 200 samples analysis) Dilute 2.7598 g of NaH2PO4·H2O in 190 mL of purified water. Adjust pH to 7.0 by adding 1 M NaOH (see Recipe 16). Transfer the solution to a beaker and complete the solution to 200 mL using purified water. Store in a volumetric flask. Note: Solution stable for undetermined time at 2–8 °C. 1 M hydrogen peroxide (H2O2) Add 970 μL of hydrogen peroxide to 9.03 mL of purified water. Note: Solution must be freshly prepared. After use, discard remaining solution. Acknowledgments The authors thank the Research Support Center (RSC) of Botucatu Medical School, São Paulo State University (UNESP) for their valuable contribution in study design and statistical analysis. This work was supported by FAPESP/Brazil (Grant number 2016/25207-5) under the coordination of Prof. Dr. Débora Cristina Damasceno and scholarship to Yuri Karen Sinzato by the CAPES (Coordination of Superior Level Staff Improvement) [Finance Code 001]. Competing interests The authors have no competing interests to declare. Ethics All experimental procedures and animal handling were performed according to guidelines provided by the Brazilian College of Animal Experimentation in agreement with the National Institutes of Health guide for the care and use of Laboratory animals. The local Ethical Committee for Animal Research (Protocol number: 832-2010) approved all procedures. References Agarwal, A., Aponte-Mellado, A., Premkumar, B. J., Shaman, A. and Gupta, S. (2012). The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 10: 49. Agarwal, A., Gupta, S. and Sharma, R. K. (2005). Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 3: 28. van Assendelft, O. W. and Zijlstra, W. G. (1975). Extinction coefficients for use in equations for the spectrophotometric analysis of haemoglobin mixtures. Anal Biochem 69(1): 43-48. Birben, E., Sahiner, U. M., Sackesen, C., Erzurum, S. and Kalayci, O. (2012). Oxidative stress and antioxidant defense. World Allergy Organ J 5(1): 9-19. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254. Buege, J. A. and Aust, S. D. (1978). Microsomal lipid peroxidation. Methods Enzymol 52: 302-310. Burton, G. J. and Jauniaux, E. (2011). Oxidative stress. Best Pract Res Clin Obstet Gynaecol 25(3): 287-299. Ćebović, T., Marić, D., Nikolić, A. and Novakov Mikic, A. (2013). Antioxidant Status in Normal Pregnancy and Preeclampsia upon Multivitamin-Mineral Supplementation in the Region of Vojvodina. Int J Biosci Biochem Bioinforma 3: 138-144. De Souza Mda, S., Sinzato, Y. K., Lima, P. H., Calderon, I. M., Rudge, M. V. and Damasceno, D. C. (2010). Oxidative stress status and lipid profiles of diabetic pregnant rats exposed to cigarette smoke. Reprod Biomed Online 20(4): 547-552. Del Rio, D., Stewart, A. J. and Pellegrini, N. (2005). A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 15(4): 316-328. Fruscione, F., Sturla, L., Duncan, G., Van Etten, J. L., Valbuzzi, P., De Flora, A., Di Zanni, E. and Tonetti, M. (2008). Differential role of NADP+ and NADPH in the activity and structure of GDP-D-mannose 4,6-dehydratase from two chlorella viruses. J Biol Chem 283(1): 184-193. Fujii, J., Iuchi, Y. and Okada, F. (2005). Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system. Reprod Biol Endocrinol 3: 43. Kumar, P. and Maurya, P. K. (2013). L-cysteine efflux in erythrocytes as a function of human age: correlation with reduced glutathione and total anti-oxidant potential. Rejuvenation Res 16(3): 179-184. Marklund, S. and Marklund, G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47(3): 469-474. Noble, R. W. and Gibson, Q. H. (1970). The reaction of ferrous horseradish peroxidase with hydrogen peroxide. J Biol Chem 245(9): 2409-2413. Pisoschi, A. and Negulescu, G. (2012). Methods for Total Antioxidant Activity Determination: A Review. Biochem Anal Biochem 1: 1-10. Riddles, P. W., Blakeley, R. L. and Zerner, B. (1983). Reassessment of Ellman's reagent. Methods Enzymol 91: 49-60. Tentori, L. and Salvati, A. M. (1981). Hemoglobinometry in human blood. Methods Enzymol 76: 707-715. Wang, H. and Joseph, J. A. (1999). Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27(5-6): 612-616. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). How to cite Category Biochemistry > Protein > Activity Biochemistry > Lipid > Lipid measurement Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays Tobias Schachinger [...] Peter L. Turecek Sep 5, 2024 340 Views Measurement of the Activity of Wildtype and Disease-Causing ALPK1 Mutants in Transfected Cells With a 96-Well Format NF-κB/AP-1 Reporter Assay Tom Snelling Nov 20, 2024 272 Views Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays Tom Snelling Nov 20, 2024 257 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,627	https://bio-protocol.org/en/bpdetail?id=4627&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Amplification and Quantitation of Telomeric Extrachromosomal Circles NR Nathaniel J. Robinson William P. Schiemann Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4627 Views: 641 Reviewed by: Gal HaimovichShyam Srivats Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Science Signaling Jun 2021 Abstract Telomeres are structures that cap the ends of linear chromosomes and play critical roles in maintaining genome integrity and establishing the replicative lifespan of cells. In stem and cancer cells, telomeres are actively elongated by either telomerase or the alternative lengthening of telomeres (ALT) pathway. This pathway is characterized by several hallmark features, including extrachromosomal C-rich circular DNAs that can be probed to assess ALT activity. These so-called C-circles are the product of ALT-associated DNA damage repair processes and simultaneously serve as potential templates for iterative telomere extension. This bifunctional nature makes C-circles highly sensitive and specific markers of ALT. Here, we describe a C-circle assay, adapted from previous reports, that enables the quantitation of C-circle abundance in mammalian cells subjected to a wide range of experimental perturbations. This protocol combines the Quick C-circle Preparation (QCP) method for DNA isolation with fluorometry-based DNA quantification, rolling circle amplification (RCA), and detection of C-circles using quantitative PCR. Moreover, the inclusion of internal standards with well-characterized telomere maintenance mechanisms (TMMs) allows for the reliable benchmarking of cells with unknown TMM status. Overall, our work builds upon existing protocols to create a generalizable workflow for in vitro C-circle quantitation and ascertainment of TMM identity. Keywords: Alternative lengthening of telomeres ALT C-circles Extrachromosomal DNA Telomere Telomere maintenance mechanism Telomeric Qpcr Background To avoid the loss of genetic information and the acquisition of potentially lethal genomic aberrations during DNA replication, linear chromosomes possess nucleoprotein cap structures known as telomeres. Conversely, active telomere extension imparts cells with replicative immortality, which is essential during embryonic development (L. Liu et al., 2007), as well as during malignant transformation and cancer progression (Robinson and Schiemann, 2016; Robinson et al., 2019). In most developmental and tumorigenic contexts, telomere extension is carried out by the reverse transcriptase telomerase. However, telomeres are also extended in a telomerase-independent fashion through a mechanism known as alternative lengthening of telomeres (ALT), which is active in embryonic stem cells and early embryogenesis (L. Liu et al., 2007; Pickett and Reddel, 2015). Moreover, approximately 15% of tumors are reliant upon ALT for telomere synthesis, with some cancer types showing evidence of ALT in more than half of patients (Sung et al., 2020). Given the growing recognition of the prevalence and functional significance of ALT in both development and disease, establishing robust methods for monitoring ALT activity is of critical importance. Telomere lengthening via ALT is mediated by homologous recombination (HR) between adjacent telomere templates, followed by DNA synthesis through a mechanism similar to break-induced replication (BIR) (Dilley et al., 2016; Roumelioti et al., 2016). Importantly, telomere HR and BIR generate small circular DNAs containing telomeric DNA sequences (T. Zhang et al., 2019).These telomeric extrachromosomal circles, termed C-circles, act as specific markers that can be used to quantitate ALT activity in biological samples (Henson et al., 2009). More broadly, multiple methods exist to assay telomere maintenance mechanism (TMM) identity. For example, HR of contiguous telomeres results in telomere sister chromatid exchange (T-SCE), a hallmark of ALT (Londono-Vallejo et al., 2004) that can be visualized using chromosome orientation fluorescence in situ hybridization (CO-FISH) (Cesare et al., 2015). In addition, ALT-associated HR and DNA synthesis are facilitated by the promyelocytic leukemia protein (PML) within structures known as ALT-associated PML bodies (ABPs) (Yeager et al., 1999; J. M. Zhang et al., 2019). APBs can be detected and quantified using a combined immunofluorescence/FISH approach (Henson et al., 2005). Lastly, mutational loss (Lovejoy et al., 2012) or downregulation (Robinson et al., 2020) of the ATRX/DAXX chromatin remodeling complex is widespread in ALT-driven cell lines and tumors and can be easily ascertained using sequencing or quantitative gene expression methods. Despite the established relationships between these molecular markers and ALT, they suffer from drawbacks that restrict their experimental utility. For instance, T-SCE can occur during telomeric DNA damage repair (Mao et al., 2016; H. Liu et al., 2018). Moreover, loss of ATRX and DAXX is not uniformly distributed in ALT-positive specimens (Lovejoy et al., 2012) and can be found in ALT-negative specimens as well (de Nonneville and Reddel, 2021). The variable nature of these markers necessitates a combinatorial approach to TMM classification that includes a sensitive and reliable method for assessing ALT. Because of the high degree of specificity of C-circles for ALT (Henson et al., 2009) and their relationship to clinical features of ALT-driven cancers (Grandin et al., 2019), measuring and quantifying C-circle abundance has become the preferred method for assessing ALT activity, motivating the continual development of improved methods for C-circle quantitation. Furthermore, the diversity of biological contexts in which telomeres are actively maintained and the differences in telomere dynamics across organisms (Monaghan et al., 2018; Wright and Shay, 2000) create demand for generalized approaches to the interrogation of TMM identity. Here, we describe a protocol for C-circle quantitation, based upon previous reports (Lau et al., 2013; Henson et al., 2017), consisting of (i) rapid isolation and spectroscopic quantification of genomic and extrachromosomal DNA using the Quick C-circle Preparation (QCP) method, (ii) enrichment of extrachromosomal circular DNAs via rolling circle amplification (RCA), and (iii) determination of C-circle abundance using quantitative polymerase chain reaction (qPCR). We have applied this protocol across an array of biological and experimental contexts (Robinson et al., 2020 and 2021), asserting it as a generalizable platform for C-circle detection and, when paired with additional approaches, the assignment of TMM identity. Materials and Reagents 1.5 mL Eppendorf tube 96-well microplate, flat bottom, clear (Greiner Bio-One, catalog number: 655101) MultiplateTM 96-well PCR plates, low profile, clear (Bio-Rad, catalog number: MLL9601) Cell line(s) of interest and appropriate media/culture reagents ALT-positive cell line for source of C-circle positive control/standard curve DNA [e.g., U2OS (ATCC, catalog number: HTB-96), Saos-2 (ATCC, catalog number: HTB-85)] Telomerase-positive cell line for source of C-circle negative control DNA [e.g., HeLa (ATCC, catalog number: CCL-2), HCT116 (ATCC, catalog number: CCL-247)] Tris base (Fisher Scientific, catalog number: BP152) Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366) Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: M35) NP-40 (also known as IGEPAL® CA-630) (Sigma-Aldrich, catalog number: I3021) Tween 20 (also known as Polysorbate 20) (Fisher Scientific, catalog number: BP337) Deionized, nuclease-free H2O [purified using Q-Gard® 2 purification cartridge (Sigma-Aldrich, catalog number: QGARD00D2)] Protease (7.5 AU) (QIAGEN, catalog number: 19155) QuantiFluor® ONE dsDNA system (Promega, catalog number: E4871) Φ29 DNA polymerase [includes 10× Φ29 reaction buffer and 20 mg/mL bovine serum albumin (BSA); New England Biolabs, catalog number: M0269) dNTP set (100 mM each dNTP) (Thermo Fisher Scientific, catalog number: R0181) UltraPureTM dithiothreitol (Invitrogen, catalog number: 15508013) iQTM SYBR® Green Supermix (Bio-Rad, catalog number: 1708880) C-circle primers (human and mouse) Forward: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′ Reverse: 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′ 36B4 primers (human) Forward: 5′-CAGCAAGT GGGAAGGTGTAATCC-3′ Reverse: 5′-CCATTCTATCATCAACGGGTACAA-3′ 36B4 primers (mouse) Forward: 5′-ACTAATCCCGCCAAAGCAACC-3′ Reverse: 5′-GTAGCGGTTTTGCTTTTTCATCCT-3′ Quick C-Circle Preparation (QCP) lysis buffer (1×) (see Recipes) 1 M Tris-HCl, pH 8.5 (see Recipes) Φ29 DNA polymerase master mix (2.16×) (see Recipes) Equipment Plate reader (Promega GloMax® Explorer Multimode Microplate Reader; catalog number: GM3500) Thermal cycler for RCA (MJ MiniTM Personal Thermal Cycle; Bio-Rad, catalog number: PTC1148) Thermal cycler for qPCR [Bio-Rad C1000 TouchTM Thermal Cycler Chassis (Bio-Rad, catalog number: 1841100) equipped with CFX96 Optical Reaction Module for Real-Time PCR (Bio-Rad, catalog number: 1845097)] Software CFX Maestro Software for CFX Real-Time PCR Instruments (Bio-Rad; https://www.bio-rad.com/en-us/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments?ID=OKZP7E15) Microsoft Excel 2019 (v16.0) Procedure The following protocol is optimized for 2 × 105 cells; however, we have successfully used this protocol to isolate and quantify C-circles with up to 5 × 105 cells. If adjusting cell numbers, all volumes should be scaled appropriately. We have successfully quantified C-circle abundance in ALT-positive human (Robinson et al., 2021) and mouse (Robinson et al., 2020) cells isolated from distinct 2D and 3D culture conditions, and from cells subjected to an array of genetic and pharmacologic manipulations. Thus, in our hands, this represents a generalizable protocol for C-circle quantitation across diverse in vitro conditions. Therefore, specific details regarding cell lines and culture conditions are not included here, except where noted as experimental controls. Note: Established ALT- and telomerase-positive cell lines (see Materials and Reagents) should be cultured in parallel with cell line(s) of interest. These serve as a source of DNA for standard curve generation (ALT) as well as positive (ALT) and negative (telomerase) controls for RCA and C-circle quantification. In our experience, results are best when all DNA (including controls) is isolated simultaneously. Doing so prevents degradation of DNA during storage or repeated freeze-thaw cycling of samples that are shared between experiments (e.g., positive and negative controls). DNA isolation from cultured cells using Quick C-circle Preparation (QCP) Pre-warm 1× QCP lysis buffer (see Recipe 1) in a 1.5 mL Eppendorf tube in a heat block or water bath set to 56 °C. Best results are achieved when QCP lysis buffer is made fresh for each experiment. Volume of QCP lysis buffer needed = 50 μL × (number of samples + 2) (included excess to account for pipetting error or evaporative loss). If lyophilized, resuspend protease (7.5 AU) in 7 mL of deionized, nuclease-free H2O. Mix thoroughly and place on ice until use. If previously resuspended, remove from -20 °C storage and thaw slowly on ice. Note: QIAGEN protease is preferred to other proteases because it can be heat-inactivated at 65 °C. Liberate cells from culture plates (e.g., trypsinization, nonenzymatic digestion from 3D culture) or remove cells from frozen storage and place on ice. At this stage, samples should be cell pellets rather than suspensions. Add protease to pre-warmed QCP lysis buffer at a ratio of 1:20 (i.e., 2.5 μL of protease to 50 μL of QCP lysis buffer). Mix thoroughly by vortexing and collect by microcentrifugation. This should be done immediately prior to cell resuspension. Resuspend each cell pellet in 50 μL of QCP lysis buffer supplemented with protease. Mix by vortexing at 2,000 rpm for 15 s. Suspensions are viscous and may require additional mixing by pipetting up and down. Be careful not to introduce bubbles during resuspension. Note: C-circles mixed with genomic DNA (i.e., in whole-cell lysates) are somewhat protected from shear forces compared to pure circular DNAs. While mixing samples by vortexing is acceptable and used throughout this protocol, repeated vortexing results in small but appreciable C-circle loss (<1% per 15 s vortex). If yield appears to be low during quantitation, vortex speed or frequency may be adjusted to minimize C-circle loss. Incubate cell suspensions at 56 °C for 1 h on a heat block. Samples should be mixed by vortexing for 15 s every 15–20 min. Increase temperature of heat block to 70 °C and incubate for 20 min to inactivate protease. Samples do not need to be mixed during this step. Allow samples to cool slowly to room temperature on a benchtop. Once cooled, mix by vortexing at 2,000 rpm for 15 s and collect any evaporated liquid by microcentrifugation. Samples may be used immediately for DNA quantitation and RCA. Otherwise, flash-freeze on dry ice and store at -80 °C. Quantitation of total cellular DNA Perform serial dilutions of QuantiFluor® ONE Lambda DNA (400 ng/μL) (from QuantiFluor® ONE dsDNA system) to generate dsDNA standards according to the manufacturer’s instructions. Final DNA concentrations are: 400, 200, 50, 12.5, 3.1, 0.8, and 0.2 ng/μL. In a 96-well microplate, transfer 200 μL of QuantiFluor® ONE dsDNA dye to each well that will contain standards, experimental samples, or blanks (i.e., background fluorescence). Each standard, sample, and blank should be analyzed in duplicate. Using a P2 pipettor, transfer 1 μL of each experimental sample or dsDNA standard prepared in Step B1 to the corresponding wells. For blank wells, add 1 μL of 1× TE buffer. Mix the plate thoroughly using a plate shaker or by pipetting the contents of each well using a multichannel pipette. If using a pipette, do not pipette up the entire volume of each well, and eject slowly to avoid introducing air bubbles, which can interfere with fluorescence detection. Incubate plate for 5 min at room temperature, protected from light. Measure fluorescence using a plate reader. If using the GloMax® system, select the preloaded protocol “QuantiFluor ONE dsDNA System.” Otherwise, use the following excitation and emission parameters: 504 and 531 nm, respectively. To calculate the DNA concentration of each experimental sample, first subtract the blank fluorescence from that of each standard and sample. Use these corrected values for the dsDNA standards to generate a standard curve of fluorescence intensity vs. DNA concentration (i.e., DNA concentration on x-axis and fluorescence on y-axis). Determine the DNA concentration of each sample using the standard curve equation (Figure 1). Figure 1. Representative standard curve of QuantiFluor® ONE Lambda DNA. Standard curve equation (top left) is used to calculate DNA abundance in experimental samples using measured absorbance. Each DNA concentration was analyzed and graphed in duplicate. Rolling circle amplification (RCA) of extrachromosomal DNA Make up Φ29 DNA polymerase master mix (see Recipe 3) and store aliquots at -20 °C if not used immediately. We have used frozen master mix up to one month after initial freezing with no appreciable impact on RCA efficiency. If thawing QCP samples, place on ice to thaw slowly. Once thawed, mix by vortexing at 2,000 rpm for 15 s and collect by microcentrifugation. Keep samples and master mix on ice while setting up RCA reactions. For samples isolated using QCP (including ALT-positive and ALT-negative controls), add 1 μL to 9 μL of 10 mM Tris, pH 7.6 in two 0.2 mL PCR tubes. Depending on the concentration of each sample, they may be diluted in 10 mM Tris, pH 7.6 to avoid pipetting small volumes. However, the total quantity of QCP buffer in each reaction must be the same and should not exceed 1 μL. Aliquot 9.25 μL of Φ29 master mix for each reaction (plus excess, i.e., enough for two extra reactions) into an Eppendorf tube. Remove Φ29 DNA polymerase from -20 °C storage and keep on ice or in a portable cooling block. Add 0.75 μL of polymerase per reaction (plus excess) to tube containing master mix to generate the final reaction mix. Mix by gently flicking the tubes and place on ice. Note: Excess polymerase is used relative to template to ensure linear amplification of circular DNAs (rather than logarithmic, which could confound comparative analysis). Other polymerases (e.g., Klenow fragment of DNA polymerase I) may be used if they possess processivity and strand displacement capability as Φ29 polymerase. Repeat step C4, aliquoting into a new Eppendorf tube. Add 0.75 μL of Milli-Q H2O per reaction to this tube. This serves as the final reaction mix for Φ29-deficient reactions, which are performed for each sample. Note: Sample-specific background can be corrected by running a Φ29-deficient RCA reaction in parallel for each sample. This is important for C-circle quantitation by qPCR because denaturation during PCR enables the detection of linear telomeric DNA. In addition, the inclusion of Φ29-deficient controls is particularly useful for samples with low or unknown ALT activity, as these may exhibit relatively high background signal. Add 10 μL of final reaction mix (Φ29-containing and Φ29-deficient) to one tube containing each QCP sample. Mix by gently vortexing (<1,500 rpm) for 5 s. Incubate tubes in a thermal cycler according to the following thermal profile: 30 °C for 4–8 h (RCA). Note: Increasing amplification time increases sensitivity at the expense of reduced linearity. If absolute C-circle abundance is known or expected to be high, 4 h is sufficient for amplification. If C-circle abundance is low or unknown, incubation should be increased to 8 h to maximize sensitivity. Reactions should not be extended beyond 8 h, as amplification becomes nonlinear at that time. 70 °C for 20 min (Φ29 inactivation). 4 °C for ∞. Typically, we perform RCA for 8 h and allow to run overnight. Samples are stable at 4 °C until convenient to collect the following morning. Generally, we use RCA reactions immediately for C-circle quantitation. However, reactions may also be stored at -20 °C for up to two weeks or at -80 °C for up to one month without appreciable DNA loss. Determination of C-circle abundance using qPCR Thaw SYBR® Green Supermix and primers from -20 °C storage on ice. Protect SYBR® Green Supermix from light. Each sample will be used to quantify C-circle abundance as well as the abundance of the single-copy gene ribosomal protein lateral stalk subunit P0 (also known as 36B4; see Materials and Reagents). Single-copy gene abundance provides a measure of total DNA content, which can be used to normalize C-circle abundance and enable comparative analysis (see Data Analysis). Thus, all subsequent steps should account for the fact that both targets will be measured (e.g., making sufficient master mix). Dilute DNA isolated from an ALT-positive cell line (e.g., U2OS, Saos-2) by QCP to obtain samples for generating a standard curve. For example, for a starting DNA concentration of 3.2 ng/μL, perform five two-fold serial dilutions for a final dilution series containing 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 ng/μL DNA. To account for differences in PCR efficiency between telomeric DNA and 36B4, each sample will be analyzed in duplicate for each target. Keep diluted samples on ice until ready to load PCR plate. Note: RCA is not required prior to dilution and standard curve generation. Assemble qPCR master mixes according to the recipe outlined in Table 1. In addition to Φ29-containing and Φ29-deficient RCA reactions, include a no-template control (using H2O in place of template DNA) to account for DNA contamination of PCR reagents. The Ct values for this no-template control should be at least four cycles greater than the Ct value of the lowest concentration on the standard curve. Table 1. Recipe for qPCR master mix. Reagent Final concentration Volume (μL) 2× SYBR® Green Supermix 1× 12.5 Forward primer (10 mM) 500 nM 1.25 Reverse primer (10 mM) 500 nM 1.25 RNase-free H2O - 6 Dilute each RCA sample to 0.5 ng/μL in 10 mM Tris, pH 7.6, using the concentrations calculated in Section B. Note: qPCR is inhibited by both Φ29 polymerase and the inorganic pyrophosphate byproduct of RCA. To avoid qPCR inhibition, limit the RCA reaction that is added to each qPCR reaction or pretreat each RCA sample with a pyrophosphatase. Given the sensitivity of qPCR, it is generally best to limit the quantity of undiluted RCA reaction that is added to <5% of the qPCR reaction volume. Add 4 μL (i.e., 2 ng) of template DNA (Φ29-containing or Φ29-deficient) or RNase-free H2O (for no-template control) to pre-designated wells of a 96-well PCR plate. Each sample should be analyzed in triplicate. Add 21 μL of qPCR master mix to each well containing DNA or H2O. Mix by gently vortexing the plate for 5 s and collect volume by centrifugation. Incubate plate in a real-time thermal cycler according to the following thermal profile: 95 °C for 15 min 95 °C for 15 s 54 °C for 2 min 72 °C for 1 min Repeat steps b–d 34 times Data analysis To construct the telomeric DNA and single-copy gene standard curves, plot Ct (y-axis) vs. DNA concentration (x-axis; log scale) for each duplicate of the six standard DNA concentrations in Microsoft Excel. Draw a standard curve by fitting a best-fit line to the data (Figure 2). There should be good linearity of the standard curve (R2 > 0.98, p < 0.05). In addition, the CFX Maestro software can be used to draw a standard curve to calculate qPCR efficiency, which describes how much of a target amplicon is being produced during each PCR cycle. To construct a standard curve in the CFX Maestro software, change designation of wells containing standard samples (i.e., from ALT-positive cell line; see step D3) to “Standard” on the plate setup (Settings → Plate Setup → View/Edit Plate). In addition, manually enter the DNA concentration in each standard well. The standard curve, along with the calculated efficiency, will now appear under the “Quantification” tab. For C-circle abundance (CCA) quantitation, efficiency should be close to 100% (± 5%). Note: Examine all Ct values for the single-copy gene to identify any abnormally high or low values, which could indicate unexpected copy number loss or gain, respectively. Discrepancies secondary to copy number alteration may complicate quantitative assessment of CCA and the assignment of ALT status and necessitate repeating the qPCR with another single-copy gene. Figure 2. Representative standard curve of Saos-2 DNA. Ct values were obtained using telomere-specific qPCR primers. Standard curve equation (bottom left) was used to calculate DNA abundance in experimental samples using sample-specific Ct values and was repeated for single-copy gene. Each DNA concentration was analyzed and graphed in duplicate. Determine the relative telomeric content (TC) and relative single-copy content (SC) for each replicate using the appropriate standard curve equation, using the Ct values obtained by qPCR. This calculation will provide the total TC (i.e., chromosomal and extrachromosomal) rather than specifically C-circle content. Calculate the mean (μ) TC and SC and standard deviation (σ) for each sample. Use these values to calculate the coefficient of variation (CV) for each sample according to the following: CVs for TC (Φ29-containing) should be <15%, while CVs for TC (Φ29-deficient) and SC should be <10%. Calculate normalized telomere content (NTC) by dividing the average TC by the average SC from the same sample. The CV for NTC is estimated as the sum of the CVs for TC and its matched SC for a given sample. Calculate CCA in each sample according to the following: CCA=NTCΦ29-NTCNo Φ29 Calculate the mean NTC for each experimental condition. Construct error bars for CCA using these mean values and the NTC CVs calculated in step 4, as follows: Upper error=(μΦ29+CVΦ29)-(μNo Φ29-CVNo Φ29)= (μΦ29-μNo Φ29)+(CVΦ29+CVNo Φ29) Lower error=(μΦ29-CVΦ29)-(μNo Φ29+CVNo Φ29)= (μΦ29-μNo Φ29)-(CVΦ29+CVNo Φ29) Plot CCA mean (step 5) and error (step 6) for each condition on a bar graph. If comparing groups, use the appropriate statistical test for comparison of means (i.e., Student’s t-test for two means, ANOVA for more than two means, or nonparametric equivalents). Assign TMM identity to each experimental group. A group is considered ALT-positive if (i) its CCA mean is >1% of the ALT-positive control; and (ii) its CCA lower error is nonnegative. Recipes Quick C-Circle Preparation (QCP) lysis buffer (1×) Stock reagent Final concentration Volume (per 100 mL) 1 M Tris-HCl, pH 8.5 10 mM 1 mL 5 M KCl 50 mM 1 mL 1 M MgCl2 2 mM 200 μL NP-40 0.5% (v/v) 500 μL Tween 20 0.5% (v/v) 500 μL Milli-Q H2O - 96.8 mL 1 M Tris-HCl, pH 8.5 For 100 mL, dissolve 12.11 g of Tris base in 80 mL of Milli-Q H2O. Adjust pH to 8.5 by adding concentrated HCl dropwise with continuous stirring. Bring volume to 100 mL with Milli-Q H2O. Φ29 DNA polymerase master mix (2.16×) Stock reagent Final concentration Volume (per 925 μL) 10× Φ29 reaction buffer 2.16× 200 μL 20 mg/mL BSA 432 μg/mL 20 μL Tween 20 (10% v/v in H2O) 0.216% (v/v) 20 μL 100 mM dATP 2.16 mM 20 μL 100 mM dGTP 2.16 mM 20 μL 100 mM dTTP 2.16 mM 20 μL 100 mM dCTP 2.16 mM 20 μL 1 M dithiothreitol 8.65 mM 8 μL Milli-Q H2O - 597 μL Acknowledgments We are grateful to all authors (M. Miyagi, J.A. Scarborough, J.G. Scott, and D.J. Taylor) of the original research paper from which this protocol is derived (Robinson et al., 2021) for their contributions to that work, including the design and implementation of this protocol. We also thank members of the Schiemann laboratory for their assistance in crafting and critically appraising this manuscript. Funding support was provided by the National Institutes of Health to W.P.S. (CA236273) and N.J.R. (F30 CA213892). Additional support was graciously provided to W.P.S. by the Case Comprehensive Cancer Center’s Research Innovation Fund, which is supported by the Case Council and Friends of the Case Comprehensive Cancer Center. Competing interests The authors declare that they have no compe ting interests. References Cesare, A. J., Heaphy, C. M. and O'Sullivan, R. J. (2015). Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques. Curr Protoc Cytom 73: 12 40 11-12 40 31. de Nonneville, A. and Reddel, R. R. (2021). Alternative lengthening of telomeres is not synonymous with mutations in ATRX/DAXX.Nat Commun 12(1): 1552. Dilley, R. L., Verma, P., Cho, N. W., Winters, H. D., Wondisford, A. R. and Greenberg, R. A. (2016). Break-induced telomere synthesis underlies alternative telomere maintenance.Nature 539(7627): 54-58. Grandin, N., Pereira, B., Cohen, C., Billard, P., Dehais, C., Carpentier, C., Idbaih, A., Bielle, F., Ducray, F., Figarella-Branger, D., et al. (2019). The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.Acta Neuropathol Commun 7(1): 175. Henson, J. D., Cao, Y., Huschtscha, L. I., Chang, A. C., Au, A. Y., Pickett, H. A. and Reddel, R. R. (2009). DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity. Nat Biotechnol 27(12): 1181-1185. Henson, J. D., Hannay, J. A., McCarthy, S. W., Royds, J. A., Yeager, T. R., Robinson, R. A., Wharton, S. B., Jellinek, D. A., Arbuckle, S. M., Yoo, J., et al. (2005). A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin Cancer Res 11(1): 217-225. Henson, J. D., Lau, L. M., Koch, S., Martin La Rotta, N., Dagg, R. A. and Reddel, R. R. (2017). The C-Circle Assay for alternative-lengthening-of-telomeres activity.Methods 114: 74-84. Lau, L. M., Dagg, R. A., Henson, J. D., Au, A. Y., Royds, J. A. and Reddel, R. R. (2013). Detection of alternative lengthening of telomeres by telomere quantitative PCR.Nucleic Acids Res 41(2): e34. Liu, H., Xie, Y., Zhang, Z., Mao, P., Liu, J., Ma, W. and Zhao, Y. (2018). Telomeric Recombination Induced by DNA Damage Results in Telomere Extension and Length Heterogeneity. Neoplasia 20(9): 905-916. Liu, L., Bailey, S. M., Okuka, M., Munoz, P., Li, C., Zhou, L., Wu, C., Czerwiec, E., Sandler, L., Seyfang, A., Blasco, M. A. and Keefe, D. L. (2007). Telomere lengthening early in development. Nat. Cell Biol 9(12): 1436-1441. Londono-Vallejo, J. A., Der-Sarkissian, H., Cazes, L., Bacchetti, S. and Reddel, R. R. (2004). Alternative lengthening of telomeres is characterized by high rates of telomeric exchange.Cancer Res 64(7): 2324-2327. Lovejoy, C. A., Li, W., Reisenweber, S., Thongthip, S., Bruno, J., de Lange, T., De, S., Petrini, J. H., Sung, P. A., Jasin, M., et al. (2012). Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway.PLoS Genet 8(7): e1002772. Mao, P., Liu, J., Zhang, Z., Zhang, H., Liu, H., Gao, S., Rong, Y. S. and Zhao, Y. (2016). Homologous recombination-dependent repair of telomeric DSBs in proliferating human cells.Nat Commun 7: 12154. Monaghan, P., Eisenberg, D. T. A., Harrington, L. and Nussey, D. (2018). Understanding diversity in telomere dynamics. Philos Trans R Soc Lond B Biol Sci 373(1741): 20160435. Pickett, H. A. and Reddel, R. R. (2015). Molecular mechanisms of activity and derepression of alternative lengthening of telomeres. Nat Struct Mol Biol 22(11): 875-880. Robinson, N. J., Miyagi, M., Scarborough, J. A., Scott, J. G., Taylor, D. J. and Schiemann, W. P. (2021). SLX4IP promotes RAP1 SUMOylation by PIAS1 to coordinate telomere maintenance through NF-kappaB and Notch signaling. Sci Signal 14(689): eabe9613. Robinson, N. J., Morrison-Smith, C. D., Gooding, A. J., Schiemann, B. J., Jackson, M. W., Taylor, D. J. and Schiemann, W. P. (2020). SLX4IP and telomere dynamics dictate breast cancer metastasis and therapeutic responsiveness.Life Sci Alliance 3(4): e201900427. Robinson, N. J. and Schiemann, W. P. (2016). Means to the ends: The role of telomeres and telomere processing machinery in metastasis.Biochim Biophys Acta 1866(2): 320-329. Robinson, N. J., Taylor, D. J. and Schiemann, W. P. (2019). Stem cells, immortality, and the evolution of metastatic properties in breast cancer: telomere maintenance mechanisms and metastatic evolution.J Cancer Metastasis Treat 5. Roumelioti, F. M., Sotiriou, S. K., Katsini, V., Chiourea, M., Halazonetis, T. D. and Gagos, S. (2016). Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication. EMBO Rep 17(12): 1731-1737. Sung, J. Y., Lim, H. W., Joung, J. G. and Park, W. Y. (2020). Pan-Cancer Analysis of Alternative Lengthening of Telomere Activity.Cancers (Basel) 12(8): 2207. Wright, W. E. and Shay, J. W. (2000). Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology. Nat Med 6(8): 849-851. Yeager, T. R., Neumann, A. A., Englezou, A., Huschtscha, L. I., Noble, J. R. and Reddel, R. R. (1999). Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res 59(17): 4175-4179. Zhang, J. M., Yadav, T., Ouyang, J., Lan, L. and Zou, L. (2019). Alternative Lengthening of Telomeres through Two Distinct Break-Induced Replication Pathways. Cell Rep 26(4): 955-968 e953. Zhang, T., Zhang, Z., Shengzhao, G., Li, X., Liu, H. and Zhao, Y. (2019). Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination. PLoS Genet 15(2): e1007925. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Cancer Biology > Replicative immortality > Cell biology assays Molecular Biology > DNA > DNA quantification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols DNase I Chromatin Accessibility Analysis Brook S. Nepon-Sixt and Mark G. Alexandrow Dec 5, 2019 4434 Views Transcervical Mouse Infections with Chlamydia trachomatis and Determination of Bacterial Burden Karthika Rajeeve and Rajeeve Sivadasan Feb 5, 2020 4619 Views Isolation and Quantification of Extracellular DNA from Biofluids Ľubica Janovičová [...] Peter Celec Aug 20, 2020 3589 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,628	https://bio-protocol.org/en/bpdetail?id=4628&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris AT Adam Turner * DL Dylan M. Lanser * AG Angie Gelli (*contributed equally to this work) Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4628 Views: 1396 Reviewed by: Neha NandwaniRitu Gupta Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in ACS Infectious Diseases Jan 2020 Abstract Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia’s low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract Keywords: Metalloprotease Mpr1 Secreted proteins Heterologous expression Pichia pastoris Background The production of active recombinant proteases at concentrations that allow for their use in downstream applications can be particularly challenging (Colige, 2020). The yeast Pichia pastoris has been successfully used as a eukaryotic expression system for foreign proteins (Macauley-Patrick et al., 2005). Unlike mammalian cells, Pichia does not require complex media or growth conditions, which makes its use cost-effective and scalable since high cell densities can be achieved in minimal media (Macauley-Patrick et al., 2005). Pichia strains are also genetically tractable, and the variety of available promoters and selectable markers facilitate gene expression (Daly and Hearn, 2005). Moreover, the presence of a eukaryotic protein synthesis pathway in Pichia allows high levels of posttranslational modification including glycosylation, proteolytic processing, and disulfide bond formation—processes that are limited in prokaryotic expression systems (Macauley-Patrick et al., 2005; Ahmad et al., 2014; Macek et al., 2019). Generating large quantities of recombinant proteins is achievable with bioreactors; however, this is not feasible for most and, in general, recombinant proteins are produced in shaking flasks, similar to our approach (Schwettmann and Tschesche, 2001; Fernandez et al., 2013). The relatively small volumes of culture and limited cell densities that are ultimately achieved with this approach have made the isolation of recombinant proteases in large quantities difficult. Our goal was to develop a standardized protocol that would allow for the routine expression and isolation of a proteolytically active secreted metalloprotease, with high purity and in large enough quantities to facilitate downstream applications. We made use of the AOX1 methanol-induced promoter in Pichia that ensures that transcription is tightly regulated by a repression/derepression mechanism (Ahmad et al., 2014). Carbon sources such as glucose or glycerol, which are nutritional requirements for Pichia’s cell growth, cannot be used during the induction phase since both would repress transcription. The challenge arises during induction with methanol, since growth is severely inhibited preventing high levels of biomass production, which can ultimately negatively impact protein production. We found that the addition of sorbitol, a non-repressible and non-fermentable carbon source in yeast, during the methanol-induction phase, significantly improved cell viability and biomass production. The inclusion of sorbitol boosted protein production to milligram quantities. Although implementing non-repressing carbon sources such as alanine, mannitol, trehalose, and sorbitol had previously been explored, it has not been readily adopted (Inan and Meagher, 2001; Celik et al., 2009). Here, we provide a detailed protocol for the expression and isolation of a proteolytically active metalloprotease from a neurotropic fungal pathogen, which has been applied to our work involving protein-nanoparticle conjugates as drug-delivery platforms for the central nervous system (Aaron and Gelli, 2020). Materials and Reagents 96-well assay plate with black wall and clear, flat bottom (Corning, catalog number: 3904) PYREX® Delong shaker Erlenmeyer flask with extra-deep baffles, 250 and 1,000 mL (Corning, catalog numbers: 4450-250, 4446-1L) 1.5 mL microcentrifuge tubes (USA Scientific, catalog number: 1615-5500) 15 mL centrifuge tubes, conical, sterile, polypropylene (VWR, catalog number 89039-664) Serological pipettes, sterile wrapped (Corning, catalog numbers: 4487, 4488, 4489, 4490) 250 mL centrifuge tube (Corning, catalog number: 430776) 10 kDa NMWL Ultra-4 centrifugal filter unit (Amicon, catalog number: UFC803008) Peptone (Research Products International, catalog number: P20250); store at room temperature Yeast extract (Thermo Scientific, catalog number: J23547-A1); store at room temperature Dextrose (VWR, BDH Chemicals, catalog number: BDH9230); store at room temperature Sorbitol (Sigma-Aldrich, CAS 50-70-4); store at room temperature Methanol (Sigma-Aldrich, CAS 67-56-1); store at room temperature Bacto agar (VWR, Life Sciences, catalog number: J637); store at room temperature G418 sulfate (Fisher Bioreagents, CAS 108321-42-2); store at 4 °C PureCube INDIGO Ni-MagBeads (magnetic beads) (Cube Biotech, catalog number: 75225) Magnet, >835 KA/m (Magneto Inc., Amazon) Pierce fluorescent protease activity kit (Thermo Fisher Scientific, catalog number: 23266) FTC-Casein (5 mg/mL in ultrapure water) TPCK-trypsin (50 mg/mL in tris-buffered saline) Tris-buffered saline (25 mM Tris, 150 mM NaCl, pH 7.2) Yeast media (see Recipes) BMGY BMMY YPD YPD + G418 (4 mg/mL) agar plate Buffers (see Recipes) Binding buffer pH 8.0 Wash buffer pH 8.0 Elution buffer pH 8.0 Phosphate-buffered saline, pH 7.4 Solutions (see Recipes) 10% glycerol 5% methanol 1 M potassium phosphate buffer, pH 6.0 10× YNB 500× biotin Strains Pichia pastoris GS115 (Invitrogen, catalog number: C18100) Pichia pastoris GS116 <Cn MPR1> (Aaron and Gelli, 2020) Equipment -80 °C freezer -20 °C freezer 4 °C refrigerator pH meter Vortex (Scientific Industries, SKU: SI-0236 or equivalent) Swing-bucket centrifuge (Beckman Coulter, model: Allegra X-15R; 208 V, 60 Hz) Innova 4200 incubator shaker (New Brunswick) or equivalent temperature-controlled shaker Pipetman (Eppendorf, P20, P200, P1000) Power supply (Bio-Rad, model: PowerPac Universal Power Supply) Protein gel electrophoresis equipment (Bio-Rad, model: Mini-PROTEAN) Microplate reader (Molecular Devices, model: SpectraMax M5e) Procedure The steps in the following segments were done using a methylotrophic yeast, Pichia pastoris, for the expression of recombinant proteins. The recombinant Mpr16XHIS protein of focus was isolated from a strain of P. pastoris transformed with a construct composed of MPR1 cDNA isolated from Cryptococcus neoformans KN99 with a 6XHIS tag at the C-terminus in a pPIC9K backbone, as described in the main article (Aaron and Gelli, 2020). This plasmid confers resistance to G418, which was added to agar plates (4 mg/mL) in both the original selection of transformed P. pastoris and while expanding frozen (-80 °C) stocks for the current protocol. We handled open cultures in a sterile field under an open flame throughout this protocol, checking for bacterial contamination at least once every 24 h by brightfield microscopy. The volumes and concentrations described below were optimal for our purposes but may need to be scaled for other experiments. The protocol below can be divided into three stages: (1) growth, induction, and expression; (2) isolation and purification; and (3) verification and storage. Expression of recombinant Mpr1 protein Streak the Pichia pastoris GS116 <Cn MPR1> strain (Aaron and Gelli, 2020) on a YPD + G418 (4 mg/mL) agar plate. Incubate the plate at 30 °C for 72 h. Note: Any gene of interest can be subcloned in expression vectors for P. pastoris. Select a single colony from the G418 plate and transfer directly to a 250 mL baffled Erlenmeyer flask containing 50 mL of BMGY media (see Recipes) using a flame-sterilized inoculating loop. Note: Using a baffled Erlenmeyer flask greatly increases biomass production compared to a non-baffled Erlenmeyer flask. Incubate the P. pastoris culture at 30 °C for 24 h, shaking at 300 rpm. Cover the flasks loosely with aluminum foil allowing for gas exchange. After 24 h, decant cultures into 50 mL centrifuge tubes and spin down at 3,724 × g for 5 min. Note: The culture is easily contaminated due to the nutrient-rich BMGY medium; thus, examine the culture for any possible contamination after every 24 h period. In our case, the cultures were examined by brightfield microscopy to rule out contamination. Discard the supernatant and wash the pellet 1–3 times using 25 mL of BMMY (see Recipes) and vortex the pellet until it is fully resuspended. Spin down the pellet at 3,724 × g between washes. Resuspend cells in BMMY and measure the optical density at 600 nm (OD600). Note: Following steps 1–5, we typically see an OD600 of approximately 25 in 20 mL of BMMY. Add the cell mixture to the desired volume of BMMY, in order to dilute the culture to a final OD600 = 1. Cover the flask with sterile cheesecloth, allowing for appropriate gas exchange. Incubate the induced culture at 30 °C, shaking, for 24 h. Add sorbitol and methanol to the culture to a final concentration of 50 g/L and 0.5%, respectively. Incubate at 30 °C, shaking, for 24 h. Note: Sorbitol is a non-repressing carbon source and we found that a co-feeding regime of methanol with sorbitol increases biomass production (Figures 1 and 2). Figure 1. Visualization of Pichia biomass production. A. Initiating induction in BMMY with OD600 = 1.0. B. Following 24 h induction in BMMY and just prior to co-feeding with 50 g/L sorbitol and 0.5% methanol. C. Following 24 h of co-feeding with sorbitol and methanol. At this stage, measurements of OD600 are typically well over 18. Figure 2. Optical densities of Pichia cultures supplemented with sorbitol. A comparison of the optical densities (OD600) of cultures with 0.5% methanol (MeOH) or co-fed with 0.5% MeOH and 50 g/L of sorbitol or 2% MeOH and 50 g/L of sorbitol. The cultures were co-fed following 24 h inoculation of the Pichia strain in BMMY media at a starting OD600 of 1. The OD600 measurements of the cultures were recorded 24 h after the co-feeding was initiated. Isolation and purification of recombinant Mpr1 protein For PureCube INDIGO Ni-MagBeads, mix and aliquot beads in a 15 mL conical centrifuge tube and briefly centrifuge the beads; then, place the aliquot near a strong magnet in order to remove and replace the storage buffer supernatant with phosphate-buffered saline, pH 7.4. Keep the beads on ice and see protocols provided by the manufacturer (PureCube) for further information. Note: If inducing a 250 mL culture, use 750 µL of beads from storage. This equals approximately 188 µL of beads (25% of storage solution contains beads with binding capacity of 70 mg/mL). Autoclave the beads for 15 min on liquid cycle. Allow beads to cool before proceeding. Measure the final OD600 of the P. pastoris culture. Decant the culture into centrifuge bottles. Note: The addition of sorbitol with cultures induced with 0.5% methanol (MeOH) significantly increased the optical density (OD600) of the Pichia cultures from ~8 to >20 (Figure 2). Centrifuge at 3,724 × g for 5 min at 30 °C. Decant and keep the supernatant. Add 1.5 mL of 25% (v/v) Ni-MagBeads per 500 mL of collected culture supernatant. Incubate at 30 °C, shaking at 250 rpm, for 30 min. Collect the Ni-MagBeads from the supernatant using a strong magnet. Decant the supernatant into a fresh vessel, then remove the magnet and resuspend the beads in 6 mL of binding buffer. Transfer resuspended beads in a 15 mL conical tube. Repeat at least twice. Wash with 10 mL of binding buffer until all beads have been collected. Place the collected beads in a centrifuge tube and centrifuge at 3,724 × g for 5 min at 4 °C. Magnetically pellet the MagBeads and remove binding buffer. Add 10 times the bead volume of wash buffer to the beads. Remove the magnet and vortex for at least 2 min. Note: Each 250 mL culture volume, receiving 188 µL of beads initially, is washed with 1.88 mL wash buffer at this step. We found that washing once was sufficient to remove any foreign matter, but still washed thrice without appreciable loss of MagBeads. Briefly centrifuge and collect beads at the bottom of the tube with a magnet to facilitate the exchange of wash buffer for elution buffer. Use twice the initial bead volume of elution buffer. Vortex beads for 5 min and centrifuge at 3,724 × g for 5 min at 4 °C. Note: Multiple elution steps can be performed at this stage to enrich for Mpr1; however, we found that one elution step was sufficient. Transfer the protein-containing elution buffer supernatant to a 10 kDa NMWL Ultra-4 centrifugal filter unit. Centrifuge and concentrate the sample at 4,000 × g for 15 min at 4 °C. Measure protein concentration by the method of Bradford (Bradford, 1976). Note: Timing for this step may vary depending on desired final concentration and quantity of elution buffer added. In our case, the above conditions yielded approximately 300 µL of protein suspended in elution buffer. PBS was exchanged for elution buffer following step 11, centrifuging for 8 min instead of 15. On average, 2.5 mg of Mpr1 was extracted from a 500 mL BMMY culture (Figure 3). Figure 3. Mpr1 production in media supplemented with sorbitol. A comparison of the quantity of Mpr1 recombinant protein extracted from cultures grown with 0.5% methanol (MeOH), 0.5% MeOH supplemented with 25 g/L of sorbitol, or 0.5% MeOH with 50 g/L of sorbitol. Data represent Mpr1 extractions from four different cultures. The addition of sorbitol boosted Mpr1 protein production when MeOH concentrations were maintained at 0.5%. Assess the purity of the recombinant protein by SDS-PAGE electrophoresis. Note: Coomassie or silver-stained SDS-PAGE polyacrylamide gels will visualize isolated recombinant proteins. Silver protein stains are an ultra-sensitive method of protein detection, which could be used to examine whether contaminating proteins were present in the isolation. Following step 11, examination of Mpr1 recombinant protein by silver-stained SDS-PAGE electrophoresis yielded a single polypeptide band (Figure 4). Activity, purity, and storage of Mpr1 After saving a portion of the Mpr1 recombinant protein for proteolytic activity and other assays, flash freeze the remainder in liquid nitrogen and store at -80 °C. Note: Proteolytic activity determination requires 50 µL or approximately 10% of the final eluted volume. We conducted proteolytic activity assays under various storage conditions (data not shown) and found that cryopreservation in liquid nitrogen produced only a small loss of proteolytic activity compared to protein that had not been subjected to freeze-thaw. Program the microplate reader to measure fluorescence (485/538 nm excitation/emission) at 5 min intervals over at least one hour, shaking between reads. Sensitivity should be set to medium or low. Following the manufacturer’s instructions, thaw aliquots of FTC-casein (5 mg/mL) and TPCK-trypsin (50 mg/mL) gradually at 4 °C. Dilute FTC-casein in tris-buffered saline to 10 µg/mL and store temporarily at 4 °C. Note: 10 mL of 10 µg/mL FTC-Casein is sufficient for a 96-well plate. Dilute Mpr1 recombinant protein in TBS to 200 µg/mL and 20 µg/mL, such that there is at least 350 µL of each dilution when finished. Set aside 350 µL of the TBS used in these dilutions to serve as a blank. Dilute a thawed TPCK-trypsin aliquot to 1 µg/mL in TBS. Add 100 µL of 200 µg/mL Mpr1, 20 µg/mL Mpr1, 1 µg/mL TPCK-trypsin, and TBS blank to triplicate wells in an opaque-walled (black or white), clear-bottom 96-well plate. Add 100 µL of 10 µg/mL FTC-casein to each well. Note: This is best done with a multichannel pipette due to the time-sensitivity of this step, although a serological pipette may suffice. Remove the lid on the plate and place it in the plate reader. Begin the program at 5 min post FTC-casein addition. Proteolytic activity is measured by the decrease in fluorescence resonance energy transfer signal (Figure 5). Data analysis Figure 4. Purification of recombinant Mpr1 metalloprotease. SDS-PAGE analysis of isolated Mpr1 recombinant protein with (A) Coomassie-stained and (B) silver-stained 10% SDS-page gel run under reducing conditions with protein molecular marker (right lane, kDa). Bold arrow indicates recombinant Mpr1 protein as a single band (left lane). Figure 5. Proteolytic activity of Mpr1 at 100 (dark blue) and 10 µg/mL (light blue) compared with 0.5 µg/mL trypsin (red). Proteolytic activity was measured by the decrease in fluorescence resonance energy transfer (FRET) signal using a Pierce fluorescent proteases assay kit. The average fluorescence intensity in control wells containing only the proteolytic substrate (FITC-conjugated casein) was subtracted at each time point. Each data point represents the average of three readings (bars: SD). This assay was repeated with Mpr1 protein from five different extractions with similar results. Mpr1 retained proteolytic activity after flash-freezing and several weeks in storage (not shown). Notes Co-feeding optimization: We optimized sorbitol and methanol concentrations for production of our protein of interest, a metalloprotease with potentially unusual properties. The optimal concentration of sorbitol and methanol for other proteins may well differ from what we report here. Sterility considerations: The media used during the initial growth of P. pastoris can easily become contaminated; hence, good sterile technique is vital for the success of your protein extraction. Variability: This protocol is robust to minor deviations. For instance, the duration of incubation periods was not always exactly 24 h. Environmental impact: Significant plastic waste was generated in the maintenance of sterile conditions throughout this protocol. Alternatives may be available, although such alternatives may have their own environmental drawbacks (e.g., the use of caustic chemicals in cleaning procedures). Recipes Yeast media BMMY media (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 5% biotin, 0.5% methanol) Mix 20 g of peptone and 10 g of yeast extract and add deionized H2O to 700 mL. Sterilize media by autoclave (liquid cycle 121 °C, 20 min). Under sterile conditions, add 100 mL potassium phosphate, pH 6.0, 100 mL of 10× YNB, 100 mL of 5% methanol, and finally 2 mL of 500× biotin. BMGY media (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 5% biotin, 1% glycerol) Mix 20 g of peptone and 10 g of yeast extract and add deionized H2O to 700 mL. Sterilize media by autoclave (liquid cycle 121 °C, 20 min). Under sterile conditions, add 100 mL 1 M potassium phosphate, pH 6.0, 100 mL of 10× YNB, 100 mL of 10% glycerol, and finally 2 mL of 500× biotin. YPD (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) Mix 20 g of peptone and 10 g of yeast extract and add deionized H2O to 950 mL. Sterilize media by autoclave (liquid cycle 121 °C, 20 min). For liquid media: add 50 mL of sterile 40% (w/v) glucose and mix. For agar plates: While stirring the autoclaved mixture on a magnetic stir plate, add 50 mL of sterile 40% glucose per liter of YPD media. While still warm, pour media into plastic Petri dishes (9 cm diameter) Allow agar mixture to cool and solidify. Buffers Binding buffer pH 8.0 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole Wash buffer pH 8.0 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole Elution buffer pH 8.0 (50 mM NaH2PO4, 300 mM NaCl, and 500 mM imidazole) Solutions 10% glycerol Mix 100 mL of glycerol with 900 mL of deionized H2O. Sterilize by autoclaving, store at room temperature. 5% methanol Mix 50 mL methanol with 950 mL of deionized H2O. Filter-sterilize and store at 4 °C. 1 M potassium phosphate buffer, pH 6.0 Mix 132 mL of 1 M K2HPO4 with 868 mL of 1 M KH2PO4. Adjust to pH 6.0 with KOH. Sterilize by autoclaving, store at room temperature. 10× YNB Weigh 34 g of YNB without ammonium sulfate and amino acids. Then, mix with 100 g of ammonium sulfate and fill up to 1 L of deionized H2O. Filter-sterilize and store at 4 °C. 500× biotin Dissolve 20 mg of biotin in 100 mL of deionized H2O. Filter-sterilize and store at 4 °C. Acknowledgments We are acknowledging the original research paper (Aaron and Gelli, 2020) published by our lab from which this protocol was derived. We are grateful to The Hartwell Foundation for their support and to members of the Gelli lab for useful suggestions. Competing interests The authors declare that there are no financial or other competing interests. References Aaron, P. A. and Gelli, A. (2020). Harnessing the Activity of the Fungal Metalloprotease, Mpr1, To Promote Crossing of Nanocarriers through the Blood-Brain Barrier. ACS Infect Dis 6(1): 138-149. Ahmad, M., Hirz, M., Pichler, H. and Schwab, H. (2014). Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12): 5301-5317. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2):248-254. Celik, E., Calik, P. and Oliver, S. G. (2009). Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol. Yeast 26(9): 473-484. Colige, A. C. (2020). Challenges and Solutions for Purification of ADAMTS Proteases: An Overview. Methods Mol Biol 2043: 45-53. Daly, R. and Hearn, M. T. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18(2): 119-138. Fernandez, D., Russi, S., Vendrell, J., Monod, M. and Pallares, I. (2013). A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus. Acta Crystallogr D Biol Crystallogr 69(Pt 10): 1946-1957. Inan, M. and Meagher, M. M. (2001). Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 92(6): 585-589. Macauley-Patrick, S., Fazenda, M. L., McNeil, B. and Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4): 249-270. Macek, B., Forchhammer, K., Hardouin, J., Weber-Ban, E., Grangeasse, C. and Mijakovic, I. (2019). Protein post-translational modifications in bacteria. Nat Rev Microbiol 17(11): 651-664. Schwettmann, L. and Tschesche, H. (2001). Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin. Protein Expr Purif 21(1): 65-70. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Microbiology > Heterologous expression system > Non-model species Biochemistry > Protein > Isolation and purification Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Isolation of Mitochondria from Ustilago maydis Protoplasts Juan Pablo Pardo [...] Lucero Romero-Aguilar Jan 5, 2022 1625 Views Lysate-to-grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging Ian Cooney [...] Peter S. Shen Jan 20, 2023 1364 Views Purification of Human Cytoplasmic Actins From Saccharomyces cerevisiae Brian K. Haarer [...] Jessica L. Henty-Ridilla Dec 5, 2023 521 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy
4,629	https://bio-protocol.org/en/bpdetail?id=4629&type=0	# Bio-Protocol Content Improve Research Reproducibility A Bio-protocol resource Peer-reviewed Visualization of Lipid Droplets in the Alveolar Macrophage Cell Line MH-S with Live-cell Imaging by 3D Holotomographic Microscopy (Nanolive) AP Andrea Pérez-Montero OZ Oscar Zaragoza Alfonso Luque Sonsoles Hortelano PA Paloma Acebo Published: Vol 13, Iss 5, Mar 5, 2023 DOI: 10.21769/BioProtoc.4629 Views: 628 Reviewed by: David PaulVasiliki Koliaraki Anonymous reviewer(s) Download PDF Ask a question Favorite Cited by Original Research Article: The authors used this protocol in Frontiers in Cellular Infection Microbiology Feb 2022 Abstract Lipid droplets (LD), triglycerides and sterol esters among them, are well known for their capacity as lipid storage organelles. Recently, they have emerged as critical cytoplasmic structures involved in numerous biological functions. LD storage is generated de novo by the cell and provides an energy reserve, lipid precursors, and cell protection. Moreover, LD accumulation can be observed in some pathologies as obesity, atherosclerosis, or lung diseases. Fluorescence imaging techniques are the most widely used techniques to visualize cellular compartments in live cells, including LD. Nevertheless, presence of fluorophores can damage subcellular components and induce cytotoxicity, or even alter the dynamics of the organelles. As an alternative to fluorescence microscopy, label-free techniques such as stimulated Raman scattering and coherent anti-stokes Raman scattering microscopy offer a solution to avoid the undesirable effects caused by dyes and fluorescent proteins, but are expensive and complex. Here, we describe a label-free method using live-cell imaging by 3D holotomographic microscopy (Nanolive) to visualize LD accumulation in the MH-S alveolar macrophage cell line after treatment with oleic acid, a monounsaturated fatty acid that promotes lipid accumulation. Keywords: Lipid droplet Alveolar macrophages Oleic acid Metabolism 3D holotomographic microscopy Lung pathologies Background Lipid droplets (LD) are dynamic cytoplasmic organelles that serve as intracellular energy storage, mainly in the form of triglycerides and cholesterol esters (van Dierendonck et al., 2022). In addition to their role in lipid storage and transport, LD are increasingly recognized to be involved in the regulation of other cellular processes, including inflammation, cell activation, and metabolism (Xu et al., 2018; Agudelo et al., 2020). Accumulation of LD is also related to several pathologies as obesity, diabetes, atherosclerosis, or lung diseases (Xu et al., 2018; Agudelo et al., 2020). Alveolar macrophages (AM) are the first line of defense against respiratory pathogens and play key roles in lung lipid metabolism (Agudelo et al., 2020). Excessive amounts of intracellular lipids in AM have been described in lung pathologies such as chronic obstructive pulmonary disease, acute lung injury, idiopathic pulmonary fibrosis, or pulmonary alveolar proteinosis, among others (Agudelo et al., 2020). Nevertheless, the mechanisms responsible for LD accumulation are not fully elucidated. Fluorescence-based live-cell imaging is the most widely used technique for LD studies, even though the phototoxicity of fluorescent dyes may disturb LD dynamics. Here, we present a protocol for live imaging of the LD accumulation without the addition of fluorescent labels, using 3D holotomographic microscopy (Nanolive). This label-free microscopy method reports the changes of the refractive indices (RIs) in three dimensions at high spatial and temporal resolution, allowing label-free analysis of organelle biology and kinetics studies with a low level of phototoxicity (Sandoz et al., 2019). We have performed this protocol in the MH-S alveolar macrophage cell line, grown in the presence of oleic acid to induce LD formation. Using this live-cell approach, foam cell formation can be analyzed under a physiological context, excluding the artefacts induced by the addition of different chemicals. Materials and Reagents µ-dish cell culture imaging dish, 35 mm, high (Ibidi, catalog number: 81156) 1,000, 200, and 20 µL pipette tips (pre-sterile w/ filter, hinged racks) (ExpellPlus, catalog numbers: 5030150C, 5030090C, 5130062C) 15 mL centrifuge tubes (Corning, catalog numbers: 430791) Corning® Costar® Stripette® serological pipettes, individually paper/plastic wrapped, 5 and 10 mL (Corning, catalog numbers: CLS4487, 4488) Falcon® 100 mm TC-treated cell culture dish (Corning, catalog number: 353003) RPMI 1640 media (Lonza, catalog number: BE 12-115F) Fetal bovine serum (FBS) (GibcoTM, catalog number: 10270106) Penicillin-streptomycin mixture (Lonza, catalog number: DE17-603E) Dulbecco's phosphate buffered saline (PBS) (10×), 95 mM (PO4) without calcium or magnesium (Lonza, catalog number: BE17-515Q) Bovine serum albumin (BSA)-oleate monounsaturated fatty acid complex (5 mM) (Cayman Chemical, catalog number: 29557) BSA control for BSA-fatty acid complexes (5 mM) (Cayman Chemical, catalog number: 29556) Trypsin/EDTA solution (Lonza, catalog number: CC-5012) Complete RPMI growth medium (see Recipes) Starving medium (see Recipes) Biological materials Cell line: MH-S (ATCC number: CRL-2019TM) Equipment Rainin Pipet-Lite XLS (Mettler Toledo, models: SL1000 and SL200) Forma Direct heat CO2 incubator 184 L digital model (Thermo Scientific, model: 311; TC 230) Biosafety cabinet (Telstar, model: Bio II Advance Plus IV) Centrifuge Sorvall ST 16R (Thermo Scientific, model: 75004380) 3D Cell Explorer microscope (Nanolive, Ecublens, Switzerland) CellDropTM FL automated cell counter (DeNovix) Laboratory water bath (Memmert, model: WNB 14) Software Steve software v1.6.3496 (Nanolive, Ecublens, Switzerland) ImageJ (FIJI) software (https://imagej.net/software/fiji/downloads) Procedure Cell culture Grow MH-S cells inside of a humidified incubator with 5% CO2 at 37 °C in complete RPMI growth medium (see Recipes) to 90% confluency in a 100 mm dish. Remove the culture media and wash the cells with 10 mL of PBS 1×. After removing the PBS, add 2 mL of trypsin/EDTA solution to detach cells, incubate in a humidified incubator with 5% CO2 for 5 min at 37 °C, and add 8 mL of complete RPMI growth medium to stop the reaction. Note: Temper the growth medium and trypsin solution at 37 °C in a water bath. Collect the cells and centrifuge at 423 × g for 5 min. Remove supernatant and resuspend the pellet in 5 mL of complete RPMI growth medium. Calculate the cell concentration using an automated cell counter. Seed MH-S cells in 35 mm μ-dishes (3 × 105 cells per dish) in 1.5 mL of complete RPMI growth medium. Note: Remember to seed additional dishes for control condition (see step B3). Incubate cells at 37 °C in a humidified incubator with 5% CO2 overnight. Temper the starving medium (see Recipes) at 37 °C in a water bath. Change the cells to tempered starving medium and incubate under the same culture conditions for approximately 3 h. BSA-oleate treatment Defrost an aliquot of BSA-oleate monounsaturated fatty acid complex (5 mM) in the water bath for 2 min. Prepare the BSA-oleate working solution at 100 µM in starving medium. Note: For 1.5 mL of medium, you need 60 µL of BSA-oleate monounsaturated fatty acid complex. Change the starving medium to starving medium + BSA-oleate (100 µM) or starving medium + BSA for the control. Prewarm the stage of the 3D Cell Explorer microscope to 37 °C at least 30 min before starting the acquisition. Place the plate in the 3D Cell Explorer microscope stage where the incubation conditions (37 °C and 5% CO2) will be maintained during the rest of the experiment. Note: Other holotomographic microscopes can also be used. Image acquisition This protocol is customized for a 3D Cell Explorer microscope equipped with a 60× magnification dry objective (λ = 520 nm, sample exposure 0.2 mW/mm2) and a depth of field of 30 μm. Proceed to capture images every minute for 18 hours, using the Steve software that controls the microscope. Each image provides a reconstruction of 30 μm in the z-axis, which corresponds to 100 images. Most of the images from the 100-image stacks are in fact out of focus, because samples are only visualized in a small range in the z-axis (around 5–10 stacks). From the whole z-stack, select at each time point the z-image in which the focus and resolution of the cells is best observed. LD are observed as intracellular spheres that have a high RI. Export the image using Steve software to transform RI volumes into .tiff format. Pictures are exported by default as 32-bits images. Process the exported image (.tiff format) using the FIJI software for performance purposes. Transform images to 8-bits and adjust gamma, brightness, and contrast identically for compared image sets using Fiji software. An example of time-lapse refractive images of LD in alveolar macrophages (MH-S cells) is shown in Figure 1. Figure 1. Time-lapse refractive images of lipid droplets (LD) in alveolar macrophages (MH-S cells). Images show LD accumulation in MH-S cells treated with oleic acid (green) compared to control condition (blue). Close-up images shown at the right column correspond to the position indicated in the small squares. Recipes Complete RPMI growth medium RPMI 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin. Starving medium RPMI 1640 media supplemented with 2% FBS and 1% penicillin-streptomycin. Acknowledgments We are grateful to Instituto de Salud Carlos III for financial support to S.H.(PI20CIII/00018). A. Pérez-Montero is supported by Comunidad de Madrid (PEJ-2020-AI/BMD-17651). Competing interests The authors declare no competing interests. References Agudelo, C. W., Samaha, G. and Garcia-Arcos, I. (2020). Alveolar lipids in pulmonary disease. A review. Lipids in health and disease 19(1): 122-122. van Dierendonck, X., Vrieling, F., Smeehuijzen, L., Deng, L., Boogaard, J. P., Croes, C. A., Temmerman, L., Wetzels, S., Biessen, E., Kersten, S., et al. (2022). Triglyceride breakdown from lipid droplets regulates the inflammatory response in macrophages. Proc Natl Acad Sci U S A 119(12): e2114739119. Xu, S., Zhang, X. and Liu, P. (2018). Lipid droplet proteins and metabolic diseases. Biochim Biophys Acta Mol Basis Dis 1864(5, Part B): 1968-1983. Sandoz, P. A., Tremblay, C., van der Goot, F. G. and Frechin, M. (2019). Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux. PLoS Biol 17(12): e3000553. Article Information Copyright © 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). How to cite Category Biophysics > Microscopy Cell Biology > Cell imaging > Live-cell imaging Do you have any questions about this protocol? Post your question to gather feedback from the community. We will also invite the authors of this article to respond. Write a clear, specific, and concise question. Don’t forget the question mark! 0/150 Tips for asking effective questions + Description Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images. Tags (0/5): Post a Question 0 Q&A Related protocols Acutely Modifying Phosphatidylinositol Phosphates on Endolysosomes Using Chemically Inducible Dimerization Systems Wei Sheng Yap [...] Maxime Boutry Oct 5, 2024 320 Views Preparing Chamber Slides With Pressed Collagen for Live Imaging Monolayers of Primary Human Intestinal Stem Cells Joseph Burclaff and Scott T. Magness Nov 20, 2024 280 Views Versatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells Angélique Pipier and David Monchaud Feb 5, 2025 199 Views News Become a Reviewer FAQs Other Resources Bio-protocol Exchange Bio-protocol Preprint Repository Bio-protocol Webinars © 2025 Bio-protocol LLC. ISSN: 2331-8325 Terms of Service Privacy Policy

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.