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4,930 | https://bio-protocol.org/en/bpdetail?id=4930&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
A Protocol for Custom Biomineralization of Enzymes in Metal–Organic Frameworks (MOFs)
ZA Zoe Armstrong *
DJ Drew Jordahl *
AM Austin MacRae
QL Qiaobi Li
ML Mary Lenertz
PS Patrick Shen
AB Anastasiia Botserovska
LF Li Feng
AU Angel Ugrinov
ZY Zhongyu Yang
(*contributed equally to this work)
Published: Vol 14, Iss 3, Feb 5, 2024
DOI: 10.21769/BioProtoc.4930 Views: 709
Reviewed by: Salim GasmiNeha Nandwani Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in ACS Applied Materials & Interfaces Nov 2022
Abstract
Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal–organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal–ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields.
Key features
• A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal–organic frameworks (MOFs) via co-crystallization.
• Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes.
• Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed.
• The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes.
Graphical overview
Keywords: Biomineralization MOF Enzyme immobilization Co-crystallization Aqueous phase coprecipitation
Background
Enzyme immobilization is receiving increased interest in both research and industry due to the (potential) promise of enhanced cost efficiency and catalytic performance control [1–4]. The biggest challenge is still maintaining enzymatic function without disturbance to the enzyme itself [5,6]. Metal–organic frameworks (MOFs) are extended 3-dimensional crystalline networks formed by the coordination bonds between certain metal ions and ligands. MOFs typically contain supreme porosity and highly tunable properties, given the high diversity in metal ions and ligands that can form such a 3D network. MOFs are advanced enzyme immobilization platforms but are mostly limited to smaller enzymes and substrates [6–13]. Co-crystallization of enzymes and certain metal ions/ligands is a unique way to host enzymes in MOF crystal scaffolds, being adaptable to large enzymes and enzyme clusters [14,15]. This strategy also allows for a small portion of enzymes to be implanted at the surface of MOF crystals, thus being partially exposed to the reaction medium, allowing contact with substrates larger than MOF apertures [16]. This strategy can therefore be applied to biocatalytic reactions—involving large substrates such as proteins/polypeptides, polysaccharides, and cells—to be carried out while reusing/recycling immobilized enzymes [17–25].
Commonly, the co-crystallization process is performed in an organic solvent such as methanol, which is a challenging condition for most enzymes [15]. There is a natural co-crystallization process called biomineralization, which is essentially co-crystallization but takes place in water phase under ambient conditions [26–28]. Although the reaction can be slow in nature, the formed biominerals are sufficiently stable with immobilized and possibly functional proteins/enzymes. Inspired by nature, biomineralization has also been carried out on lab benches [29,30]. Although nature might find its own sophisticated way to biomineralize proteins or other biomacromolecules, on-bench biomineralization in a reasonable timeframe for enzymes needs additional planning. Furthermore, although natural biomineralization could generate enzyme@MOF composites with distinct morphology and crystallinity, certain considerations are needed to optimize the on-bench strategy in order to better preserve enzyme activity and reusability and the stability of co-crystals. Lastly, aqueous phase co-crystallization may generate imperfect crystals but retains enzyme activity and reusability, which would still improve biocatalysis and thus be worth pursuing. Focusing on aqueous phase co-crystallization, we have acquired extensive experience in immobilizing various enzymes on MOFs [16,20,21]. Our recent work has revealed a combination of 10 metal ions/ligands to carry out biomineralization for effective enzyme immobilization; broadening the spectrum of available metal/ligand pairs allows for custom immobilization of enzymes according to their characteristics and stability (in certain metal ions and ligands) [19]. However, there is a lack of detailed experimental protocols to carry out such a sophisticated mission.
In this protocol, we detail the biomineralization procedures, highlight the precautions, potential pitfalls, and suggested solutions, and summarize the assessment of successful enzyme biomineralization based on our recent experience. We will cover MOF preparation in aqueous phase (metal/ligand selection and preparation and criteria of a useful MOF) to obtain a background without enzymes, enzyme@MOF composite formation (enzyme preparation and activity assessment on MOFs), and additional experimental conditions that may help the formation of co-crystals without damaging the enzymes. The strategy and methods can be applicable to immobilizing other enzymes and searching for other metal ion/ligand combinations for custom immobilization of other enzymes.
Materials and reagents
Reagents
Terephthalic acid (BDC, 98%) (Millipore Sigma, catalog number: 185361-100G)
4,4’-Biphenyldicarboxylic acid (BPDC) (CHEM-IMPEX INT’L Inc., catalog number: 26841)
Bicinchoninic acid (BCA) kit for protein determination (Millipore Sigma, catalog number: BCA1)
MES (Millipore Sigma, catalog number: 475893-100GM)
Sodium acetate (Millipore Sigma, catalog number: S8750)
HEPES (Millipore Sigma, catalog number: PHG0001-100G)
Glycine (Millipore Sigma, catalog number: 410225)
Ethanol (EtOH) (standard resources—could be obtained from any commercial resources)
Acetic acid (standard resources)
Sodium hydroxide (NaOH) (standard resources)
Concentrated hydrochloric acid (HCl) (standard resources)
Double-distilled water (standard resources)
Thin test tubes (Wiretrol® II, catalog number: 5-000-2020)
Solutions
MES buffer 0.5 M, pH 6 (10 mL) (see Recipes)
Acetate buffer 0.05 M, pH 4.6 (40 mL) (see Recipes)
HEPES buffer 0.2 M, pH 6.8 (40 mL) (see Recipes)
Glycine buffer 0.05 M, pH 9 (40 mL) (see Recipes)
Recipes
MES buffer 0.5 M, pH 6 (10 mL)
8 mL of double-deionized water (ddH2O)
0.976 g of MES
pH to 6 using HCl
Dilute to 10 mL
Acetate buffer 0.05 M, pH 4.6 (40 mL)
30 mL of ddH2O
82.272 mg of sodium acetate
0.057 mL of glacial acetic acid
pH to 4.6 using HCl
Dilute to 40 mL
HEPES buffer 0.2 M, pH 6.8 (40 mL)
30 mL of ddH2O
1.91 g of HEPES
pH to 6.8 using HCl
Dilute to 40 mL
Glycine buffer 0.05 M, pH 9 (40 mL)
30 mL of ddH2O
150 mg of glycine
10 mg of sodium hydroxide
pH to 9 with NaOH
Dilute to 40 mL
Equipment
Centrifuge (Thermo Fisher, model: Sorvall Legend Micro 21R)
Thermogravimetric analyzer (TA instruments Water, model: TGA-Q500)
XRD–Brucker’s Single Crystal Diffractometer, Apex 2 Duo with Cu IμS X-ray Source (Bruker Corporation, model: Apex 2 Duo)
Field-emission scanning electron microscope (STEM, model: JEOL JSM-7600F)
Millipore concentrators (Merck KGaA, catalog number: UFC500396)
Mortar and pestle setup (Millipore Sigma, catalog number: Z136077)
Spectrometer (Thermo Scientific, model: NanoDrop 2000)
Procedure
To minimize mineral impact on our MOF preparation, all water in this procedure is double-deionized water (ddH2O) and all solutions are prepared using ddH2O. The pH in all buffers is adjusted with 1 M HCl or NaOH. Enzyme activity often requires specific pH; however, certain MOFs are unstable in certain pHs. In this case, alternative buffers in the same pH range are provided in the Recipes section. All enzymes and enzyme@MOF composites should be stored at 4 and delivered on ice prior to activity tests. Enzyme@MOF composites do not need to be freshly prepared unless left in the fridge for over a month. All involved equipment operation and data analysis should follow the corresponding users’ manuals/guidance and will only be briefly covered here. A general overview of this protocol is shown in Figure 1.
Figure 1. An overview of the procedures involved in this protocol
Metal ion/ligand selection and aqueous phase MOF preparation
It is necessary to prepare MOFs without enzyme because these can serve as the background or control signals for characterization. The best pool to select metal ions and ligands are the published MOFs prepared via solvothermal or hydrothermal methods [6–8], bearing in mind that the same pair may not form the same crystal structures in the aqueous phase under ambient conditions or may not form crystal at all in water. A few summative lists of commonly seen combinations of metal ions and ligands can be found in the literature [8,14,31]. Enzyme stability and functionality in the presence of excess metal ions and ligands, solubility of ligands, and potential toxicity should be considered during the selection and scanning of metal ion–ligand combinations [32–35]. It is common to scan a number of metal ions and ligands but only find a few combinations that can form crystals [19]. Normally, +2 oxidation state of metal ions is preferred according to our experience, although +3 and +4 are possible in certain circumstances. Once a combination is found as judged by direct visualization, proceed with the following.
Optimize MOF forming conditions. This step is necessary because the optimal metal:ligand ratio often deviates from the content ratio in a MOF. We have found that the quality of co-crystals formed this way is dependent on the reaction volume even under the same metal and ligand concentrations. In rare cases, the anions of the metal ion stock can affect the formation and stability of co-crystals [36]. We found the following steps useful for a new metal/ligand combination:
Improve ligand solubility in water. Organic linkers/ligands can have a low solubility in water. To be able to reach the concentration needed for co-crystallization, a typical operation is to react the ligand with NaOH to prepare a salt-based ligand as detailed in our recent work. In brief, 50 mM NaOH reacting with 25 mM ligand in water under vigorous stirring at room temperature for 1–2 h should be sufficient, although the concentration and reaction time may vary depending on ligands. Taking terephthalic acid (BDC) and 4,4’-Biphenyldicarboxylic acid (BPDC) as example ligands, the following procedures were proved effective.
To prepare the more soluble disodium terephthalate (BDC-Na2), terephthalic acid (4.16 g, 25 mM) and NaOH (2.0 g, 50 mM) were mixed by stirring in 20 mL of ddH2O at room temperature until transparent (~1 h). Then, the mixture was precipitated in 400 mL of cold isopropanol by mixing. Precipitate was washed via centrifugation with isopropanol until filtrate reached pH 7 and dried overnight at 75 °C in an oven.
Disodium BPDC (BPDC-Na2) was synthesized by adding 6 g (22.2 mM) of BPDC along with 2.65 g of NaOH in 100 mL of ddH2O. The mixture was stirred at 95 °C for 3 h until transparent and then precipitated, washed, and dried in a similar fashion to the BDC-Na2 synthesis above.
Metal ion selection. Typical salts containing the needed metal ions with a low charge are preferred, as high negative charges may interference with the formation of MOFs [i.e., Zn(NO3)2, Ca(NO3)2, Al(NO3)2].
Caution: In water co-crystallization, the anion of the metal ion stock solution may affect the formation of the co-crystals. A typical example is ZIF: Zn(AoC)2 forms more stable, smaller ZIF, yet Zn(NO3)2 forms larger ones. When choosing commercial resources for metal ions, the anions should be carefully selected.
Forming MOF. We typically start with 1 mL of water containing 25 mM metal ions and 50 mM of ligands under gentle nutation at room temperature. MOFs can be formed from minutes to hours depending on metal and ligand selection.
Caution: We found it more effective to add the metal first followed by the ligand.
Washing off unreacted species. We suggest centrifuging the composites (13,000× g for 5 min) and removing the water from the supernatant as much as possible. Then, 1 mL of EtOH or MeOH should be used to wash additional reaction residuals as water may crush the formed crystals, which can be weak given the formation condition. Usually, three washes are sufficient. The obtained co-crystals should be stored at 4 for further use.
Caution: We suggest avoiding extensive washes with water as it may disrupt the crystal lattice. In addition, organic solvents are easier to dry out for crystal characterization.
MOF characterization. All characterization techniques are well-established with standard operation procedures. Here, we only highlight the differences/precautions when dealing with MOFs formed in the aqueous phase.
Powder X-ray diffraction (PXRD). Upon removal of EtOH via drying, a PXRD sample should be prepared by loading the powder to the bottom of a thin test tube (Figure 2). The sample height should be ~1.6 mm with a ~2.5 mm seal. If a single crystal is formed (which may happen in aqueous phase too), then the crystal should be directly loaded into the X-ray diffractometer for data acquisition. For powder samples, it is very important to finely grind the particles in order to obtain a high-quality diffraction pattern. Usually, we use a regular mortar and pestle setup. Once data are acquired, we typically compare the pattern from 4–70° of 2θ to those reported in the literature on the same metal/ligand. If no match can be found, there is a chance that we are forming multi-phase co-crystals or a completely new crystal, either of which can be resolved with a powder X-ray diffractometer and proper analysis.
Figure 2. Preparing a powder sample for powder X-ray diffraction (PXRD) data acquisition by loading the powder to the bottom of a thin test tube
Scanning electron microscope (SEM). SEM is needed to confirm the morphology and size of the formed co-crystals. Regular operation on SEM data acquisition is applicable here without special cautions (~2 mg of dried sample is needed).
Thermalgravimetric analysis (TGA). TGA needs special caution because the sample holders can be sensitive and thus be damaged during data acquisition for MOFs made of Al, Zn, Ni, Fe, etc. Our typical suggestion is to select ceramic holds for these MOFs and regular holds for the rest (to save the cost). In most cases, 10 mg of sample is needed.
Caution: It is very important to completely dry all samples.
N2 isotherm. This is necessary to confirm the porosity, which is needed for substrate diffusion and enzyme activity. N2 isotherm measurements require a high amount of sample and high crystallinity. A collapsed N2 isotherm plot often indicates poor crystallinity and porosity [19]. Typical data analysis reported in the literature is applicable here. We found 10 mg of sample is needed, with typical porosity of 0.05–0.5 cm3/g being the normal range for an acceptable MOF.
Caution: Avoiding washing with water can reduce the potential crystal damage during wash. MeOH or EtOH are good options.
pH stability. Due to the potential interaction between metal ions and anions in buffers, certain buffers can disassemble certain MOFs. PBS buffer is a typical example, wherein the highly negatively charged phosphate group could coordinate with cations and disassemble the MOF scaffolds. We found citrate buffer also capable of disassembling MOFs. For low pH ranges, we typically use acetate and MES buffer; for near-neutral pH, we found HEPES buffer the best; at high pH range, glycine buffer is optimal. These buffers (usually at 50 mM concentration) also do not significantly affect the activities of most enzymes and thus should be tested on the formed MOFs in order to guide the pH stability for the next steps. Once soaked in a buffer, we monitor the turbidity at 450 nm over time using a spectrometer. If no turbidity changes over a certain time (one day for example), the pellets will then be subjected to PXRD and SEM studies to confirm the presence of co-crystals.
Thermal stability. For thermal stability testing, we usually place the co-crystals in an oven and set the temperature to the target temperature. For most biological applications, 37–45 is the typical temperature range. The turbidity is measured approximately every hour to confirm the presence of crystals. After 1–2 days, if a crystal is still present, then PXRD and SEM should be applied to confirm, as broken crystals can also show turbidity. The same test should be carried out for crystals stored at 4 over a long period of time to document the long-term stability of the formed crystals.
Enzyme@MOF composite preparation
Enzymes may participate in the co-crystallization of metal ions and ligands by serving as the nuclei, which can affect the kinetics of co-crystal formation and even the structure. Thus, all data on enzyme@MOF composites should be compared to those on MOFs alone. Because enzymes’ properties differ significantly, the following procedures were only based on our experience on a few enzymes. Specific enzymes’ biomineralization should be dealt with in a case-by-case manner.
Prepare enzymes and control experiments. This step is necessary to retain enzyme functionality and validate the measured enzyme activity in the next steps.
Enzyme preparation. If an enzyme is ordered in the powder form, then an appropriate buffer or ddH2O should be used to dissolve it under the manufacturer’s instructions if available. If no instructions are given, we typically start with HEPES buffer. The typical enzyme stock concentration should follow the ones that are suggested in the literature, as high enzyme concentration can cause enzyme precipitation. We suggest storing enzymes below 1 mM at 4 . If possible, it is also ideal to store enzymes in the buffers that are favored by MOF (see above). If an enzyme is ordered or expressed/purified in the solution form in a low concentration, then a buffer exchange via dialysis or centrifugation-concentrators is needed to maximize the effectiveness of MOF encapsulation.
Caution: Minimize enzyme loss by optimizing concentration and buffer selection during buffer exchange.
Positive control of enzyme activity. Typical activity assays should be carried out to confirm that the enzymes are active. Usually, results are compared to the activity data from a reliable resource with known activity. Often, the dependence of activity as a function of enzyme concentration is needed in order to confirm that enzymes are active.
Negative controls of activity. It is necessary to confirm that metal ions, ligands, and MOFs alone do not show activity using the typical activity assays. Should a metal/ligand combination influence the activity of the target enzyme, an alternative MOF should be used to biomineralize this specific enzyme. Enzymes physically mixed with MOFs after washing (on ice with sonication under 50% duty cycle with medium power) should also be subjected to activity tests to confirm no physical adsorption of enzymes. This is important because physical adsorption can result in enzyme leaching and poor reusability after multiple rounds of activity tests. Only enzymes implanted in MOFs are needed.
Caution: Although metal ions and ligands often do not affect enzyme activity measurements, depending on the activity kit and mechanism certain metal ions and ligands may affect the reading of the involved equipment. Thus, negative controls are necessary and have to be carefully designed.
Prepare enzyme@MOF composites.
Typical recipe: usually, mixing 1 mL of ddH2O, 25 μL of 0.5 M metal salt solution, 1 mM enzyme, and 0.5–0.25 M ligand (total volume is usually ~1.1 mL after mixing) depending on the ligand can form the needed composites. The concentrations are mostly applicable to the MOFs reported in our recent work. Should a new combination of metal and ligand be needed, the concentrations of both metal ion and ligand need to be optimized.
Caution: Avoiding high enzyme concentrations can reduce enzyme loss.
Reaction at room temperature overnight under nutation.
Wash with EtOH at 4 as described above (step A.1.d). The low temperature is needed for retaining enzyme activity.
The prepared enzyme@MOF composites can be stored at 4 .
Enzyme@MOF characterization. All characterization techniques are well-established with standard operation procedures. Here, we only highlight the differences/cautions when dealing with enzyme@MOF composites.
PXRD. Upon removal of EtOH via drying, a similar loading procedure and analysis should be applied as described above (step A.2.a). Example PXRD data is shown in Figure 3A.
Figure 3. Example powder X-ray diffraction (PXRD) (A), scanning electron microscope (SEM) (B), thermalgravimetric analysis (TGA) (C), and lipase activity (D) data when lipase was immobilized in a model metal–organic framework (MOF), Zn-BDC [19]. The close PXRD patterns in the absence and presence of enzyme indicate that the enzyme did not cause significant alterations to the crystal structure (A). SEM images displayed the general shape of the enzyme@Zn-BDC biocomposites (A). TGA data suggested the loading capacity (~1%; C). The activity assay indicates that lipase is active in Zn-BDC (D).
SEM. SEM is needed to confirm the morphology and size of the formed co-crystals. Regular operation on SEM data acquisition is applicable here without special cautions (2 mg of the composite sample is needed). Example SEM data is shown in Figure 3B.
TGA. TGA needs special precautions because the sample holders can be sensitive and thus damaged during data acquisition for MOFs made of Al, Zn, Ni, Fe, etc. Our typical suggestion is to select ceramic holds for these MOFs and regular holds for the rest (to save the cost). In most cases, 10 mg of sample is needed. The TGA of enzyme@MOF composites should be compared to that of MOF alone, which should highlight the weight loss due to enzyme encapsulation. Example TGA data is shown in Figure 3C.
N2 isotherm. Typical data analysis reported in the literature is applicable here. We found 10 mg of sample is needed. The N2 isotherm of enzyme@MOF composites should be compared to that of MOF alone, which should highlight the porosity loss due to enzyme encapsulation. The typical porosity loss range is 0.05–0.3 cm3/g. Most N2 isotherm instruments directly provide digital numbers of porosity.
pH and thermal stability. A similar procedure to test the pH and thermal stability should be carried out for the enzyme@MOF composites as well.
Enzyme activity on MOFs. Enzyme@MOF composites should be subjected to the activity assays mentioned above to confirm the functionality of the encapsulated enzymes. Also, if enzyme performance needs to be compared among different MOFs, then the same loading capacity should be used (or at least normalized) for comparison. This is typically done via BCA assay. In detail, the formation and reduction of Cu2+ to Cu+ with the help of specific amino acids (cystine, tryptophan, tyrosine, etc.) is proportional to the amount of protein present. The commercially available BCA working reagent is light green and turns purple-blue in the presence of protein. Approximately 0.1 mL of protein sample is mixed with 2 mL of working BCA reagent and incubated for 30 min at 37 °C. Absorbance of standard solutions and samples are measured at 562 nm. Under the same enzyme quantity, a fair comparison can be carried out to determine which MOF best reserves enzyme activity. Example activity data when lipase is immobilized in Zn-BDC is shown in Figure 3D.
If multiple combinations of metal ions and ligands are all able to immobilize enzymes with acceptable activity remaining, usually large crystalline MOFs are favorable in biocatalysis applications, although smaller particles may improve the catalytic efficiency against large-size substrates.
Data analysis
The typical data analysis procedure is to compare the characterization data with published ones. For example, the PXRD pattern of a MOF with and without enzyme immobilization should be compared to the literature on the same MOF without enzymes (for examples, see Figure 4).
Figure 4. Simulation of the powder X-ray diffraction (PXRD) pattern of Ni-BDC (A) and Ni-BPDC (B) based on published structures upon overlapping with the experimental data [37,38]. Simulation was done using the freeware Mercury developed by the Cambridge Crystallographic Data Centre, which is accessible to most public academic users. Based on the simulation, we were able to propose the possible crystal structure of our biomineralization products.
It is possible to find multiple PXRDs for a certain MOF in the literature. The obtained MOFs could be a combination of several PXRD patterns, indicating the presence of multiple crystal phases as in the case shown in Figure 5, wherein Al-BDC MOF synthesized in the aqueous phase is most likely a multi-phase MOF with at least two possible structures. It is not uncommon to see multi-phase MOF when the synthesis is carried out in water under ambient conditions.
Figure 5. Simulation of the powder X-ray diffraction (PXRD) pattern of Al-BDC (black) based on two published structures (inset) upon overlapping with the experimental data (purple) [39]. Simulation was done using the freeware Mercury developed by the Cambridge Crystallographic Data Centre, which is accessible to most public academic users. Our PXRD patterns do not match either simulated spectrum, suggesting the possibility of multiple phases coexisting in our products.
The activity assay of the enzyme in solution and upon immobilization in MOFs should also be compared. Depending on the enzyme being studied, different data analysis and interpretation could be carried out. For example, for lysozyme enzyme, we typically compare the drop in OD450 and compare the slope to that of the free enzyme in the lysozyme activity assay. For lipase, we compare the slope of increase in optical density to assess the efficiency of catalysis by lipase. Details of the comparison are shown in our recent work [19].
Validation of protocol
The whole procedure is validated in our recent work and supplemental information [19].
General notes and troubleshooting
All procedures assumed normal conditions. Once an enzyme is immobilized on various MOFs, additional considerations could be worth mentioning to better utilize the formed composites.
Cause of activity difference. For biomaterials/biocatalyst development purposes, molecular-level details of the performance of enzymes are often needed in order to understand the functionality and guide the rational design of future MOF platforms. Depending on enzyme and metal/ligand selection, multiple reasons could cause the differences in enzyme activity on different MOFs even under the same loading quantity. For example, different MOFs may present distinct hydrophilicity and thus result in different enzyme intrinsic dynamics (most enzymes are hydrophilic and could be less active in a hydrophobic scaffold). Smaller ligands may present smaller gaps/pores and thus tight restrictions to enzymes, which would also reduce the activity. For large-substrate enzymes, the amount of active site being exposed to the solution is directly related to the activity. These structure/dynamic details of enzymes upon biomineralization in MOFs can be probed using our recently developed techniques [40,41].
Disassemble MOFs to release enzymes. This is a common practice to confirm the loading capacity and enzyme functionality after MOF encapsulation. Most MOFs are unstable under either acidic or basic pHs as well as specific buffers (e.g., PBS buffer). Thus, it is possible to disassemble MOFs to release the enzyme and double-check the integrity using circular dichroism and activity assay. This is also an effective approach to confirm the loading capacity.
Other synthetic conditions of enzyme@MOF composites. We found it to be practically useful if slightly higher temperatures (< 60 ) can help the formation of co-crystals without damaging some enzymes. It is also possible to use some modulators to adjust the rate of crystal formation.
Precautions on metal/ligand selection according to the target enzyme. Metalloenzymes should receive additional care when being biomineralized this way because the endogenous metal binding site may be occupied by the metal ions required for MOF formation. Our typical suggestion would be to use MOFs with charges different from the endogenous metal. For example, to immobilize human Cu/Zn superoxide dismutase (SOD1) [42,43], Al3+ should be used as the metal center of MOF.
Imperfect crystallinity and low porosity of MOFs formed in the aqueous phase. High crystallinity definitely enhances stability and substrate diffusivity. However, if an amorphous or multi-phase crystal is formed when the enzyme is co-crystallized with certain metal ions and ligands, which can be easily and quickly confirmed by PXRD, we still suggest testing the reusability of enzymes on these MOFs. It is likely that the imperfect crystals are still able to immobilize enzymes and retain enzyme activity and thus be useful for biocatalysis applications. It is especially useful when only specific metal ions can be applied to immobilize a metalloenzyme and imperfect crystals are the only option.
Stability and reusability. The enzyme@MOF composites are generally stable after interacting with substrates under reaction conditions. This has been confirmed with our reusability tests in the key reference [19].
Acknowledgments
This work is supported by the National Science Foundation (NSF: MCB 1942596 and DMR 2306137). We appreciate Dr. Peter G. Fajer for generously donating the Bruker ECS-106 to our institution (North Dakota State University) and Dr. Wayne Hubbell for generously providing the EPR data analysis software.
Competing interests
The authors declare no competing interests.
Ethical considerations
No human subjects are involved in this work.
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4,931 | https://bio-protocol.org/en/bpdetail?id=4931&type=0 | # Bio-Protocol Content
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Streamlined Adeno-Associated Virus Production Using Suspension HEK293T Cells
AK Aditi A. Kulkarni
AS Austin G. Seal
CS Corinne Sonnet
KO Kazuhiro Oka
Published: Vol 14, Iss 3, Feb 5, 2024
DOI: 10.21769/BioProtoc.4931 Views: 1309
Reviewed by: Neha NandwaniXiaozhe DingVictor Tse
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Abstract
Recombinant adeno-associated viruses (rAAVs) are valuable viral vectors for in vivo gene transfer, also having significant ex vivo therapeutic potential. Continued efforts have focused on various gene therapy applications, capsid engineering, and scalable manufacturing processes. Adherent cells are commonly used for virus production in most basic science laboratories because of their efficiency and cost. Although suspension cells are easier to handle and scale up compared to adherent cells, their use in virus production is hampered by poor transfection efficiency. In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent. The established protocol allows AAV production from transfection to quality analysis of purified AAV within two weeks. Typical vector yields for the described suspension system followed by iodixanol purification range from a total of 1 × 1013 to 1.5 × 1013 vg (vector genome) using 90 mL of cell suspension vs. 1 × 1013 to 2 × 1013 vg using a regular adherent cell protocol (10 × 15 cm dishes).
Key features
• Adeno-associated virus (AAV) production using serum-free-media-adapted HEK293T suspension cells.
• Efficient transfection with VirusGEN.
• High AAV yield from small-volume cell culture.
Graphical overview
Keywords: AAV Suspension cells Serum-free media Transfection reagent Iodixanol density gradient
Background
The adeno-associated virus (AAV) is a non-enveloped virus belonging to the Parvoviridae family that was discovered as a contaminant in adenovirus preparations (Atchison et al., 1965). The AAV has icosahedral protein capsids of approximately 26 nm in diameter containing a single-stranded DNA genome of approximately 4.7 kb (Wang et al., 2019). Recombinant AAV (rAAV) primarily forms non-integrating episomes and sustains long-term transgene expression. It is considered to be a non-pathogenic virus and a leading viral gene transfer vector for human gene therapy (Pupo et al., 2022). Due to the discovery of numerous naturally occurring variants (> 200) and the small size of the Cap gene, capsid engineering to confer new properties to the vector has become a hot research area (Wang et al., 2019; El Andari and Grimm, 2021). Improving rAAV production that can easily be adapted to engineered serotypes is increasingly important to meet clinical demand as well as basic science needs.
Most laboratories commonly use adherent HEK293 or HEK293T cells as producer cells because transfection of adherent cells is more efficient than that of suspension cells. However, suspension cells are easier to handle and scale up, which is of particular interest to Good Manufacturing Practice (GMP) facilities. Scalable production methods include transfection of Sf9 (Mietzsch et al., 2014), HEK293 (Grieger et al., 2016), or HEK293T cells (Zhao et al., 2020). Although the insect cell-based method is more robust (Mietzsch et al., 2014), baculoviruses must be prepared before AAV production. Therefore, transfection of mammalian cells using three plasmids (a helper AdΔF6 plasmid, a Rep/Cap plasmid, and the transgene-containing AAV transfer plasmid) is more versatile and easier to adapt to newly engineered capsids. The most common transfection reagent for suspension cells is polyethyleneimine (PEI) because it is inexpensive and effective (Grieger et al., 2016; Blessing et al., 2019; Zhao et al., 2020). However, PEI is cytotoxic. Most protocols using PEI require changing media (Zhao et al., 2020; Challis et al., 2019) or diluting transfection reagent (Grieger et al., 2016; Blessing et al., 2019), while VirusGEN, a newly developed transfection reagent, is less toxic and does not require media change. We found that AAV production by transfecting HEK293T suspension cells by VirusGEN is superior to PEI and its efficiency is comparable to that of transfecting adherent cells by iMFectin poly (Deng and Oka, 2020).
There are several methods available for the purification of AAV (El Andari and Grimm, 2021). A commonly used method involves either cesium chloride (CsCl) (Ayuso et al., 2010) or iodixanol density gradient (Zolotukhin et al., 1999), which offers an advantage in separating empty and full capsids based on their density regardless of the serotype. This method allows the simultaneous purification of multiple small-scale preparations. However, scaling up or automating this process presents challenges. Another purification method is liquid chromatography. In this approach, ion exchange columns or affinity columns are connected to fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) (Nass et al., 2018; Joshi et al., 2021; Florea et al., 2023). This method is robust and preferred for clinical-grade AAV, since it can be automated and is suitable for large-scale production. However, the affinity column does not differentiate between empty and full capsids. Generally, a second column, such as an anion exchange (AEX) column, is employed to separate empty and full capsids based on the charge difference brought on by the vector genome. However, the chromatographic purification method requires adjustments and optimizations for each serotype. The choice of purification method(s) depends on various factors, including the specific AAV serotype, the downstream application, available resources, cost, and desired purity level.
Purified AAV can be characterized by several methods for quality control (QC). The most important QC assay is genome titer, which can be standardized among laboratories using reference standard material (Ayuso et al., 2014). The most popular method for this purpose is quantitative PCR (qPCR), with digital PCR (dPCR) being the most recent advancement of qPCR technology (Quan et al., 2018). Although dPCR measures absolute numbers without a standard, dPCR has a narrow dynamic range compared to qPCR due to the limited number of partitions. The conventional assay to determine empty capsids in AAV preparations is transmission electron microscope (TEM) (Grieger et al., 2006). Nonetheless, FPLC- or HPLC-AEX (Lock et al., 2012; Khatwani et al., 2021) and HPLC coupled with size exclusion column and multi-angle light scattering (McIntosh et al., 2021) have gained popularity for their simplicity and high throughput. Analytical ultracentrifugation, mass spectrometry, and charge detection mass spectrometry (Werle et al., 2021; Ebberink et al., 2022) are used to analyze empty and full capsids, including those containing partial genomes. For detecting minor DNA contaminants, such as host DNA contamination and plasmid DNA used for transfection, next-generation sequencing (Lecomte et al., 2015; Guerin et al., 2020) is employed, which can also assess vector genome integrity. The integrity of capsid proteins can be assessed by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which detects major capsid proteins (VP1, VP2, and VP3) in a 1:1:10 ratio and other protein contaminants. However, the heterogeneity of capsid proteins has been reported. Another important QC parameter that may influence transgene expression is the infectivity of AAV. Different serotypes use different cellular docking sites, making a universal method difficult to achieve. The most accurate assay relying on AAV biological activities is the infectious center assay, while the most widely used is the median tissue infective dose (TCID50) assay, which determines genome replication by qPCR (Zen et al., 2004). Neither assay addresses cell type-specific infectivity. Although measuring intracellular vector genomes upon cell infection may not directly reflect biological activities (François et al., 2018), this method is simple and broadly applicable to any target cell. Therefore, the choice of QC methods is again dictated by the same factors described for purification methods.
In this protocol, a subclone of HEK293T cells adapted for serum-free media is transfected in suspension and AAV9 is purified by iodixanol density gradient (Zolotukhin et al., 1999). The critical determinants for transfection are cell viability and cell density. The combination of suspension cells and effective transfection reagents allows high-yield vector production [> 1 × 1013 vector genome of purified AAV9 from 90 mL of culture vs. 1 × 1013 to 2 × 1013 vg from a comparable adherence cell culture described in Deng and Oka (2020)]. In addition, hands-on time is drastically shortened compared with conventional adherent cell protocols. AAVs produced by this protocol have been characterized by qPCR, SDS-PAGE, TEM, and infectious titer assay. The quality of AAVs of different serotypes and transgenes varies and additional QCs may be required in some experiments. Using the same protocol and QCs to standardize AAVs for experiments is recommended.
Materials and reagents
Biological materials
HEK293T (ATCC, catalog number: CRL-3216) adapted for serum-free media; alternatively, request BalanCD HEK293 serum-free-media-adapted HEK293T cells (1F11S) from the corresponding author
pAdΔF6 helper plasmid (Addgene, catalog number: 112867)
pAAV2/9 Rep/Cap plasmid (Addgene, catalog number: 112865)
AV0-EF1-N-cG (control AAV transfer plasmid) (Addgene, catalog number: 192888) or AV0-EF1-N-tdT (Addgene, catalog number: 192889)
DNase I (Sigma-Aldrich, catalog number: DN25-1G)
RNase A (Thermo Fisher Scientific, catalog number: BP25391)
Forward qPCR primer such as WPRE or other vector specific primer (Sigma-Aldrich, WPRE-172 nucleotide sequence: TTTATGAGGAGTTGTGGCCC)
Reverse qPCR primer such as WPRE or other vector specific primer (Sigma-Aldrich, WPRE-392 nucleotide sequence: CAACACCACGGAATTGTCAG)
Reagents
BalanCD HEK293 media liquid or powder (Fujifilm Irvine Scientific, catalog number: 91165 or 94137, respectively)
GlutaMAX (Thermo Fisher Scientific, catalog number: 35050061)
100× Pluronic F-68 (Thermo Fisher Scientific, catalog number: 24040032)
VirusGEN AAV Transfection kit (Mirus Bio, catalog number: MIR 6750)
1 M Tris-HCl pH 8.0 (Thermo Fisher Scientific, catalog number: AAJ22638AP)
NaCl (Sigma-Aldrich, catalog number: BP35810)
MgCl2 Sigma-Aldrich, catalog number: 442611-500GM)
KCl (Sigma-Aldrich, catalog number: 1049360250)
1 M DTT (Thermo Fisher Scientific, catalog number: 11-101-3992)
Glycerol (Thermo Fisher Scientific, catalog number: J62399.AP or equivalent)
Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750)
HEPES (Sigma-Aldrich, catalog number: H3784)
Sarcosyl (VWR, catalog number: D719-500G)
EDTA (Sigma-Aldrich, catalog number: E5134)
PEG8000 (Sigma-Aldrich, catalog number: P2139-2KG)
OptiPrep Density Gradient (Thermo Fisher Scientific, catalog number: NC1174452)
Phenol red (Sigma-Aldrich, catalog number: P0290-100ML)
DPBS (Gendepot, catalog number: CA008-300 or equivalent)
HyPure molecular biology grade water (Thermo Fisher Scientific, catalog number: SH3053802 or equivalent)
SYBR Green SuperMix (VWR, catalog number: 101414-168)
Sodium bicarbonate (Sigma-Aldrich, catalog number: S6014-500G)
Trypan blue (Thermo Fisher Scientific, catalog number: 15250061)
Solutions
Complete BalanCD HEK293 media (c-BalanCD HEK293) (see Recipe 1)
DNase I solution (see Recipe 2)
RNase A solution (see Recipe 3)
2 M MgCl2 (see Recipe 4)
5 M NaCl (see Recipe 5)
2.5 M NaCl (see Recipe 6)
40% PEG8000/2.5 M NaCl (see Recipe 7)
TMN (see Recipe 8)
HBS (see Recipe 9)
5% sodium deoxycholate (see Recipe 10)
PBS-MK (see Recipe 11)
PBS-NMK (see Recipe 12)
DPBS/Pluronic F-68 (see Recipe 13)
BalanCD HEK293 media—powder reconstituted (see Recipe 14)
Recipes
c-BalanCD HEK293 media
Reagent Final concentration Quantity
BalanCD HEK293 [or BalanCD HEK293—powder reconstituted (see Recipe 14)] n/a 97 mL
100× GlutaMAX 4 mM 2 mL
100× Pluronic F-68 1× 1 mL
Total n/a 100 mL
Store this solution at 2–4 °C. See General Note 1 for alternatives to this recipe.
DNase I solution
Reagent Final concentration Quantity
1 M Tris-HCl pH 7.6 20 mM 2 mL
NaCl 50 mM 0.2922 g
Glycerol 50% (v/v) 50 mL
1 M DTT 1 mM 100 µL
DNase I 10 mg/mL 1 g
ddH2O n/a Add up to 100 mL
Total n/a 100 mL
Adjust pH to 7.6 (see General Note 2). See General Note 3 for more information about ddH2O. Aliquot this solution into 1.5 mL microcentrifuge tubes and store at -20 °C.
RNase A solution
Reagent Final concentration Quantity
1 M Tris-HCl pH 7.5 10 mM 1 mL
NaCl 15 mM 87.66 mg
RNase A 10 mg/mL 1 g
ddH2O n/a Add up to 100 mL
Total n/a 100 mL
Adjust pH to 7.5. Aliquot this solution into 1.5 mL microcentrifuge tubes and store at -20 °C.
2 M MgCl2
Reagent Final concentration Quantity
MgCl2 2 M 95.211 g
ddH2O n/a Add up to 500 mL
Total 2 M 500 mL
Filter with a 0.22 µm pore size filter. Store this solution at room temperature.
5 M NaCl
Reagent Final concentration Quantity
NaCl 5 M 292.2 g
ddH2O n/a Add up to 1,000 mL
Total 5 M 1,000 mL
Filter with a 0.22 µm pore size filter. Store this solution at room temperature.
2.5 M NaCl
Reagent Final concentration Quantity
NaCl 2.5 M 73.05 g
ddH2O n/a Add up to 500 mL
Total 2.5 M 500 mL
Store this solution at room temperature.
40% PEG 8000/2.5 M NaCl
Reagent Final concentration Quantity
5 M NaCl (see Recipe 5) 2.5 M 500 mL
PEG8000 40% 400 g
ddH2O n/a Add up to 1,000 mL
Total n/a 1,000 mL
Prime a 0.22 µm pore size filter with 2.5 mL of 2.5 M NaCl before filtering the 40% PEG 8000/2.5 M NaCl solution. The filtering will take over an hour due to the viscosity of the solution. Store at room temperature.
TMN
Reagent Final concentration Quantity
1 M Tris-HCl pH 8.0 50 mM 50 mL
2 M MgCl2 5 mM 2.5 mL
NaCl 150 mM 8.77 g
ddH2O n/a Add up to 1,000 mL
Total n/a 1,000 mL
Adjust solution to pH 8.0. Filter with a 0.22 µm pore size filter. Store this solution at room temperature.
HBS
Reagent Final concentration Quantity
HEPES 50 mM 2.980 g
NaCl 150 mM 2.192 g
Sarcosyl 1% 2.5 g
EDTA 20 mM 1.46 g
ddH2O n/a Add up to 250 mL
Total n/a 250 mL
Adjust solution to pH 8.0. Filter with a 0.22 µm pore size filter. Store this solution at room temperature.
5% sodium deoxycholate
Reagent Final concentration Quantity
Sodium deoxycholate 5% 28.2 g
ddH2O n/a Add up to 500 mL
Total 5% 500 mL
Filter with a 0.22 µm pore size filter. Store this solution at room temperature. Protect from light.
PBS-MK
Reagent Final concentration Quantity
MgCl2 2.7 mM 263 mg
KCl 2 mM 149.1 mg
DPBS n/a Add up to 1,000 mL
Total n/a 1,000 mL
Filter with a 0.22 µm pore size filter. Store this solution at 2–4 °C.
PBS-NMK
Reagent Final concentration Quantity
NaCl 1 M 29.2 g
MgCl2 2.7 mM 131.5 mg
KCl 2 m 74.55 mg
DPBS n/a Add up to 500 mL
Total n/a 500 mL
Filter with a 0.22 µm pore size filter. Store this solution at 2–4 °C.
DPBS/0.001% Pluronic F-68
Reagent Final concentration Quantity
1× DPBS n/a 500 mL
100× Pluronic F-68 0.001% 50 µL
Total n/a 500 mL
Store this solution at 2–4 °C.
BalanCD HEK293 media—powder reconstituted
Reagent Final concentration Quantity
BalanCD HEK293 powder n/a 21.32 g
Sodium bicarbonate 2.20 g
ddH2O n/a Add up to 1,000 mL
Total n/a 1,000 mL
Ensure that the pH of the solution is between 6.7 and 7.4 and the osmolality is between 280 and 320 mOsm/kg. Filter with a 0.22 µm pore size filter. Store this solution at 2–4 °C for up to one year.
Laboratory supplies
Pipet-X pipette controller (Rainin, catalog number: 17011733 or equivalent)
20 µL pipette (Rainin, catalog number: 17008650 or equivalent)
200 µL pipette (Rainin, catalog number: 17008652 or equivalent)
1000 µL pipette (Rainin, catalog number: 17008653 or equivalent)
Filtered 20 µL pipette tips (Rainin, catalog number: 30389274 or equivalent)
Filtered 200 µL pipette tips (Rainin, catalog number: 30389276 or equivalent)
Filtered 1000 µL pipette tips (Rainin, catalog number: 30389272 or equivalent)
125 mL baffled Erlenmeyer flasks (Sigma-Aldrich, catalog number: CLS431404-1EA)
250 mL baffled Erlenmeyer flasks (VWR, catalog number: 75993-572)
0.6 mL microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-120)
1.5 mL microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-129)
0.5 mL microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 02-707-357)
Microtube racks (Thermo Fisher Scientific, catalog number: 22-313630)
Countess cell counting chamber slides (Thermo Fisher Scientific, catalog number: C10228)
24-well polystyrene microplates (Thermo Fisher Scientific, catalog number: 08-772-1)
15 mL polypropylene centrifuge tubes (VWR, catalog number: 89039-666)
15 mL steel wire racks (Thermo Fisher Scientific, catalog number: 3422306)
50 mL steel wire racks (Thermo Fisher Scientific, catalog number: FB147916A)
250 mL polypropylene centrifuge tubes (Thermo Fisher Scientific, catalog number: 05-538-53)
0.22 µm bottle top filters (Genesee Scientific, catalog number: 25-235)
10 mL serological pipettes (VWR, catalog number: 29443-047)
5 mL serological pipettes (VWR, catalog number: 29442-422)
Amicon centrifugal filter units (Sigma-Aldrich, catalog number: UFC910024)
5 mL sterile syringes (Thermo Fisher Scientific, catalog number: 14955458)
20 G sterile hypodermic needles (Thermo Fisher Scientific, catalog number: 14-817-209)
Thinwall polypropylene centrifuge tubes (Beckman Coulter, catalog number: 326823)
96-well qPCR plates (Genesee Scientific, catalog number: 27-105)
Strip caps for qPCR plate (VWR, catalog number: ST401425)
Fine-tipped markers (Thermo Fisher Scientific, catalog number: 19-166-600)
Alcohol wipes (Thermo Fisher Scientific, catalog number: 22-037790)
Equipment
Biosafety cabinet (NuAire LabGard, Class II, type A2, catalog number NU-540-400UB10 or equivalent)
CO2 resistant shaker platform (Thermo Scientific, catalog number: 88881103 or equivalent)
Agilent qPCR Machine (Agilent, model: MX30005P or equivalent)
Countess automated cell counter (Invitrogen, catalog number: C10281 or equivalent)
Reach-in CO2 incubator (Cell IQ, catalog number: MCO-80ICL-PA or equivalent)
Avanti J-15R benchtop centrifuge (Beckman Coulter, catalog number: B99517 or equivalent)
JS-4750 swinging-bucket rotor (Beckman Coulter, catalog number: B77580 or equivalent)
60 mm diameter bottle adaptors (Beckman Coulter, catalog number: 392079 or equivalent)
18 mm diameter tube adaptors (Beckman Coulter, catalog number: 359473 or equivalent)
Avanti JXN-26 centrifuge (Beckman Coulter, catalog number: B38619 or equivalent)
JA-25.50 fixed-angle rotor (Beckman Coulter, catalog number: 363058 or equivalent)
JS-5.3 swinging-bucket rotor (Beckman Coulter, catalog number: 368690 or equivalent)
Optima XPN-90 ultracentrifuge (Beckman Coulter, catalog number: A99842 or equivalent)
SW32Ti swinging-bucket rotor package (Beckman Coulter, catalog number: 369694 or equivalent)
-80 °C freezer (PHCbi, model: MDF-DU502VHA-PA)
Water bath (Thermo Fisher Scientific, catalog number: FSGPD10 or equivalent)
Microfuge 18 centrifuge (Beckman Coulter, catalog number: BE-M18C or equivalent)
Test tube rocker (Thermo Fisher Scientific, catalog number: 12-815-6Q or equivalent)
(Optional) Millipore Biopak Polisher (Sigma-Aldrich, catalog number: CDUFBI001)
(Optional) Millipak Express 20 filter (Sigma-Aldrich, catalog number: MPGP02001)
Software and datasets
MxPro—Mx3005P (version 4.10, 2/15/23)
Procedure
Defrosting and cell maintenance
Defrost cells into 30 mL of warm c-BalanCD HEK293 media (see Recipe 1) into a 125 mL baffled Erlenmeyer flask.
Note: If you are using HEK293T cells and adapting to serum-free media yourself, please view Supplementary information 1 for more information on how to adapt your cells to c-BalanCD HEK293 media.
Place the flask onto the shaker platform in the 37 °C and 5% CO2 incubator at 120 rpm.
Count cell density every other day and dilute cells with c-BalanCD HEK 293 media as needed to maintain the cell density between 3 × 105 live cells/mL and 3 × 106 live cells/mL. Dilution will likely need to occur at least twice a week (dilute into a new, clean 125 mL baffled Erlenmeyer flask). Follow the subsequent steps to count cells and check cell viability with a Countess automated cell counter (or equivalent equipment):
Obtain a 0.6 mL microcentrifuge tube (MCT).
Aliquot 10 µL of trypan blue into the 0.6 mL MCT.
Remove the 125 mL baffled Erlenmeyer flask from the shaker platform.
Swirl the flask before taking a 10 µL aliquot of cells from the 125 mL baffled Erlenmeyer flask.
Add the 10 µL aliquot from the 125 mL baffled Erlenmeyer flask to the 10 µL of trypan blue in the 0.6 mL MCT.
Reflux the cell/trypan blue mixture.
Pipette 10 µL of the cell/trypan blue mixture onto a Countess cell counting chamber slide.
Insert the chamber slide into the automated cell counter and note the live cell count and cell viability.
Note: It is wise to systematically document these cell counts and cell viabilities so that you can monitor the growth and robustness of the cells throughout the cell maintenance process.
Pause point: The cells can be continually maintained in this way until you are ready to inoculate and transfect.
Inoculate flask (Day 1)
Warm c-BalanCD HEK293 media at 37 °C in a water bath.
Inoculate a 250 mL baffled Erlenmeyer flask with 1 × 106 live cells/mL in a total of 90 mL of c-BalanCD HEK293 media.
Note: The cells are being expanded from the initial 30 mL maintenance volume to a larger 90 mL volume suitable for transfection.
Place the 250 mL baffled Erlenmeyer flask on the shaker platform in the 37 °C and 5% CO2 incubator at 120 rpm.
Transfection (Day 2)
Ensure that the cell density of the previously inoculated 250 mL baffled Erlenmeyer flask reaches 1.0–2 × 106 live cells/mL (1 day after inoculation). Follow the method in step A3h to determine cell counts.
Transfer 0.5 mL of the cell suspension to a 24-well plate as a negative control.
Note: This is optional for your AAV transfer plasmids that contain a fluorescent marker.
Add 9 mL of complex formation solution and enhancer (CFSE) from the VirusGEN AAV Transfection kit .to a 15 mL centrifuge tube.
Add the appropriate quantity of plasmid DNA to the CFSE in the 15 mL centrifuge tube and gently reflux to combine for 10 s. Table 1 shows the DNA quantities needed for each component of the transfection mixture.
Table 1. DNA quantities (Challis et al., 2019)
Plasmid For 1 mL of cells For 90 mL of cells
Helper plasmid (AdΔF6) 0.57 µg 51.3 µg
AAV Rep/Cap plasmid 1.13 µg 101.7 µg
AAV shuttle vector 0.30 µg 27 µg
Total DNA 2 µg 180 µg
Add 270 µL of TransIt-VirusGen Transfection Reagent (3 µL per 1 mL of cells) to the CFSE/DNA mixture and gently reflux to mix well.
Let the transfection mixture incubate at room temperature for 30 min.
After the incubation period is over, gently reflux the transfection mixture well and add directly to the 250 mL flask while manually swirling the flask.
Put the 250 mL baffled Erlenmeyer flask back on the shaker platform in the 37 °C and 5% CO2 incubator at 120 rpm.
Harvesting after 72 h (Day 5)
(Optional) Transfer 0.5 mL of cell suspension to a 24-well plate and observe fluorescent protein expression if it exists. See Figure 1 for what a typical transfection efficiency may look for a transgene plasmid that had a fluorescent marker.
Figure 1. Transfection efficiency gauged by fluorescence. The image was taken using GFP filter (left panel) and was overlayed with a bright field image (right panel).
Transfer the entire contents of the 250 mL baffled Erlenmeyer flask (this should be approximately 100 mL of cell/media suspension) to a newly labeled 250 mL centrifuge tube.
Wash the flask with 24 mL of DPBS and combine with cell/media suspension.
Centrifuge cell/media suspension at 335× g for 10 min at room temperature. See Figure 2 for what this may look like.
Figure 2. Centrifuged cell pellet and supernatant 72 h after transfection with AV0-EF1-N-tdT
Digestion (Day 5)
Pour supernatant carefully into a newly labeled 250 mL bottle, being careful not to disturb the cell pellet.
Note: The supernatant and cell pellet are treated separately in parallel as both contain AAV. The steps during this digestion phase (step E2 for the supernatant and step E3 for the cell pellet) will serve to withdraw the virus from either the cell pellet or the supernatant. After having been treated in parallel, the crude virus from both the cell pellet and the supernatant will ultimately be combined and loaded onto one OptiPrep gradient for further purification/concentration, thus necessitating their simultaneous handling/treatment.
For the supernatant, complete the following steps:
To the new 250 mL bottle with the supernatant in it, add 150 µL of DNase I solution (see Recipe 2), 150 µL of RNase A solution (see Recipe 3), and 1 mL of 2 M MgCl2 (see Recipe 4).
Invert the centrifuge tube to mix and incubate for 1 h in the 37 °C and 5% CO2 incubator.
After 1 h, add 25 mL of 40% PEG 8000/2.5 M NaCl (see Recipe 7) for every 100 mL of digested supernatant.
Invert the bottle at least 20 times to mix and store at 4 °C until purification.
Pause point: The supernatant can stay at 4 °C for a maximum of two weeks before it is purified.
For the cell pellet, complete the following steps:
Resuspend the cell pellet in 8 mL of TMN (see Recipe 8).
Loosen the pellet by tapping the bottom of the centrifuge tube against the metal surface of the cell culture hood.
Resuspend the cell pellet in the TMN using a pipette controller and transfer the mixture to a 15 mL centrifuge tube.
Add 10% by volume of 5% sodium deoxycholate (see Recipe 3) and rock for 30 min at room temperature.
Add 100 µL of 2 M MgCl2, 150 µL of DNase I solution, and 150 µL of RNase A solution.
Mix and incubate for 1 h in a 37 °C water bath. Vortex the tube every 15 min.
Freeze at -80 °C until purification.
Pause point: The cell pellet suspension can remain at -80 °C before it is purified.
Preparation for purification (Day 6)
When ready to purify, complete the following steps for the supernatant:
Remove the 250 mL centrifuge tube from the 4 °C refrigerator and centrifuge it at 410× g for 30 min at 4 °C.
Aspirate the supernatant carefully, ensuring to leave the pellet intact at the bottom.
Add 2.5 mL of HBS (see Recipe 9) to the pellet.
Loosen the pellet by tapping the bottom of the centrifuge tube against the metal surface of the cell culture hood.
Resuspend the pellet in the HBS using a pipette controller and transfer the mixture to a 15 mL centrifuge tube.
Note: The pellet may stick to the bottom of the 250 mL centrifuge tube when homogenizing with HBS. Do your best to completely pick up the pellet and ensure that no portion of the pellet sticks to/within the serological pipette.
Vortex the HBS/pellet homogenate well.
Note: The pellet will not homogenize completely within the HBS at this point and may remain clumpy. This is acceptable as the pellet should dissolve in the HBS in the next step.
Place the 15 mL centrifuge tube on a rocker and rock until the pellet dissolves completely in the HBS. This will take at least 2 h, but likely longer.
When ready to purify, complete the following steps for the cell pellet:
Remove the cell pellet suspension from the -80 °C freezer and thaw completely in a 37 °C water bath.
Once thawed, add 100 µL of DNase I solution and 100 µL of RNase A solution.
Incubate the suspension in a 37 °C water bath for 1 h.
Centrifuge the cell pellet suspension at 410× g for 10 min at 4 °C.
Carefully combine the cleared cell lysate solution with the dissolved HBS/pellet mixture. Be careful not to touch any of the cell debris collected at the bottom of the 15 mL centrifuge tube in which the cell pellet solution was originally in. You should now have one 15 mL centrifuge tube that has the cleared cell pellet suspension solution combined with the HBS mixture.
Place this solution into the 4 °C refrigerator momentarily until the purification gradient is made, as described in the next step.
Purification (Day 6)
Prepare four solutions for the different densities that will be used to create the OptiPrep purification density gradient. Table 2 describes the quantities of OptiPrep, PBS-MK (see Recipe 11) or PBS-NMK (see Recipe 12), and phenol red needed for these solutions.
Note: Be very cautious to use PBS-NMK for the 15% OptiPrep solution and PBS-MK for the 25% OptiPrep and 40% OptiPrep solutions.
Table 2. OptiPrep gradient components
% OptiPrep 60% OptiPrep Buffer Phenol red
15% 4 mL 12 mL PBS-NMK n/a
25% 6.7 mL 9.3 mL PBS-MK 40 µL
40% 10 mL 5 mL PBS-MK n/a
60% 10 mL n/a 25 µL
Into a sterile thin-wall polypropylene centrifuge tube, start by adding 7 mL of 15% OptiPrep (see Table 2) to the bottom of the tube.
Carefully underlay 5 mL of 25% OptiPrep (see Table 2) beneath the 15% OptiPrep layer.
Caution: Pipette carefully so that no bubbles disturb the preexisting layer.
Carefully underlay 5 mL of 40% OptiPrep (see Table 2) beneath the 25% OptiPrep layer. See Figure 3 for how this underlay should look.
Caution: Pipette carefully so that no bubbles disturb the preexisting layers.
Figure 3. OptiPrep underlay. The left panel illustrates how the 25% OptiPrep is underlayed below the 15% OptiPrep layer. The right panel shows the underlaying of 40% Optiprep below 25% OptiPrep layer.
Finally, carefully underlay 4 mL of 60% OptiPrep (see Table 2) beneath the 40% OptiPrep layer. See Figure 4 for what a successfully created gradient should look like.
Caution: Pipette carefully so that no bubbles disturb the preexisting layers.
Figure 4. Complete gradient. Discrete separation of each layer should be visible. Top→Bottom: 15%, 25%, 40%, and 60%
Outline the top and bottom interfaces of the 40% OptiPrep layer with a fine-tipped marker for a reference point during extraction.
Remove the cleared cell pellet suspension solution combined with the HBS/pellet mixture from the 4 °C refrigerator.
Centrifuge the combined solutions’ 15 mL centrifuge tube at 5,000× g for 10 min at 4 °C.
Carefully overlay the combined solution over the OptiPrep gradient. Be careful not to pipette any cellular debris that may have accumulated at the bottom of the 15 mL centrifuge tube. See Figure 5 for what the gradient should look like with the combined solution overlayed on top.
Caution: Do not disturb the density gradient you created. Adjust the speed of your pipetting to ensure that you do not see any disturbance at the interface between the topmost 15% OptiPrep layer and the combined solution you are overlaying.
Figure 5. Gradient with crude virus before ultracentrifugation. This image shows the loaded density gradient tube before ultracentrifugation. AAV containing lysate in top layer remains separate from the 15% layer.
Transfer the thin-wall polypropylene centrifuge tube to the SW32Ti swinging-bucket rotor in the ultracentrifuge.
Centrifuge at 160,713× g for 15 h at 20 °C (overnight centrifugation).
Note: Ensure that you have adequately balanced the centrifuge for proper purification and to prevent equipment malfunction/potential injury.
Band extraction (Day 7)
After the ultracentrifuge has stopped, remove the thin-wall polypropylene centrifuge tube from the rotor.
Remove the thin-wall polypropylene centrifuge tube from the adaptor and place it into a holder. See Figure 6 for what the gradient should look like after ultracentrifugation.
Figure 6. Gradient after ultracentrifugation. This image shows the centrifuge tubes after ultracentrifugation. The separation of each layer is no longer discrete.
Prepare a 50 mL centrifuge tube in a rack.
Attach a 20 G sterile hypodermic needle to a 5 mL sterile syringe.
Use an alcohol wipe to disinfect the puncturing location (the bottom interface of the 40% OptiPrep layer that you previously marked).
Apply even pressure and puncture the thin-wall polypropylene centrifuge tube at the previously marked bottom interface of the 40% OptiPrep layer. See Figure 7 for guidance on where to puncture the tube.
Caution: Even pressure is important. Nothing should leak from the tube as you will be losing virus, nor should you puncture with such force that you accidentally puncture another part of the thin-wall polypropylene centrifuge tube.
Figure 7. Band extraction. This image illustrates where the needle should puncture the tube. This represents the 40%–60% interface where AAV should band.
Extract 4–4.5 mL of the OptiPrep solution, being very careful not to accidentally extract any debris that may have sedimented at a different density in the gradient.
Empty the contents of the syringe into the pre-prepared 50 mL centrifuge tube. Be sure to squeeze out as much of the extracted solution as possible before disposing of the needle and syringe in the appropriate sharps container.
Add 45 mL of DPBS/0.001% Pluronic F-68 (see Recipe 13) up to the 50 mL mark of the 50 mL centrifuge tube and place at 4 °C.
Concentration of virus (Day 7)
Add 15 mL of DPBS/0.001% Pluronic F-68 to an Amicon centrifugal filter unit and let it equilibrate for 15 min at room temperature.
Centrifuge the Amicon filter at 2,096× g for 2 min at 20 °C. Most, but not all, of the DPBS/0.001% Pluronic F-68 should have flowed through to the collection reservoir.
Note: It is of the utmost importance that the Amicon filter does not dry out. Otherwise, the solution will not pass through the filter and the virus will fail to concentrate.
Empty the collection reservoir into a waste bucket.
Add the diluted AAV in the 50 mL centrifuge tube to the Amicon filter and centrifuge the Amicon filter at 2,096× g for 2 min at 20 °C.
Empty the flowthrough from the collection reservoir before adding any more diluted AAV.
Repeat the previous steps I3 and I4. Add diluted AAV to the Amicon filter. Centrifuge at 2,096× g for 2 min at 20 °C until the entirety of the diluted sample has been applied to the Amicon filter.
Note: You will have to adjust the centrifuge run time based on how quickly the solution filters through. Do not allow the Amicon filter to dry out.
When the diluted AAV is exhausted, add 30 mL of DPBS/0.001% Pluronic F-68 to the 50 mL centrifuge tube to rinse it out.
Add the rinse solution from the 50 mL centrifuge tube to the Amicon filter and centrifuge at 2,096× g for 2 min at 20 °C until the rinse solution is exhausted as well.
Centrifuge at 2,096× g for 1 min at 20 °C as many times as needed until the liquid level in the Amicon tube reaches 200 µL.
Transfer the entire 200 µL of purified AAV from the Amicon filter to another tube and store at 4 °C for up to six months. For longer storage, aliquot the purified AAV into smaller quantities and store at -80 °C.
QPCR for genome titer (Day 8)
Prepare a serial dilution of AV0-EF1-N-cG working standard (1 ng/µL) with molecular biology grade water to cover 0.1 ng/µL (-1 dilution) to 0.00001 ng/µL (-5 dilution).
Prepare a serial dilution of AAV with molecular biology grade water starting at 1:100 (-2 dilution) and ending at 1:100,000 (-5 dilution).
Prepare a primer mix with 80 µL of molecular biology grade water, 10 µL of forward primer, and 10 µL of reverse primer. Vortex well.
Prepare the qPCR mix for projected numbers of wells using the following proportions shown below in Table 3.
Table 3. qPCR reaction mix components
Reagent For one reaction
2× SYBR Green SuperMix 10 µL
Primer mix 1 µL
Molecular biology grade water 4 µL
Total 15 µL
Pipette 5 µL of H2O for non-template control in duplicates.
Pipette 5 µL of the standard in duplicates.
Pipette 5 µL of each AAV virus dilution.
Pipette 15 µL of qPCR reaction mix to each well.
Place the strip cap lids over each well. Ensure that the lids have a good seal.
Centrifuge the qPCR plate at 1,087× g for 2 min to remove any air bubbles (see General Note 4).
Place the PCR plate into the qPCR machine.
Run qPCR cycle.
Note: For this analysis, the qPCR cycle used by the Agilent qPCR Machine is a standard preprogrammed method of 40 cycles. Each cycle consists of the following: 95 °C for 30 s (denaturation), 65 °C for 1 min (annealing), and 72 °C for 1 min (extension).
Data analysis
After the qPCR cycle has been run, you should analyze the data. Within the MxPro software, first ensure that the RSq of the standard curve is as close as possible to 1.000. If not, remove standard wells that skew the RSq further away from 1.000 until the value is as close to 1.000 as it can be. Export the text data and calculate the physical titer using the formula found in Deng and Oka (2020) in Section 3.5.1 (Step 8): Titer (vg/mL) = [1 × 1012 × (Concentration in ng/µL from qPCR software) × (dilution factor)]/3.222 ng. See General Note 5 for how this formula will change based on the size of your standard plasmid. See Supplementary information 2 for more details.
Validation of protocol
We followed the above protocol for five independent preparations of AAV9. Table 4 below shows relevant information for each of those preparations.
Table 4. AAV9 preparations
Vector name Serotype Cell density at time of transfection (live cells/mL) Viability at time of transfection Titer from 90 mL cell suspension culture (vg/mL) Total particles from 90 mL cell suspension culture (vg)
AV0-EF1-N-cG AAV9 1.6 × 106 97% 8.24 × 1013 1.65 × 1013
AV0-EF1-N-cG AAV9 1.3 × 106 97% 1.04 × 1014 2.08 × 1013
AV0-EF1-N-cG AAV9 1.3 × 106 97% 6.15 × 1013 1.23 × 1013
AV0-EF1-N-tdT AAV9 1.4 × 106 86% 6.18 × 1013 1.23 × 1013
AV0-EF1-N-tdT AAV9 1.1 × 106 87% 6.23 × 1013 1.25 × 1013
As can be noted from this table, each prep titers at approximately the 5 × 1013 to 1 × 1014 vg/mL range, with total particles ranging from approximately 1.2 × 1013 to 2.1 × 1013 vg.
Additional quality control assays
Protocol 1: SDS-PAGE to analyze capsid proteins
Laboratory supplies
NuPAGE 4–12%, Bis-Tris gel, 1.5 mm × 15 well (Thermo Fisher, catalog number: NP0336BOX)
PageRuler Plus prestained protein ladder, 10–250 kDa (Thermo Scientific, catalog number: Pl26620)
NuPAGE LDS sample buffer (4×) (Thermo Fisher, catalog number: NP0007)
NuPAGE MES SDS Buffer kit (for Bis-Tris gels) (Thermo Fisher, catalog number: NP0060)
SimplyBlue SafeStain (Thermo Fisher, catalog number: LC6060)
2-Mercaptoethanol (Thermo Fisher, catalog number: 21985023)
Equipment
Invitrogen PowerEase Touch 120W power supply and mini gel tank (Thermo Fisher, catalog number: PSC120MB) or equivalent
Invitrogen iBright FL 15000 imaging system or any other gel imaging system
Procedure
Preparation of samples (Day 1)
Pipette 5 µL of 4× Laemmli buffer supplemented with β-mercaptoethanol (1:20 dilution, 0.25 µL) into a 0.5 mL microcentrifuge tube.
Add AAV samples (a total of 1.0 × 1011 vg) up to 15 µL.
Make up to 20 µL with H2O.
Heat the samples at 100 °C for 5 min. This can be done using any PCR machine.
Set up gel tank and run electrophoresis
Prepare running buffer by diluting 20-fold with H2O.
Remove the comb from the precast gel and rinse the wells with H2O or running buffer.
Remove the tape from the bottom of the gel.
Insert the gel into the slot in the gel tank, leaving the integrated upper buffer chamber facing the center of the cell.
Load the samples.
Run the gel at room temperature at a constant 120 V for 110 min or until the dye front runs off the gel.
Gel staining
After electrophoresis, remove the gel into a container and wash the gel three times for 5 min with deionized water to remove SDS and buffer salt.
Add a sufficient volume (~20 mL) of SimplyBlue SafeStain to cover the entire gel and incubate for 1 h at room temperature with gentle shaking or using a rocker platform.
Aspirate or decant staining solution and replace with 100 mL of deionized water plus 20 mL of 20% NaCl (w/v) for 1 h. The gel can be left overnight in this destaining solution.
Take the image (see an example in Figure 8) with any image analyzer.
Figure 8. SDS-PAGE. 1. AAV9-EF1-N-cG produced 1F11 adherent cells. 2. AAV9-EF1-N-cG produced by 1F11S suspension cells. Prominent VP1, VP2, and VP3 bands should be visible. However, minor bands presumably derived from capsid proteins, ferritin, or of unknown origin could be observed (Sen et al., 2013; Wang et al., 2016; Nass et al., 2018; Zolotukhin et al., 1999).
Protocol 2: Infectious assay
Infectious AAV particles can be determined by measuring the presence of the intracellular vector genome after infection. Although this method does not accurately reflect biological activity (François et al., 2018), it can be adapted to any type of cultured cells and serotypes.
Laboratory supplies
HEK293T (ATCC, catalog number: CRL-3216)
Dulbecco’s modification of Eagle’s medium (DMEM), high glucose (VWR, catalog number: MT10013CM)
Fetal bovine serum (FBS) (VWR, catalog number: MT35010CV)
Note: Each lot requires testing for cell growth.
Trypsin-EDTA (1×) (GenDEPOT, catalog number: CA014-100)
Antibiotic-Antimycotic (×100) (GenDEPOT, catalog number: CA002-100)
E.Z.N.A Tissue DNA kit (Omega, catalog number: D3396-01)
24-well cell culture plate (Falcon, catalog number: 353047)
15-cm tissue culture dish (Falcon, catalog number: 353025)
Equipment
NanoDrop spectrophotometer or equivalent that can measure OD of DNA in a small volume
Procedure
Cells are maintained in a 15 cm dish with the growth media composed of DMEM/10% FBS/1× antibiotic-antimycotic in a humidified CO2 incubator (5% CO2) at 37 °C. Cells are split twice a week.
Preparation of cells for infection (Day 1)
Aspirate the media.
Rinse the cells with 10 mL of PBS.
Aspirate the media and overlay 1 mL of trypsin.
Incubate for 1–2 min or until all cells are rounded.
Gently tap the side of the dish to detach cells.
Add 9 mL of growth media.
Measure cell density using a cell counter (see section A of the main protocol).
Dilute cells to 200,000 cells/mL with the growth media and aliquot 0.5 mL/well into a 24-well plate in duplicate or triplicate.
Note: Do not forget control wells (no infection control).
Infection (Day 2)
Add 2 µL of non diluted AAV to each well.
Mix by rocking the plate.
Harvest cells for DNA extraction
Twenty-four hours later, count the number of cells in the control wells and determine the total number of cells/well.
Note: HEK293T cells loosely attach to the culture dish. They are easily detached by pipetting in and out. If not, consider using trypsin to detach cells as outlined in step A.
Transfer cells into a 1.5 mL MCT.
Rinse the well with 0.5 mL of DPBS and combine.
Centrifuge cells at 335× g for 1 min.
Aspirate the supernatant and wash the cells with 1 mL of DPBS twice.
Resuspend the cells with 0.2 mL of PBS.
Extract DNA from cells using an E.Z.N.A. Tissue DNA kit according to the manufacturer’s instructions.
Elute DNA with 100 µL of elution buffer.
Measure OD260nm to determine DNA concentrations.
Perform qPCR using 5 µL of eluate as described in section J of the main protocol to determine infectivity.
Data analysis
The amount of one copy of vector DNA spiked in 1 ng of genomic DNA = 1 ng × 6,424 bp (AV0-EF1-N-cG used as standard)/6.37 × 109 bp (female genomic DNA) (Piovesan et al., 2019) = 1.0085 × 10-6 ng. Although AAV is a single-stranded DNA virus, the second strand is synthesized after AAV is taken up by cells.
Calculate the infectious titer using the following formula:
Infectious titer (ivg/mL) = (copy number/genome) × (number of cells 24 h after infection) × 0.5 (2 µL of AAV was used) × 1,000 µL
= (Concentration in ng/µL from qPCR software)/(1.0085 × 10-6 ng × DNA concentration in ng/µL) × (number of cells) × 500.
Note: Infectious titer of various AAV serotypes varies on HEK293T cells because of serotype-specific attachment sites (Weinmann et al., 2022). Generally, infection of cells with 2 µL of purified AAV is sufficient to estimate infectious titer. A typical ratio of AAV9 particle (genome titer): infectious particle (infectious titer) on HEK293 cells is 5,000–10,000:1.
Protocol 3: Electron microscopy to analyze empty and full AAV particles
Reagents, Laboratory supplies, and Equipment
Pelco EasiGlow discharge set (Ted Pella, Inc., catalog number: 9100S)
Quantifoil 2/2 200Cu + 2 nm ThinC grids (Quantifoil Micro Tools GmbH, Jena, Germany)
120 kV JOEL 1230 electron microscope (JOEL Ltd, Japan) or equivalent
Desiccator (any)
2% uranyl acetate (Fisher Scientific, catalog number: NC1085517)
Sample preparation
Dilute AAV (~0.5–1 × 109 vg/µL) with DPBS.
Turn on the Pelco EasiGlow for 10 s with a current value of 15 µA and a vacuum of ~200 psig.
Deposit 5 µL of sample onto glow discharge Quantifoil 2/2 200Cu + 2 nm ThinC grids.
Incubate the carbon layer for 3 min.
Wick the excess buffer using Whitman 541 filter paper.
Wash the grids and blot twice with 20 µL of H2O.
Wash and blot with 20 µL of 2% uranyl acetate.
Drop 20 µL of 2% uranyl acetate and incubate for 1 min before blotting.
Dry the grids for a minimum of 2 h or overnight in a desiccator.
Imaging
Place the grids into an electron microscope.
Randomly select at least 10 fields.
Data analysis
Download the NIH ImageJ https://imagej.net/ij/index.html.
Convert native .dm3 files to .tiff files.
Count the number of full and empty capsids. AAV capsids containing vector genomes (full capsid) appear as particles without darkly stained centers, while empty capsids have stained centers (Figure 9).
Figure 9. Negative stain transmission electron microscopy images. A. AAV9-EF1-N-cG produced by suspension 1F11S cells. B. AAV9-EF1-N-cG produced by adherent 1F11 cells. The black circle indicates a full capsid having no darkly stained center. The red circle indicates an empty particle showing a dark stain in the particle center. The white circle indicates impurities previously identified as ferritin (Grieger et al., 2016). Scale bar: 100 nm.
General notes and troubleshooting
General notes
If it is not possible to obtain the preformulated BalanCD HEK293 media from Fujifilm Irvine Scientific, it is acceptable to utilize their BalanCD HEK293 powder. This powder will require reconstitution yourself; the recipe may be found in Recipe 14.
The preferred method for pH balancing solutions is utilizing 3 M NaOH to increase pH and 3 M HCl to decrease pH.
Ensure that you utilize high quality ddH2O. Our preferred system of water purification is a Millipore system with a Millipore Biopak Polisher and a Millipak Express 20 filter. However, you may consider using an equivalent system under the condition that it provides high-quality water filtration.
If bubbles persist in the qPCR plate wells even after centrifugation, use your finger to lightly flick the bottom of the wells to dissipate those bubbles. After doing so, put the plate back into the centrifuge at 1,087× g for 1 min before running the qPCR cycle.
Depending on what you use for your AAV standard for the qPCR, the formula to calculate titer may change slightly. The titer formula’s divisor is dependent on the size of the AAV standard plasmid. In our case, the plasmid vector AV0-EF1-N-cG's genome size is 6,424 bp long (3.88 × 106 Da). Therefore, to determine the divisor of the titer formula, you would carry out the following equation (you can input your own genome size into this equation): Mass of 1 × 109 molecules of AAV ssDNA = (3.88 × 106 Da × 1 × 109/(6.02 × 1023 × 0.5) = 3.222 ng.
Troubleshooting (Table 5)
Table 5. Troubleshooting
Issue Resolution
Clumping/aggregation of cells Switch baffled Erlenmeyer flasks and change media completely. Monitor cell growth/clumping for 3–4 days. If not improved, abandon cells and defrost new cells.
Formation of a ring of cells inside the baffled Erlenmeyer flask Transfer cells/media to new baffled Erlenmeyer flask. However, this ring has not shown to be detrimental in production quality, so you may choose to continue culturing those cells if you desire.
Slow cell growth Monitor cell growth every day and change media if necessary. If the cells do not recover, abandon cells and defrost new cells.
Cells grow too fast Dilute cells often with c-BalanCD HEK293 media. Always maintain the cell density between 3 × 105 live cells/mL and 3 × 106 live cells/mL.
Contamination of cells in baffled Erlenmeyer flask (indicated by excessive turbidity of cells and/or unnaturally fast cell growth) Abandon cells and defrost new ones. Make sure to also appropriately discard any old media suspected of contamination and use completely new baffled Erlenmeyer flasks. You may consider adding 1× antibiotic/antimycotic to the flask but be advised that this may inhibit cell growth; proper aseptic technique should suffice in preventing contamination.
Empty capsid contamination The protocol is optimized for maximizing the titer of full capsids. If the presence of empty capsids is deemed unacceptable for your specific application, a conventional plasmid ratio (helper plasmid: Rep/Cap plasmid: AAV shuttle vector = 2:1:1) can be used to mitigate this concern.
Acknowledgments
This work was supported by the general fund of Baylor College of Medicine and the Advanced Technology Cores. TEM data was collected at the Baylor College of Medicine CryoEM ATC, which includes equipment purchased under support of CPRIT Core Facility Award RP190602. We acknowledge Fujifilm Irvine Scientific for providing BalanCD HEK293 media and technical assistance in suspension cell adaptation alongside MirusBio for providing transfection reagents. We would also like to thank Dr. William Lagor for his comments on this manuscript and Issac Foresster for technical assistance in EM.
Competing interests
The authors declare no competing financial interests.
References
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Supplementary information
The following supporting information can be downloaded here:
Supplementary information 1
Supplementary information 2
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4,932 | https://bio-protocol.org/en/bpdetail?id=4932&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Use of the Fluorescent Dye Thioflavin T to Track Amyloid Structures in the Pathogenic Yeast Candida albicans
TM Thierry Mourer §
Cd Christophe d'Enfert
SB Sophie Bachellier-Bassi
(§ Technical contact: [email protected])
Published: Vol 14, Iss 3, Feb 5, 2024
DOI: 10.21769/BioProtoc.4932 Views: 524
Reviewed by: Alba BlesaLucy Xie Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in NPJ Biofilms and Microbiomes Jan 2023
Abstract
The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm.
Key features
• Study of amyloid properties of fungal proteins.
• Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy.
• Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation.
• Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.
Graphical overview
Keywords: Candida albicans Biofilm Cell wall Adhesion Amyloid peptides Fungal amyloid fibers Thioflavin T assays Protein biochemistry Fluorescence microscopy UV-VIS spectroscopy
Background
Under certain circumstances (e.g., broad-spectrum antibiotics, neutropenia, abdominal surgery, or central venous catheter), the human fungal pathogen Candida albicans can be translocated through the gastrointestinal mucosa to reach the bloodstream and colonize organs such as kidneys or the brain (Pfaller and Diekema, 2007). Upon host colonization, C. albicans can form a highly structured microbial community named biofilm (Cabral et al., 2014). Biofilm formation starts with adhesion of cells on biotic or abiotic surfaces (Nobile and Johnson, 2015). Then, hyphae are extruded and elongated from the mother cells before the synthesis of the extracellular matrix. Once the matrix covers the fungal community, yeast cells are released from the biofilm, allowing new areas to be colonized. C. albicans biofilms are difficult to cure, as such structures reduce both sensitivity to antifungal agents and pathogen recognition by the host immune system (Brown et al., 2012). The cell wall plays a critical role in fungal biofilm establishment by allowing C. albicans cells to interact with their environment as well as maintaining the biofilm integrity. Over the past decade, multiple reports have shown that amyloid structures present in the microorganisms’ cell wall create adhesion forces between cells that promote biofilm formation (Ho et al., 2019). We have recently uncovered that Pga59, a cell wall protein, forms amyloid structures to promote cell–cell adhesion forces and hence contribute to biofilm formation in C. albicans (Mourer et al., 2023). To draw these conclusions, we took advantage of the amyloid-specific fluorescent dye thioflavin T (ThT). Specific binding of ThT to amyloid structures results in a shift of its emission wavelength and in an increase of its quantum yield. Hence, ThT is a powerful tool used to detect amyloid components in biological specimens. Regarding fungal biofilms, some cell wall proteins have been positively stained by ThT, thus demonstrating their ability to adopt the shape of amyloid structure (Mourer et al., 2023; Ramsook et al., 2010). However, the link between in vitro experiments and the fact that amyloid properties of proteins are important for biofilm formation or establishment is often missing. We developed a pipeline to explore the amyloid properties of target proteins as well as their relevance for fungal cell adhesion and biofilm formation. We used ThT to successively stain octapeptides, recombinant proteins, amyloid structures enriched from fungal cells, adherent cells treated or not with amyloid peptides, and finally intact biofilms. Using this pipeline can formally confirm or reject that 1) the target protein is able to form an amyloid structure and 2) this amyloid is physiologically relevant for biofilm formation. This protocol has the potential to be applied to other microorganisms that can form biofilms like the bacteria Pseudomonas aeruginosa or Staphylococcus epidermidis and hence, create new knowledge regarding the impact of amyloid proteins on biofilm formation.
Materials and reagents
Biological materials
Candida albicans BWP17 (Wilson et al., 1999)
Saccharomyces cerevisiae BY4742 (Winston et al., 1995)
Reagents
Thioflavin T (Sigma-Aldrich, catalog number: T3516-5g)
Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855-250 mL)
M-280 Dynabeads, tosyl-activated magnetic beads (Thermo Fisher Scientific, catalog number: 14204)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418)
Concanavalin A, Alexa FluorTM 594 conjugate (Thermo Fisher Scientific, catalog number: C11253)
RNase A solution (Promega, catalog number: A7973)
TritonTM X-100 (Sigma-Aldrich, catalog number: X100-100ML)
Sucrose (Sigma-Aldrich, catalog number: S0389-500G)
10% CriterionTM XT Bis-Tris protein gel (Bio-Rad, catalog number: 3450112)
Bio-Rad protein assay dye reagent concentrate (Bradford) (Bio-Rad, catalog number: 5000006)
Poly-L-lysine solution (Sigma-Aldrich, catalog number: P8920)
Aclar® embedding film (TED PELLA, INC, catalog number: 10501-10)
GibcoTM RPMI 1640 medium (Thermo Fisher Scientific, catalog number: 10379144)
Dulbecco’s phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 11590476)
Ethanol absolute (Merck, catalog number: 1009831011)
Magnesium chloride (Sigma-Aldrich, catalog number: 208337-100G)
Nonidet P-40 (Sigma-Aldrich, catalog number: I8896)
cOmplete, EDTA-free protease inhibitor tablets (Roche, catalog number: 11873580001)
Phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher Scientific, catalog number: 36978)
Glycerol (Sigma-Aldrich, catalog number: G5516-100ML)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 62862)
Dithiothreitol (Sigma-Aldrich, catalog number: D0632-1G)
Tris hydrochloride (Thermo Fisher Scientific, catalog number: 15893661)
Sodium chloride (Sigma-Aldrich, catalog number: S9888-500G)
Bromophenol blue (Sigma-Aldrich, catalog number: B0126-25G)
Bradford reagent (Bio-Rad, catalog number: 500205)
BactoTM yeast extract (Thermo Fisher Scientific, catalog number: 212750)
BactoTM peptone (Thermo Fisher Scientific, catalog number: 211677)
Dextrose (Sigma-Aldrich, catalog number: D9434-1KG)
Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626-1KG)
Glucose (Sigma-Aldrich, catalog number: G8270-1KG)
Arginine (Sigma-Aldrich, catalog number: A5006-100G)
Uridine (Sigma-Aldrich, catalog number: U3750-25G)
Histidine (Merck, catalog number: H3911)
Yeast synthetic drop-out medium supplements without uracil (Sigma-Aldrich, catalog number: Y1501-20G)
Solutions
Thioflavin T solution (ThT) (see Recipes)
Thioflavin T buffer (see Recipes)
Lysis buffer (see Recipes)
Amyloid-Prion resuspension buffer (see Recipes)
Amyloid-Prion buffer R (see Recipes)
YPD medium (see Recipes)
SD complete medium (see Recipes)
SC-Ura medium (see Recipes)
Recipes
Thioflavin T solution (3.3 mM)
Reagent Final concentration Quantity
Thioflavin T 3.3 mM 10.53 mg
Absolute ethanol n/a 10 mL
Total n/a 10 mL
Protect the thioflavin T solution from light with aluminum foil and store the solution at -20 °C up to six months.
Thioflavin T buffer
Reagent Final concentration Quantity
Tris hydrochloride (1 M, pH 8.0) 20 mM 200 μL
Sodium chloride (5 M) 150 mM 300 μL
Thioflavin T (3.3 mM) 40 μM 121 μL
Distilled H2O n/a 9.4 mL
Total n/a 10 mL
Prepare the thioflavin T buffer freshly before each experiment.
Lysis buffer
Reagent Final concentration Quantity
Tris hydrochloride (1 M, pH 7.5) 50 mM 750 μL
Sodium chloride (5 M) 150 mM 450 μL
Magnesium chloride (1 M) 5 mM 75 μL
Nonidet P-40 0.1% 15 μL
PMSF (200 mM) 1 mM 75 μL
cOmplete protease inhibitor n/a 2 tablets
Distilled H2O n/a 13.6 mL
Total n/a 15 mL
Store the lysis buffer without protease inhibitors (PMSF and cOmplete protease inhibitor tablets) at 4 °C for two months maximum. Add protease inhibitors freshly before each experiment.
Amyloid-Prion resuspension buffer
Reagent Final concentration Quantity
Tris hydrochloride (1 M, pH 7.5) 50 mM 350 μL
Sodium chloride (5 M) 100 mM 140 μL
SDS (20%) 2% 700 μL
Dithiothreitol (1 M) 5 mM 35 μL
Glycerol 5% 350 μL
cOmplete protease inhibitor n/a 1 tablet
Distilled H2O n/a 5.4 mL
Total n/a 7 mL
Store the Amyloid-Prion resuspension buffer for eight weeks at 4 °C without dithiothreitol and protease inhibitor. Both the dithiothreitol and the protease inhibitor tablet should be added just before the start of the experiment.
Amyloid-Prion buffer R
Reagent Final concentration Quantity
Tris hydrochloride (1 M, pH 7.5) 10 mM 100 μL
SDS (20%) 0.4% 200 μL
Dithiothreitol (DTT, 1 M) 5 mM 50 μL
Distilled H2O n/a 9.7 mL
Total n/a 10 mL
The amyloid-prion buffer R can be kept at room temperature for three months. Add the dithiothreitol just before starting the experiment.
YPD medium
Reagent Final concentration Quantity
BactoTM yeast extract 1% 10 g
BactoTM peptone
Dextrose
Distilled H2O
2%
2%
n/a
20 g
20 g
q.s. to 1 L
Total n/a 1 L
SD complete medium
Reagent Final concentration Quantity
Yeast nitrogen base without amino acids 0.67% 6.7 g
Glucose
Arginine (2 mg/mL)
Uridine (4 mg/mL)
2%
20 mg/mL
40 mg/mL
20 g
10 mL
10 mL
Histidine (2 mg/mL) 20 mg/mL 10 mL
Distilled H2O n/a q.s. to 1 L
Total n/a 1 L
SC-Ura medium
Reagent Final concentration Quantity
Yeast nitrogen base without amino acids 0.67% 6.7 g
Glucose 2% 20 g
Yeast synthetic drop-out medium supplements without uracil 0.1% 1 g
Distilled H2O n/a q.s. to 1 L
Total n/a 1 L
Laboratory supplies
1.5 mL Eppendorf tubes (Eppendorf, catalog number: 0030125207)
50 mL FalconTM tubes (Fisher Scientific, catalog number: 10203001)
TPP® 96-well plate (Merck, catalog number: Z707902-108EA)
Culture plate 12 wells TPP (Dutscher, catalog number: 109212)
1.5 mL screw-cap micro tubes (Fisher Scientific, catalog number: 11549924)
Glass beads 0.5 mm, acidic wash (Sigma-Aldrich, catalog number: G8772-100 G)
Petri dishes 35 mm (Greiner, catalog number: P5112)
Ultracentrifuge polypropylene tubes (Beckman-Coulter, catalog number: 331372)
Glass slide (Merck, catalog number: S9027-1CS)
Coverslip (VWR, catalog number: 43210.KG)
Centrifuge bottles (Beckman-Coulter, catalog number: C31600)
Plastic cuvettes BRANDTM (Fisher Scientific, catalog number: 10566581)
Equipment
Microplate reader (TECAN, model: Infinite® 200 PRO)
Microcentrifuge (Eppendorf, catalog number: Z606235)
Ultracentrifuge OPTIMA XPN (Beckman-Coulter, catalog number: A94468)
Upright confocal microscope (Zeiss, model: LSM700)
ThermoMixer® C (Eppendorf, catalog number: 5382000015)
Sorvall RC-5B centrifuge (Thermo Fisher Scientific, catalog number: sorvall-RC-5B)
Vortex mixer (VWR, catalog number: 444-1372)
Centrifuge 5810 (Eppendorf, catalog number: 5810000010)
Microscope (Zeiss, model: Epifluorescence ApoTome)
Spectrophotometer (GE UltrospecTM 2100 pro, catalog number: GE80211221)
Bullet Blender Storm Pro (Next Advance, catalog number: 152081)
2 L Erlenmeyer flask (VWR, catalog number: 10545-844)
Electrophoresis power supply (Bio-Rad, model: 200/2.0)
Mini trans-blot electrophoretic transfer cell (Bio-Rad, catalog number: 1703930)
Water bath (Cole-Parmer, catalog number: WB-300-15)
Benchtop tube rotator RotoFlex (Cole-Parmer, catalog number: 120VAC)
Multitron shaker (Infors HT)
Software and datasets
Fiji ImageJ software (https://imagej.net/software/fiji/downloads)
Procedure
Identification and validation of amyloid-forming regions present in a protein of interest
Download the complete amino acid sequence of the target protein from the Uniprot database (https://www.uniprot.org/).
Copy and paste the primary sequence of the studied protein on the TANGO amyloid prediction tool (http://tango.crg.es/). The software will predict the presence of amyloid-forming regions in the primary sequence and, therefore, the potential of the protein to be assembled as an amyloid structure.
Note: TANGO provides a β-aggregation potential for every amino acid of the sequence. The higher the β-aggregation potential for an amino acid, the more likely that it is involved in the formation of an amyloid structure. This step will allow the identification of regions within the primary sequence of the protein that could be involved in amyloid formation. In our study, we have selected a region with a β-aggregation potential equal or better than 30%.
Order peptides corresponding to regions identified in step A2. All peptides were ordered from Thermo Fisher Scientific.
Note: All peptides must have acetyl and amide group at the N-terminus and C-terminus, respectively. These modifications avoid introduction of charged groups in the peptide as well as its degradation by exopeptidases.
Centrifuge the peptides at 18,000× g for 1 min to ensure that all powder is at the bottom of the tubes. Resuspend each peptide at a concentration of 100 μM in DMSO (stock solution).
Pause point: The protocol could be stopped at this step and the peptide solution stored at 4 °C for up to a month.
Note: Make sure that the powder is well resuspended either by pipetting or vortexing.
Dilute stock solutions of each peptide at a final concentration of 5 μM in the thioflavin T buffer. Distribute in triplicate 100 μL of diluted peptides in a 96-well plate. A solution composed of DMSO, 20 mM Tris-HCl, 150 mM NaCl, and 40 μM thioflavin T should be added to the plate as a negative control.
Note: Thioflavin T is resuspended at 3.3 mM in absolute ethanol.
Incubate the 96-well plate at 37 °C in a Tecan Infinite plate reader for 16 h.
Note: Set up the parameters to record thioflavin T fluorescence every hour with an excitation wavelength of 440 nm and emission wavelength of 496 nm.
Evaluation of amyloid structure assembly on recombinant full-length protein
Express and purify the recombinant protein of interest.
Critical point: Once purified, the recombinant protein should be used as soon as possible to avoid degradation of the polypeptide chain and/or random aggregation.
Notes:
As numerous methods exist to express and subsequently purify recombinant proteins, we cannot recommend a unique path to produce the protein of interest with high purity. Protein expression and purification correspond to a trial-and-error process. If the behavior of the protein is unknown, several expression systems (Escherichia coli, Pichia pastoris, Baculovirus expression system, Chinese hamster ovary mammalian cell line) should be tested for their ability to produce the required quantity (1 mg/mL) of recombinant protein. We used pET28a to express His6-Pga59 in E. coli SHuffle strain (Lobstein et al., 2012).
The appropriate chromatography strategy should be carefully designed to reach a protein purity as high as 95%. In our case, proteins were solubilized from inclusion bodies with a solution containing 6 M guanidium hydrochloride, refolded by dialysis, and purified by affinity chromatography (α-HIS tag). It is highly recommended to assess the purity of proteins using a MALDI-TOF mass spectrometry analysis.
Make a 10-time dilution of the recombinant protein in the dialysis buffer (final volume: 100 μL). Transfer the diluted protein in a quartz cuvette and measure its concentration using UV-spectrophotometry at 280 nm.
Caution: As the recombinant protein is purified under the monomeric form, the protein concentration must be kept low (5–10 mM) to avoid unwanted random aggregation.
Note: The concentration is determined by applying the Beer-Lambert’s law A = ϵlc, where A is the absorbance at 280 nm, ϵ the molar extinction coefficient, l the length of the optical path, and c the concentration. ϵ can be calculated by inserting the primary sequence of the protein in the Expasy ProtParam tool (https://web.expasy.org/protparam/). If the protein has no aromatic residues (tryptophan, histidine, tyrosine, and phenylalanine), the absorbance should be measured at 214 nm (absorbance of peptidic bonds).
Dilute recombinant proteins at 5 μM in 300 μL of thioflavin T buffer and distribute triplicates (3 × 100 μL) of each mix in 96-well plates.
Critical point: Before starting the thioflavin T staining assay, the recombinant protein must be present under the monomeric form. Starting the amyloidogenesis with preexisting aggregates in the mixture will impair amyloid assembly and hence the results of the thioflavin T staining.
As described in step A6, incubate the plate at 37 °C in a Tecan Infinite plate reader for 16 h. Measure the fluorescence every hour to monitor amyloidogenesis.
Thioflavin T staining of amyloid structures on intact fungal cells upon adhesion
Isolated colonies of C. albicans are inoculated in 4 mL of YPD medium and incubated under shaking (220 rpm) overnight at 30 °C.
Note: According to the protein of interest, the appropriate genetic background should be constructed to perform experiments for section C (e.g., knockout cells).
The same day as C. albicans precultures, wash 200 μL of magnetic beads twice with PBS.
Resuspend the beads in 1 mL of heat-denatured BSA (1 mg/mL) and incubate at 37 °C overnight on a benchtop rotator.
Note: To denature BSA, incubate the solution for 1 h in a water bath at 70°C.
The day after, dilute 20 μL of the culture in 980 μL of YPD. Transfer in a 1 mL plastic cuvette and measure the optical density OD at 600 nm (OD600).
Caution: If OD600 of cell dilution is above 1, it is likely that it is outside of the linearity zone of the spectrophotometer. Therefore, to be precise about cells’ quantity, samples should be diluter further to have an OD600 between 0.1 and 1.
Dilute the overnight culture to an OD600 of 0.3 in 5 mL of fresh YPD medium. Then, incubate cells for four additional hours under shaking (220 rpm) at 30 °C.
Note: Cells should be at mid-logarithmic phase before starting the ThT staining (OD600 around 0.8 or 1).
Centrifuge 108 yeast cells at 956× g for 4 min and wash them twice with sterile PBS.
Note: The quantity of cells is determined by measuring the OD600 with a spectrophotometer. One milliliter of C. albicans culture at OD600 of 1 corresponds to 2 × 107 fungal cells.
Resuspend fungal cells in 1 mL of SD complete medium containing 25 μM of ThT and 106 magnetic beads treated with heat-denatured BSA. Incubate the cells at room temperature on a benchtop rotator for 1 h.
Note: At this step, the impact of amyloid peptides on fungal adhesion can be assessed on a wild-type strain of C. albicans. For this, proceed exactly as described in steps C1–C5, but at step C6 add 5 μM of the peptide of interest in the SD medium.
Drop 10 μL of fungal cells on a glass slide and add a coverslip on top of the droplet. Observe the presence of amyloid structure by recording the ThT signal with the blue filter of an epifluorescence microscope.
Heterologous expression of amyloidogenic proteins in S. cerevisiae
Note: If too much redundancy arises between the protein of interest and other proteins from the host organism, it could be very difficult to observe phenotypes using ThT staining. One alternative would be to heterologously express the protein in another microorganism such as S. cerevisiae.
Inoculate S. cerevisiae from isolated colonies in 4 mL of SC-Ura medium and incubate overnight at 30 °C. Prepare magnetic beads as described in step C2.
The day after, dilute 20 μL of the overnight culture in 980 μL of YPD, transfer the entire volume in a plastic cuvette, and measure the OD600 nm.
Dilute the preculture of S. cerevisiae cells to an OD600 of 0.5 in 4 mL of fresh SC-Ura medium. Incubate cells with agitation (220 rpm) at 30 °C for 4 h.
Centrifuge yeast cells at 956× g for 3 min and resuspend the resulting pellet in 1 mL of SC-Ura medium.
Then, treat the cell suspension simultaneously with 106 BSA-coated magnetic beads and 25 μM of ThT. Incubate the mixture on a benchtop rotator at room temperature for 1 h.
Note: At this step, fluorescent dyes targeting specific cellular compartments can be added to perform colocalization experiments with the signal generated by the ThT. As our amyloids of interest are assembled in the cell wall, we used Concanavalin A Alexa Fluor 594-conjugate as cell wall marker.
Centrifuge cells at 956× g for 3 min and wash the pellet twice with 1 mL of PBS.
Add a droplet of fungal cells (10 μL) on a glass slide and cover with a coverslip. Monitor the ThT signal using epifluorescence microscopy with the blue filter.
Isolation of amyloid material from fungal cells
Note: To study the effect of various stimuli on amyloidogenesis in fungal cells, whole amyloid material could be isolated by cell fractionation (Kryndushkin et al., 2017) and subsequently stained with thioflavin T. We used this approach to show the formation of amyloids in C. albicans upon adhesion.
Inoculate fungal cells in 50 mL of YPD medium and incubate the flasks at 30 °C overnight under agitation (220 rpm).
Note: Prepare a solution of beads with heat-denatured BSA as described in step C2.
The day after, measure the OD600 of the preculture as described in step C3.
Dilute overnight culture to an OD600 of 0.3 in 1 L of YPD medium. Incubate cells at 30 °C for 4 h with shaking (220 rpm).
Caution: To ensure good oxygenation of fungal cells, the 1 L YPD culture should be performed in two flasks of 2 L (2 × 500 mL).
Transfer the cultures in 500 mL centrifuge bottles and centrifuge (Sorvall centrifuge) fungal cells at 956× g for 10 min.
Resuspend the pellet in 50 mL of SD medium and transfer the cell suspension in a 50 mL Falcon tube. Add 1.25 × 107 BSA-coated beads to the mixture, except for the negative control, and insert Falcon tubes on a benchtop rotator. Incubate cells for 1 h at room temperature.
Centrifuge fungal cells at 956× g for 4 min (Eppendorf 5810 R centrifuge). Discard the supernatant and store pellets at -80 °C until needed.
Pause point: The frozen pellet can be stored at -80 °C for months before being processed for amyloids extraction.
Thaw pellets on ice and wash once with cold PBS. Centrifuge at 956× g for 4 min and discard the supernatant.
Resuspend pellets with 4 mL of lysis buffer and distribute the entire volume in 1.5 mL screw-cap tubes (600 μL per tube). To each screw-cap tube, add 200 μL of 0.5 mm glass beads.
Mechanically disrupt yeast cells with a Bullet Blender bead beater using a cycle of six times for 1 min at full speed. Centrifuge the protein mixture at 800× g for 5 min at 4 °C in a tabletop centrifuge. Take out the supernatant that contains amyloid materials and transfer it in a clean 1.5 mL Eppendorf tube.
Note: Between each cycle, incubate the lysates on ice for 1 min.
Incubate protein extracts with RNase A (0.1 mg/mL) at room temperature for 10 min. Add Triton X-100 at a final concentration of 0.5% and incubate on ice for 10 min. Centrifuge the lysate at 2,000× g for 10 min at 4 °C. Discard the pellet and transfer the supernatant in a new 1.5 mL Eppendorf tube.
Load the supernatant from the previous step on a 2 mL sucrose layer (40%). Centrifuge samples at 200,000× g for 2 h at 4 °C.
Note: Make sure to use an ultracentrifuge tube for this step to avoid plastic collapsing at 200,000× g.
Resuspend the pellet in 200 μL of amyloid-prion resuspension buffer by pipetting up and down with a pipette. Incubate the mix for 10 min at 37 °C in a ThermoMixer device without shaking.
Centrifuge the solution at 5,000× g for 10 min in a tabletop centrifuge. Save the resulting supernatant in a 1.5 mL Eppendorf tube and discard the pellet.
For each 100 μL of amyloid preparation, add 0.01% of bromophenol blue and 5% of glycerol. Mix well and load the solution on a 10% polyacrylamide gel. Run the gel first for 15 min at 70 V and then switch to 200 V for 40 min. Use as many wells as required according to your sample volume.
Wash the polyacrylamide gel in distilled water once and cut out the top part (3 mm) of the stacking gel with a scalpel. Pool all gel sections per sample in a 1.5 mL Eppendorf tube and freeze gel squares at -20 °C for at least 20 min.
To each tube, subsequently add 200 μL of amyloid-prion buffer R, vortex for 10 s, and incubate for 15 min at 98 °C. Then, vortex and centrifuge at 4,000× g for 1 min. Take out the supernatant and drop it in a new 1.5 mL tube. Repeat this step twice.
Note: All supernatants must be pooled.
Quantify proteins in each sample using a classical Bradford assay.
Pause point: At this point, the protocol can be stopped, and amyloids stored at -20 °C until thioflavin T staining assays.
Dilute the amyloid-enriched fraction at a final concentration of 100 μM in 300 μL of thioflavin T buffer. Distribute 100 μL of amyloid fractions in a 96-well plate in triplicate. A solution composed of DMSO, 20 mM Tris-HCl, 150 mM NaCl, and 40 μM thioflavin T should be used as a negative control.
Incubate the 96-well plate at 37 °C in a Tecan Infinite plate reader for 16 h. Record ThT fluorescence every hour.
Track the presence of amyloid structures in intact biofilms of C. albicans
Note: C. albicans biofilms can be grown on a plastic surface (Aclar film) before being stained with ThT and then imaged with a confocal microscope to observe the presence of amyloid structures.
Inoculate C. albicans cells in 4 mL of YPD medium and incubate overnight at 30 °C.
Coat the Aclar film with a freshly made poly-L-lysine solution (0.1%) for 30 min at 37 °C. Wash the Aclar film twice with distilled water, cut it in small squares (1 cm × 1 cm), and finally sterilize each side under UV light for 15 min.
The day after, wash yeast cells twice with sterile PBS and dilute fungal cells at 1.106 cells/mL in RPMI medium.
Note: The RPMI medium should be prewarmed at 37 °C before use.
Place sterile Aclar film squares in wells from a 12-well tissue culture plate. Add 3 mL of diluted cells to each well.
Incubate fungal cells for 1 h at 37 °C with moderate shaking (110 rpm) in an Infors HT Multitron to allow adhesion of C. albicans to the plastic surface. Then, remove non-adherent cells by washing the wells twice with 1 mL of sterile PBS and fill up with 3 mL of prewarmed RPMI medium (37 °C).
Incubate the culture plates at 37 °C for 48 h at 110 rpm.
Wash mature biofilms once with PBS and stain with 2 mL of PBS containing 25 μM of ThT.
Caution: In order to prevent the biofilm to be detached from the Aclar film, the fungal community should be washed gently.
Wash biofilms two times with PBS and transfer them in a 35 mm Petri dish. Cover C. albicans biofilms with 2 mL of PBS and image using the blue channel of a confocal microscope.
Critical point: The objective of the microscope will be immersed in the culture medium. To avoid any damage, make sure to use an immersion objective.
Note: We record Z-stacks on a LSM700 upright using a 40× immersion objective. We then reconstruct the volume using Fiji ImageJ software.
Data analysis
For sections A–E, an example of each experiment has been published in Mourer et al. (2023). The step-by-step protocol has been summarized in Figure 1.
I) Regarding in vitro assays with peptides, recombinant proteins, or amyloid-enriched fractions from C. albicans (sections A, B, and E), the formation or the presence of amyloid structures is monitored by the ThT fluorescence over time. The higher the ThT fluorescence, the more amyloid structures are formed in the wells. If possible, add a peptide or a protein that is unable to form amyloids as an additional negative control to the blank. At the end of each experiment:
(i) Average the thioflavin T intensity value of triplicates for each sample.
(ii) Perform the statistical analysis to check if differences are relevant. Repeat this experiment three times, with three technical replicates for each peptide/protein. Samples are compared two by two using a Student’s t-test.
Figure 1. Summary of the protocol steps that must be followed successively to assess the propensity of a protein to form amyloids. The protocol will also assess the relevance of amyloids in vivo as well as their involvement in biofilm formation.
II) For in vivo staining (sections C, D, and F), the presence of amyloid structures is indicated by the blue fluorescence emitted by the ThT. According to the protein studied or the genotype of the yeast strains, the ThT pattern could change drastically. We suggest following this routine for data analysis:
(i) Observe fungal cells first at a rather low magnification using a 40× objective to evaluate the impact of the loss of certain proteins on amyloid formation, using ThT fluorescence intensity as a readout of amyloid content at the population level; indeed, the fluorescence levels correlate with the quantity of amyloids produced in the cells.
(ii) Record pictures of at least 100 cells at low magnification (40×).
(iii) Count the number of positive cells in each condition.
(iv) Repeat experiments at least three times in an independent manner and perform statistical analysis (compare two by two using a Student’s t-test).
(v) Draw a conclusion on the relevance of a target protein for amyloid formation in vivo and biofilm formation.
(vi) Then, switch to a 100× objective to observe the precise localization of amyloids within the cell. Fluorescent markers specific for cellular compartments could be used to assess colocalization with the ThT fluorescence as an indication of the presence of amyloid material in this specific compartment. Experiments must be carried out with three independent biological replicates.
III) Peptides encompassing the amyloid-forming region of candidate proteins can also be used for in vivo ThT staining on wild-type strains (section C). Follow the same routine as described in II.
The impact of wild-type and mutant peptides will be monitored by the ThT fluorescence intensity. If a peptide stimulates amyloid assembly, the ThT fluorescence will be higher in treated cells than in the untreated control. On the contrary, treating cells with peptides that inhibit amyloid assembly, for instance mutated peptides, will result in a lower ThT fluorescence than in the untreated control.
Finally, by using this protocol, we can also assess the relevance of amyloid structures for biofilm establishment. C. albicans biofilms can be easily stained with ThT and subsequently imaged with confocal microscopy to record Z-stacks of complete biofilms. According to the genotype of yeast cells, both the biofilm shape and thickness can be linked to the amyloid content of yeast cells that constitute the biofilm. Comparing the biofilm shape, thickness, and ThT fluorescence intensity to those of a wild-type strain biofilm will reveal if given mutations affect amyloid formation and hence biofilm establishment. An example of biofilm staining with ThT is presented in Figure 2, where we can see the shape, height, and amyloids (ThT staining in white). The experiments should be repeated three times with independent biological replicates. At this step, it is important to know how to record Z-stacks on the confocal microscope available in your institute or university. Also, users need to know how to reconstruct 2D images with Z-stacks using the Fiji ImageJ software.
Figure 2. Thioflavin T (ThT) staining of intact fungal biofilm. C. albicans cells were allowed to attach on an Aclar film for 1 h. Fungal biofilm was then grown at 37 °C in RPMI under gentle shaking. After 48 h, amyloid structures were stained with a solution of PBS containing 25 mM of ThT. Z-stacks of biofilms were then recorded in the blue channel with a confocal microscope. Top view (left part) and side view (right part) of the biofilm are shown. Scale bars: 10 mm.
Validation of protocol
This protocol was developed to assess if a fungal candidate protein can be self-assembled to form an amyloid structure both in vivo and in vitro. If so, the physiological relevance of the protein for biofilm establishment or maintenance could be assessed by following section F of the present protocol. Except for ThT staining of intact biofilms, all experimental procedures were validated in Figure 1C, 2C, 3C, 4A and B, 5B, and 7 of Mourer et al., 2023 (doi: 10.1038/s41522-023-00371-x). For in vivo ThT staining of intact biofilm of C. albicans, see Figure 2 of the present manuscript.
General notes and troubleshooting
General notes
The culture conditions to perform ThT staining on fungal cells should be carefully determined. Indeed, C. albicans undergoing planktonic growth produces virtually no amyloid structures. Cells should be cultivated in conditions where amyloid assembly is active, like for instance cell adhesion triggered by magnetic beads. Otherwise, all attempts to stain amyloid fiber on fungal cells with ThT will be a failure.
The ThT fluorescence will be recorded in the blue channel either on epifluorescence or confocal microscopes. The signal of the resulting images will be weak and sometimes difficult to analyze. To visualize it better, use the Fiji ImageJ software to switch the color of images from blue to yellow using the channel tools option. Apply this change to all microscopic images.
Troubleshooting
Make sure that the buffer used to purify recombinant proteins is not autofluorescent in the blue channel (section B). This would generate erroneous results.
The concentration of recombinant proteins during amyloidogenesis should be kept low (section B). High concentration of proteins will interfere with the amyloid assembly and generate anarchic aggregates.
The purification of proteins that tend to aggregate under the monomeric form is a tedious process that could lead to project failure by lack of biological samples to perform experiments. If experimenters face difficulties to produce protein monomers, the purification product could be treated with hexafluoroisopropanol (HFIP). This compound will suppress the formation of amorphous aggregates and hence keep the protein in the monomeric form before starting in vitro thioflavin T assays (section B).
The ThT concentration required to stain amyloids in vivo can change depending on the cellular model (section C). According to the microorganism used, the amyloid content could change, and hence, the ThT concentration should be adjusted for efficient staining.
A low quantity of amyloid materials is extracted from fungal cells in section E. According to the growth conditions, C. albicans cells may be difficult to disrupt, and hence, a small amount of protein is extracted from cells during the lysis step. If this happens, do not skip the freezing step at -80 °C (step E6). The freezing will generate ice crystals in the membranes, which will positively impact cell lysis. Also, to increase cell lysis efficiency, add more glass beads on cells (400 μL instead of 200 μL).
Some amyloid structures can be sensitive to buffers that contain SDS, leading to a poor amount of amyloids recovery after the extraction protocol (section E). If amyloid structures are sensitive to SDS treatment, the detergent should be changed in amyloid-prion resuspension buffer as well as the amyloid-prion R buffer. We suggest replacing SDS by 1% Sarkosyl in both buffers.
C. albicans cells do not attach well on the plastic surface, making the imaging by confocal microscopy complicated. To solve this problem, two solutions are available: (i) increase the quantity of Poly-L-Lysine in step F2 or (ii) choose another extracellular matrix protein (e.g., Fibronectin) to coat the Aclar film (step F2).
Acknowledgments
T.M. was a recipient of the Pasteur-Cantarini postdoctoral fellowship from the Institut Pasteur. This work was supported by the French Government's Investissement d’Avenir program (Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases, ANR-10-LABX-62-IBEID). The graphical overview and Figure 1 were created with BioRender.com. This protocol has been used in Mourer et al. (2023), doi: 10.1038/s41522-023-00371-x.
Competing interests
The authors declare no competing interests.
References
Brown, G. D., Denning, D. W., Gow, N. A. R., Levitz, S. M., Netea, M. G. and White, T. C. (2012). Hidden Killers: Human Fungal Infections. Sci. Transl. Med. 4(165): e3004404.
Cabral, V., Znaidi, S., Walker, L. A., Martin-Yken, H., Dague, E., Legrand, M., Lee, K., Chauvel, M., Firon, A., Rossignol, T., et al. (2014). Targeted Changes of the Cell Wall Proteome Influence Candida albicans Ability to Form Single- and Multi-strain Biofilms. PLoS Pathog. 10(12): e1004542.
Ho, V., Herman-Bausier, P., Shaw, C., Conrad, K. A., Garcia-Sherman, M. C., Draghi, J., Dufrene, Y. F., Lipke, P. N. and Rauceo, J. M. (2019). An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion. mBio 10(5): e01766–19.
Kryndushkin, D., Pripuzova, N. and Shewmaker, F. P. (2017). Isolation and Analysis of Prion and Amyloid Aggregates from Yeast Cells. Cold Spring Harb. Protoc. 2017(2): pdb.prot089045.
Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P. and Berkmen, M. (2012). SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb. Cell Fact. 11(1): e1186/1475–2859–11–56.
Mourer, T., El Ghalid, M., Pehau-Arnaudet, G., Kauffmann, B., Loquet, A., Brûlé, S., Cabral, V., d’Enfert, C. and Bachellier-Bassi, S. (2023). The Pga59 cell wall protein is an amyloid forming protein involved in adhesion and biofilm establishment in the pathogenic yeast Candida albicans. npj Biofilms Microbiomes 9(1): e1038/s41522–023–00371–x.
Nobile, C. J. and Johnson, A. D. (2015). Candida albicans Biofilms and Human Disease. Annu. Rev. Microbiol. 69(1): 71–92.
Pfaller, M. A. and Diekema, D. J. (2007). Epidemiology of Invasive Candidiasis: a Persistent Public Health Problem. Clin. Microbiol. Rev. 20(1): 133–163.
Ramsook, C. B., Tan, C., Garcia, M. C., Fung, R., Soybelman, G., Henry, R., Litewka, A., O'Meally, S., Otoo, H. N., Khalaf, R. A., et al. (2010). Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences. Eukaryotic Cell 9(3): 393–404.
Wilson, R. B., Davis, D. and Mitchell, A. P. (1999). Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions. J. Bacteriol. 181(6): 1868–1874.
Winston, F., Dollard, C. and Ricupero‐Hovasse, S. L. (1995). Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11(1): 53–55.
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SUrface SEnsing of Translation (SUnSET), a Method Based on Western Blot Assessing Protein Synthesis Rates in vitro
MP Marie Piecyk
JF Joëlle Fauvre
CD Cédric Duret
CC Cédric Chaveroux
CF Carole Ferraro-Peyret
Published: Vol 14, Iss 3, Feb 5, 2024
DOI: 10.21769/BioProtoc.4933 Views: 2210
Reviewed by: Chiara AmbrogioMayank GautamRupkatha Banerjee
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Original Research Article:
The authors used this protocol in Molecular Oncology Jul 2023
Abstract
As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to the cellular microenvironment. Consequently, variations in protein synthesis rates occur under various physiological and pathological conditions, enabling an adaptive response by the ce•ll. For example, global protein synthesis increases upon mitogenic factors to support biomass generation and cell proliferation, while exposure to low concentrations of oxygen or nutrients require translational repression and reprogramming to avoid energy depletion and cell death. To assess fluctuations in protein synthesis rates, radioactive isotopes or radiolabeled amino acids are often used. Although highly sensitive, these techniques involve the use of potentially toxic radioactive compounds and require specific materials and processes for the use and disposal of these molecules. The development of alternative, non-radioactive methods that can be easily and safely implemented in laboratories has therefore been encouraged to avoid handling radioactivity. In this context, the SUrface SEnsing of Translation (SUnSET) method, based on the classical western blot technique, was developed by Schmidt et al. in 2009. The SUnSET is nowadays recognized as a simple alternative to radioactive methods assessing protein synthesis rates.
Key features
• As a structural analogue of aminoacyl-transfer RNA, puromycin incorporates into the elongating peptide chain.
• Detection of puromycin-labeled peptides by western blotting reflects translation rates without the need for radioactive isotopes.
• The protocol described here for in vitro applications is derived from the SUnSET method originally published by Schmidt et al. (2009).
Keywords: SUnSET Protein synthesis Translation rates Puromycin Alternative to radioactivity
Background
As a structural analogue of aminoacyl-tRNA, the aminonucleoside antibiotic puromycin is incorporated through non-hydrolysable peptide bounding into the growing peptide chain along the elongation process (Nathans, 1964). While high concentrations block the elongation phase and hence translation, at low doses the overall translation rate of protein synthesis remains unchanged. Consequently, the rate of formation of puromycin-labeled peptides mirrors the rate of protein translation. Taking advantage of this property, 3H-puromycin labeling was first used in 1979 to assess the rate of protein synthesis in various tissues in vivo under nutrient- and protein-poor diets (Nakano and Hara, 1979). The SUnSET (SUrface SEnsing of Translation) technique was developed 30 years later by Schmidt and colleagues to detect variations in protein synthesis rates in cultured cells by western blotting (Schmidt et al., 2009). This method was then coupled with other techniques (fluorescence-activated cell sorting or immunohistochemistry) to assess protein synthesis at different scales. Further developments, notably based on the Clik-it technology combined with O-propargyl puromycin (OP-Puro), a puromycin analogue, enable visualization of puromycilated proteins and assessment of elongation rates in tissues (Morral et al., 2020). The main advantage of using puromycin and its analogues is that it does not require radioactive isotopes such as 35S-methionine, historically used to measure protein synthesis rates, with comparable analytical performance (Schmidt et al., 2009). Because proteostasis defects are associated with a variety of chronic diseases (cancer, tissue fibrosis, inflammatory syndromes, etc.) or aging, it is necessary to monitor the changes in protein synthesis rates in response to a variety of stressors in order to better understand the translational reprogramming underlying cell adaptation. The following protocol describes a SUnSET method (Figure 1) suitable for assessing protein synthesis rates in lysates from cells grown in vitro by conventional western blot analysis.
Figure 1. Principle of the SUnSET assay. During the elongation step, addition of puromycin leads to its incorporation into the A site of the ribosome. The transfer and linkage of the polypeptide to the puromycin cause the termination of translation releasing puromycilated proteins that can subsequently be detected by western blotting against the puromycin.
Materials and reagents
Biological materials
Cell lines of interest obtained from the American Type Culture Collection (ATCC). In this study, we used the colon cancer cell line HCT116.
Reagents
Puromycin (Sigma-Aldrich, catalog number: P9620)
PBS (Sigma-Aldrich, catalog number: D1408)
Complete anti-protease (Roche, catalog number: 11836145001)
Dry milk (Régilait, catalog number: 304934416704)
Bovine serum albumin (BSA) (Roche, catalog number: 10735094001)
DC Protein Assay kit (Bio-Rad, catalog number: 5000111)
Prestained protein ladder (Euromedex, catalog number: 06P-0111)
Immobilion Forte western substrate (Merck Millipore, catalog number: WBLUF0500)
Ponceau red solution (Sigma-Aldrich, catalog number: 141194)
Mouse anti-puromycin antibody (clone 12D10) (Merck Millipore, catalog number: MABE343)
Mouse anti-tubulin antibody (clone DM1A) (Sigma, catalog number: T6199)
HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology, catalog number: 7076)
Stripping buffer (Thermo Scientific, catalog number: 21059)
Solutions
RIPA protein lysis buffer 2× (see Recipes)
Tris-buffered saline-Tween (TBS-T) (see Recipes)
TBS-T 5% BSA (see Recipes)
TBS-T 5% dry milk (see Recipes)
Laemmli 6× (see Recipes)
Recipes
RIPA protein lysis buffer 2×
Reagent Final concentration
Tris-HCl pH 7.2 100 mM
NaCl 300 mM
EDTA 10 mM
Sodium deoxycholate 2%
SDS 20% 0.1%
Triton 100× 2×
Na3VO4 4 mM
β glycerophosphate 20 mM
NaF 20 mM
Complete anti-protease 2×
Dilute to 1× in H2O.
Tris-buffered saline-Tween (TBS-T)
Reagent Final concentration
Tris-HCl pH 7.5 50 mM
NaCl
Tween
150 mM
0.1%
Add 5% of BSA or dry milk for obtaining TBS-T 5% BSA or 5% dry milk, respectively.
Laemmli 6×
Reagent Final concentration
Tris-HCl pH 6.8 0.5 M
Glycerol 1%
SDS 20% 2%
DTT 0.6 M
Bromophenol blue 0.4%
Laboratory supplies
6-well or 10 cm diameter tissue culture plates
Sterile scrapers
Microcentrifuge tubes
Plastic wrap
Equipment
Tissue culture apparatus (tissue culture hood, CO2 incubator, etc.)
Pipettes and micropipettes
Vacuum pump
Centrifuge (4 °C)
Cold room
Heat block
Western blotting apparatus (SDS-PAGE running cassette, power supply, shaker, transfer cassette, nitrocellulose membrane, etc.)
ChemiDoc imaging system (Bio-Rad, catalog number: 12003153)
Software and datasets
Fiji (National Institutes of Health)
Procedure
Puromycin incorporation in vitro
Seed HTC116 cells one day before the experiment and maintain at 37 °C and 5% CO2 to allow attachment to the tissue culture plate. In the experiment presented in Figure 2, 200,000 cells per well were seeded in a 6-well plate.
On the day of the experiment, apply the studied treatment on cells for the desired time.
Before harvesting and 15 min before the end of the treatment, expose cells to 5 μg/mL puromycin directly diluted in the media. All samples must be incubated with the same concentration of puromycin for an equal period.
Protein extracts
At the end of the 15-min incubation with puromycin, remove media and rinse cells once with cold PBS.
Put the tissue culture plate on ice and incubate with 1× RIPA protein lysis buffer (see Recipes) containing proteases and phosphatases inhibitors (1 volume of 1× RIPA for 1 volume of cell pellet) for 20 min.
Scrape the cells and collect in a microcentrifuge tube.
Centrifuge at 13,000× g for 20 min at 4 °C to get rid of cellular debris.
Collect the supernatant in a new microcentrifuge tube and add the appropriate volume of Laemmli 6× (see Recipes).
Dose the amounts of extracted proteins using the DC Protein Assay kit according to manufacturer’s instructions.
Add the appropriate volume of Laemmli 1× to normalize protein concentrations in all samples and denaturate by heating at 95 °C for 5 min.
Western blotting
Load 20 μg of proteins for all studied conditions on a 10% SDS-PAGE gel. We recommend adding 5 μL of protein ladder to estimate the molecular weight of visualized proteins.
When separated, transfer proteins onto nitrocellulose membranes as a standard western blot protocol. Stain with Ponceau red solution to check equal protein amount loading before electrophoresis.
Block the membrane with TBS-T 5% dry milk for 1 h at room temperature with gentle shaking.
Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
Incubate overnight at 4 °C on a gentle shaker with puromycin antibody diluted at 1/10,000 into TBS-T 5% BSA.
Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
Incubate at room temperature for 1 h with the HRP-conjugated anti-mouse secondary antibody (1/10,000 dilution) into TBS-T 5% dry milk.
Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
Gently dry the membrane using paper towels and place it face up and flat on a sheet of plastic wrap.
Directly add the Immobilion Forte western substrate onto the whole membrane.
Detect chemiluminescence with the ChemiDoc imaging system (Figure 2). Intensity of the smear depends on cell ability to incorporate puromycin and is thus representative of protein synthesis rate.
Rinse the membrane in TBS-T and transfer in 5 mL of stripping buffer.
Protect from the light and put on thorough agitation for 15 min. Check that all bound antibodies have been detached by verifying that no chemiluminescent signal is detected on the ChemiDoc imaging system.
Block the membrane with TBS-T 5% dry milk for 30 min at room temperature on gentle shaking.
Incubate at room temperature for 1 h with the anti-tubulin antibody diluted into TBS-T 5% dry milk.
Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
Incubate at room temperature for 1 h with the HRP-conjugated anti-mouse secondary antibody (1/10,000 dilution) into TBS-T 5% dry milk.
Wash with TBS-T for 5 minutes on a shaker and repeat the operation two times.
Gently dry the membrane using paper towels and place it face up and flat on a sheet of plastic wrap.
Directly add 1 mL of the Immobilion Forte western substrate in order to cover the whole membrane.
Detect chemiluminescence with the ChemiDoc imaging system (Figure 2). Intensity of the bands is proportional to protein loading before electrophoresis.
Quantification of the puromycin and tubulin signals can be performed using the Fiji software (refer to Data analysis section).
Figure 2. SUnSET assay demonstrating a repression of protein synthesis upon treatment with GCN2 kinase inhibitor. HCT116 cells were treated or not for 24 h with a GCN2 kinase inhibitor (GCN2i also named TAP20) and exposed to puromycin (5 μg/mL) 15 min before protein extraction. Protein translation rates were assessed by western blot analysis using an anti-puromycin antibody (Puro). Tubulin is presented here as a loading control. The corresponding molecular weights are indicated on the right of the western blots. Results extracted from Piecyk et al. (2023). Quantification of the data on the left represents the mean ± SEM (n = 3). Unpaired two-tailed t-test with p-value (** p < 0.01).
Data analysis
Open the Fiji software.
Click on the File menu to seek for the ChemiDoc images of the SUnSET experiment.
Use the Rectangular tool to graph a frame around the first smear to quantify and define the region of interest.
In the Analyze menu, select Gels and Select First Lane.
Move the section to the next lane and select Gels and Select Next Lane in the Analyze menu.
Repeat the operation for each lane.
Select Plot Lanes in the Analyze/Gels menu to create each lane profile plot.
Define a closed area for each lane plot using the Straight Line Selection Tool to draw a baseline for each peak.
Measure each peak area clicking inside with the Wand tool.
Report area measurements in a Results sheet.
Repeat this process for tubulin signal quantification.
Normalize: for each lane, calculate the ratio SUnSET area/Tubulin area.
Compare the ratio observed in each lane to assess the impact of studied conditions on protein synthesis rate.
Validation of protocol
This protocol was adapted from the original article: Schmidt et al. (2009). The SUnSET method was performed and validated for in vitro applications in several articles from Dr. Chaveroux’s group (Sarcinelli et al., 2020; Piecyk et al., 2021 and 2023) and other teams in the literature (e.g., Martineau et al., 2014; Mesclon et al., 2017; Arioka et al., 2020; Fong et al., 2021). The SUnSET principle has been adapted for in vivo and ex vivo applications (Goodman et al., 2011; Morral et al., 2020) and at the single-cell level coupled to flow cytometry for energy metabolism assessment by Dr. Pierre’s group (Argüello et al., 2020).
Acknowledgments
This work was supported by the Cancéropôle CLARA (CVPPRCAN000174, CVPPRCAB000180 and CV-2021-039), Region Auvergne Rhone-Alpes (19-010898-01), Institut National Du Cancer (PLBIO22-227), Projets Fondation and Aide doctorale (R16173CC, ARCMD-Doc22021020003295) from ARC, Ligue Nationale contre le Cancer (R17167CC, R19007CC), Institut Convergence François Rabelais (17IA66ANR-PLASCAN-MEHLEN), and the IPR (Innovation Pharmaceutique et Recherche) program. Figure 1 was created with BioRender.com. This protocol was adapted from the original article: Schmidt et al., 2009.
Competing interests
The authors declare no competing interests.
References
Argüello, R. J., Combes, A. J., Char, R., Gigan, J.-P., Baaziz, A. I., Bousiquot, E., Camosseto, V., Samad, B., Tsui, J., Yan, P., et al. (2020). SCENITH: A Flow Cytometry-Based Method to Functionally Profile Energy Metabolism with Single-Cell Resolution. Cell Metab. 32(6): 1063–1075.e7. https://doi.org/10.1016/j.cmet.2020.11.007
Arioka, Y., Shishido, E., Kushima, I., Suzuki, T., Saito, R., Aiba, A., Mori, D. and Ozaki, N. (2020). Chromosome 22q11.2 deletion causes PERK-dependent vulnerability in dopaminergic neurons. EBioMedicine 63: 103138. https://doi.org/10.1016/j.ebiom.2020.103138
Fong, M. Y., Yan, W., Ghassemian, M., Wu, X., Zhou, X., Cao, M., Jiang, L., Wang, J., Liu, X., Zhang, J., et al. (2021). Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis. EMBO Rep. 22(2): e51239. https://doi.org/10.15252/embr.202051239
Goodman, C. A., Mabrey, D. M., Frey, J. W., Miu, M. H., Schmidt, E. K., Pierre, P. and Hornberger, T. A. (2011). Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 25(3): 1028–1039. https://doi.org/10.1096/fj.10-168799
Martineau, Y., Azar, R., Müller, D., Lasfargues, C., El Khawand, S., Anesia, R., Pelletier, J., Bousquet, C. and Pyronnet, S. (2014). Pancreatic tumours escape from translational control through 4E-BP1 loss. Oncogene 33(11): Article 11. https://doi.org/10.1038/onc.2013.100
Mesclon, F., Lambert-Langlais, S., Carraro, V., Parry, L., Hainault, I., Jousse, C., Maurin, A.-C., Bruhat, A., Fafournoux, P. and Averous, J. (2017). Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells. Oncotarget 8(16): 27440–27453. https://doi.org/10.18632/oncotarget.15828
Morral, C., Stanisavljevic, J., Hernando-Momblona, X., Mereu, E., Àlvarez-Varela, A., Cortina, C., Stork, D., Slebe, F., Turon, G., Whissell, G., et al. (2020). Zonation of Ribosomal DNA Transcription Defines a Stem Cell Hierarchy in Colorectal Cancer. Cell Stem Cell 26(6): 845–861.e12. https://doi.org/10.1016/j.stem.2020.04.012
Nakano, K. and Hara, H. (1979). Measurement of the protein-synthetic activity in vivo of various tissues in rats by using [3H]Puromycin. Biochem. J. 184(3): 663–668. https://doi.org/10.1042/bj1840663
Nathans, D. (1964). PUROMYCIN INHIBITION OF PROTEIN SYNTHESIS: INCORPORATION OF PUROMYCIN INTO PEPTIDE CHAINS. Proc. Natl. Acad. Sci. U.S.A. 51(4): 585–592. https://doi.org/10.1073/pnas.51.4.585
Piecyk, M., Triki, M., Laval, P.-A., Dragic, H., Cussonneau, L., Fauvre, J., Duret, C., Aznar, N., Renno, T., Manié, S. N., et al. (2021). Pemetrexed Hinders Translation Inhibition upon Low Glucose in Non-Small Cell Lung Cancer Cells. Metabolites 11(4): 198. https://doi.org/10.3390/metabo11040198
Piecyk, M., Triki, M., Laval, P.-A., Duret, C., Fauvre, J., Cussonneau, L., Machon, C., Guitton, J., Rama, N., Gibert, B., et al. (2023). The stress sensor GCN2 differentially controls ribosome biogenesis in colon cancer according to the nutritional context. Mol. Oncol.. https://doi.org/10.1002/1878-0261.13491
Sarcinelli, C., Dragic, H., Piecyk, M., Barbet, V., Duret, C., Barthelaix, A., Ferraro-Peyret, C., Fauvre, J., Renno, T., Chaveroux, C., et al. (2020). ATF4-Dependent NRF2 Transcriptional Regulation Promotes Antioxidant Protection during Endoplasmic Reticulum Stress. Cancers (Basel) 12(3): 569. https://doi.org/10.3390/cancers12030569
Schmidt, E. K., Clavarino, G., Ceppi, M. and Pierre, P. (2009). SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6(4): Article 4. https://doi.org/10.1038/nmeth.1314
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4,934 | https://bio-protocol.org/en/bpdetail?id=4934&type=0 | # Bio-Protocol Content
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Addressing the Role of Conventional CD8αβ+ T Cells and CD4+ T Cells in Intestinal Immunopathology Using a Bone Marrow–Engrafted Model
AA Amneh Aoudi
OL Ossama Labiad
RI Ramdane Igalouzene
OM Ousséma Mejri
MS Maxime Sanchez
SS Saïdi Soudja
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4934 Views: 796
Reviewed by: Andrea GramaticaMarco Di Gioia Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Clinical Investigation Apr 2022
Abstract
Inflammatory bowel disease (IBD) is characterized by an aberrant immune response against microbiota. It is well established that T cells play a critical role in mediating the pathology. Assessing the contribution of each subset of T cells in mediating the pathology is crucial in order to design better therapeutic strategies. This protocol presents a method to identify the specific effector T-cell population responsible for intestinal immunopathologies in bone marrow–engrafted mouse models. Here, we used anti-CD4 and anti-CD8β depleting antibodies in bone marrow–engrafted mouse models to identify the effector T-cell population responsible for intestinal damage in a genetic mouse model of chronic intestinal inflammation.
Key features
• This protocol allows addressing the role of CD4+ or CD8αβ+ in an engrafted model of inflammatory bowel disease (IBD).
• This protocol can easily be adapted to address the role of other immune cells or molecules that may play a role in IBD.
Keywords: T cell CD8αβ+ T-cell depletion in vivo Gut inflammation CD4+ T cells Bone marrow–engrafted model
Background
Inflammatory bowel disease (IBD) is a chronic and recurring inflammation in the gastrointestinal tract that affects more than 1.3 million people in Europe (Zhao et al., 2021). IBD includes ulcerative colitis and Crohn’s disease. It is common for patients with ulcerative colitis and Crohn's disease to experience diarrhea, rectal bleeding, abdominal pain, fatigue, and weight loss. Furthermore, bearers of this debilitating disease are at high risk of inflammation-associated cancers such as colorectal cancer (Kim and Chang, 2014). Although it is widely accepted that IBD is characterized by an excessive immune response to microbiota, the etiology of IBD remains a mystery, being crucial to create novel models that can unravel the intricate microbial, molecular, and cellular interactions contributing to the onset of IBD.
Several immune-regulatory mechanisms prevent IBD. Particularly, SMAD4 in T cells, a significant downstream component of the cytokine TGF-β pathway, prevents mice from developing chronic intestinal inflammation. Indeed, researchers have demonstrated that ablation of SMAD4 specifically in T cells using a CD4-CRE system leads to severe chronic intestinal inflammation (Hahn et al., 2011; Kim et al., 2006). This inflammation typically emerges around 5–7 months of age. T cells deficient in SMAD4 contribute to intestinal inflammation. However, the model employed to illustrate this is based on the CD4-CRE system. As both CD4+ and CD8αβ+ T cells undergo a stage expressing both CD4 and CD8 markers during their generation in the thymus, the deletion of SMAD4 occurs in both CD4+ and CD8αβ+ T cells. It is crucial to specify which subset of T cells is responsible for mediating the observed pathology in the context of the immunopathology observed in IBD.
At present, no animal model entirely recapitulates Crohn’s disease or ulcerative colitis. Models such as the SMAD4 model present the benefit of not using chemical agents such as dextran sulfate sodium (DSS) and closely mimicking the chronicity of the inflammation without external intervention, in contrast to chemical-induced colitis models. Although useful, colitis models based on the transfer of naïve T cells into mice lacking adaptive immunity (SCID or RAG mice) do not recapitulate the entire onset of IBD. Indeed, those models lack an adaptive immune system. The SMAD4 model provides some interesting insights that closely resemble what happens in humans, such as impairment on TGFβ signaling pathway (Monteleone et al., 2001).
The present protocol offers an approach to address the implication of different populations of T cells in IBD. It utilizes a bone marrow–engrafted mouse model and specific anti-CD4 and anti-CD8β depleting antibodies to eliminate conventional CD8αβ+ and CD4+ T cells, sparing unconventional T cells such as TCRγδ T cells (Figure 1). This protocol can be adapted for the study of other cellular and molecular mechanisms potentially implicated in IBD.
Figure 1. Schematic model of the procedures. This figure was created with BioRender.com.
Materials and reagents
Biological materials
Animals
All mice were on the C57BL/6J background and were maintained in PPAC, a specific pathogen-free animal facility of the Cancer Research Center of Lyon (CRCL), France. RAG2-KO mice were obtained from Charles River Laboratories and bred and maintained in the animal facility (specific pathogen-free). Mice were older than 8 weeks when used for the experiment. CD4-Cre+ Smad4floxed/floxed (SKO) mice and littermate CD4-Cre-Smad4floxed/floxed (WT) mice were the donors of bone marrow cells. In terms of gut inflammation development, no discernible difference was observed between males and females. However, the mice used should be of the same age and sex in the same experiment.
Materials
Pipette tips 10 μL, 20 μL, 200 μL, and 1 mL (Starlab)
5 mL sterile syringe (BD, catalog number: 3095971)
50 mL centrifuge tube (Falcon, catalog number: 352070)
15 mL conical centrifuge tube (Falcon, catalog number: 352097)
Petri dishes (100 × 20 mm) (FALCON, catalog number 353003)
10 mL serological pipettes (SARTSTEDT, catalog number: 86.1254.001)
25 G needle (BD, catalog number: 300600)
BD U-100 insulin syringe with a 29 G needle (BD, catalog number: 324891)
Neubauer chamber (KOVA international, catalog number: 87144 F)
Tubes 1.5 mL, autoclaved (STARLAB, catalog number: S1615-5500)
Nylon meshes with a mesh size of 70 μm (SEFAR NITEX, catalog number: 03-80/29), sterilized by autoclaving
Reagents
Surface disinfectants (Anios, SURFA’SAFE)
LD columns (Miltenyi Biotec, catalog number: 130-042-901)
Anti-CD8α BV605 (clone 53-6.7) (BioLegend, catalog number: 100748)
Anti-CD8b Alexa 700 (clone YTS156.7.7) (BioLegend, catalog number: 126618)
Anti-CD4 Brilliant Violet 711 (clone RM4.5) (BD, catalog number: 563726)
Anti-CD3e Brillant Violet 650 (clone 17A2) (BioLegend, catalog number: 100229)
Anti-CD45 APC-Cy7 (clone 30-F11) (BioLegend, catalog number: 103116)
Anti-CD103 e450 (clone 2E7) (eBioscience, catalog number: 48-1031-82)
Anti-Ly6g PE/Dazzle594 (clone 1A8) (BioLegend, catalog number: 127648)
Anti-CD11b APC (clone M1/70) (BioLegend, catalog number: 553312)
Anti-TCRgd FITC (clone GL3) (BD, catalog number: 553177)
LIVE/DEAD™ Fixable Yellow Dead Cell Stain kit (Life technologies, catalog number: L34968)
InVivoMAb anti-mouse CD8β (Lyt 3.2) (Clone 53-5.8) (BioXcell, catalog number: BE0223)
InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase (Clone HRPN) (BioXcell, catalog number: BE0088)
InVivoMAb anti-mouse CD4 (Clone GK1.5) (BioXcell, catalog number: BE0003-1)
InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin (Clone LTF-2) (BioXcell, catalog number: BE0090)
Fetal bovine serum (FBS) (Gibco®, catalog number: A5256701)
RPMI-1640 medium, GlutaMAX™ supplement (Life Technologies, Gibco, catalog number: 61870036)
HEPES buffer solution, 1 M (Gibco, catalog number: 15630-080)
Hank’s buffered salt solution (HBSS) (Gibco, catalog number: 14175-053)
Phosphate buffered saline (PBS 10×) (Life Technologies, Gibco, catalog number: 14200-059)
Ethanol 70% (Alcool modifié COOPER®) used as an antiseptic
Ethanol absolute (VWR Chemicals, catalog number: 20821.365)
CD3ϵ microbead kit, mouse (Miltenyi Biotec, catalog number: 130-094-973)
Trypan blue solution, 0.4% (Thermo Fisher, catalog number: 15250061)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906)
EDTA, pH 8.0 (EUROMEDEX, catalog number: EU0084)
Isoflurane
Gentamicin (50 mg/mL) (Gibco, catalog number: 15750-037)
Penicillin-Streptomycin (Life Technologies, catalog number: 15140-122)
Solutions
RPMI complete media (see Recipes)
Intra-epithelial lymphocyte enrichment buffer (see Recipes)
MACS buffer (see Recipes)
Recipes
RPMI complete media
RPMI-1640 enriched with 10% heat-inactivated FBS, 10 mM HEPES, and penicillin-streptomycin (1,000 U/mL).
MACS buffer
PBS 1× with 2 mM EDTA and 2% BSA
Equipment
Irradiator (Elekta Synergy® or any alternate irradiator)
Sterile cell culture hood (Thermo, model: HERAsafe KS 12 1.2m/41489710/2013)
QuadroMACS magnetic cell separator (Miltenyi Biotec, catalog number: 130-090-976)
Microscope (Zeiss, model: Primovert)
Serological pipettes [SARSTEDT, catalog number: 86.1253.001 (5 mL), 86.1254.001 (10 mL), and 86.1685.001 (25 mL)]
Pipette controller (INTEGRA, model: pipetboy)
Surgical scissors and forceps (Dutscher, catalog number: 005064, 005065, 711198, 005072)
Ice bucket (Dutscher, catalog number: 830116)
Anesthesia chamber (Tem Sega® or any alternate instrument)
Weigh balance (OHAUS®, model: CS200)
Pie-shape chambers (model equivalent of pie cage for X-ray irradiation from Bioseb Lab instrument®)
Animal transfer box [InnoVive, model: M-BTM (bottom) and MSX4 (lid)]
LSRII flow cytometer (BD Biosciences, model: LSR Fortessa HTS)
Centrifuge (Thermo Fisher, model: Heraeus Megafuge)
Software
FlowJoTM software (BD Biosciences) (v. 10.9, 2023)
GraphPad Prism (v. 10.1.2, 2023)
Procedure
Isolation of bone marrow cells for engraftment into recipient mice
Euthanize the donor mouse using an approved method; here, we used CO2 inhalation. Generally, 8–10 mice can be reconstituted from a single donor.
Disinfect the surgical area with 70% ethanol.
With scissors, make a small incision in the skin overlying the femur and tibia. Collect and place the femurs and tibias in RPMI complete media (see Recipes).
Under the sterile culture hood, initiate the process by immersing the bone within a Petri dish filled with 70% ethanol in order to eliminate potential contaminations (30 s). Try to meticulously remove a substantial portion of muscle and connective tissues from the bone. Leaving bones in ethanol for an extended period of time while flushing each bone is possible, but caution is advised because ethanol may penetrate the bone, potentially compromising viable cells. Subsequently, relocate the bone to a fresh Petri dish containing approximately 15 mL of RPMI complete media.
With sterile scissors, cut both ends of the femur and tibia to expose the bone marrow cavity.
After exposing the bone marrow cavity, carefully grasp the bone with forceps. Load a 5 mL syringe with complete media, using a 25 G needle, for the extraction process. Gently insert the needle into the bone marrow cavity and push the syringe plunger to flush out bone marrow cells into the Petri dish. Repeat this process iteratively until the bone transitions from its initial red hue to a paler, whitish appearance, indicating that the bone marrow cells have been extracted (Figure 2).
Figure 2. Bone marrow cell extraction. Utilize a 5 mL syringe with a 25 G needle filled with RPMI complete media for extraction of bone marrow cells (A). Insert the needle into the bone marrow cavity, push the syringe plunger to flush out cells into the Petri dish, and repeat until the bone shifts from red to a whitish appearance. Photo of mouse tibias (B). Figure 2A was created with BioRender.com.
Thoroughly resuspend the bone marrow cells using a p1000 pipette. This step is crucial to ensure the bone marrow cells do not form any aggregate.
Using a 70 μm sterile nylon mesh, filter the solution into a 50 mL tube to eliminate any possible clumps. Then, centrifuge the cells at 600× g for 5 min at 4 °C.
Discard the supernatant, resuspend the pellet into 500 μL of MACS buffer (see Recipes), and add 50 μL of CD3ϵ-biotin antibody. Incubate for 15 min at 4 °C. CD3e biotin antibodies will bind to T cells, permitting their specific elimination in the subsequent step of the protocol. After the incubation period, retrieve the tube and add 5 mL of MACS buffer, and spin down the cells by centrifugation at 600× g for 5 min at 4 °C.
Resuspend the pellet with 500 μL of MACS buffer and add 100 μL of anti-biotin microbeads. Mix well and incubate at 4 °C for 15 min.
After incubation, add 5 mL of MACS buffer and load the cell suspension to a LD column placed on the QuadroMACS magnetic cell separator. Using a 15 mL Falcon tube, collect the unlabeled cells that pass through.
Centrifuge the collected cells at 600× g for 5 min at 4 °C. Resuspend the pellet in 1 mL of PBS 1× buffer. Count the viable cells using 0.4% Trypan blue solution, a Neubauer chamber, and an optical microscope.
Adjust the cell concentration such that 2 million cells in 200 μL of PBS 1× can be engrafted into each mouse. Alternatively, if there are insufficient cells, 1 million cells may be engrafted. Optionally, to prevent potential contamination, gentamicin (50 μg/mL) can be added to the preparation. Then, keep cells at 4 °C until they are ready to be used.
Irradiation and T cell–depleted bone marrow transplantation
The day before the irradiation, weigh the recipient mice to obtain the starting weight. Tag the mice (earmark) prior to the irradiation to identify them and try, as much as possible, to place the control and future treated mice in the same cage, in order to minimize cage effects.
On the day of irradiation, place mice in a pie cage and transfer to the irradiator. This process must be conducted under the hood and all materials must be sterilized. In order to comply with the facility's specific pathogen-free conditions, mice should be transferred in a sterile transfer box to the irradiator.
To ablate the hematopoietic system of recipient mice, administer a single dose of sub-lethal irradiation. This is accomplished by irradiating 8–12-week-old RAG2KO mice with a total body irradiation dose of 6 Gy (600 rads).
After irradiation, replace the recipient mice in their cage with caution to maintain the specific pathogen-free conditions of the animal facility (e.g., the outside of the box is disinfected with Anios for at least 10 min).
After irradiation, reconstitute mice within 6–12 h. For the reconstitution of irradiated mice, inject 2 million T cell–depleted bone marrow cells from WT or SKO mice in 200 μL of PBS intravenously via the retro-orbital vein with a BD U-100 insulin syringe. To minimize mouse stress and discomfort, anesthetize mice with isoflurane in an anesthesia chamber.
Post-irradiation, as the mice undergo substantial immunosuppression and are consequently susceptible to infections, these animals should be housed within a sterile environment. For reconstituted mice, regular observation is particularly crucial during the first 14 days following irradiation to identify any indicators of pain or distress. In cases where animals exhibit signs of suffering for any reason (loose feces, rectal bleeding, or constant hunched posture), the ethical decision is to euthanize them to alleviate pain and distress. It is important to highlight that the reconstituted mice are intentionally not administered antibiotics, as this can adversely influence the development of IBD in SMAD4-deficient reconstituted mice (Igalouzene et al., 2022).
Wait 21 days after bone marrow reconstitution to allow the donor hematopoietic cells to establish within the recipient and then proceed to the T-cell depletion.
Weigh the mice twice a week to determine whether they are losing weight relative to their initial weight. This constitutes an objective indication that the mice are suffering from chronic intestinal inflammation.
CD8αβ+ and CD4+ T-cell depletion in vivo
On day 21 after bone marrow reconstitution, intraperitoneally inject each mouse with 150 μg of one of the following antibodies in 200 μL of PBS 1×: anti-CD8β antibody (clone 53-5.8), anti-CD4 antibody (clone GK1.5), rat IgG1 isotype control antibody (clone HRPN), or rat IgG2b isotype control antibody (clone LTF-2) (Figure 3).
Figure 3. Experimental groups. This figure was created with BioRender.com.
Repeat the injection once per week until the SMAD4-deficient mice lose approximately 15%–20% of their original weight and must be euthanized and analyzed. After irradiation and reconstitution, the SKO-reconstituted mice generally become ill 5–7 weeks later, exhibiting loose stools, diarrhea, and rectal bleeding, as well as a hunched-over appearance and a weight loss of 15%–20%. The colon, small intestine, and mesenteric lymph nodes can be harvested for histology or flow cytometry analysis.
As well as monitoring the body weight two times per week, monitor the health status of the mice at least three times per week to intervene promptly and ethically. Carefully check mice for signs of discomfort such as social isolation, hunched posture, immobility, blood in feces, loose feces, or ruffled or unkempt appearance.
Data analysis
Weight loss is indicative of the severity of gut inflammation. Throughout the experiment, mice are weighed twice a week, and the percentage of weight loss is calculated in comparison to the initial weight. Readers are directed to Igalouzene et al. (2022) and Figure 2 of the present manuscript to see how the elimination of CD8αβ+ T cells prevent bone marrow–engrafted SKO mice from developing severe intestinal inflammation. It is important to remind that, for ethical reasons, mice experiencing a 20% loss of their initial body weight are euthanized within 24 h with the corresponding controls. Following euthanasia, the colon, small intestine, and mesenteric lymph nodes are collected for further analysis.
The colon length is also a parameter that indicates the severity of the chronic inflammation. Using a ruler, the colon length is determined. Shorter colons relative to mice with the same age and sex are associated with severe gut inflammation (Igalouzene et al., 2022 and Figure 5).
Figure 4. Validation of specific deletion within the colon and small intestine. Representative flow cytometry plots showing the frequency of CD4+ and CD8αβ+ T cells and CD8αα+ T cells within the colon and small intestine of irradiated RAG2KO mice reconstituted with WT or SMAD4-deficient bone marrow cells and injected with isotype or anti-CD8β (clone 53-5.8) or anti-CD4 (clone GK1.5) depleting antibodies.
Figure 5. Analysis of gut inflammation in bone marrow–engrafted mice: colon length. Photo of the colon of irradiated RAG2KO mice reconstituted with WT or SMAD4-deficient bone marrow cells and injected with isotype or anti-CD8β or anti-CD4 depleting antibodies. The reduction of the size of the colon is a sign of a severe chronic gut inflammation.
Histological assessment of inflammation can also be performed as proposed by Erben et al. (2014). Briefly, the colon and small intestine were treated with 2% formaldehyde (VWR) for fixation, followed by embedding in paraffin and sectioning. Tissues sections were subjected to hematoxylin and eosin staining. We evaluated intestinal inflammation in a blinded manner using a systematic scoring system based on the following criteria: colon length, extent and distribution of inflammatory cell infiltration, crypt hyperplasia, presence of neutrophils within crypts, occurrence of crypt abscesses, erosion, formation of granulation tissues, and villous blunting. Please refer to Igalouzene et al. (2022) to see some examples of images and Erben et al. (2014) if you are eager to learn more on the procedure. In Igalouzene et al. (2022), histological examination demonstrated a reduction in immune cell infiltration and absence of hyperplasia and crypt abscesses in SKO BM/engrafted mice treated with anti-CD8β (Igalouzene et al., 2022, Figure 2D and 2E, and Supplemental Figure 4B).
To validate the effectiveness of specific T-cell depletion and assess inflammation levels, flow cytometry analysis can be performed. Use distinct antibody clones from those applied in the in vivo depletion process. Specifically, anti-CD8β (clone YTS156.7.7) and anti-CD4 (clone RM4.5) can be employed to label the cells. For the analysis of neutrophil infiltration, indicative of inflammation, use anti-Ly6g and anti-CD11b APC antibodies. Acquire the cells using a flow cytometry machine. Gate the events based on FSC-A and SSC-A, followed by gating on FSC-A and FSC-H to exclude doublets. Eliminate dead cells by gating on cells negative for LIVE/DEAD fixable staining. Then, focus on CD45+ cells, which correspond to immune cells. For instance, CD45+CD8αβ+ T cells represent CD8αβ+ T cells. Successful depletion of CD8αβ T cells is evidenced by the complete absence of CD8αβ T cells in mice treated with anti-CD8β (see Figure 2). Importantly, the elimination of other populations (TCRγδ+ and CD8aa+ T cells) is not observed, indicating the specificity of the depletion (Figure 4).
By using this protocol, we identified CD8αβ+ T cells as an important effector driving intestinal severe inflammation in SMAD4-deficient mice (Igalouzene et al., 2022), providing new insights into the role of T-cell populations in this complex disease. The depletion of conventional CD8αβ+ T cells alleviates the onset of inflammation (weight loss, colon length reduction) evidenced by the reduction of neutrophils (CD11b+Ly6g+) recruitment within the epithelium and the reduction in colon length (Figure 5–6) (see Igalouzene et al. 2022 for further details). Listed below are a few examples of how one can analyze the effect of T-cell depletion on inflammation; these examples are not exhaustive.
Figure 6. Analysis of gut inflammation in bone marrow–engrafted mice: neutrophil infiltration. Representative flow cytometry plots showing the frequency of neutrophils CD11b+Ly6g+ mobilized within the epithelium of the colon and the small intestine.
Validation of protocol
For representative data including weight loss, colon length, histological scoring, and cytometry, we refer readers to Igalouzene et al., (2022), Figures 2 and 8 as well as Supplemental Figure 2, 3, and 4.
General notes and troubleshooting
General notes
All solutions, media, materials, and buffers should be sterile and prepared according to standard protocols. It is essential to perform the isolation procedure under sterile conditions to avoid contamination. Additionally, work fast and always keep buffers and cell suspensions at 4 °C to prevent cell death.
The use of a bone marrow–engrafted mouse model accelerates the intestinal pathologies, allowing researchers to address questions without the need of long-term treatments. This approach significantly minimizes potential side effects associated with prolonged antibody treatment, as the pathology emerges in short term; therefore, there is no need to treat the mice for a long period. Additionally, this facilitates the synchronization of the chronic inflammation development in mice of similar sex and age, thereby enhancing the precision and comparability in investigation.
The elimination of T cells from the donor bone marrow cells is performed to prevent the transfer of T cells that have been conditioned in the donor, potentially accelerating and exacerbating the pathology by bypassing the natural initiation of inflammation in the engrafted mice. Novel T cells will be generated from the stem cells present in the donor bone marrow cells within the reconstituted mice, thereby providing a more faithful natural recapitulation of the onset of inflammation.
Notably, for the precise depletion of conventional CD8αβ T cells while sparing other cell types that express the common marker CD8α, researchers can take advantage of using of CD8β depleting antibodies (clone: 53-5.8) as performed in this protocol, in contrast to other studies using CD8α depleting antibodies that deplete a lot of unspecific immune populations (Figure 4).
Acknowledgments
The protocol described here was adapted from our previously published work (Igalouzene et al., 2022). This work was supported by the Association pour la Recherche sur le Cancer (ARC) (PJA20181207928-PJA20151203509-ARCDOC42020070002550). We would like to thank the P-PAC animal platform for taking care of the mice and the flow cytometer platform of the CRCL.
Competing interests
The authors declare no competing financial interest.
Ethical considerations
The experiments were conducted in accordance with the guidelines for animal care of the European Union (ARRIVE) and were validated by the local Animal Ethic Evaluation Committee (CECCAPP) and the French Ministry of Research.
References
Erben, U., Loddenkemper, C., Doerfel, K., Spieckermann, S., Haller, D., Heimesaat, M.M., Zeitz, M., Siegmund, B., and Ku¨hl, A.A. (2014). A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int. J. Clin. Exp. Pathol. 7, 4557.
Hahn, J. N., Falck, V. G. and Jirik, F. R. (2011). Smad4 deficiency in T cells leads to the Th17-associated development of premalignant gastroduodenal lesions in mice. J. Clin. Invest. 121(10): 4030–4042.
Igalouzene, R., Hernandez-Vargas, H., Benech, N., Guyennon, A., Bauché, D., Barrachina, C., Dubois, E., Marie, J. C. and Soudja, S. M. (2022). SMAD4 TGF-β–independent function preconditions naive CD8+ T cells to prevent severe chronic intestinal inflammation. J. Clin. Invest. 132(8): e1172/jci151020.
Kim, B. G., Li, C., Qiao, W., Mamura, M., Kasperczak, B., Anver, M., Wolfraim, L., Hong, S., Mushinski, E., Potter, M., et al. (2006). Smad4 signalling in T cells is required for suppression of gastrointestinal cancer. Nature 441(7096): 1015–1019.
Kim, E. R., and Chang, D. K. (2014). Colorectal cancer in inflammatory bowel disease: the risk, pathogenesis, prevention and diagnosis. World J. Gastroenterol. 20(29): 9872–9881.
Monteleone, G., Kumberova, A., Croft, N. M., McKenzie, C., Steer, H. W. and MacDonald, T. T. (2001). Blocking Smad7 restores TGF-β1 signaling in chronic inflammatory bowel disease. J. Clin. Invest. 108(4): 601–609.
Zhao, M., Gönczi, L., Lakatos, P.L., and Burisch, J. (2021). The Burden of Inflammatory Bowel Disease in Europe in 2020. J. Crohn’s Colitis 1573–1587.
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4,935 | https://bio-protocol.org/en/bpdetail?id=4935&type=0 | # Bio-Protocol Content
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CoCoNat: A Deep Learning–Based Tool for the Prediction of Coiled-coil Domains in Protein Sequences
MM Matteo Manfredi
CS Castrense Savojardo
PM Pier Luigi Martelli
RC Rita Casadio
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4935 Views: 1066
Reviewed by: Prashanth N Suravajhala Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Bioinformatics Aug 2023
Abstract
Coiled-coil domains (CCDs) are structural motifs observed in proteins in all organisms that perform several crucial functions. The computational identification of CCD segments over a protein sequence is of great importance for its functional characterization. This task can essentially be divided into three separate steps: the detection of segment boundaries, the annotation of the heptad repeat pattern along the segment, and the classification of its oligomerization state. Several methods have been proposed over the years addressing one or more of these predictive steps. In this protocol, we illustrate how to make use of CoCoNat, a novel approach based on protein language models, to characterize CCDs. CoCoNat is, at its release (August 2023), the state of the art for CCD detection. The web server allows users to submit input protein sequences and visualize the predicted domains after a few minutes. Optionally, precomputed segments can be provided to the model, which will predict the oligomerization state for each of them. CoCoNat can be easily integrated into biological pipelines by downloading the standalone version, which provides a single executable script to produce the output.
Key features
• Web server for the prediction of coiled-coil segments from a protein sequence.
• Three different predictions from a single tool (segment position, heptad repeat annotation, oligomerization state).
• Possibility to visualize the results online or to download the predictions in different formats for further processing.
• Easy integration in automated pipelines with the local version of the tool.
Graphical overview
Keywords: Coiled-coil segments Oligomerization Deep learning Prediction Protein Language Models Web server
Background
Coiled-coil domains (CCDs) are structural motifs where α‐helices pack together in an arrangement called knobs-into-holes [1], by which residues from one helix (the knobs) pack into holes formed by side chains in the other helices participating in the domain. CCDs have been observed in different kinds of proteins sequenced from all the kingdoms of life [2] and perform a great number of diverse functions.
Canonical CCDs include the interaction of two or more α‐helices, each characterized by the repetition of a seven-residue motif called heptad repeat. The positions of the heptad repeat are referred to as registers and are labeled with the letters a–g. CCDs can be classified into different oligomerization states, depending on the number (dimers, trimers, tetramers, and higher orders) and orientation (parallel or antiparallel) of the involved α‐helices.
Methods such as SOCKET [3] or SamCC-Turbo [4] can annotate CCDs starting from the experimental 3D structure of a protein. In the absence of structural information, several tools have been proposed over the years to perform automatic annotations on protein sequences, each addressing different tasks of CCD prediction (i.e., segment localization, heptad repeat annotation, oligomerization state classification).
Recently, the development of protein language models (PLMs) introduced a novel way of generating embeddings to encode protein sequences for downstream predictive tasks. We proposed CoCoNat [5], a deep learning–based approach that exploits two different and complementary PLMs, ProtT5 [6] and ESM2 [7], to produce a predictive pipeline for the complete ab initio annotation of CCDs.
CoCoNat processes input sequences with three cascading networks, each trained independently to solve a specific task. The first step adopts a deep architecture based on convolutional and recurrent layers to identify the presence of coil-coiled segments along the sequence. The second step adopts a probabilistic graphical model to assign registers to each residue in the segment. Finally, the third step adopts a neural network to predict the oligomerization state of each segment. Each prediction is also complemented with the probabilities computed by the network, allowing users to assess their reliability.
CoCoNat is trained on a dataset comprising 2,198 proteins annotated with 4,342 helices and 9,062 proteins without CCD. When tested on a non-redundant benchmark dataset, comprising 400 proteins annotated with 863 helices and 318 proteins without CCD, CoCoNat outperforms other methods on all three predictive tasks included in the pipeline. Specifically, it achieves a 0.54 per-residue F1 score and a 0.49 per-segment F1 score on the identification of segment boundaries over the sequence (first step in the Graphical overview), a Matthew's Correlation Coefficient (MCC) between 0.83 and 0.84 for each type of register in the annotation of the heptad repeats (second step in the Graphical overview), and an average MCC of 0.58 for the 4-class classification of the oligomerization state (third step in the Graphical overview).
Moreover, the adoption of PLMs to encode the input allows CoCoNat to be extremely time efficient. When tested on the virtual machine hosting the web server (AMD EPYC 7301 12-Core Processor, 48 GB RAM, no GPU), CoCoNat requires an average running time of 330 s (5.5 min) to predict 100 sequences of length comprised between 100 and 200 residues. The same computation takes approximately 2.5 h with CoCoPRED [8], a similar tool based on canonical multiple sequence alignments.
Here, we illustrate in detail how CoCoNat can be adopted as a web server or as a standalone tool, allowing for easy integration into any computational pipeline. As a test case, we select one of the proteins belonging to our benchmark dataset, the Mating-type switching protein swi5 from the organism Schizosaccharomyces pombe (UniProt accession: Q9UUB7). This protein presents two coiled-coil segments organized as a parallel dimer that CoCoNat identifies. Both the registers and the oligomerization classes are correctly assigned. The only difference between the putative and the real annotations is the length of the segments.
Supplementary File S1 reports a schema of the web server compliant with the Minimum Information About Bioinformatics investigation (MIABi) guidelines [9].
Equipment
Computer with internet access and a web browser
(Only for online execution) CoCoNat web server (https://coconat.biocomp.unibo.it/)
(Only for local execution) Machine with a macOS or Linux operating system and at least 4 CPU cores and 48 GB of RAM
The local release is not suitable to be executed directly on a machine with a Windows operating system. In this case, the adoption of a virtual machine or the Windows Subsystem for Linux (WSL) is recommended.
Software and datasets
Docker Engine, installed (https://docs.docker.com/engine/install/debian/)
Miniconda, installed (https://docs.conda.io/projects/miniconda/en/latest/miniconda-install.html)
As long as Miniconda is installed, Python 3 and pip (both mentioned in the protocol) do not need to be installed separately, as they will be included in the environment generated by Miniconda.
Procedure
Online single sequence prediction
Prepare your sequence in FASTA format.
Open the homepage of the web server (https://coconat.biocomp.unibo.it/).
Paste your sequence in the form or upload the FASTA file.
The web server will validate the format of your input. If the limitations are too strict for your target sequence, we suggest following Procedure C and running CoCoNat locally.
Valid FASTA format.
Sequence length between 40 and 700 residues.
Contains only 20 standard amino acids, 3 non-standard residues (UZB), or undetermined character (X).
(Optional) Provide precomputed coiled-coil segments.
Tick the box Provide precomputed coiled-coil segments (predict only oligomerization state).
Paste sequence of states. Use the letter i to indicate a residue that is not part of a coiled-coil segment; use the letters a–g to indicate the registers of the residues in the coiled-coil segment.
If you only know the segments but not the registers, skip this step and run the full prediction.
Press Submit (Figure 1).
Figure 1. Online single sequence submission page. Homepage of the CoCoNat web server, where users can submit a target sequence in FASTA format. It is possible to load an example sequence (1) or to upload a file from the computer (2). Optionally, it is possible to provide precomputed coiled-coil segments. A button loads an example (3) showing the correct format. At the end of the page, it is possible to clear the forms (4) or to submit the job to start the prediction (5).
Bookmark the page that loads after job submission. This page will be updated with the results as soon as they are ready.
Graphically visualize the results (Figure 2).
Under Predicted coiled-coil segments, you will see a list of all predicted segments, including the following information:
i. Segment start.
ii. Segment end.
iii. Length of segment.
iv. Predicted registers.
v. Predicted oligomerization state.
Figure 2. Online single sequence result page (full prediction). Results are visualized after submitting the example input to the web server, without precomputed segments. From the results page, it is possible to copy the link (1) to access them at a later date and to download the results in tab-separated values (TSV) format (2) or in JSON format (3). For each predicted segment, clicking on the magnifying glass (4) will zoom in the corresponding region on the sequence feature viewer. The viewer itself offers the possibility to change visualization by zooming in or out, resetting the zoom, and moving along the sequence length (5). Finally, it is possible to save a screenshot of the selected portion of the sequence (6).
Under Sequence feature view, you will see four lines visualizing from top to bottom the following data:
i. Residue sequence.
ii. Predicted coiled-coil regions (colored depending on the predicted oligomerization state).
iii. Predicted registers (colored depending on the register type).
iv. Coiled-coil probability, showing how confident the model is at each position.
(Optional) If you provided precomputed coiled-coil segments (see step A5), the coiled-coil probabilities (see steps A8b–8d) will be all equal to 1, and only the oligomerization state will be predicted by the web server (Figure 3).
If needed, a button is available to take screenshots of the selected region of the sequence.
Figure 3. Online single sequence result page (precomputed segments). Results are visualized after submitting the example input to the web server. In this case, only the oligomerization state was predicted, and the precomputed segments are visualized with an associated probability equal to 100%. The functionalities of the web server are the same as detailed in Figure 2.
Download results with the buttons at the top of the page.
If you select the tab-separated format (TSV), the generated file will contain a line for each residue (plus the header) organized in 14 columns, containing information about:
i. ID: ID of the protein.
ii. RES: Residue type.
iii. CC_CLASS: Predicted coiled-coil class. The coiled-coil class is i if the residue does not belong to a coiled-coil segment; otherwise, it is a letter from a to g that indicates the predicted register.
iv. OligoState: Predicted oligomerization state (P, A, 3, or 4 for parallel dimer, antiparallel dimer, trimer, or tetramer, respectively).
v. Pi, Pa-Pg, PH: nine columns for the computed probabilities for each possible coiled-coil class (the higher probability will determine the content of the third column).
vi. pOligo: Computed probability of the predicted oligomerization state reported in point iv (the higher the probability, the higher the confidence of the model).
If you select the JSON format, the generated file will contain one entry with 10 fields:
i. accession: ID of the protein.
ii. res: FASTA sequence.
iii. labels: Sequence of predicted coiled-coil classes (see step A9a.iii).
iv. prob: List of probabilities assigned by the predictor to the most likely coiled-coil class for each residue.
v. oligo: Sequence of predicted oligomerization classes (see step A9a.iv).
vi. prob_oligo: List of probabilities assigned by the predictor to the most likely oligomerization class for each residue.
vii. segments: List with one item for each predicted segment reporting summary information about it.
viii. length: Length of the protein.
A typical computation for a single sequence prediction will require approximately 90 s (1.5 min).
For further help and details, please reference the Help page of the web server (https://coconat.biocomp.unibo.it/help/).
Online batch prediction
Prepare your sequences in FASTA format.
Open the Homepage of the web server and go to the tab batch submission (https://coconat.biocomp.unibo.it/batch/).
Upload the FASTA file.
(Optional) Provide an email address. When results are ready, the link to access them will be sent to you.
The web server will validate the format of your input. If the limitations are too strict for your target sequences, we suggest following Procedure C and running CoCoNat locally.
Valid FASTA format.
No more than 500 sequences.
All sequences with length between 40 and 700 residues and total number of residues not greater than 80,000.
Each sequence contains only 20 standard amino acids, 3 non-standard residues (UZB), or undetermined character (X).
Press Submit.
Bookmark the page that loads upon job submission. This page will be updated with the results as soon as they are ready. If you have provided an email address, you will receive the link to this page via email once the results are ready.
Download results with the buttons at the top of the page.
See Procedure A, step 9a for a description of the TSV format.
See Procedure A, step 9b for a description of the JSON format. Mind that, in this case, the JSON file will contain one entry for each sequence in the input file.
The time needed for computing the outputs will depend on the number and length of the input sequences. A job containing 100 sequences with lengths between 100 and 200 residues should take approximately 330 s (5.5 min).
For further help and details please reference the Help page of the web server (https://coconat.biocomp.unibo.it/help/).
Local prediction
Note: All lines of code reported in this section are meant to be executed inside a terminal.
Prepare the environment (see Figure 4).
Create a conda environment with Python 3.
conda create -n coconat python
conda activate coconat
Install dependencies using pip.
pip install docker absl-py
Clone locally the GitHub repository in your current working directory; then, cd (change directory) to the newly created package directory.
git clone https://github.com/BolognaBiocomp/coconat
cd coconat
Build the Docker image.
docker build -t coconat:1.0.
Download the required PLMs (e.g., in the Home directory).
cd
wget https://coconat.biocomp.unibo.it/static/data/coconat-plms.tar.gz
tar xvzf coconat-plms.tar.gz
Figure 4. Configuration of local installation. The screenshot displays the content of the CoCoNat folder after downloading the GitHub repository in a local folder. The word “coconat” between parenthesis at the beginning of the prompt (in white) appears only if the conda environment is properly set up.
Run CoCoNat (see Figure 5).
Prepare your sequences in FASTA format. Input requirements are the same as the online predictions but without limits on the number and length of the sequences. Please remember that the maximal number of sequences and the maximal length of each sequence that the model will be able to process together will depend on the specifics of your machine.
Run the run_coconat_abinitio_docker.py script, providing paths to the input FASTA file, the output TSV file, and the models downloaded at step C1e.
cd coconat
python run_coconat_abinitio_docker.py \
--fasta_file=example-data/example.fasta \
--output_file=example-data/example.tsv --plm_dir=${HOME}/coconat-plms
(Optional) Run instead the run_coconat_state_docker.py script, providing an additional input file, to run predictions with precomputed coiled-coil segments. The input segment file must be a TSV containing a row for each precomputed segment and four columns:
i. ID of the protein containing the segment.
ii. Segment start.
iii. Segment end.
iv. Registers sequence of the segment.
cd coconat
python run_coconat_state_docker.py \
--fasta_file=example-data/example.fasta \
--seg_file=example-data/example-seg.tsv
--output_file=example-data/example.tsv --plm_dir=${HOME}/coconat-plms
See step A9a for a description of the output file generated by the program.
Figure 5. Local execution. The screenshot displays the execution of an example contained in the GitHub repository. At the top, the content of the input files is displayed, including a FASTA file and a segment file. The third command is the execution of the CoCoNat script, followed by the expected messages given during a normal execution. Finally, the last command shows the first 10 lines of the output file in tab-separated format. The meaning of each column is detailed in step A9a.
For further help and details please reference the README provided in the GitHub repository (https://github.com/BolognaBiocomp/coconat).
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Madeo et al. (2023). CoCoNat: a novel method based on deep learning for coiled-coil prediction. Bioinformatics (Tables 3, 4, 5).
Acknowledgments
The work was supported by the European Union—NextGenerationEU through the Italian Ministry of University and Research under the projects “Consolidation of the Italian Infrastructure for Omics Data and Bioinformatics” (ElixirxNextGenIT)” (Investment PNRR-M4C2-I3.1, Project IR_0000010, CUP B53C22001800006) and "HEAL ITALIA" (Investment PNRR-M4C2-I1.3, Project PE_00000019, CUP J33C22002920006).
This protocol describes the method presented in Madeo et al. (2023).
Competing interests
There are no conflicts of interest or competing interests.
References
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Walshaw, J. and Woolfson, D. N. (2001). Socket: a program for identifying and analysing coiled-coil motifs within protein structures. J. Mol. Biol. 307(5): 1427–1450. https://doi.org/10.1006/jmbi.2001.4545
Szczepaniak, K., Bukala, A., da Silva Neto, A. M., Ludwiczak, J. and Dunin-Horkawicz, S. (2021). A library of coiled-coil domains: from regular bundles to peculiar twists. Bioinformatics 36(22–23): 5368–5376. https://doi.org/10.1093/bioinformatics/btaa1041
Madeo, G., Savojardo, C., Manfredi, M., Martelli, P. L. and Casadio, R. (2023). CoCoNat: a novel method based on deep learning for coiled-coil prediction. Bioinformatics 39(8). https://doi.org/10.1093/bioinformatics/btad495
Elnaggar, A., Heinzinger, M., Dallago, C., Rihawi, G., Wang, Y., Jones, L., Gibbs, T., Feher, T., Angerer, C., Steinegger, M., et al. (2020). ProtTrans: Towards Cracking the Language of Life’s Code Through Self-Supervised Deep Learning and High Performance Computing. arXiv. Retrieved from http://arxiv.org/abs/2007.06225
Lin, Z., Akin, H., Rao, R., Hie, B., Zhu, Z., Lu, W., Smetanin, N., Verkuil, R., Kabeli, O., Shmueli, Y., et al. (2022). Evolutionary-scale prediction of atomic level protein structure with a language model. bioRxiv https://doi.org/10.1101/2022.07.20.500902
Feng, S.-H., Xia, C.-Q. and Shen, H.-B. (2022). CoCoPRED: coiled-coil protein structural feature prediction from amino acid sequence using deep neural networks. Bioinformatics 38(3): 720–729. https://doi.org/10.1093/bioinformatics/btab744
Tan, T., Tong, J., Khan, A. M., de Silva, M., Lim, K. and Ranganathan, S. (2010). Advancing standards for bioinformatics activities: persistence, reproducibility, disambiguation and Minimum Information About a Bioinformatics investigation (MIABi). BMC Genomics 11: S27. https://doi.org/10.1186/1471-2164-11-s4-s27
Supplementary information
The following supporting information can be downloaded here:
File S1.
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4,936 | https://bio-protocol.org/en/bpdetail?id=4936&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies
KD Katarina Stoklund Dittlau
AC Abinaya Chandrasekaran
KF Kristine Freude
LB Ludo Van Den Bosch
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4936 Views: 2307
Reviewed by: Marina Sánchez PetidierAnthony Flamier Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Neurodegeneration Jan 2023
Abstract
Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.
Key features
• This protocol requires preexisting experience in hiPSC culturing for a successful outcome.
• The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points.
• The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.
Graphical overview
Keywords: Astrocyte Human induced pluripotent stem cell Neurodegeneration Amyotrophic lateral sclerosis Small-molecule differentiation
Background
Neurodegenerative diseases affect millions of people worldwide and as the average population age increases, there is a corresponding rise in the number of patients. Amyotrophic lateral sclerosis (ALS) is one of these neurodegenerative diseases. ALS, the most prevalent motor neuron disorder among adults, affects approximately 2 out of 100,000 individuals across a wide age range, encompassing cases from teenagers to the elderly [1]. Ten percent of cases are caused by inherited familial mutations, while 90% have no family history and are therefore classified as sporadic [2]. Hallmarks of ALS include toxic protein aggregations, axonal transport impairments, DNA damage, and glial reactivity, leading to extensive motor neuron death [3–7]. This causes muscle atrophy, paralysis, and death of patients typically within 2–5 years after symptom onset, and currently, there is no cure [1]. As with many other neurodegenerative diseases, the focus has been on unraveling the underlying disease mechanisms behind the apparent (motor)neuronal cell death; however, the widespread glial reactivity has recently resulted in a shift from the neurocentric perspective towards increased appreciation of the role of glial cells. Astrocytes are shown to be key players in neurodegeneration [8]. As one of the most abundant glial cell types in the central nervous system, astrocytes govern the support and homeostatic maintenance of neurons and their surroundings [9]. Under physiological conditions, astrocytes have many functions including neurotransmitter modulation, nutritional distribution, ion, pH, and water homeostasis, blood–brain barrier regulation, and trophic support [9,10]. However, in ALS and other neurodegenerative diseases, astrocytes lose these supportive characteristics and take on a more toxic reactive role [10]. Considerable knowledge about the pathophysiology of ALS involving astrocytes has been gained through the use of animal models. However, it is important to acknowledge that, like all models, they come with their inherent limitations [10]. Animal models often rely on overexpression of human mutant genes, which despite showing various disease-relevant mechanisms, often fail to translate to a human context [11]. Importantly, overexpression models also exclude the large and important group of sporadic patients. Furthermore, human astrocytes exhibit larger sizes, more intricate branching structures, and a greater extent of synapse interactions compared to their rodent counterparts [12–14]. As a result, the field of animal research necessitates reinforcement from human in vitro models, and human-induced pluripotent stem cells (hiPSCs) present as highly promising candidates. With their ability for self-renewal and indefinite proliferation, as well as their possibility to generate any cell type, they hold significant potential. Several protocols for generating hiPSC-derived astrocytes exist, but many of the protocols are complex and require long timelines to reach the state of full astrocyte differentiation [15–21]. Our protocol is based on a 25-day-long differentiation followed by a 4+ week maturation. The differentiation is based on a dual inhibition of SMAD signaling pathway with the introduction of 3D culturing [22] to generate neural progenitor cells (NPCs) and a modified astrocyte differentiation protocol from Shaltouki et al. [23] to generate astrocytes [24,25]. After four weeks of maturation, > 95% of the hiPSC-derived astrocyte population is positive for typical astrocyte markers (S100β, AQP4, SOX9, and ALDH1L1) [24]. Importantly, the hiPSC-derived astrocytes retain their morphology, marker expression, and functionality, when cocultured with hiPSC-derived motor neurons [24].
Materials and reagents
Biological materials
Human induced pluripotent stem cells (hiPSCs) (generated in-house [5])
Reagents
Essential 8TM Flex medium kit (E8 Flex medium) (Thermo Fisher Scientific, Gibco, catalog number: A28583-01)
GeltrexTM Matrix (Geltrex) (Thermo Fisher Scientific, Gibco, catalog number: A1413301)
DMEM/F-12 +Lglut, +HEPES (DMEM/F-12) (Thermo Fisher Scientific, Gibco, catalog number: 11330032)
Penicillin-Streptomycin (Pen/Strep) (5,000 U/mL) (Thermo Fisher Scientific, Gibco, catalog number: 15070063)
RevitaCellTM supplement (100×) (Thermo Fisher Scientific, Gibco, catalog number: A2644501)
DPBS, no calcium, no magnesium (Thermo Fisher Scientific, Gibco, catalog number: 14190144)
Collagenase type IV powder (Thermo Fisher Scientific, Gibco, catalog number: 17104019)
SB431542 (Tocris Bioscience, catalog number: 1614; product format: 10 mM in ethanol)
LDN193189 (Stemgent, catalog number: 04-0074-02; product format: 10 mM in solution)
NeurobasalTM medium (Thermo Fisher Scientific, Gibco, catalog number: 21103049)
B-27TM supplement minus vitamin A (50×) (Thermo Fisher Scientific, Gibco, catalog number: 12587-010)
N-2 Supplement (100×) (Thermo Fisher Scientific, Gibco, catalog number: 17502048)
L-Glutamine (200 mM) (Thermo Fisher Scientific, Gibco, catalog number: 25030024)
Aqua ad iniectabilia (injectable water) (B. Braun, catalog number: 2351744)
Recombinant murine FGF-basic (FGF) (Peprotech, catalog number: 450-33; product format: 10 µg/mL in DPBS)
Recombinant human epidermal growth factor (EGF) (ProSpec, catalog number: CYT-217; product format: 100 µg/mL in injectable water)
Accutase® solution (Sigma-Aldrich, catalog number: A6964)
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco, catalog number: 10270106)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)
MEM non-essential amino acids solution (NEAA) (100×) (Thermo Fisher Scientific, Gibco, catalog number: 11140050)
L-Ascorbic acid (Sigma-Aldrich, catalog number: A4403; product format: 200 µM in injectable water)
Recombinant human IGF-1 (IGF) (Peprotech, catalog number: 100-11; product format: 100 µg/mL in injectable water)
Human Activin A recombinant protein (A) (Thermo Fisher Scientific, Gibco, catalog number: PHC9564; product format: 10 µg/mL in DPBS)
Recombinant human Heregulin β-1 (H) (Peprotech, catalog number: 100-03, product format: 250 µg/mL in injectable water)
Sodium pyruvate (100 mM) (Thermo Fisher Scientific, Gibco, catalog number: 11360070)
Trypan blue solution, 0.4% (Thermo Fisher Scientific, Gibco, catalog number: 15250061)
Ethanol absolute ≥ 99.8% (VWR, catalog number: 20821.296)
Isopropanol, 99.5% (Thermo Fisher Scientific, catalog number: 184130010)
Solutions
E8 Flex medium (see Recipes)
Geltrex coating (see Recipes)
Collagenase type IV (10× and 1× solutions) (see Recipes)
Neural induction medium (NIM) (see Recipes)
Neural maturation medium (NMM) (see Recipes)
Astrocyte differentiation medium (ADM) (see Recipes)
Astrocyte maturation medium (AMM) (see Recipes)
Recipes
E8 Flex medium
Thaw the frozen Essential 8 TM Flex supplement from the Essential 8TM Flex medium kit at room temperature for approximately 1 h or at 2–8 °C overnight. Protect the supplement from light, as it is light sensitive. Mix the thawed supplement by gently inverting the vial a couple of times and then aseptically transfer the entire contents of the Essential 8TM Flex supplement to the bottle of Essential 8 TM Flex basal medium. Swirl the bottle to mix. E8 Flex medium can be stored at 2–8 °C for up to two weeks.
Reagent Final concentration Volume
Essential 8TM Flex basal medium 98% 500 mL
Essential 8TM Flex supplement (50×) 2% 10 mL
Total 100% 510 mL
Geltrex coating
It is important to keep all components at ≤ 2–8 °C during the preparation of the Geltrex coating to prevent premature solidification. To ensure this, first prepare 24.75 mL of 2–8 °C DMEM/F-12 in a 50 mL conical tube and then collect the Geltrex aliquot from -20 °C. Transfer approximately 0.5 mL of 2–8 °C DMEM/F-12 from the prepared 50 mL conical tube to the Geltrex aliquot, pipette up and down to dissolve the frozen aliquot, and transfer approximately 0.75 mL of the solution back to the 50 mL conical tube. Repeat the process a few times to transfer the entire content of the Geltrex aliquot to the 50 mL conical tube. Mix well. Geltrex coating can be stored at 2–8 °C for one week.
Reagent Final concentration Volume
DMEM/F-12 99% 24.75 mL
Geltrex 1% 250 µL
Total 100% 25 mL
Collagenase type IV (10× and 1× solutions)
First, prepare a stock concentration (10×): Dilute 1 g of collagenase type IV powder in 100 mL of DMEM/F-12 and filter sterilize. Aliquot the 10× solution in 10 mL/aliquot. Next, prepare the working concentration (1×): Dilute 10 mL of the stock concentration (10×) with 90 mL of DMEM/F-12 to make a 1× solution and filter sterilize. Aliquot the 1× solution in 12.5 mL/aliquot. Both 10× and 1× aliquots can be stored at -20 °C for ≤ 6 months. Bring collagenase type IV (1×) to room-temperature before use.
Reagent Final concentration Quantity or Volume
DMEM/F-12 100% 100 mL
Collagenase type IV (powder) 10 mg/mL (10×) 1 g
DMEM/F-12 90% 90 mL
Collagenase type IV (10×) 1 mg/mL (1×) 10 mL
Neural induction medium (NIM): d0-d6
Prepare ~500 mL of bulk solution of E8 Flex medium and Pen/Strep and filter sterilize. E8 Flex + Pen/strep can be stored at 2–8 °C for two weeks. To prepare NIM, make an aliquot of the required volume for the day of E8 Flex + Pen/Strep solution and add SB431542 and LDN193189 fresh on the day of use. Filter sterilize and bring the NIM solution to room temperature before use.
Reagent Final concentration Volume
E8 Flex medium 100% 500 mL
Pen/Strep 1% 5 mL
SB431542 10 µM see note
LDN193189 0.1 µM see note
Neural maturation medium (NMM): d7-d15
Prepare > 200 mL of bulk solution of basic medium (DMEM/F12, neurobasal medium, Pen/Strep, B-27 minus vitamin A, N-2, and L-Glutamine) and filter sterilize. Basic medium can be stored at 2–8 °C for four weeks. To prepare NMM, make an aliquot of the required volume for the day of basic medium solution and add SB431542, LDN193189, FGF, and EGF fresh on the day of use. Filter sterilize and bring the NMM solution to 37 °C before use.
Reagent Final concentration Volume (for 200 mL)
DMEM/F-12 47.5% 95 mL
Neurobasal medium 47.5% 95 mL
Pen/Strep 1% 2 mL
B-27 minus vitamin A 2% 4 mL
N-2 1% 2 mL
L-Glutamine 1% 2 mL
SB431542 10 µM see note
LDN193189 0.1 µM see note
FGF 10 ng/mL see note
EGF 10 ng/mL see note
Astrocyte differentiation medium (ADM): d16-d25
Prepare >200 mL of bulk solution of basic medium (neurobasal medium, Pen/Strep, N-2, NEAA, and L-Ascorbic acid) and filter sterilize. Basic medium can be stored at 2–8 °C for four weeks. To prepare ADM, make an aliquot of the required volume for the day of basic medium solution and add FGF, IGF, A, and H fresh on the day of use. Filter sterilize and bring the ADM solution to 37 °C before use.
Reagent Final concentration Volume (for 200 mL)
Neurobasal medium 97% 194 mL
Pen/Strep 1% 2 mL
N-2 1% 2 mL
NEAA 1% 2 mL
L-Ascorbic acid (200 µM) 0.8 µM 800 µL
FGF 10 ng/mL see note
IGF 200 ng/mL see note
A 10 ng/mL see note
H 10 ng/mL see note
Astrocyte maturation medium (AMM): d25+
Prepare 500 mL of bulk solution of basic medium (DMEM/F12, neurobasal medium, Pen/Strep, N-2, NEAA, L-Ascorbic acid, L-Glutamine, sodium pyruvate, and FBS) and filter sterilize. Basic medium can be stored at 2–8 °C for four weeks. To prepare AMM, make an aliquot of the required volume for the day of basic medium solution and add IGF, A, and H fresh on the day of use. Filter sterilize and bring the AMM solution to 37 °C before use.
Reagent Final concentration Volume (for 500 mL)
DMEM/F-12 46.3% 231.5 mL
Neurobasal medium 46.3% 231.5 mL
Pen/Strep 1% 5 mL
N-2 1% 5 mL
NEAA 1% 5 mL
L-Ascorbic acid (200 µM) 0.8 µM 2 mL
L-Glutamine 1% 5 mL
Sodium pyruvate 1% 5 mL
FBS 2% 10 mL
IGF 200 ng/mL see note
A 10 ng/mL see note
H 10 ng/mL see note
Laboratory supplies
Cell culture flasks, 25 cm2, cell-repellent surface (T25 non-adherent flask) (Greiner Bio-One, catalog number: 690980)
Cell culture multi-well plates (6-well plate) (Greiner Bio-One, catalog number: 657160)
25 mL sterile reservoirs (Thermo Fisher Scientific, catalog number: 95128095) or 50 mL sterile reservoirs (InvitroLab, catalog number: IV-6002)
Cell scrapers (TH Geyer, catalog number: 7696760)
15 mL conical tubes (TH Geyer, catalog number: 7696714)
50 mL conical tubes (Greiner Bio-One, catalog number: 227261)
150 mL vacuum filtration devices, pore 0.22 µm (Jet Biofil, catalog number: FCF010004)
500 mL vacuum filtration devices, pore 0.22 µm (Jet Biofil, catalog number: FPE204500)
2 mL cryovials (Maxxline, catalog number: MLC2B)
5 mL serological pipettes (Greiner Bio-One, catalog number: 606180)
10 mL serological pipettes (Greiner Bio-One, catalog number: 607180)
25 mL serological pipettes (Greiner Bio-One, catalog number: 760160-TRI)
Sterile PES syringe filters (Thermo Fisher Scientific, catalog number: 15206869)
50 mL 3-part syringes (Chirana T. Injecta, catalog number: CH03050LL)
10 µL pipette tips (TH Geyer, catalog number: 7695881)
20 µL pipette tips (TH Geyer, catalog number: 7695882)
200 µL pipette tips (TH Geyer, catalog number: 7695884)
1,250 µL pipette tips (TH Geyer, catalog number: 7695887)
CountessTM cell counting chamber slides (Thermo Fisher Scientific, Invitrogen, catalog number: C10283)
Equipment
NordicSafe® Class II biological safety cabinet (ESCO, catalog number: NC2-L)
CellXpert® C170i CO2 incubator (Eppendorf, catalog number: 6734)
EVOSTM XL Core inverted microscope (objectives: 4×, 10×, 20×) (Thermo Fisher Scientific, catalog number: AMEX1000)
Laboratory centrifuge with rotors for 15 and 50 mL conical tubes (Biosan, catalog number: LMC-3000)
Water bath (37 °C) (Julabo, catalog number: TW8)
FinnpipetteTM F2 GLP pipetting kit 2 (Thermo Fisher Scientific, catalog number: 11835850)
Pipetboy acu 2 (Integra, catalog number: 1550179)
Mr. FrostyTM freezing container (Thermo Fisher Scientific, catalog number: 5100-0001)
CountessTM II FL automated cell counter (Thermo Fisher Scientific, catalog number: AMQAF1000)
Racks
Liquid nitrogen (N2) tank
Freezer (-20 °C)
Refrigerator (2–8 °C)
Procedure
Neural induction
Plate hiPSCs on Geltrex-coated 6-well plates (Table 1) in 2 mL/well of E8 Flex medium and expand according to standard protocol. See Recipes 1 and 2.
Note: The use of Geltrex can be replaced by Matrigel during the entire protocol.
After a minimum of seven days as iPSCs, cell lines are prepared for neural induction when reaching 70%–90% confluence (day 0).
Notes:
Use a full 6-well plate per cell line to start one differentiation.
To avoid excessive weekend work, start day 0 (d0) on a Monday.
Remove spent E8 Flex medium, wash cells once with 1 mL/well of DPBS, and incubate with 1 mL/well of room-temperature collagenase type IV (1×) for 10–20 min at 37 °C with 5% CO2 to dissociate the colonies. See Recipe 3.
Note: When ready, iPSC colonies will lift and curl up around the borders. Larger colonies might require longer incubation time. After 10 min incubation, check under light microscope every 5 min. Maximum collagenase type IV (1×) incubation time: 60 min.
After incubation, remove the spent collagenase and add 1 mL/well of room-temperature E8 Flex medium, gently scrape the loosened colonies with a cell scraper, and use a P1000 pipette to transfer the cell suspension to individual 15 mL conical tubes.
Notes:
Use one 15 mL conical tube per 6-well plate.
Avoid excess pipetting to sustain clumps of colonies.
If needed, use 1 mL/well of fresh E8 Flex medium to gently flush around the borders of the well to collect remaining cells.
Incubate the conical tube at 37 °C with 5% CO2 for 15 min to allow the clusters to sediment.
After incubation, remove supernatant.
Carefully dissolve cell pellet in 10 mL of room-temperature NIM (see Recipe 4) by pipetting up and down a few times and transfer the cell suspension to a T25 non-adherent flask.
Critical: Do not pipette up and down too much to sustain cell clumps.
Note: Mark as passage 0 (P0).
Check the cell density under a light microscope and incubate the flask at 37 °C with 5% CO2.
Perform a medium change with 10 mL/flask of room-temperature NIM on day 1 (d1), d2, and d4.
When changing medium, place the T25 non-adherent flask in an upright position in a 25 or 50 mL sterile reservoir in the incubator and allow cells to sediment for 5–10 min (Figure 1A). Carefully transfer the flask in its upright position into the biological safety cabinet and remove approximately 9 mL of spent medium to allow the cells to remain covered in a small volume of medium. Add 10 mL of fresh room-temperature NIM per flask.
Notes:
Three to four days after the start of induction, embryoid bodies (EBs) are clearly visible (Figure 1B). EBs appear irregular around their borders for the first few days but take on a rounder form during the induction phase.
A cell shaker is not required during neural induction. If the EBs begin to adhere to each other, perform a gentle pipetting during medium changes.
See Troubleshooting if EBs attach to the bottom of the flask.
Figure 1. Cell morphology during the astrocyte differentiation protocol. A. Image illustrating the use of a sterile reservoir for flask support to allow embryoid body (EB) sedimentation during medium changes. B. Brightfield images of EBs, cell morphologies, and optimal confluence during the astrocyte differentiation protocol. Scale bar: 200 µm. d+ refers to days of astrocyte maturation. The use of patient fibroblasts for the generation of hiPSCs was approved by the ethics committee of University Hospital Leuven (number S50354 and S63792).
Table 1. Differentiation step and assay overview
Differentiation step/assay Plate format Seeding density Optimal seeding time point Medium change volume Geltrex-coating volume Dissociation/fixation reagent volume
(e.g., accutase)
Start of differentiation 6-well plate 70%–90% confluent hiPSCs Day 0 (d0) 2 mL/well 1 mL/well 1 mL/well
Neural induction T25 non-adherent flask NA Day 0 (d0) 10 mL/flask NA NA
Neural maintenance 6-well plate NA Day 7 (d7) 2 mL/well 1 mL/well 1 mL/well
Neural expansion/thawing 6-well plate 70%–100% confluence See Procedure C and D 2 mL/well 1 mL/well 1 mL/well
Astrocyte differentiation 6-well plate 90%–100% confluence Day 16 (d16) 2 mL/well NA NA
Astrocyte maturation/expansion/thawing 6-well plate 70%–100% confluence See Procedure F–H 2 mL/well 1 mL/well 1 mL/well
Immunocytochemistry 24-well plate 30–50,000 cells/cm2 Two days before fixation 0.5 mL/well 0.5 mL/well 0.5 mL/well
Transmission electron microscopy 24-well plate 50,000 cells/cm2 >2 weeks before experiment 0.5 mL/well 0.5 mL/well 0.5 mL/well
Protein extraction 6-well plate 50,000 cells/cm2 >2 weeks before experiment 2 mL/well 1 mL/well 1 mL/well
RNA extraction 6-well plate 50,000 cells/cm2 >2 weeks before experiment 2 mL/well 1 mL/well 1 mL/well
Metabolic assays 12-well plate 32,000 cells/cm2 2–7 days before experiment 1 mL/well 0.75 mL/well 0.75 mL/well
Neural maintenance
On d7, plate EBs for neural rosette formation: prepare one full Geltrex-coated 6-well plate (Table 1) per T25 non-adherent flask and incubate at 37 °C with 5% CO2 for at least 30 min.
Note: Geltrex-coated plates can be prepared the day before and incubated at 37 °C with 5% CO2 overnight.
After 30 min, remove spent Geltrex and add 1 mL/well of NMM (see Recipe 5). Incubate the plates at 37 °C with 5% CO2 to be ready for use.
Transfer EBs in spent media to individual 15 mL conical tubes (one conical tube per T25 non-adherent flask) and incubate upright at 37 °C with 5% CO2 for 5–10 min to allow sedimentation of EBs.
After the incubation, aspirate spent medium and add 1 mL/well of 37 °C NMM to the conical tube without pipetting up and down.
Transfer 1 mL/well of EB suspension to the prepared Geltrex-coated 6-well plates with 1 mL/well of NMM so that each well contains 2 mL of NMM. Incubate the plate at 37 °C with 5% CO2.
Note: Make sure to evenly distribute the EBs among the different wells and finish by carefully rocking the plate to facilitate even EB dispersal.
Change medium daily with 37 °C NMM until d10.
Note: At d8–d9, small colonies, each with 1–10 neural rosettes containing NPCs, will be visible (Figure 1B).
On d10, neural rosettes are clearly visible and ready for passaging (P1) and NPC expansion.
Prepare one Geltrex-coated 6-well plate per 6-well plate with cells and incubate at 37 °C with 5% CO2 for at least 30 min.
Note: Geltrex-coated plates can be prepared the day before and incubated at 37 °C with 5% CO2 overnight.
After 30 min, remove spent Geltrex and add 1 mL/well of NMM + 5 µL/mL RevitaCellTM supplement. Incubate the plates at 37 °C with 5% CO2 to be ready for use.
Incubate neural rosettes with 1 mL/well of room-temperature accutase for 4 min at 37 °C with 5% CO2 to detach the neural rosettes containing NPCs.
After the incubation, add 1 mL/well of 37 °C NMM to inactivate the accutase.
Gently scrape the cells with a cell scraper and transfer cell suspension to a 15 mL conical tube.
Note: As it is not recommended to centrifuge with volumes below 3 mL for 15 mL conical tubes, increase the volume above 3 mL by adding more NMM if needed.
Centrifuge at 145× g for 4 min at room temperature.
Remove supernatant, add 1 mL/well of 37 °C NMM + 5 µL/mL RevitaCellTM supplement, and carefully pipette up and down to dissolve the cell pellet.
Transfer 1 mL/well of cell suspension to the prepared Geltrex-coated 6-well plates with 1 mL/well of NMM + 5 µL/mL RevitaCellTM supplement so that each well contains 2 mL of NMM + 5 µL/mL RevitaCellTM supplement.
Note: To evenly distribute the NPCs, carefully rock the plate from side to side.
Examine cell distribution under the light microscope and incubate the plate at 37 °C with 5% CO2.
Perform medium change with 2–6 mL/well of 37 °C NMM on d11, followed by every second day until day 16.
Note: NPCs are usually 100% confluent on d11 (Friday), but do not require passaging. Weekend work can be avoided by giving 4–6 mL/well of 37 °C NMM on d11 (Friday), followed by passaging (P2) and cryopreservation on d14 (Monday). Standard medium changes are 2 mL/well every second day.
Pause point: On d12–d14, NPCs are passaged (P2) and/or cryopreserved one time. If desired, the protocol can be paused and restarted at this time point in the differentiation. Always change the medium the day after passaging/thawing NPCs.
Critical: Make sure the NPCs are 90%–100% confluent when changing to ADM (see Recipe 6) on d16 (Figure 1B). ADM is harsh on the cells and causes extensive cell death. The NPCs require cell–cell contact to survive day 16–25 (d16–d25/d+0) of the differentiation protocol.
NPC expansion
On d12–d14, passage NPCs 1:3–1:6 once to enhance cell expansion (P2).
Note: Passage ratio depends on cell confluence and growth rate, as it is important to have 90%–100% confluent cells by d16. Of a full 6-well plate, authors recommend passaging one well 1:3–1:6 and cryopreserving the remaining five wells (one cryovial/well).
Prepare Geltrex-coated 6-well plates (Table 1) and incubate at 37 °C with 5% CO2 for at least 30 min.
Note: Geltrex-coated plates can be prepared the day before and incubated at 37 °C with 5% CO2 overnight.
After 30 min, remove spent Geltrex and add 1 mL/well of NMM + 5 µL/mL RevitaCellTM supplement. Incubate the plates at 37 °C with 5% CO2 to be ready for use.
Remove spent NMM, wash cells once with DPBS, and incubate with 1 mL/well of room-temperature accutase for 4 min at 37 °C with 5% CO2.
After the incubation, gently tap the side of the plate to check if cells readily detach.
Note: If cells do not detach easily, incubate for one more minute and repeat step C5.
Add 1 mL/well of 37 °C NMM to inactivate the accutase.
Gently flush along the sides of the well to detach remaining NPCs and transfer cell suspension to a 15 mL conical tube.
Notes:
If not all cells detach after 5 min incubation with accutase, use a cell scraper to collect remaining cells.
As it is not recommended to centrifuge with volumes below 3 mL for 15 mL conical tubes, increase the volume above 3 mL by adding more NMM if needed.
Centrifuge at 145× g for 4 min at room temperature.
Remove supernatant and continue with step C10 for passaging or step C11 for cryopreservation.
For passaging:
Add 1 mL/well of 37 °C NMM + 5 µL/mL RevitaCellTM supplement and carefully pipette up and down to dissolve the cell pellet.
Transfer 1 mL/well of cell suspension to the prepared Geltrex-coated 6-well plates with 1 mL/well of NMM + 5 µL/mL RevitaCellTM supplement, so that each well contains 2 mL of NMM + 5 µL/mL RevitaCellTM supplement.
To evenly distribute the NPCs, carefully rock the plate from side to side.
Examine cell distribution under the light microscope and incubate the plate at 37 °C with 5% CO2.
The following day, perform a medium change with 2 mL/well of 37 °C NMM followed by every second day until d16.
For cryopreservation:
Add 1 mL/vial of room-temperature freezing medium (90% FBS and 10% DMSO) and carefully pipette up and down to dissolve the cell pellet.
Transfer 1 mL of cell suspension to prelabeled cryovials, transfer vials to a Mr. Frosty with isopropanol, and incubate at -80 °C overnight.
The following day, transfer vials to liquid N2 storage for long-term cryopreservation.
Thawing NPCs
Cryopreserved NPCs (d12–d14) should be thawed and continued on the same day of the differentiation protocol.
Note: For example, cryopreserved d14 NPCs are thawed and continued on day 14 of the protocol.
Prepare Geltrex-coated 6-well plates (Table 1) (one well/cryovial) and incubate at 37 °C with 5% CO2 for at least 30 min.
Note: Geltrex-coated plates can be prepared the day before and incubated at 37 °C with 5% CO2 overnight.
Per cryovial, prepare 15 mL conical tubes with 9 mL of 37 °C basic NMM (without growth factors).
After 30 min, remove spent Geltrex and add 1 mL/well of NMM (with growth factors) + 5 µL/mL RevitaCellTM supplement. Incubate the plates at 37 °C with 5% CO2 to be ready for use.
Remove the cryovial from liquid N2 storage and thaw for approximately 1 min in a 37 °C water bath until a small pea-sized ice clump is left.
Transfer ~0.5 mL of 37 °C basic NMM (without growth factors) from the prepared 15 mL conical tube to the cryovial, pipette up and down to dissolve remaining ice, and transfer ~1 mL of cell suspension back to the 15 mL conical tube. Repeat the process a few times to transfer the entire content of the cryovial to the 15 mL conical tube.
Centrifuge at 145× g for 4 min at room temperature.
Remove supernatant and add 1 mL/well of 37 °C NMM (with growth factors) + 5 µL/mL RevitaCellTM supplement and carefully pipette up and down to dissolve the cell pellet.
Transfer 1 mL/well of cell suspension to the prepared Geltrex-coated plates with 1 mL/well of NMM (with growth factors) + 5 µL/mL RevitaCellTM supplement, so that each well contains 2 mL of NMM (with growth factors) + 5 µL/mL RevitaCellTM supplement. To evenly distribute the NPCs, carefully rock the plate from side to side.
Examine cell distribution under the light microscope and incubate the plate at 37 °C with 5% CO2.
Perform a medium change with 2 mL/well of 37 °C NMM (with growth factors) the next day, followed by every second day until d16.
Astrocyte differentiation
On d16, change medium to 2 mL/well of 37 °C ADM (see Recipe 6) to commence the conversion of NPCs to astrocyte progenitor cells (APCs) and induce astrocyte differentiation (Figure 1B).
Change the ADM every second day until day 25 (d25/d+0), when a glial switch to astrocytes is expected to occur.
Critical: Do not passage cells during this period to ensure a sustained confluent layer of cells.
Notes:
Medium changes can be avoided in the weekend if giving 4–6 mL/well of 37 °C ADM on a Friday.
Potential intracellular vacuolization and/or increased cell death are expected during this period.
See Troubleshooting if complete cell death occurs during d16–d25.
Astrocyte maturation
On d25, astrocyte maturation is commenced (d+0). Several options are available at this time point:
Pause point: On day 25 (d25/d+0), astrocytes can be cryopreserved. If desired, the protocol can be paused and restarted at this time point. To commence, follow Procedure G.
If large cell samples are desired (such as for protein and RNA extraction), passage and plate some of the cells in specified cell densities (Table 1) in AMM (see Recipe 7) to allow maturation of astrocytes for 4 weeks. Astrocytes are not passaged during this period and therefore become very confluent (~3D). Adjust AMM volume accordingly: 4–6 mL/well with every medium change (Monday, Wednesday, and Friday). Remaining d25/d+0 astrocytes can be plated for expansion and/or cryopreserved. To commence, follow Procedure G.
Create an astrocyte cell bank: Passage d25/d+0 astrocytes 1:6 for maturation and expansion. Every 4–7 days, passage astrocytes 1:6 over the time course of 14 days. After two weeks (d+14), cryopreserve astrocytes for future experiments (1–3 cryovials per confluent well of a 6-well plate). The protocol can be paused and restarted any day during the maturation. For future experiments, thaw d+14 astrocytes, expand for one week, and plate according to recommended cell densities (Table 1). Change medium every second day. To commence, follow Procedure G.
Notes:
i. For immunocytochemistry experiments, plate cells two days before fixation to avoid excessive cell proliferation. Change medium the day after plating.
ii. Authors have observed astrocyte proliferation until week 6 of maturation.
iii. Astrocyte markers increase over the time course of 3–4 weeks of maturation. Approximately 95% of astrocytes at positive for astrocyte markers (SOX9, S100β, ALDH1L1, and AQP4) at week 4 of maturation (d+28). See Figure 2.
Figure 2. Astrocyte maturation verification with immunocytochemistry. A. Representative confocal images of human induced pluripotent stem cell (hiPSC)-derived astrocytes at week 1–4 of maturation stained with astrocyte-specific markers S100β, AQP4, ALDH1L1, GFAP, SOX9, and neuronal marker MAP2. Nuclei stained with DAPI (blue). Scale bar: 75 μm. B. Quantification of the number of antibody-positive cells during week 1–4 of maturation. Mean ± S.E.M. of three biological replicates (n = 15 images). The figure is modified from Stoklund Dittlau et al. [24] with permission under the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).
Passaging and cryopreservation of astrocytes
Prepare Geltrex-coated plates (for recommended plate format see Table 1) and incubate at 37 °C with 5% CO2 for at least 30 min.
Note: Geltrex-coated plates can be prepared the day before and incubated at 37 °C with 5% CO2 overnight.
After 30 min, remove spent Geltrex and add half of the volume of AMM + 5 µL/mL RevitaCellTM supplement (for required volumes see Table 1). Incubate the plates at 37 °C with 5% CO2 to be ready for use.
Remove spent ADM/AMM, wash cells once with DPBS, and incubate with room-temperature accutase (for required volumes see Table 1) for 4 min at 37 °C with 5% CO2.
After the incubation, gently tap the side of the plate to check if cells readily detach.
Note: If cells do not detach easily, incubate for one more min and repeat step G4.
Add 1:1 volume of 37 °C AMM to inactivate the accutase.
Gently flush along the sides of the well to detach remaining astrocytes and transfer cell suspension to a 15 mL conical tube.
Notes:
If not all cells detach after 5 min incubation with accutase, use a cell scraper to collect remaining cells.
As it is not recommended to centrifuge with volumes below 3 mL for 15 mL conical tubes, increase the volume above 3 mL by adding more AMM if needed.
Centrifuge at 145× g for 4 min at room temperature.
Remove supernatant.
For passaging:
Add the remaining half volume of 37 °C AMM + 5 µL/mL RevitaCellTM supplement (for required volumes see Table 1) and carefully pipette up and down to dissolve the cell pellet.
Transfer cell suspension to the prepared Geltrex-coated plates with AMM + 5 µL/mL RevitaCellTM supplement.
To evenly distribute the astrocytes, carefully rock the plate from side to side.
Examine cell distribution under the light microscope and incubate the plate at 37 °C with 5% CO2.
Perform medium change with 37 °C AMM the next day followed by every second day.
For cryopreservation:
Add 1 mL/vial of room-temperature freezing medium (90% FBS and 10% DMSO) and carefully pipette up and down to dissolve the cell pellet.
Transfer 1 mL of cell suspension to prelabeled cryovials, transfer vials to a Mr. Frosty with isopropanol, and incubate at -80 °C overnight.
The following day, transfer vials to liquid N2 storage for long-term cryopreservation.
Thawing astrocytes
Critical: When thawing astrocytes, allow at least one week of recovering, passaging, and expansion in 6-well plates before plating for experiments.
Note: Authors recommend to passage cells 1–2 times every 3–4 days before plating for experiments. Passaging enhances viability and culture purity.
Prepare Geltrex-coated 6-well plates (Table 1) (one well/cryovial) and incubate at 37 °C with 5% CO2 for at least 30 min.
Note: Geltrex-coated plates can be prepared the day before and incubated at 37 °C with 5% CO2 overnight.
Per cryovial, prepare 15 mL conical tubes with 9 mL of 37 °C basic AMM (without growth factors).
After 30 min, remove spent Geltrex and add 1 mL/well of AMM (with growth factors) + 5 µL/mL RevitaCellTM supplement. Incubate the plates at 37 °C with 5% CO2 to be ready for use.
Remove cryovial from liquid N2 storage and thaw for approximately 1 min in a 37 °C water bath until a small pea-sized ice clump is left.
Transfer ~0.5 mL of 37 °C basic AMM (without growth factors) from the prepared 15 mL conical tube to the cryovial, pipette up and down to dissolve remaining ice, and transfer ~1 mL of cell suspension back to the 15 mL conical tube. Repeat the process a few times to transfer the entire content of the cryovial to the 15 mL conical tube.
Centrifuge at 145× g for 4 min at room temperature.
Remove supernatant and add 1 mL/well of 37 °C AMM (with growth factors) + 5 µL/mL RevitaCellTM supplement and carefully pipette up and down to dissolve cell pellet.
Transfer 1 mL/well of cell suspension to the prepared Geltrex-coated plates with 1 mL/well of AMM (with growth factors) + 5 µL/mL RevitaCellTM supplement, so each well contains 2 mL of AMM (with growth factors) + 5 µL/mL RevitaCellTM supplement.
Note: To evenly distribute the astrocytes, carefully rock the plate from side to side.
Examine cell distribution under the light microscope and incubate the plate at 37 °C with 5% CO2.
Perform medium change with 2 mL/well of 37 °C AMM (with growth factors) the next day followed by every second day.
Validation of protocol
This protocol has been used and validated in the following research article:
Stoklund Dittlau et al. [24]. FUS-ALS hiPSC-derived astrocytes impair human motor units through both gain-of-toxicity and loss-of-support mechanisms. Molecular Neurodegeneration (Figures 1, 2, 4–7, Supplemental figures 1–4 and 6–8, and Additional files 3–6).
The protocol is an optimized version of our previous hiPSC-derived astrocyte protocol, which was used and validated in the following research article:
Chandrasekaran et al. [25] Astrocyte reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3. Stem Cell Reports (Figures 1–5 and Supplemental figures 1–5).
General notes and troubleshooting
General notes
Weekend work can be avoided by increasing medium volumes during medium changes on Fridays (4–6 mL/well for 6-well plates, 2–3 mL/well for 12-well plates, and 1 mL/well for 24-well plates). No addition of medium is required for flasks.
Always change the medium the day after passaging and thawing cells.
Increase passage number upon passaging, cryopreserving, and thawing.
Troubleshooting
Problem 1: EBs attach to the bottom of the T25 low-attachment flask around d4–d7.
Possible cause: EBs might be slightly too large or the T25 low-attachment flask might have a flaw in its surface treatment.
Solution: Transfer EBs in fresh NIM to a new T25 low-attachment flask. When transferring, pipette up and down 2–3 times to decrease the size of the EBs.
Problem 2: Complete cell death during d16–d25.
Possible cause: Cell density at d15 is too low.
Solution: Aim for >90% cell confluence at d15.
Acknowledgments
The authors would like to thank the VIB, KU Leuven, the Agency for Innovation by Science and Technology, the “Fund for Scientific Research Flanders” (FWO-Vlaanderen), Target ALS, the ALS Liga België, the Belgian Government (Interuniversity Attraction Poles Program P7/16 initiated by the Belgian Federal Science Policy Office), the Thierry Latran Foundation and the “Association Belge contre les Maladies neuro-Musculaires” (ABMM). The graphical abstract was created with Biorender.com. This protocol is adapted from Stoklund Dittlau et al. [24], which has been modified from Chandrasekaran et al. [25] and Shaltouki et al. [23].
Competing interests
The authors declare that they have no competing interests.
Ethical considerations
Written informed consent was obtained from all subjects who provided tissue samples. The use of patient fibroblasts for the generation of hiPSCs was approved by the ethics committee of University Hospital Leuven (number S50354 and S63792).
References
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Stoklund Dittlau, K. and Van Den Bosch, L. (2023). Why should we care about astrocytes in a motor neuron disease?. Front. Mol. Med. 3: 1047540. https://doi.org/10.3389/fmmed.2023.1047540
Petrov, D., Mansfield, C., Moussy, A. and Hermine, O. (2017). ALS Clinical Trials Review: 20 Years of Failure. Are We Any Closer to Registering a New Treatment? Front. Aging Neurosci. 9: 68. https://doi.org/10.3389/fnagi.2017.00068
Oberheim, N. A., Takano, T., Han, X., He, W., Lin, J. H. C., Wang, F., Xu, Q., Wyatt, J. D., Pilcher, W., Ojemann, J. G., et al. (2009). Uniquely Hominid Features of Adult Human Astrocytes. J. Neurosci. 29(10): 3276–3287. https://doi.org/10.1523/jneurosci.4707-08.2009
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Bushong, E. A., Martone, M. E., Jones, Y. Z. and Ellisman, M. H. (2002). Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains. J. Neurosci. 22(1): 183–192. https://doi.org/10.1523/jneurosci.22-01-00183.2002
Birger, A., Ben-Dor, I., Ottolenghi, M., Turetsky, T., Gil, Y., Sweetat, S., Perez, L., Belzer, V., Casden, N., Steiner, D., et al. (2019). Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity. eBioMedicine 50: 274–289. https://doi.org/10.1016/j.ebiom.2019.11.026
Hedegaard, A., Monzón-Sandoval, J., Newey, S. E., Whiteley, E. S., Webber, C. and Akerman, C. J. (2020). Pro-maturational Effects of Human iPSC-Derived Cortical Astrocytes upon iPSC-Derived Cortical Neurons. Stem Cell Rep. 15(1): 38–51. https://doi.org/10.1016/j.stemcr.2020.05.003
Mulica, P., Venegas, C., Landoulsi, Z., Badanjak, K., Delcambre, S., Tziortziou, M., Hezzaz, S., Ghelfi, J., Smajic, S., Schwamborn, J., et al. (2023). Comparison of two protocols for the generation of iPSC-derived human astrocytes. Biol. Proced. Online 25(1): 26. https://doi.org/10.1186/s12575-023-00218-x
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A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson’s Disease
RC Rahul Chaurasia *
MA Mohamad Ayajuddin *
GR Girish S. Ratnaparkhi
SL Shashidhara S. Lingadahalli
SY Sarat C. Yenisetti
(*contributed equally to this work)
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4937 Views: 713
Reviewed by: Alessandro DidonnaSara Bagnoli Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Frontiers in Neuroscience Jun 2023
Abstract
Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories.
Key features
• Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body.
• Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.
Graphical overview
Keywords: Drosophila Dopaminergic neurodegeneration Fluorescence intensity Tyrosine hydroxylase Tissue imaging Fluorescence microscope
Background
Degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta and noradrenergic neurons in the locus coeruleus region of the human brain is the characteristic pathological feature of Parkinson’s disease (PD) subjects. Tyrosine hydroxylase (TH) is the marker protein for DAergic neurons, as it is the rate-limiting enzyme in the synthesis of dopamine. Hence, demonstration of the DAergic degeneration phenotype using TH immunostaining is critical in the development and validation of animal models of PD. For the first time, in an α-synuclein Drosophila model of PD, Feany and Bender [1] demonstrated a progressive, age-dependent locomotor dysfunction similar to the PD subjects accompanied by a loss of DAergic neurons. Since then, many labs have been using the fly to model PD [2–13].
One prime feature of Drosophila PD models is the DAergic cell loss, as claimed by several independent labs [1,2,6,12,14–19]; however, some other labs found no loss in neuronal numbers [7,9,20–23]. Further neurotoxins such as paraquat and rotenone have been used to develop the Drosophila model of PD. In most cases, cluster-specific loss of DAergic neurons [6,24–27] or no variation in the number of DAergic neurons [3,7,13,21,22,28] were observed. TH immunostaining and green fluorescent protein (GFP) reporter-based methods have typically been adopted to quantify DAergic neurons in the whole Drosophila brain. The reduction in fluorescence intensity (FI) of TH immunostaining or GFP signal has been termed neuronal dysfunction [7].
Here, we describe a sensitive fluorescence microscopy–based assay developed in our laboratory [28], less expensive and more user friendly than cumbersome confocal microscopy, to characterize DAergic neuronal dysfunction even in the absence of loss of neuronal cell bodies. Through this assay, it is feasible to characterize the early DAergic neurodegeneration, which will be of great support in following the progression of the disease; the same can be employed to understand the neuroprotective efficacy of small molecules/nutraceuticals/drugs. With appropriate modifications, this method can also be used for identifying other cell type–specific neurodegeneration in fly models of different neurodegenerative disease(s).
Materials and reagents
Drosophila melanogaster [Oregon K (OK) flies, procured from the National Drosophila Stock Center, Mysore University, Mysuru, India, were raised in food media containing sucrose, yeast, agar-agar, and propionic acid, and maintained in a fly environment chamber and used in the current study [3,10,13,28]]. Depending on the nature of the experiment, flies with and/or without neurotoxicant treatment (sporadic PD model) and transgenic fly model(s) can be employed.
Sterilized 1.5 mL centrifuge tubes (Tarsons, catalog number: 500010)
ParafilmTM wrapping film (Bemis, catalog number: PM996)
Conical flask (Borosil, catalog number: 5100)
Magnetic stirrer bar #8 mm × 40 mm (Tarsons, catalog number: 4113)
SPINNOTTM digital magnetic stirrer hotplate (Tarsons, catalog number: 6090)
Sterilized micro tips (Tarsons, catalog number: 521010)
Freshwrapp aluminum foil 9–11 μm (Hindalco, catalog number: HV2241)
Glass plate (Suwimut, catalog number: B08FRB2NTM)
Fingernail polish (FacesCanada, catalog number: CC4403)
Glass spacer (Borosil, catalog number: 9115S01)
Microscopy slides #76 mm × 26 mm (ReliGlas, catalog number: 7101)
Gold-seal coverslips (22 mm2) (Electron Microscopy Sciences, catalog number: 63765-01)
Sucrose (SRL, catalog number: 84973)
Agar-agar (HiMedia, catalog number: GRM666)
Sugar-tolerant yeast (Angel, catalog number: 5331A201908)
Propionic acid (Merck, catalog number: 80060505001730)
WhatmanTM filter paper (GE Healthcare, catalog number: 1001917)
Paraformaldehyde (PFA) pH 7.4 (Sigma-Aldrich, catalog number: I58127)
Phosphate buffered saline (PBS) pH 7.4 (HiMedia, catalog number: ML023)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
Normal goat serum (NGS) (Vector Labs, catalog number: S1000)
Rabbit anti-tyrosine hydroxylase (anti-TH) polyclonal primary ab (Millipore, catalog number: Ab152)
Goat anti-rabbit IgG H&L (TRITC-labeled) polyclonal secondary ab (Abcam, catalog number: Ab6718)
VECTASHIELD® mounting medium (Vector Labs, catalog number: H1000)
Solutions
4% PFA solution (50 mL) (see Recipes)
0.1% PBST (phosphate buffered saline and Triton X-100) (50 mL) (see Recipes)
0.5% PBST (50 mL) (see Recipes)
5% NGS blocking buffer solution (1 mL) (see Recipes)
Anti-TH polyclonal primary ab solution (see Recipes)
TRITC-labeled polyclonal secondary ab solution (see Recipes)
Recipes
4% PFA solution (50 mL)
PFA 2 g
1× PBS 50 mL
Add PFA in 1× PBS in a conical flask, cover it with parafilm, and shake it thoroughly for 10 min.
Transfer the flask with a magnetic stirrer on the hotplate for heating/boiling with a temperature ranging from 80 °C to 110 °C with moderate stirring at 150 rpm.
Keep the flask on the hotplate until the cloudy solution becomes transparent.
After this, switch off the hotplate but keep the stirring for 15 min. Allow the solution to cool down, aliquot it in a 1.5 mL centrifuge tube, and store it at -80 °C.
Critical: Do not store the solution for more than a week.
Caution: PFA is a potential carcinogen; hence, the whole process should be done under a fume hood. Wear hand gloves and a lab coat during handling and preparation of PFA solution.
0.1% PBST (phosphate buffered saline and Triton X-100) (50 mL)
10× PBS 5 mL
Autoclaved enzyme-free water 45 mL
Triton X-100 50 μL
Add 5 mL of 10× PBS in 45 mL of autoclaved enzyme-free water.
Mix 50 μL of Triton X-100 and vortex it for 10 seconds. The solution can be stored at room temperature for one week.
0.5% PBST (50 mL)
10× PBS 5 mL
Autoclaved enzyme-free water 45 mL
Triton X-100 250 μL
Add 5 mL of 10× PBS in 45 mL of autoclaved enzyme-free water.
Mix 250 μL of Triton X-100 and vortex it for 10 s. The solution can be stored at room temperature for one week.
5% NGS blocking buffer solution (1 mL)
NGS 50 μL
0.5% PBST 950 μL
Add 50 μL of NGS in 950 μL of 0.5% PBST and mix it properly by vortexing for 10 s. The solution can be stored at room temperature for 1–2 h.
Anti-TH polyclonal primary ab solution
Anti-TH polyclonal primary ab 5 μL
5% NGS blocking buffer 1,245 μL
Take 1,245 μL of 5% NGS blocking buffer and add 5 μL of anti-TH polyclonal primary ab (1:250 dilution). Mix it gently by inverting the tube slowly and place it on the ice until used.
TRITC-labeled polyclonal secondary ab solution
TRITC-labeled polyclonal secondary ab 5 μL
5% NGS blocking buffer 1,245 μL
Take 1,245 μL of 5% NGS and add 5 μL of TRITC-labeled polyclonal secondary ab (1:250 dilution). Mix it gently by inverting the tube slowly and store it on ice until used.
Equipment
Fly head capsule handling items e.g., needles #31 G × 6 mm (Tentabe BD, catalog number: 324902)
Dissecting fine forceps (EMS, catalog number: 78620-4B)
Brush (TEYUP, model number: SR-1013)
Delicate task Kim wipers (KIMTECHTM, catalog number: 370080)
Micropipette i.e., 1,000 μL, 50 μL, 10 μL, 2 μL (Gilson, catalog number: 30040)
Frost-free refrigerator (Whirlpool, model number: FF26 4S)
pH/mV meter (Hanna Instruments, model: HI2211-02)
-20 °C ES Series refrigerator (Thermo Scientific, model: 50616100444443250)
-80 °C ultra-low temperature freezer (New Brunswick Innova, model: U101-86)
Stereo zoom microscope (Carl Zeiss, model: Stemi 305)
Stereo zoom microscope (Leica, model: E24)
Fume hood (BIOMATRIX, Telangana, India)
BOD incubator (Percival, model: DR-36VL)
Test tube rotator (Tarsons, Rotospin, catalog number: 3070) and disk for 24 × 1.5 mL tube (Tarsons, catalog number: 3071)
Axio Imager M2 fluorescence microscope fitted with 100 W Mercury lamp (Carl Zeiss, catalog number: 430004-9902-000)
AxioCam ICm1 monochromatic camera (Carl Zeiss, catalog number: 426553-9901-000)
Software and datasets
ZEN 2012 SP2 blue edition, version 2.0.14283.302 (Carl Zeiss, Jena, Germany)
Microsoft Office Excel Worksheet 2007 (Microsoft Inc., WA, USA)
GraphPad Prism, March 7, 2007, version 5.00 (GraphPad Inc., MA, USA)
Procedure
Anti-TH immunostaining of the whole Drosophila brain
Fix the fly whole head tissue in 1 mL of 4% PFA (pH 7.4) containing 0.5% Triton X-100 in a sterilized 1.5 mL centrifuge tube for 2 h through mixing by using a test tube rotator with constant velocity (10 rpm) at room temperature (RT).
Remove PFA after 2 h of fixation by washing the fly head tissue with 1 mL of 0.1% PBST three times for 15 min each at RT.
Carry out dissection of brains in 1× PBS (pH 7.4) under a stereo zoom microscope at RT using fine forceps and needles to remove the head capsule and connecting tissues.
Then, wash the brains with 0.1% PBST five times for 15 min each at RT.
Block the brains with 5% NGS blocking buffer solution for 120 min at RT.
Then, incubate/probe brains with anti-TH polyclonal primary ab solution for 72 h at 4 °C through mixing by using a test tube rotator at constant velocity (10 rpm).
Wash off excess primary antibodies by 0.1% PBST five times for 15 min each at RT.
Incubate brains with TRITC-labeled polyclonal secondary ab solution for 24 h in the dark (Critical: Cover centrifuge tube containing brains with aluminum foil) by thorough mixing with a test tube rotator at a constant velocity (10 rpm) at RT.
To eliminate excess secondary antibodies, wash brains again with 0.1% PBST five times for 15 min each at RT.
Mount brains in VECTASHIELD® mounting medium and then top with cover glass.
Critical: Glass spacers were placed around the VECTASHIELD® mounting medium to protect brains from being crushed by a coverslip.
Critical: Brains were scanned in a dorsoventral orientation.
Use clear fingernail polish to seal the edges.
The sample is ready for image acquisition.
Image acquisition
Steps for the acquisition of Drosophila brain image(s) for quantification of DAergic neurons and FI using a fluorescence microscope with ZEN 2012 SP2 software are as follows:
Observe stained brains under a fluorescence microscope equipped with a 40× objective lens (Figure 1).
Figure 1. Scanning of the whole brain of Drosophila. Scan the anti-TH immunostained Drosophila brain using Carl Zeiss, Axio Imager M2 (40× objective lens) with ZEN 2012 SP2 software that interactively controls image acquisition, image processing, and analysis of the images.
Scan and take images using a monochromatic camera with a Rhodamine fluorescence filter (Figure 2).
Figure 2. Image acquisition and performing the red dot test. For image acquisition, select a monochromatic camera with a Rhodamine filter. Perform a red dot test for visibility of dopaminergic (DAergic) neurons and assessing saturation using a brain, reusing the same exposure time for other samples.
Perform a red dot test in the acquisition panel (select range indicator from Dimensions and set exposure from Acquisition parameter) for visibility of DAergic neurons and to assess the signal saturation during the image acquisition. Reuse the same exposure time for all brain samples (Figure 2).
Then, perform Z-stack programming with constant intervals of 1.08 μm for each image (Figure 3).
Figure 3. Selection of images and Z-Stacking
To generate a 2D image, on the method column apply Ortho and Maximum intensity projection (MIP) from Ortho display with X–Y Plane (Figure 4).
Figure 4. Creation of 2D image. For creating a 2D merged image, on the Method column, select Maximum intensity projection (MIP) with X-Y Plane.
Export the 2D image of the brain in .jpg format for presentation (Figure 5).
Figure 5. Export of 2D brain image to the required format
Method for quantification of DAergic neurons
Identify clusters from the images/scans obtained through Z-stack programming with constant intervals (Figure 6).
Enlarge the image to reveal the cell body/structure (Figure 6).
Figure 6. Quantification of dopaminergic (DAergic) neuronal number and fluorescence intensity (FI). For the quantification of DAergic neuronal number and FI, select 3D images/scans of Z-Stack with brain regions; PAL, PPL1, PPL2, PPM1/2, and PPM3 (PAL: Protocerebral anterior lateral; PPL: Protocerebral posterior lateral; PPM: Protocerebral posterior medial).
Determine/count the number of DAergic neurons in each cluster in an unbiased manner.
Method for characterization of FI of DAergic neurons
From 3D scan images, chose the PAL, PPL1, PPL2, PPM1/2, PPM3, and VUM (quantifiable DAergic neuronal clusters) regions of the fly brains (Figure 6).
Enlarge brain images to see the clear neurites (Figure 7).
Figure 7. Details of the quantification of the fluorescence intensity (FI). Enlarge the images to see clear neurites, select appropriate tools, draw spline contour from graphics and draw a line around the neuron, and display intensity mean value and area.
Select the appropriate graphics tool, draw spline contour, and draw a line to encircle the neuron giving intensity mean and area (Figure 7).
Select More measurement options and chose intensity sum by right-clicking inside the neuron (Figure 8).
Figure 8. Measurement of fluorescence intensity (FI) sum. Select intensity sum by opting for more measurement options (software provides the pixel value upon right-clicking on the neuron).
From the Measurement tab on the left side of the panel, select List, All views and Create document (Figure 9).
Figure 9. Fluorescence intensity (FI) compilation. From the measurement option select list, All views, and create document.
Record the area and FI sum for each image of a neuron in .xml format (Figure 10).
Figure 10. Measuring the FI sum for each scan of a neuron in .xml format
For quantification of FI of a single neuron, we considered a total of 11 scans with an interval of 1.08 μm for each scan (cumulative of 11.88 μm width) (Figure 11).
Figure 11. Compilation of fluorescence intensity (FI) of a single neuron and all the neurons of a cluster. For the characterization of FI of a single neuron, a total of 11 scans with an interval of 1.08 μm for each scan (cumulative 11.88 μm width) were considered. Take the average and find the standard error. Follow the same method/step(s) for all the dopaminergic (DAergic) neurons. The intensity sum of all the neurons in a specific cluster gives the total FI of that particular region (cluster-wise). The total FI is the sum of the FI of all the neurons belonging to all the DAergic neuronal clusters.
The intensity sum of all the neurons in a cluster gives the total FI of that particular region (cluster-wise).
Total FI is the sum of the FIs of all the neurons belonging to all the DAergic neuronal clusters.
Precautions and recommendations
Head tissue fixation is critical and should be properly done [use the test tube rotator with constant velocity (10 rpm) for fixing the head tissue gently and thoroughly]. If any issue arises with the working of antibodies (primary ab), the worker should first check if fixation was done thoroughly and properly before investing time in figuring out the efficiency of the antibody.
Incubation with primary and secondary antibodies should be done using a test tube rotator with constant velocity (10 rpm).
Critical: To prevent brains from being crushed, glass spacers should be used while mounting the brains with a coverslip.
Coverslip edges should be thoroughly sealed with nail polish to avoid drying up of the brains.
Image acquisition should be done on the same day to avoid bleaching.
Care should be taken to capture images from the samples with the same orientation.
The red dot test should be carried out carefully.
The same setting should be reused to capture the images/scans of different samples.
Care should be taken while performing Z-stack programming so that no neuron is left out of scanning.
Data analysis
Analysis of Z-stack scans/images for quantification of DAergic neurons and characterization of FI in Drosophila brain
The DAergic neurons were determined/counted from the Z-stack images/scans. The number of DAergic neurons in each cluster was counted in an unbiased manner. A minimum of 10 brains were analyzed for DAergic neuronal number quantification. A merged fly brain 2D image was used for presentation (Figure 12).
Figure 12. Demonstration of dopaminergic (DAergic) neuronal clusters in the whole brain of a Drosophila. A. Cartoon of Drosophila brain showing the position of different clusters of DAergic neurons. B. Whole-brain mount of Drosophila captured and analyzed using ZEN software of Carl Zeiss fluorescence microscope using fluorescently labeled secondary antibody targeted against the primary anti-TH antibody. The brain of Drosophila has approximately 140 DAergic neurons in each hemisphere, which are arranged into different clusters. Some of them are PAL (4–5 neurons), PPL1 (11–12 neurons), PPL2 (6/7 neurons), PPM1/2 (8/9 neurons), PPM3 (5–6 neurons), and VUM (3 neurons) (PAL: Protocerebral anterior lateral; PPL: protocerebral posterior lateral; PPM: protocerebral posterior medial). Figures 1–12 adapted with modification from Ayajuddin et al. [28].
For the characterization of FI of a single neuron, a total of 11 scans with an interval of 1.08 μm for each (cumulative 11.88 μm width) were considered. Then, average and standard error were obtained. The same method/step(s) were followed for all the DAergic neurons. The intensity sum of all neurons in a specific cluster gives the total FI of that particular region (cluster-wise). The total FI is the sum of the FI of all the neurons belonging to all the DAergic neuronal clusters.
Data was arranged and plotted using GraphPad Prism for total DAergic neurons and FI of all the DAergic neurons (Figure 13).
Figure 13. Data analysis and representation of results. A. Data was arranged and plotted using GraphPad Prism for total dopaminergic (DAergic) neurons and fluorescence intensity (FI) of all the neurons. B. Graphical representation of DAergic neurons. C. Graphical representation of total FI of secondary ab tagged against the primary ab (anti-TH) of DAergic neurons in Drosophila brain. Figures 12 and 13 were prepared using Adobe Photoshop CS 6.2 version: 13.0.1.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Ayajuddin et al. [28]. Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson’s disease. Front. Neurosci. (Figure 2, Supplementary figures 1–11).
General notes and troubleshooting
Utmost care should be exercised while dissecting the head capsule out to avoid damaging the brain, which may damage certain neuronal clusters that will affect FI quantification (may not affect quantification of neuronal number).
It is important to pay attention and mix the heads thoroughly and gently while fixing them in PFA.
Image acquisition should be done soon after the preparation of slides to minimize the signal bleaching that could influence FI quantification.
Care should be taken to scan brains that are oriented in a dorsoventral position.
Scanning different brains in different orientations will give variation in FI signal levels.
Acknowledgments
This research was supported by the Department of Biotechnology (DBT), India (R&D grant nos. BT/405/NE/U-Excel/2013; BT/PR16868/NER/95/328/2015 and BT/COE/34/SP28408/2018) and the Science and Engineering Research Board (SERB) of the Department of Science and Technology (DST) India (R&D grant no. EMR/2016/002375, 27-3-2017) awarded to SCY. Part of this work was presented at the Indian Drosophila Research Conference (InDRC)-13-17 December 2021, organized by the Indian Institute of Science Education and Research (IISER-K), Kolkota by RC. RC and MA received DBT-JRF (junior research fellowship); RC received DBT-SRF (senior research fellowship) and ICMR (Indian Council of Medical Research)-SRF.
Original research paper: Ayajuddin, M., Chaurasia, R., Das, A., Modi, P., Phom, L., Koza, Z. and Yenisetti, S.C. (2023). Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson’s disease. Front. Neurosci. 17:1158858 [28].
Competing interests
The author declares no conflicts of interest.
References
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A Versatile Pipeline for High-fidelity Imaging and Analysis of Vascular Networks Across the Body
SV Stephen Vidman
ED Elliot Dion
AT Andrea Tedeschi
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4938 Views: 1117
Reviewed by: Pilar Villacampa AlcubierreXiaokang Wu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Brain Jul 2022
Abstract
Structural and functional changes in vascular networks play a vital role during development, causing or contributing to the pathophysiology of injury and disease. Current methods to trace and image the vasculature in laboratory settings have proven inconsistent, inaccurate, and labor intensive, lacking the inherent three-dimensional structure of vasculature. Here, we provide a robust and highly reproducible method to image and quantify changes in vascular networks down to the capillary level. The method combines vasculature tracing, tissue clearing, and three-dimensional imaging techniques with vessel segmentation using AI-based convolutional reconstruction to rapidly process large, unsectioned tissue specimens throughout the body with high fidelity. The practicality and scalability of our protocol offer application across various fields of biomedical sciences. Obviating the need for sectioning of samples, this method will expedite qualitative and quantitative analyses of vascular networks. Preparation of the fluorescent gel perfusate takes < 30 min per study. Transcardiac perfusion and vasculature tracing takes approximately 20 min, while dissection of tissue samples ranges from 5 to 15 min depending on the tissue of interest. The tissue clearing protocol takes approximately 24–48 h per whole-tissue sample. Lastly, three-dimensional imaging and analysis can be completed in one day. The entire procedure can be carried out by a competent graduate student or experienced technician.
Key features
• This robust and highly reproducible method allows users to image and quantify changes in vascular networks down to the capillary level.
• Three-dimensional imaging techniques with vessel segmentation enable rapid processing of large, unsectioned tissue specimens throughout the body.
• It takes approximately 2–3 days for sample preparation, three-dimensional imaging, and analysis.
• The user-friendly pipeline can be completed by experienced and non-experienced users.
Graphical overview
Keywords: Vascular networks Vasculature tracing Tissue clearing Three-dimensional imaging Vessel segmentation AI-based convolutional reconstruction
Background
Vasculature plays a key role during the growth, maintenance, and repair of tissues throughout the body. Accumulating evidence suggests that the slow and protracted reorganization of vascular networks can promote recovery of function (Felmeden et al., 2003; Evans et al., 2021) but may also contribute to neuropathological hallmarks of injury and disease (Ostergaard et al., 2016).
A critical point to consider is that standard methods to study vasculature changes in laboratory settings have proven inaccurate and labor-intensive, lacking the inherent three-dimensional structure of the vasculature. Conventionally, visualization of blood vessels in tissues of interest often employs immunohistochemical (IHC) techniques. This approach uses thin tissue sections to detect endothelial cells through vessel wall stains. The resulting two-dimensional images do not capture the inherent three-dimensional, tortuous structure of vasculature networks (Rust et al., 2020). Furthermore, IHC is a multistep process, increasing the likelihood of introducing error, non-experimental variables (e.g., lot-to-lot variability that contributes to inconsistent results in assays using antibodies), or experimenter bias to analysis, as only a small fraction of the tissue is typically processed. Thus, quantifying the vascular response to injury or disease is often limited to the mean fluorescent intensity of IHC stains, which is highly dependent on technical consistency and subject to bias (Marien et al., 2016; Cheung et al., 2020). Methods of three-dimensional imaging of vasculature networks are currently available. However, they are niche in their applications throughout the body and are largely constrained by expensive or otherwise inaccessible imaging equipment and methodologies including tomography, magnetic resonance imaging, and photoacoustic imaging (Xiong et al., 2017; Pac et al., 2022; Menozzi et al., 2023).
To overcome these barriers, we have developed a practical and accessible pipeline that permits robust and highly reproducible qualitative and quantitative three-dimensional analysis of vascular networks that can resolve changes to the capillary level. Initially, we detail our approach for vasculature tracing and preparing unsectioned tissue samples throughout the body. Then, we present a methodology for optically clearing neuronal and non-neuronal tissues (Susaki et al., 2015) and provide the necessary steps to acquire three-dimensional images using conventional laser scanning confocal microscopy. Lastly, we detail our pipeline for vessel segmentation using AI-based convolutional reconstruction (Imaris software, paid subscription) or semi-automated three-dimensional reconstruction (ImageJ software, freely available). This framework can consistently capture subtle changes in capillary structure such as total length, branching points, branch length, and diameter of vessels, providing a robust and user-friendly approach to study vascular networks in health and disease. Additionally, this scalable method can be adapted for volumetric imaging and analysis of vasculature structures in a variety of preclinical models, thus accelerating translation of promising findings to large animals and non-human primates.
Materials and reagents
Biological materials
Adult (7–8-week-old) female and male C57BL/6J mice (The Jackson Laboratory, stock number: 000664)
Reagents
Paraformaldehyde (PFA) (Sigma, catalog number: 441244)
Ketamine hydrochloride (100 mg/mL) (Covertrus, catalog number: 071069)
Xylazine hydrochloride (100 mg/mL) (Rompun)
Albumin-tetramethylrhodamine isothiocyanate bovine (albumin-TRITC) (Sigma, catalog number: A2289)
0.9% Isotonic saline solution (B. Braun, catalog number: R5200)
Gelatin from porcine skin (Sigma, catalog number: G1890)
Phosphate buffered saline (PBS) (Gibco, catalog number: 21600-044)
Urea (Bio-Rad, catalog number: 161-0731)
Quadrol [N,N,N’,N’-Tetrakis(2-hydroxypropyl)ethylenediamine] (Sigma, catalog number: 122262)
Triton-X 100 (Sigma, catalog number: T9284)
Solutions
4% PFA (see Recipes)
80 wt% Quadrol (see Recipes)
Clearing solution (see Recipes)
Gel perfusate (see Recipes)
Ketamine/Xylazine mixture (see Recipes)
Recipes
4% PFA
Reagent Final Concentration Quantity
PFA 4% 40 g
PBS 1× 1,000 mL
Total 4% 1,000 mL
80 wt% Quadrol
Reagent Final Concentration Quantity
Quadrol 80 wt% 500 g
dH2O n/a 125 g
Total 80 wt% 625 g
Clearing solution
Reagent Final Concentration Quantity
80 wt% Quadrol 80% 156 g
Urea n/a 125 g
dH2O n/a 144 g
Triton-X 100 n/a 75 g
Total 80% 500 g
Gel perfusate
Reagent Final Concentration Quantity
Gelatin from porcine skin 2% 0.1 g/5 mL
Albumin-TRITC 0.05% 2.5 mg/5 mL
PBS 1× 5 mL/mouse
Total n/a n/a
Ketamine/Xylazine anesthetic
Reagent Final Concentration Quantity
Ketamine 150 mg/kg body weight n/a
Xylazine hydrochloride 15 mg/kg body weight n/a
Final n/a
Laboratory supplies
Aluminum foil (Fisher Scientific, Fisherbrand, catalog number: 01-213-100)
Microruler (Fine Science Tools, catalog number: 30086-30)
Fluted filter paper circles (Fisher Scientific, Fisherbrand, catalog number: 09-790-14D)
2 mL Eppendorf tube (Eppendorf, catalog number: 022363352)
50 mL conical centrifuge tube (VWR, catalog number: 89039-656)
100 mL glass beaker (Pyrex, Corning, catalog number: 1000-100)
Glass coverslip (Fisher Scientific, catalog number: 1254588)
Microscope glass slides (Fisher Scientific, Fisherbrand, catalog number: 12-550-15)
23 G needle (BD Biosciences, catalog number: 305193)
10 mL syringe (Henry Schein, catalog number: 570-2620)
1 L round media storage bottle (Pyrex, catalog number: GL45)
Equipment
Vacuum desiccator (Thermo Scientific, catalog number: 08-642-5)
Peristaltic pump (Cole-Parmer, Masterflex, catalog number: 07525)
Vortex mixer (VWR, catalog number: BV1000-GM)
Hotplate stirrer (Benchmark Scientific, catalog number: H400-HS)
Laser scanning microscope (Nikon, model: C2 plus)
Dissection microscope (Zeiss, model: Stemi 2000, catalog number: SP-STEMI2000-TS2)
Computer with 3.3 GHz CPU (Intel or AMD) 6–8 cores, 32–64 GB of RAM, NVIDA Quadro RTX 4000 (8 GB), and multiple fast hard disks or (SATA) SSDs (recommended system when using Imaris software)
Software and datasets
Imaris Microscopy Image Analysis software (Bitplane, Oxford Instruments, v10.0, RRID: SCR_007370) https://imaris.oxinst.com/products/imaris-for-neuroscientists
Free alternative software: FIJI/ImageJ (NIH, RRID: SCR_002285) https://imagej.nih.gov/ij/download.html)
Excel (Microsoft, https://www.microsoft.com/en-us/microsoft-365/p/excel/cfq7ttc0hr4r?activetab=pivot:overviewtab)
Procedure
4% PFA preparation
In a chemical fume hood, heat 600 mL of PBS to 65 °C while stirring on a hotplate stirrer.
Add 40 g of PFA and continue stirring for approximately 5 min.
While stirring, turn down the heat and add 1 M NaOH dropwise until the solution is clear.
Cool the solution immediately on ice for approximately 30 min.
Buffer the solution with 1 M HCl as needed to obtain a pH of 7.4.
Add PBS to 1 L; then, filter using a fluted filter paper into a 1 L glass bottle.
Store at 4 °C until ready to use.
Clearing solution preparation
Note: Liquids are measured by volume as well as weight. Quadrol is a highly viscous liquid; therefore, an 80 wt% working solution is used.
Prepare 80 wt% Quadrol:
Add 125 g of dH2O to 500 g of Quadrol. We suggest measuring liquids by weight during the preparation of the clearing solution.
Stir for at least 30 min. This solution can be stored at room temperature for approximately six months.
Mix 125 g of urea with 156 g of 80 wt% Quadrol in 144 g of dH2O.
Use a heated stirrer at low–medium temperature.
Once the solution is homogenous and fully dissolved, remove it from heat and continue stirring at room temperature.
Add 75 g of Triton X-100 and continue stirring for ~1 min.
Degas the solution using a vacuum desiccator at ~0.1 MPa for ~30 min or until bubbles are removed from the solution.
Store at room temperature and protected from light for up to six months.
Gel perfusate preparation
Note: The following procedure will require a minimum of 5 mL of PBS per mouse. When determining the amount of gelatin to make, it is best to factor in approximately 10%–20% to compensate for repetitive injections.
Calculate the necessary quantity of sterile PBS, gelatin powder, and albumin-TRITC conjugate for samples.
The concentration of gelatin is 2% in PBS while albumin-TRITC concentration is 0.05% of the gelatin mixture volume.
Store the measured albumin-TRITC in a 2 mL Eppendorf tube in a 4 °C refrigerator until adding it to the solution in step 7.
Wrap the tube in aluminum foil to protect the compound from light.
Prepare the 2% gelatin mixture.
Transfer PBS to a glass beaker and begin heating until simmering or lightly boiling. Cover the beaker to prevent liquid loss while heating.
Add a small stir bar and begin gradually adding the gelatin powder while stirring.
Reduce heat to a simmer and continue stirring for ~5 min or until the powder is completely dissolved.
Transfer the gelatin mixture to a 50 mL conical tube and allow the temperature to stabilize at 45 °C in the heated water bath.
Remove 1 mL of gelatin mixture from the conical tube, add it to the 2 mL Eppendorf tube containing the albumin-TRITC from step 2, mix the solution using a vortex mixer, and transfer the mixture back to the 50 mL conical tube.
Place the 50 mL conical tube in a 45 °C heated water bath.
Perfusion and vessel labeling
Weigh the mice and administer the ketamine (150 mg/kg body weight) and xylazine (15 mg/kg body weight) mixture (see Recipes) intraperitoneally. Determine if the animal is deeply anesthetized using the toe pinch reflex.
Prepare 30 mL of 1× PBS per adult mouse and 100 mL of 4% PFA in PBS (see Recipes) for perfusion, pour into glass beakers, and connect to pump (Figure 1A). Prefill tubing with 1× PBS and remove any air bubbles.
Position the mouse on a dissection board and secure limbs using small pins (Figure 1A). Locate the edge of the rib cage and make a small incision into the abdominal cavity. Without damaging the heart, open the thoracic cavity by cutting along either side of the rib cage.
Insert the 23 G needle connected to 1× PBS into the left ventricle (Figures 1B and 1C), make a small cut into the right atrium, and begin perfusion for 3 min at a rate of 10 mL/min to clear the vasculature of blood and red blood cells.
Critical: Be careful not to puncture the septum as this will result in poor/no perfusion and vasculature labeling. Also, be sure to cut the right atrium before turning on the pump to prevent capillary collapse or damage.
After flushing with PBS, pause the peristaltic pump and transfer the tubing to ice-cold 4% PFA. Resume perfusion for 10 min at a rate of 10 mL/min. The pausing prevents air bubbles from entering the tube. If performed correctly, the body will harden a few minutes after injecting the fixative (4% PFA).
Figure 1. Mouse perfusion and injection of the gel perfusate. (A) Representative images of the perfusion setup and a deeply anesthetized adult mouse fixed on a dissection board. (B) Schematic of mouse heart. The syringe needle is inserted into the left ventricle (arrow). After making a small cut into the right atrium, the peristaltic pump is turned on and mouse perfusion begins. (C) Representative images of how to insert the syringe needle into the left ventricle of an adult mouse. Right panel: higher magnification of the region indicated by the dashed line. (D) Representative image of how to insert the syringe needle during the injection of the gel perfusate.
When approaching 8–9 min, remove the fluorescent gel perfusate from the heated water bath.
Draw up 5 mL into a 10 mL syringe and place the fluorescent gel perfusate back into the water bath.
After 10 min, stop the pump and quickly remove the needle from the heart. Using a 23 G needle, slowly inject 5 mL of the fluorescent gel perfusate into the left ventricle (use the same opening as before) over 30 s to match the rate of fixative perfusion (Figure 1D).
Clamp the right atrium with a hemostat immediately after finishing.
Remove the mouse from the dissection board and place it on ice for 30 min to allow gel polymerization.
Note: Place a plastic or hydrophobic covering over the ice so that the mouse does not get wet.
Carefully remove the hemostat clamp and prepare for dissection.
Note: It may be useful to post-fix the animal(s) in approximately 50 mL of 4% PFA at 4 °C overnight, depending on the tissue of interest and quality of fixation.
Tissue isolation and optical clearing
Begin dissection of the necessary tissue.
Predetermine regions of interest before clearing tissue. Remove any debris that may obscure the view during image acquisition, as it will be difficult to see following tissue clearing.
Note: The samples can be kept in 1× PBS at 4 °C until they need to be cleared. Once the samples have been cleared, do not immerse them in another liquid. This will alter the refractive index within the tissue and impede optimal imaging.
Place the samples in the appropriate volume of clearing solution (see Recipes) and incubate according to Table 1 at 37 °C.
Table 1. Tissue clearing (neuronal and non-neuronal tissues originating from adult mice)
Tissue Incubation time Solution volume
Brain 7 days 3.0 mL
Spinal cord 24 h 1.0 mL
Dorsal root ganglion 24 h 0.5 mL
Optic nerve and chiasm 24 h 0.5 mL
Facial nerve 24 h 0.5 mL
Sciatic nerve 24 h 0.5 mL
Adrenal gland 24 h 1.0 mL
Heart 48 h 1.0 mL
Kidney 48 h 1.0 mL
Intestines 48 h 1.0 mL
Muscle 48 h 1.0 mL
Sample mounting and three-dimensional imaging
Gently remove samples from the clearing solution and place them on a microscope glass slide (Figure 2). If using a confocal microscope equipped with conventional optics with a working distance of < 4 mm, imaging of thick tissue samples (e.g., adult mouse brain) will require cutting a portion of the tissue (2–3 mm thick).
Roll two pieces of Blu-Tack into cylinders. The cylinders should be approximately 2–3 cm long and slightly thicker than the sample.
Figure 2. Tissue sample mounting for three-dimensional imaging using an upright laser scanning microscope. Representative image of a cleared mouse spinal cord mounted on a microscope glass slide and covered with a glass coverslip held in place by two pieces of modeling clay (R: rostral, C: caudal).
Mount the cylinders near the edges of the glass slide, parallel to the long axis of the slide (Figure 2).
Place the tissue sample in the middle of the slide, parallel to the Blue-Tack cylinders.
Ensure the sample is properly aligned, especially if the sample requires the use of the tiling function while imaging.
Put a small drop of clearing solution (~50 µL) onto the sample. Ensure the solution does not touch the putty.
Place a glass coverslip (thickness: No. 1) on top of the sample and gently apply pressure on top of the putty.
When pressing, ensure the pressure is firm enough that a small pool forms around the sample and the coverslip is adequately held in place. Take care not to compress the sample too much, as this will distort the sample during imaging.
Position the sample under the microscope and locate the region of interest using a low magnification, long-working distance objective. If necessary, switch to a higher magnification objective (e.g., 20× with at least 1 mm working distance and high numerical aperture) and select the imaging area.
Image albumin-TRITC-filled vascular networks using an excitation wavelength of 544 nm and an emission wavelength of 570 nm (Figures 3 and 4). TRITC is a bright-orange fluorescent dye with a 557 nm maximum excitation.
Critical: Be sure to check the specification of your laser scanning microscope to determine whether the 557 nm excitation maximum is far away from your laser line. Alternatively, consider using albumin-FITC (excitation peak of 490 nm) instead of albumin-TRITC.
Figure 3. Vasculature tracing, tissue clearing, and three-dimensional imaging enable visualization of vasculature networks in neuronal tissues. (A) Representative images of whole organs and neuronal tissue samples before and after clearing. Scale bars: 2 mm. Representative fluorescence images (maximum intensity projections) of albumin-TRITC filled vasculature networks within the mouse (B) somatosensory cortex, (C) spinal cord (low thoracic-lumbar segment), (D) lumbar dorsal root ganglion (DRG), (E) facial nerve, (F) sciatic nerve, and (G) the optic nerve and chiasm. All images were acquired using a conventional laser scanning confocal microscope equipped with a long-working distance air objective (magnification: 10×; working distance: 4 mm; numerical aperture: 0.45). Scale bars: 200 µm.
Figure 4. Vasculature tracing, tissue clearing, and three-dimensional imaging enable visualization of vasculature networks in non-neuronal tissues. (A) Before and after clearing images of whole organs and non-neuronal tissue samples. Scale bars: 2 mm. Representative fluorescence images (maximum intensity projections) of albumin-TRITC-filled vasculature networks within the mouse (B) kidney, (C) adrenal gland, (D) hindlimb muscle (gastrocnemius), (E) heart, and (F) intestine (duodenum). All images were acquired using a conventional laser-scanning confocal microscope equipped with a long-working distance air objective (magnification: 10×; working distance: 4 mm; numerical aperture: 0.45). Scale bars: 200 µm.
Select the mode Frame Scan and set the image size to 1,024 × 1,024 pixels. Adjust the laser speed, laser power, and gain to obtain high-quality, high-contrast, and low-background images. If necessary, use the range indicator to adjust acquisition parameters and avoid pixel saturation. Adjust line averaging to 2 to reduce background noise.
Select the imaging depth in the z-axis (< 1.5–2 mm depending on the tissue of interest) and adjust the step size accordingly. When using a 10× objective, the z-step should be 4–5 µm.
Three-dimensional reconstruction and analysis
Open Imaris and select Surpass > File > Open to import desired file(s). Files can also be dragged and dropped into the Arena interface.
Select Arena > Observe Folder for the software to recognize folders.
Convert file to .ims by double-clicking on file(s).
Note: This is a non-destructive process that will create a copy of the original file.
Select Surpass to see a three-dimensional view of the file.
Crop the file by selecting Edit > 3D Crop.
Determine a region of interest (ROI) for your sample and drag the ROI box over that location. The dimensions can be changed by either dragging the corners of the box or typing in dimensions as described below.
Input desired dimensions in the x, y, and z planes under the drop-down boxes titled Size.
The ROI can be moved by left-clicking and dragging the box while still maintaining the specified dimensions.
Begin surface creation by clicking on the blue globular button (fourth from the left).
Open the surface wizard by clicking on the Creation/wand button (second from left) within the surface properties window (Figure 5A). No options need to be selected in the first window.
Select the vasculature channel via the Source Channel window (Figure 5A).
Select Background Subtraction and adjust the diameter of the largest sphere to the largest diameter vessel found within the image.
To measure the diameter of vessels, select the Slice button at the top of the window (to the right of 3D view).
Left-click on each side of the largest vessel to measure the distance between the two points. The measurement can be found within the docked window on the right side under Distance.
Adjust the threshold by dragging the bar within the background subtraction threshold window until the vasculature source signal is sufficiently covered by the surface. This surface represents the volume of the new mask.
Note: There will need to be a compromise between the larger and smaller vessels, as the smaller vessels can become engorged. Ensure the desired vasculature and continuous vessels are accurately represented.
Adjust filters as needed to reduce background labeling. Surfaces can be filtered according to volume, size, or other parameters by clicking on the drop-down menu.
Allow the surface to render and then mask the surface over the vasculature channel.
Select the pencil icon (fourth from left) and select Mask All....
Select the source channel for the vasculature and then click Ok. This will create a new vasculature channel only encompassing what is occupied within the surface. There will now be two channels within the Display Adjustment window.
Figure 5. Imaris pipeline for convolutional reconstruction and analysis of vasculature networks in unsectioned organs and tissue specimens. (A) Region of interest (ROI) selection of the mouse spinal cord and three-dimensional cropping within Imaris. (B) Surface rendering (masking) step within Imaris. (C) Filament tracing machine learning step, showing predicted renderings of the spinal cord capillary network. (D) Completed three-dimensional rendering of the capillary network and steps for exporting object statistics and batch files. (E) Exporting statistics and editing batch files for multi-image batch processing.
Select the leaf icon (sixth from the left) to begin filament tracing (Figure 5B).
Select Autopath (loops) no soma and no spine (Figure 5B).
Measure the diameter of the source channel within the Slice view as described in steps 4b and 4c.
Set the Source Channel to the newly created masked vasculature channel.
Select Multiscale seeding points and input the approximate diameter of the largest and smallest vessels using Slice view for measurement (Figure 5B).
Adjust the Seed Points Threshold to have seeding points populate the midline of desired vessels and reduce seeding points within the empty volume.
Note: Entering the slicer view may help to check the accuracy of seed point placement.
Seed points may need to be manually added or removed via Ctrl + left-click.
Classify seed points as either Keep or Discard depending on how they fill and match the vessel labeling. Use the circle selection tool on the right docked window to quickly select and classify groups of seed points.
Note: To keep selected points, press K; to discard them, press D (Figure 5B).
The size of the circle selection tool can be adjusted by holding Ctrl/Cmd + scroll on the mouse.
To select multiple points, hold Ctrl/Cmd + left-click/drag over the points you want to select. After enough seed points have been classified, select Train and Predict to begin training the machine learning algorithm.
Note: We have found a minimum of 50 classified points in each category is sufficient, although this will vary depending on the signal-to-noise ratio within the image.
Classify skeleton segments as Keep or Discard as described previously for step 5f–g.
Note: The algorithm tends to bridge parallel structures that are in proximity. Check high-density regions and classify them, as necessary. The Maximum Gap Length option also helps to mitigate this issue.
Filter the filament segments to correct for errors as described in step 5e (Figure 5C). We have found that filtering out segments with a length of less than 2–4 µm aids in correcting the bridging errors; however, this will vary between images.
Identify each layer (surface or filament) that will be exported with statistics with a unique identifier specific to the file and surface or filament being generated.
Critical: The default name for a surface created is “Surface 1.” Rename this to a unique ID for each surface or filament. If this step is not completed, statistics from multiple images cannot be exported.
Batch processing (Optional) (Figure 5C):
To save the new algorithm parameters within Surpass, select the desired surface or filament rendering under the Scene folder.
Note: Algorithm parameters for batch processing should be named, saved, and run individually for each rendering, e.g., save surface creation parameters as a separate batch file from filament tracing parameters and then sequentially run the batch processes on desired files.
Select the Creation (wand) button then Store Parameter For Batch Processing. This will create a batch process file within the same folder as the image that contains the algorithm.
In Arena, ensure the folder contains only the appropriate batch files and image files for batch processing. Right-click on the batch file and select Run.
To create a new algorithm within Arena, select Batch Process to open the algorithm wizard.
Select surfaces or filaments from the top left panel.
Drag and drop an image file to the open algorithm wizard pipeline and begin training.
Note: When training the algorithm for batch processing, ensure the channels and any masked channels are identical to the training file.
To save edits to the algorithm wizard, click on the save icon (floppy disk).
Critical: Edits made in the wizard do not save automatically. If the batch is started prior to saving changes, the algorithm wizard will use the batch file created since the last save, not since the last edit was made.
Export statistics via the Arena interface.
Select all images to be analyzed and select New Plot near the top of the window (Figure 5D). Each rendering with a unique name will be displayed here.
Ensure that only the renderings being exported with statistics are selected from the list (Figure 5D).
Change the displayed statistics by selecting the dropdown menu and scrolling to the desired statistic.
Note: Branch hierarchy is not an option due to the presence of looping structures.
Select the save icon (floppy disk) to export the statistics as a .xls file (Figure 5E).
FIJI/ImageJ reconstruction and analysis of vasculature networks in unsectioned organs and tissue specimens (Optional)
Note: The SNT plugin for FIJI/ImageJ must be downloaded and installed before beginning via the following link: https://imagej.net/update-sites/neuroanatomy.
Open FIJI/ImageJ and select File > Open (Figure 6A). Select the image that will be analyzed.
Optional: If the source signal is not white, convert the signal to binary by selecting Image > Adjust > Threshold.
Using the arrow buttons or manually, input a value to the right and adjust the top (minimum) threshold bar to find an acceptable level (Figure 6B). We have found that a value of roughly 80–110 (2.8%–3.6%) works, although this will vary between images.
Figure 6. FIJI pipeline for reconstruction and analysis of vasculature networks in unsectioned organs and tissue specimens. (A) Importing 3D files into FIJI. (B) Adjusting binary threshold for improved visualization and tracing. (C) Setting a ROI and cropping the files in the x and y dimensions. (D) Cropping the image in the z dimension. (E) Tracing the vascular signal and adding branch points, depicting the Fork Point function. (F) Analyzing the vasculature and exporting statistics of interest.
Determine a ROI within the image by selecting the rectangle tool under File (Figure 6C). Then, left-click and drag to the desired size.
The ROI can be precisely changed by manually inputting dimension parameters via Edit > Selection > Specify (Figure 6C).
Select Analyze > Tools > ROI Manager to save the current ROI selection.
Crop the current image selection via Image > Crop.
To crop a file in the z dimension, select Image > Hyperstacks > Make Subset….
Enter the range of stacks that will be traced (Figure 6D). Note that this distance in the z dimension will be related to the step size selected when imaging. (e.g., an image with 4 µm step size cropped to 50 stacks will be 200 µm in the z dimension).
Select Plugins > Neuroanatomy > SNT to begin tracing the vasculature.
Select the current image under the drop-down menu titled Image.
Begin tracing the vasculature by left-clicking on the image. It may be helpful to begin with a larger diameter vessel first to keep track of the tracings and branches.
Once two points are selected, click Y to keep or N to discard.
Repeat this process until the end of the signal is reached.
When finished with a path, press F to save and add it to the path manager.
Add branches as needed by pressing Alt + left-click (Figure 6E). Ensure the path that the branch will be coming off is selected by hovering the cursor over the path and pressing G. The selected path will be green.
Export statistics via the Path Manager window by selecting Analyze > Measurements > Measure Path(s) (Figure 6F).
From the new window, select Length and No of Branch Points (Figure 6F).
A new window will provide the specified parameters. Highlight these values, copy them, and paste them into an Excel spreadsheet.
Data analysis
The practicality and scalability of our protocol offer application across various fields of biomedical sciences. The entire procedure can be conducted by a competent graduate student or laboratory technician. Group size should be estimated using power analysis and historical laboratory data. Based on our experience, 6–7 biological replicates will produce robust and reproducible results. Examples of exclusion criteria include poor mouse perfusion, inconsistent vasculature tracing, tissue damage during dissection, or excessive photobleaching. Of note, three-dimensional reconstruction and analysis of vasculature networks using Imaris and ImageJ produced similar results (Table 2). A major limitation of ImageJ vs. Imaris is that it takes significantly longer to process the same ROI for three-dimensional reconstruction. Statistical analysis of albumin-TRITC-filled vasculature (Imaris pipeline) was performed using Prism (Version 10.0.1, GraphPad) through a Mann-Whitney test (Figure 7B). Other statistical analysis software (e.g., SPSS, R, or MATLAB) can also be used. For all analyses performed, significance was defined as p < 0.05. The exact value of biological replicates and the definition of measures are shown in the corresponding figure legend. Experimental data were evaluated blind to condition.
Table. 2 Imaris vs. FIJI comparison
Imaris FIJI
Number of branch points 78 52
Total length (µm) 5,778 5,642
Comparison of using Imaris vs. FIJI for vascular tracing and analysis using the same ROI with a mouse spinal cord sample.
Validation of protocol
To verify the robustness and reproducibility of our protocol, we compared results obtained by an experienced user (> 20 years of research experience) with those from a non-experienced user (e.g., a first-year graduate student with no prior experience in mouse perfusion, tissue clearing, three-dimensional imaging, and analysis). Our analysis demonstrated consistent and reproducible results between users (Figure 7). Additionally, this protocol has been already adopted in a previous publication (Tedeschi et al., 2022), which leveraged the vascular tracing methodology to assess capillary network changes following stroke in adult mice. A similar protocol has also been described in a previous study (Di Giovanna et al., 2018). Tissue clearing methods are adaptable and highly reproducible (Ueda et al., 2020). The clearing protocol we used here is based on the advanced CUBIC protocol for whole-brain and whole-body clearing and imaging (Susaki et al., 2015).
Figure 7. Assessing the robustness and reproducibility of our vasculature tracing, imaging, and analysis pipeline by compering results from users with and without research experience. (A) Representative fluorescence images of albumin-TRITC-filled vasculature networks in the adult mouse spinal cord. Regions of interest (ROIs) are depicted as a maximum intensity projection and three-dimensional renderings (Imaris). Scale bars: 200 µm. Data were generated by an experienced user (> 20 years research experience) and a non-experienced user (e.g., a first-year graduate student with no prior experience in mouse perfusion, tissue clearing, three-dimensional imaging, and analysis). (B) Quantification of (A). Mean and SEM (Mann-Whitney test, ns: not significant; n = 6 biological replicates/user).
General notes and troubleshooting
General notes
Although each component was chosen to achieve optimal image quality and precise quantification, modifications can be made to extend the utility of this protocol. The selection of a preconjugated fluorophore can be adapted to avoid spectral overlap in the instance of additional labeling strategies such as IHC or genetic reporters. When choosing a fluorophore, key molecular properties to be considered are excitation and emission spectra, quantum yield, and fluorescence lifetime. Check the specifics of your laser scanning microscope and confirm that the maximum excitation wavelength of the selected fluorophore is within the range accepted by your imaging system. Our chosen fluorophore, TRITC, has a comparatively high quantum yield and fluorescence lifetime, while its excitation spectrum responds to red-shifted light, peaking at 544 nm. This component is crucial, as longer wavelengths are less likely to be attenuated in thick samples, providing improved signal-to-noise ratio and deeper imaging of samples (Belov et al., 2010; Im et al., 2019). Optical clearing methodologies can be adjusted to improve transparency with particular tissue compositions. The decision to use aqueous-based vs. organic solvent–based clearing methods will primarily depend on tissue thickness and composition, although some clearing methods can damage the fluorophore and should be carefully assessed. Additionally, these two classifications of tissue clearing have several variations of protocols that can be tailored to the research question at hand. Imaging offers a variety of suitable microscopy strategies including 1P and 2P laser scanning and light sheet fluorescence microscopy (Hilton et al., 2019; Tedeschi et al., 2016 and 2022; Wang et al., 2018). Lastly, three-dimensional reconstruction can be completed using either Imaris or ImageJ. Note that a benefit of ImageJ is that it is a free, open-source software and less computationally demanding. This can be leveraged for less dense vascular networks, such as those found in peripheral nerves (e.g., facial or sciatic nerves).
Further adaptations of this protocol permit the study of blood–brain barrier/blood–spinal cord barrier (BBB/BSCB) integrity in CNS injury (e.g., stroke, traumatic brain, and spinal cord injury) or disease (e.g., Alzheimer’s disease, multiple sclerosis, etc.) models. To trace vessels, we leveraged a 67 kDa albumin-fluorophore conjugate in a 2% gelatin mixture. At this molecular weight, the conjugate cannot leak out of intact capillaries but can be used to identify compromised barrier regions by adjusting the concentration of gelatin. Decreasing the percentage of gelatin to approximately 0.5% will reduce viscosity, allowing albumin-TRITC to leak through compromised regions and illustrate the extent of BBB/BSCB damage in vivo. While this is possible with other vessel tracing methods, our pipeline offers the advantage of quantitatively assessing concurrent changes in other capillary network characteristics that can accompany a loss of barrier integrity.
Troubleshooting
Table 3. Troubleshooting
Problem observed Possible cause Solution
Poor, uneven, or absence of fluorescence during image acquisition Sub-optimal perfusion or fluorophore damaged during preparation
• Ensure the needle does not puncture through the left atrial wall.
• Adjust the needle to prevent fixative from entering pulmonary circulation and expelling from the nose or mouth.
• Ensure the temperature of gelatin is stabilized at 45 °C before resuspending albumin-TRITC.
High background/noise during image acquisition Clearing incubation time too short, samples too thick, or improper clearing method for sample
• Increase time of clearing incubation.
• Determine a better-suited clearing method, considering tissue composition and size.
Imaris not showing files in Arena Overloaded cache
• Save work and restart Imaris.
Imaris frequently crashing/not responding Files too large or computer specifications not sufficient
• Reduce ROI dimensions.
• Run smaller datasets through the pipeline.
• If object-to-object statistics are not needed, uncheck the box when creating the algorithm.
Unable to connect gaps in signal during FIJI/ImageJ tracing Cursor auto-snapping enabled
• In the SNT Snapshot window, uncheck Enable snapping within: XY.
Acknowledgments
We would like to thank Dr. Wenjing Sun for critically reading and all members of the laboratory for discussion. This work was supported by the National Institute of Neurological Disorders (grant R01NS110681).
Competing interests
All authors declare no competing interests.
Ethical considerations
The methods and experimental procedures described above are designed to limit discomfort and distress to the animals that may affect the interpretation of the results. All experiments were performed following protocols approved by the Institutional Animal Care and Use Committee at The Ohio State University (Protocol #2017A00000027-R2) and complied with accepted ethical best practices.
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4,939 | https://bio-protocol.org/en/bpdetail?id=4939&type=0 | # Bio-Protocol Content
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Unlocking Bio-Instructive Polymers: A Novel Multi-Well Screening Platform Based on Secretome Sampling
SF Shirin Fateh *
RA Reem A. Alromaihi *
AG Amir M. Ghaemmaghami
MA Morgan R. Alexander
(*contributed equally to this work)
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4939 Views: 1302
Reviewed by: Geoffrey C. Y. LauSrajan Kapoor Anonymous reviewer(s)
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Abstract
Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials’ development due to the lack of underlying theory governing cell response to materials’ chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials’ HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions.
Key features
• Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O2 plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating.
• Preparations of 7 mm × 7 mm polymers employing pin printing system.
• Provision of confined area for each polymer using ProPlate® multi-well chambers.
• Compatibility of this platform was validated using adherent cells [primary human monocyte–derived macrophages (MDMs)) and non-adherent cells (primary human monocyte–derived dendritic cells (moDCs)].
• Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet®).
Graphical overview
Keywords: Biomaterials High-throughput screening HTS Secretome profiling Immune-instructive Bio-instructive 2D printing Polymer microarray
Background
Biomaterials play a pivotal role in the development of advanced medical devices, drug delivery systems, and regenerative therapies to enhance patient outcomes and quality of life [1–4]. However, current drawbacks include device-associated infection, adverse immune responses, and in-service degradation that can collectively reduce implant performance [5–7]. Rational design of bio-instructive materials remains unattainable due to our limited understanding of material–biological interactions [1]. As a result, screening is a commonly employed approach for discovering and optimising novel biomaterials with a desired biological function, such as pro- or anti-inflammatory properties. High-throughput screening (HTS) strategies have accelerated the development of biomaterials by enabling researchers to rapidly analyse a large number of samples or conditions in a systematic manner [8]. A commonly used HTS approach for biomaterials is the use of printed polymer microarrays, which have been employed to screen cell responses to materials, allowing reproducible control over cellular behaviour [9]. Key examples are screening for scalable synthetic cultureware for human pluripotent stem cells [10], polymers to modulate the foreign body response and wound healing [11,12], and a new class of bacteria-attachment-resistant materials [13]. In these studies, thousands of materials are robotically printed and in situ ultraviolet (UV)-polymerised on a single slide. This approach is used to discern specific biological interactions by culturing cells directly on the polymer array surfaces within a shared culture medium [14,15]. Despite the success of such polymer microarray platforms in biomaterials’ discovery, the secreted biochemical signals are an untapped resource in understanding cellular phenotypes. Paracrine signalling is also a worry requiring follow-up studies on individually scaled up polymers before potential hits can be verified. Understanding cell-surface interactions requires probing biochemical cues, in addition to biomechanical, topographical, and material chemistry/bio-interfacial cues. Hence, without knowledge of the secretome profile of the cells following their interaction with materials, the understanding of the functionality of the polymer is incomplete. Furthermore, polymer microarrays are limited to the assessment of cells with strong adhesion abilities. Notably, for cells such as dendritic and T cells, which possess weaker substrate adhesion tendencies, attributing their phenotypical changes to a particular polymer spot would be incredibly challenging [16–18].
In response to these limitations of the polymer microarray, we developed a novel platform that combines existing arraying technology such as contact and inkjet printing, multi-well microchambers, and high-throughput secretome profiling. The platform benefits from reusable superstructures (ProPlate®) that are mountable on the printed glass substrates. This offers the provision of confined culture volumes, each designated for a distinct polymer condition with a surface area and volume compatible with cell culture for a few days. This separation allows us to closely study the mediators released by cells, helping us understand how these materials interact with cells and the mechanisms of the biomolecules involved. In addition, this system allows for high-throughput surface characterisation such as time-of-flight secondary ion mass spectrometry, x-ray photoelectron spectroscopy, and attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR), ensuring the accurate identification and validation of controlling moieties [19]. Together, these analytical approaches have the potential to enhance the discovery of new biomaterials and improve our understanding of biomaterials–cells interface. Yet, there are constraints to the throughput and time-related aspects of this platform. In a single operational cycle, the platform demonstrates the capacity to fabricate ten slides, each accommodating 16 unique chemical compositions, resulting in the synthesis of 160 distinct polymer entities, thereby yielding 8,960 discrete polymer spots. By way of comparison, a polymer microarray system can generate 17,280 polymer deposition sites from 576 chemistries in triplicates across ten slides [20]. Nevertheless, this limited output can be ameliorated through the implementation of 64-well superstructures from GraceBio-Labs and the application of multiplexing methodologies as alternatives to conventional techniques such as enzyme-linked immunosorbent assay (ELISA). Here, we provide a step-by-step description of methodologies and troubleshooting aspects involved in developing this platform, including preparation of substrate, fabrication of multi-well chambers, and provision of confined area for each polymer condition, followed by relevant examples of the anticipated outcomes such as chemical characterisation and biological assessments of the secreted soluble mediators.
Materials and reagents
Reagents
Oxygen (O2) gas (any vendor)
Molecular sieves (4 Å) (VWR International, catalog number: 215-283-8)
Toluene (Fisher Scientific, catalog number: T290-4)
3-glycidoxypropyltrimethoxysilane (GPTMS) (Sigma-Aldrich, catalog number: 440167-500mL)
Argon gas (any vendor)
Acetone (Fisher Scientific, catalog number: A949SK-4)
Poly(2-hydroxyethyl methacrylate) (pHEMA) (Sigma-Aldrich, catalog number: P3932-25g)
Deionised distilled water (Milli-Q®) (Millipore, Sigma-Aldrich, USA)
Ethanol (Sigma-Aldrich, catalog number: 1009862500)
Ethoxyethyl acrylate (EOEA) (Sigma-Aldrich, USA, catalog number: 106-74-1)
Dimethylformamide (DMF) (Fisher Scientific, catalog number: AA22915K7)
Photoinitiator (2,2-dimethoxy-2-phenylacetophenone) (DMPA) (Sigma-Aldrich, catalog number: 19611-8)
Isopropanol (Sigma-Aldrich, catalog number: 563935)
Tween® 20 (Sigma-Aldrich, catalog number: 9005-64-5)
Phosphate buffer solution (PBS) (Sigma-Aldrich, catalog number: D8537)
Trypan blue dye (any vendor)
ToxiLightTM assay (Lonza, USA, catalog number: LT17-217)
ELISA DuoSet® (R&D Systems, USA) (TNF-α, catalog number: DY210; IL-10, catalog number: DY217; TGF-β1, catalog number: DY240; CCL-18, catalog number: DY394; IL-6, catalog number: DY206; IL-12, catalog number: DY1270)
Laboratory supplies
Glass slides (25 mm × 75 mm) (VWR, catalog number: 631-1553)
Glass beaker (any vendor)
Needles (21 G, 120 mm) (Fisher Scientific, catalog number: 10438881)
Needles (21 G, 40 mm) (Sterican® Safety Needle, catalog number: Z118044)
Plastic syringe 50 mL (any vendor)
Crystallizing dish (Pyrex, capacity 1,200 mL)
Parafilm® (Sigma-Aldrich, catalog number: P7543)
Falcon tube 50 mL (Sigma-Aldrich, catalog number: T2318)
Polypropylene 384-well plate (Corning, product number: 3656)
Glass Pasteur pipette (VWR, catalog number: 612-1702p)
Weighing boats (any vendor)
Glove box (MBRAUN, Germany)
Microarray print head 16 pins (BioDot, USA)
Plastic snap clips (GraceBio-Labs, catalog number: 204830)
4-well rectangular plate for slides (Thermo Fisher, NuncTM, catalog number: 267060)
Equipment
Glass funnel (any vendor)
Support stand (A-frame) (any vendor)
Laboratory clamp (any vendor)
Hotplate (any vendor)
Stainless steel rack for glass slides (Sigma-Aldrich, catalog number: Z710989)
Fume hood (any vendor)
Plasma etcher (Diener, model: Nano LFG40)
Vacuum oven (Thermo Scientific, model: Vacutherm)
Sonicator (any vendor)
Dip-coater (Holmarc, model: HO-TH-01 dip-coater)
Water contact angle measurement apparatus (KSV Instruments, model: CAM 100)
Polypropylene pipette tips (any vendor)
Pipette (any vendor)
Electric pipettor controller (any vendor)
Weighing scale (0.01 g and 0.0001 g sensitivity) (any vendor)
Spatula (any vendor)
Pin printing workstation (BioDot, model: XYZ3200)
Microarray ceramic pin 500 µm (LabNEXT Inc, model: XtendTM)
UV lamp (365 nm, any vendor)
O2 sensor (Cambridge Sensotec, model: rapidox 1100)
Optical profiler (KLA, model: ZetaTM-300)
Multi-well chambers (16-well ProPlate®) (GraceBio-Labs, catalog number: 244864)
Software and datasets
BioDot AxSysTM (BioDot, USA)
MicroLab expert (Agilent, USA)
Spectrus Processor (ACD lab, USA)
GraphPad Prism software (Version 10.0.2, USA)
Procedure
This method consists of three key steps: O2 plasma etching, optimisation of functionalisation process, and pHEMA dip coating. Dip coating of glass substrate with pHEMA was performed as previously described [20] with some modifications as detailed here.
Part I. Substrate preparation
O2 plasma etching
Put conventional 25 mm × 75 mm glass sides in plasma etcher chamber and subject them to O2 plasma (P = 300 mbar, 100 W) for 10 min.
To confirm that the slides are activated by excited ions, measure water contact angle (WCA) before and after plasma treatment [21]. An example is shown in Figure 1; the presence of organic contamination increases the water contact angle; therefore, its removal results in a reduction in the observed contact angle [22,23].
Functionalisation process
Add molecular sieves (4 Å) to toluene at 20% w/v for 24 h prior to the silanisation process to have anhydrous toluene. See Troubleshooting.
After the etching step, submerge glass slides immediately into 500 mL of anhydrous toluene in a crystallisation dish.
Place the reaction vessel on a hot plate set to 50 °C (see Troubleshooting).
Put the reaction under an argon atmosphere, place the funnel over the reaction vessel, and connect the argon to the neck of the funnel.
Add 10 mL of GPTMS into the anhydrous toluene solution.
Allow this reaction to proceed for 24 h to achieve completion. You can use a frame stand and clamp to secure the reaction and argon.
Cool the slides to room temperature and wash them three times in fresh acetone to remove any residue of unbound silane.
Dry the slides under vacuum (< 50 mTorr) for 24 h.
Use WCA measurement after the functionalisation step to ensure that silanisation has taken place completely. The functionalised glass slide becomes hydrophobic with a higher contact angle of 54° ± 1.11 (Figure 1). Measuring the WCA on a substrate is a commonly used method to quantify the substrate wettability and its cleanliness.
Figure 1. Quality control for the effectivity of plasma etching and silanisation process. Water contact angle (WCA) measurement of the glass substrate to validate the functionalisation. The O2 plasma–activated glass slide shows increased hydrophilicity with very low contact angle of < 10°, while after silanisation the substrate becomes hydrophobic with a higher contact angle of 54° ± 1.11.
pHEMA dip coating
Prepare 4% (w/v) pHEMA solution in ethanol (95% v/v in deionised distilled water). See Troubleshooting.
Set up the settings of the dip-coater to 9 mm/s speed and a dip duration of 2 s with a retention speed of 1 s.
Clip the glass slides to the holder and adjust the holder position to a proper height vertically.
Pour pHEMA solution in a 100 mL beaker and immerse epoxy silanised glass slides in the 4% pHEMA solution.
Repeat coating four times with enough time for drying in between the dips (see Troubleshooting). While waiting for the slides to dry, cover the beaker containing pHEMA to prevent evaporation.
Leave the pHEMA-coated slides at atmospheric conditions for three days prior to its use for arraying.
Use optical profilometry to evaluate the evenness of pHEMA coating of the glass substrates. Figure 2 illustrates the uniformity of step height results from pHEMA coating.
Figure 2. Evaluation of evenness of pHEMA coat. Optical profilometer images and height line scans (5×) of a pHEMA-coated glass slide. (A) 2D image of the pHEMA-coated glass slide. The arrow shows the measured distance; the dipping lines are apparent on the side of the glass substrate, as the glass is coated four times. (B) Graph representing the space highlighted on the 2D image (A) showing the evenness of the pHEMA coat, as the step height variation is minimal.
Troubleshooting
Plasma treatment has been proven to be a simple technique for modifying surface properties, ensuring removal of impurities and organic contaminants from the surface, which are fixed by weak electrostatic/van der Waal’s forces [24]. Bombardment of plasma excited ions to the surface of the glass slide promotes hydroxylation (OH groups) and generates the Si–OH groups.
Excess amount of water is detrimental to the extent of silanisation; therefore, use of molecular sieves in proper proportions to the water content of the solvent is critical [22].
Maintaining the reaction at 50 °C is crucial to lower the number of weakly bonded silane molecules by disrupting the hydrogen bonds in silane layer [25].
The silane provides a bind between the glass slide and the pHEMA coat, as the methoxy groups of GPTMS bind the -OH on the glass slide and the reactive epoxy group binds -CH in pHEMA [22,26].
To speed up the dissolvability of pHEMA in the intended solvent, it is useful to use smaller amounts in larger containers and sonication. For example, add 2 g of pHEMA in 50 mL of 95% ethanol/deionised distilled water, close the cap, and leave the tube in the sonicator for 24 h. Ultrasonication is often used to promote an effective and fast dissolution of pHEMA powder in ethanol.
Wet pHEMA appears cloudy on the surface of the glass slide and it takes much longer to dry between coating repetitions.
Tight sealing containers for pHEMA solutions are very important, as evaporation causes concentration change. Therefore, it is best to prepare fresh pHEMA solution for each coating session.
Coating the glass substrate with pHEMA provides an anchorage for arraying a diverse acrylate and methacrylate polymer library [4,9]. As pHEMA swells in the presence of atmospheric moisture, it provides the possibility to form an interpenetrating network between the deposited polymer spots and the pHEMA. This increases their stability in the pretreatment washing steps, sterilisation process, and cell culturing conditions.
Part II. Fabrication of polymer wells
Preparation of polymerisation solution
Degas monomers under argon for 30 min by purging method.
First, add the required amount of monomer in a scintillation glass vial and seal the cap using rubber stoppers.
Add a layer of parafilm around the cap area to ensure that the vial is airtight.
Insert a short needle (21 G, 40 mm) through the cap to enable the degassing process.
Insert a longer needle (21 G, 120 mm) to bubble argon through the monomer. Make sure that the needle attached to argon source is dipped in the monomer while the degassing needle is away from the monomer.
Weigh DMPA and make 2% (w/v) in DMF in a polypropylene or glass vial.
Prepare the polymerisation solution by mixing 50% (v/v for liquids, w/v for solids) monomer in DMF and 1% DMPA in a glove box under argon condition with O2 levels < 2,000 ppm. See Troubleshooting.
Transfer 40 μL of each polymerisation solution to a source plate (e.g., 384-well polypropylene plate).
Position the source plate in its place on printing machine stage.
Polymer deposition and in situ UV curing
Set the printing conditions inside the chamber for O2 < 2,000 ppm using argon and 30%–40% humidity. See Troubleshooting.
Wash the pin with isopropanol and insert into the microarray print head.
Close the printer chamber to keep the printing environment settings.
Initiate the software (BioDot AxSysTM) for printer stage and head. Recalibrate the X, Y, and Z positioning. Refer to Table 1 for the instrumental factors including print head travel speed and contact times.
Table 1. Instrumental settings for movements of the stage and pin holder
Loading sample from source plate
Held in monomer solution Withdrawal speed
4 s 25 mm/s
Monomer solution deposition on the substrate
Total contact time for each contact Withdrawal speed
10 ms 175 mm/s
Z-axis speed
6.531 mm/s
Vacuum wash interval
Held in vacuum Withdrawal speed
10 s (3×) 175 mm/s
Pre-spot four times on a plasma-etched glass slide prior to deposition of the polymerisation solution on pHEMA-coated substrate. The blotting pattern can be two contacts per position to remove the excess polymerisation solution from the outside of the pin.
Start the printing process. For example, for an area of 7 mm × 7 mm of pHEMA-coated glass slide to be coated by acrylate polymers, print 56 spots with 850 μm distance in the y-axis, with an alternating +850 μm/-850 μm offset in the x-axis. Figure 3A illustrates the brightfield images of the single printed polymer spots.
Polymerise deposited spots with 40 s of long wave UV (365 nm) intervals to liberate the radical ions in the DMPA, which creates the polymerisation of monomers. During this time, the pin is washed in DMF and dried three times before printing the next polymer.
After printing all the polymers, further add 20 min of UV exposure at the completion point of each printing session.
Keep the printed glass slides in < 50 mTorr vacuum for 24 h prior to the second print in between spots.
To print in between polymer spots for full coverage, increase the initiation position by 800 μm. Figure 3B shows the double-printed spots coverage.
Figure 3. Comparison of brightfield (5×) images of printing pattern. A. Single-printed polymer spots. B. Double-printed polymer surface. The visual examination demonstrates a significantly enhanced substrate coverage after the second printing.
Repeat step B6.
Lastly, keep the printed slides in < 50 mTorr vacuum for at least seven days to ensure extraction of the residual solvent and unpolymerised monomers.
Troubleshooting
Be vigilant about potential issues arising from phase separation before printing, possibly linked to solvent compatibility.
Minimise contamination in polymerisation solutions by using solvent-compatible pipetting tools. For example, avoid using polystyrene pipetting tips while using DMF as a solvent.
There are many aspects to consider in contact printing to ensure consistency of the polymer areas; some of these are addressed through optimisation of the chamber environment such as humidity, temperature, and O2 percentage. O2 higher than 2,000 ppm inhibits radical polymerisation; 30%–40% humidity reduces the static effects of printing head movement and induces swelling in pHEMA to ease the interpenetration of the formed polymer spot [9,20,27].
Even though pHEMA provides the possibility to form an interpenetrating network between the deposited polymer spots and is known to be a low fouling material [28], it has been observed that it has effects on cell biology [29,30]. This could influence the interpretation of cells’ behaviour towards the printed polymers as it decreases the signal-to-noise ratio. This can be resolved by double-printing in between the spots.
For this work, inert ceramic capillary pins were used for polymer deposition using contact printing technique. Figure 4A is the schematic of the contact printer robotic head and printing steps. Ultra-smooth surface and tubular construction of these pins eliminates cross-sample contamination and improves spot uniformity and deposition repeatability while printing a large library of chemistries [31].
Optimise the printing pattern via trialling with spot–spot spacing (refer to Figure 4B) in order to achieve a coalescence free coverage of the surface by deposited polymer spots.
Consider volatility of the polymerisation solutions to adjust timing of the printing.
It is worth mentioning that this platform has the flexibility to utilise inkjet printing as an alternative to contact printing. Trials for proof of concept have been successfully carried out, involving spot pattern adjustments, voltage, pulse, and uniformity. This approach is particularly advantageous for printing on sensitive and/or expensive surfaces.
Figure 4. Schematic representation of contact printing technique and optimising printing patterns. A) The process of contact printing: 1) picking up the polymerisation solution from the source plate; 2) movement of the robotic head to the position coordination; 3) deposition of polymerisation solution via contacting the surface of the substrate; 4) in situ polymerisation by exposure to UV; 5) washing and vacuuming of the pin for the next material. B. Optimising printing patterns to address coalescence issues. Image shows trials B, C, D, and E and achieving uniform thin polymer layers (spot F) by minimising coalescence via xy-axes adjustments.
Part III. Provision of confined area for each polymer
Preparation of the microchambers
ProPlate® are reusable and should be thoroughly cleaned before and after each experiment to remove any dust, particles, or contaminants, following the instructions provided by the manufacturer and summarised below:
Make a washing solution by adding 0.1% (v/v) Tween® 20 to PBS.
Soak the chamber and clips in a clean container for 15 minutes in the washing solution.
Rinse three times with deionised distilled water.
Soak in isopropanol overnight.
Air dry before use.
Leakage assessment
Conduct a preliminary leakage assessment to verify the integrity of the superstructure assembly, ensuring the absence of cross-contamination or leakage between the individual wells. The efficacy of the plates in retaining samples within the designated wells was qualitatively evaluated by adding trypan blue dye into the wells in an alternating manner (refer to Figure 5) and subsequently incubating them for a period of seven days or in accordance with the experimental timeline.
Figure 5. ProPlate® leakage assessment. Evaluate sample retention by adding trypan blue dye to alternating wells and incubate for seven days or as per the experiment timeline to confirm the superstructure's integrity.
Assembly of the multi-well chambers on the printed polymer slides
This platform integrates contact printing microarray technology with high-throughput microtiter plate processing. Slides with polymer squares were assembled using multi-well structures to provide enclosed areas for each polymer with a total of 16 per slide. Figure 6 represents schematically the steps for mounting the multi-well chambers.
Figure 6. Assembly steps for the multi-well chambers on printed polymer slides. Images of the ProPlate® have been adapted from GraceBio-labs website [32].
Data analysis
ATR-FTIR spectra were acquired from MicroLab expert (Agilent, USA) and analysed by Spectrus Processor. Results for viability were measured indirectly by performing a cytotoxicity assay and presented as a percentage of the control (in this work, the control was commercial TCP). The corresponding concentrations of cytokines were measured by extrapolating the colorimetric readings (O.D.) to standard curve ELISA from three independent experiments each with two technical replicates. For both viability and secretome data, GraphPad Prism software was used to plot the graphs as mean and standard deviation (SD).
Validation of protocol
Presence of polymers (fabricated using this platform) on the surface of the substrate was validated by ATR-FTIR. The applicability of this platform for cell culture was validated by culturing two different primary cell types and assessing cell viability and cytokine secretion:
Chemical characterisation: ATR-FTIR spectra was used to validate the presence of specific chemical components or functional groups on the substrate surface following contact printing and in situ UV polymerisation. Spectra were obtained by scanning the samples (254 scans) from 600 to 4,000 cm-1 with a 4 cm-1 resolution, with subtraction against background (air) after each scan. The analysis of the printed polymers' spectra reveals significant distinctions when compared to pHEMA. Specifically, the O–H stretching (3,550–3,200 cm-1) present in the pHEMA spectra is no longer evident, and distinct new peaks have emerged in the polymer spectrum in the fingerprint region (400–1,500 cm-1). For example, the ATR-FTIR analysis clearly confirms the conversion of ethoxyethyl acrylate (EOEA) monomer into a polymer (pEOEA). The disappearance of the C=C stretching peak at 1,640 cm-1, characteristic of the monomer, in the polymer spectrum, along with the presence of typical acrylate polymer peaks (e.g., C=O stretching at 1,720 cm-1), indicates successful polymerisation as shown in Figure 7A and B.
Figure 7. Attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) spectra of pHEMA and ethoxy ethyl acrylate (EOEA) monomer (red) and its corresponding printed polymer (blue). A. The spectra of pHEMA shows O–H stretching (3,550–3,200 cm-1), C–H stretching, corresponding aliphatic stretching vibration of CH2 (3,000–2,840 cm-1), and C=O stretching (1725 cm-1). B. The disappearance of the 1,640 cm-1 peak in the polymer spectrum (blue) indicates that the C=C double bond in the EOEA monomer has polymerised, leading to structural changes. Characteristic peaks at 1,720 cm-1 (C=O ester group) and 2,960/2,880 cm-1 (C-H groups) remain. Also, O–H stretching (3,550–3,200 cm-1) peak of pHEMA structure does not appear in pEOEA spectra, indicating the full coverage of the surface.
Viability assessment: Cell viability was assessed after 24 h of incubation of cells on the surface of the polymers using ToxiLightTM assay by measuring the release of adenylate kinase from damaged cells. To showcase the applicability of the platform, viability results were measured for two distinct cell types: monocyte-derived dendritic cells (moDCs) and monocyte-derived macrophages (MDMs). The polymers fabricated through this platform support cell viability equal or higher than tissue culture plastic control (TCP), as illustrated in Figure 8.
Figure 8. Viability assessment. Two distinct primary cell types were examined following a 24 h incubation with pEOEA using the microchamber platform. The graphs depict the mean ± SD results from two independent experiments, demonstrating that the polymer supports cell viability. MDMs: monocyte-derived macrophages; moDCs: monocyte-derived dendritic cells.
Secretome profiling: Even without access to a multiplexing assay, the volume of supernatant obtained following cell culture using this platform is sufficient for measuring up to seven different soluble protein mediators with replicates. Different cytokines and chemokines including TNF-α, IL-10, TGF-β1, CCL-18, IL-6, and IL-12 were assayed from the supernatants using high-binding 384-well plate and ELISA DuoSet® kits. A representative example for moDCs and MDMs is shown in Figure 9. The capability to measure various soluble factors using this platform serves as compelling evidence for the exceptional integrity of the fabricated polymer surfaces. Indeed, this indicates that the system is capable of effectively accommodating a diverse range of chemistries.
Figure 9. Secretome analysis of two primary cell types after incubation with the pEOEA. Heat maps are the representative mean values of cytokine ELISA results from three independent experiments with two technical repeats. TCP, M0, M1, and M2 are internal controls for comparisons only. TCP: tissue culture plastic control.
Data Availability Statement: The data that support the findings of this study are openly available at the University of Nottingham data repository, DOI: 10.17639/nott.7362.
Acknowledgments
The authors wish to express their gratitude to the Vice-Chancellor's Scholarship for Research Excellence (International) at the University of Nottingham and extend their appreciation for PhD scholarship provided by the Ministry of Higher Education of Saudi Arabia, Qassim University. This work was also supported by the Engineering and Physical Sciences Research Council (EPSRC) (grant number: EP/X001156/1).
Author Contributions: S.F. and R.A.A. performed the experiments and wrote the manuscript. M.R.A. and A.M.G. supervised the project and revised the manuscript. S.F. and R.A.A. contributed equally to this work.
Competing interests
There are no conflicts of interest or competing interests.
Ethical considerations
Monocyte-derived cells used for validation of protocol were generated from monocytes isolated from blood samples from healthy volunteers provided by National Blood Services (Sheffield, United Kingdom), following ethics committee approval (2009/D055).
References
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Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography
LS Landi Sun *
JM Jana Meissner *
JH Jianfeng He
LC Lihong Cui
TF Tobias Fürstenhaupt
XL Xin Liang
(*contributed equally to this work)
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4940 Views: 1034
Reviewed by: Xiaokang WuRama Reddy GoluguriChen Fan
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Aug 2023
Abstract
Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples.
Key features
• Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET).
• Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms.
• The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.
Graphical overview
Keywords: Electron tomography High-pressure freezing Serial sectioning Transmission electron microscope (TEM) Drosophila Mechanosensory organelle
Background
Drosophila type I sensory cells serve as a model for the study of mechanosensation, neuronal cell biology, and ciliogenesis due to their compatibility with functional, imaging, and structural assays [1–3]. In particular, the convenience of performing ultrastructural analysis has facilitated mechanistic studies on the above-mentioned topics, especially for understanding the molecular mechanisms of neurosensory transduction.
Mechanotransduction is the process by which mechanoreceptor cells convert physical signals in the environment into cellular signals [1,2,4]. Mechanoreceptors form a specialized functional entity called mechanosensory organelles (MOs) [5–11]. Because MOs are essentially mechanosensors, they possess a dedicated structural architecture that facilitates the detection of mechanical stimuli. To suit this purpose, MOs develop a delicate intracellular structure [5,7,8,11]. Information regarding this structure is required to understand how mechanosensory cells encode mechanical signals and are formed.
We and others have previously studied the ultrastructure of fly external mechanosensory cells using conventional transmission electron microscopy (TEM)[8,9,12–15]. These studies made a clear presentation of nanoscopic ultrastructures and provided molecular insights. However, the 3D structure of mechanosensory organelles and organization of the sensory molecules are absent. This information allows molecular interpretation of how the force-sensitive molecules transduce mechanical signals and how the specialized structures are formed. To solve these issues, we recently established a protocol for serial section electron tomography (SS-ET) [11,16,17]. Using this protocol, we successfully resolved the delicate structure and molecular organization of the MOs. Together with cell biological and modeling analysis, the structural analysis has been useful in understanding the working mechanism of fly MOs and how they are formed.
Electron tomography (ET) acquires a series of 2D projection images, known as tilt series, by incrementally varying the orientation of the sample relative to the incident electron beam. Using computational methods, the set of 2D images can be processed to yield a tomographic volume reconstruction at nanometer resolution [18,19]. However, the imaging depth of ET is restricted to a few hundred nanometers due to the limited penetration depth of electrons. Therefore, it is recommended that the thickness of plastic samples should be less than 300 nm for the most widely used TEMs. SS-ET, which merges the tomographic volume constructions from consecutive sections, compensates for this limitation to some extent [20,21].
Materials and reagents
Biological materials
Drosophila flies, maintained on a standard medium at 23 to 25 °C
Reagents
Carbon dioxide (CO2) (any supplier)
Sodium dihydrogen phosphate dihydrate (NaH2PO4 ·2H2O) (analytical grade) (e.g., Sinopharm Chemical Reagent Co., Ltd., catalog number: 20040718)
Disodium hydrogen phosphate dodecahydrate (Na2HPO4 ·12H2O) (analytical grade) (e.g., Sinopharm Chemical Reagent Co., Ltd., catalog number: 10020318)
Albumin bovine serum (BSA) (Sigma-Aldrich, catalog number: A9647)
Liquid nitrogen (any supplier)
Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19110)
Glutaraldehyde (25% aqueous solution) (Electron Microscopy Sciences, catalog number: 16220)
Uranyl acetate (Polysciences, catalog number: 21447 or Electron Microscopy Sciences, catalog number: 22400)
Lead citrate (Electron Microscopy Sciences, catalog number: 17800)
Anhydrous acetone (analytical grade) (e.g., Electron Microscopy Sciences, catalog number: 10015)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465)
Araldite/embed-812 embedding kit (Electron Microscopy Sciences, catalog number: 13940), including embed-812, araldite, DDSA, and DMP-30
Teflon (Miller-Stephenson Chemical Co., Inc., U.S.A.)
Cyanoacrylic adhesive (any supplier)
Chloroform (analytical grade) (e.g., Electron Microscopy Sciences, catalog number: 12540)
Formvar 15/95 resin (Electron Microscopy Sciences, catalog number: 15800)
Methanol (analytical grade) (e.g., RHAWN, catalog number: R007542-4L)
Gold colloid (BBI Solutions, catalog number: EM. GC15)
Solutions
Phosphate buffer (0.1 mol/L, pH 7.2) (see Recipes)
20% BSA solution (see Recipes)
10% osmium tetroxide (see Recipes)
20% uranyl acetate (see Recipes)
Freeze-substitution solution (see Recipes)
30%, 50%, 70% aqueous methanol (see Recipes)
2% uranyl acetate (see Recipes)
10 mol/L NaOH (see Recipes)
0.4% lead citrate (see Recipes)
Araldite/embed-812 embedding media (see Recipes)
1%–3% formvar casting solution (see Recipes)
Recipes
Phosphate buffer (0.1 mol/L, pH 7.2)
Reagent Final concentration Quantity or volume
NaH2PO 4·2H2O 0.437% (w/v) 0.437 g
Na2HPO4·12H2O 2.579% (w/v) 2.579 g
Ultrapure water n/a 100.0 mL
Total n/a 100.0 mL
20% BSA (wt/vol) solution
Reagent Final concentration Quantity or volume
BSA 20% (w/v) 0.2 g
Phosphate buffer (0.1 mol/L, pH 7.2) n/a 0.8 mL
Total n/a 1.0 mL
10% osmium tetroxide
Reagent Final concentration Quantity or volume
Osmium tetroxide 10% (w/v) 1.0 mg
Anhydrous acetone n/a 10 mL
Total n/a 10 mL
Caution: Osmium tetroxide is highly toxic and fatal. Handle it in a fume hood and wear personal protective equipment.
20% uranyl acetate
Reagent Final concentration Quantity or volume
Uranyl acetate 20% (w/v) 0.2 mg
Anhydrous methanol n/a 1.0 mL
Total n/a 1.0 mL
Caution: Uranyl acetate is toxic and slightly radioactive. Handle it in a fume hood and wear personal protective equipment.
Freeze-substitution solution
Reagent Final concentration Quantity or volume
10% osmium tetroxide 1% (w/v) 1.0 mL
20% uranyl acetate 0.1% (w/v) 50 μL
25% aqueous glutaraldehyde 0.5% (w/v) 200 μL
Ultrapure water 4% (v/v) 250 μL
Anhydrous acetone n/a 8.5 mL
Total n/a 10 mL
Caution: Freeze-substitution solution contains toxic and radioactive compounds. Handle them inside a fume hood and wear personal protective equipment.
30%, 50%, 70% aqueous methanol
Add 30, 50, and 70 mL of anhydrous methanol into 70, 50, and 30 mL of ultrapure water, respectively. Then, mix each aqueous methanol thoroughly by vortexing. Leave them at room temperature overnight or shake them in an ultrasonic cleaner for ~10 min to remove all air bubbles inside the aqueous methanol.
2% uranyl acetate
Note: Filter the solution using a 0.22 μm syringe filter unit or centrifuge the solution at 10,625× g for 10 min and then collect the filtrate or supernatant to use.
Reagent Final concentration Quantity or volume
Uranyl acetate 2% (w/v) 0.2 g
70% aqueous methanol n/a 10 mL
Total n/a 10 mL
10 mol/L NaOH
Note: Boil the ultrapure water for 15–30 min and then cool it to room temperature before use.
Reagent Final concentration Quantity or volume
NaOH 10 mol/L 4.2 g
Cooled boiled water n/a 10 mL
Total n/a 10 mL
0.4% lead citrate
Note: Add 0.2 g of lead citrate into 50 mL of cooled boiled water in a 50 mL centrifuge tube. Immediately turn the tubes upside down several times and add 500 μL of 10 mol/L NaOH in it. Shake the tubes on an orbital shaker for at least 30 min and place them in the fume hood for at least two days before use.
Reagent Final concentration Quantity or volume
Lead citrate 0.4% (w/v) 0.2 g
10 mol/L NaOH n/a 500 μL
Cooled boiled water n/a 50 mL
Total n/a 50 mL
Araldite/embed-812 embedding media
Reagent Final concentration Quantity or volume
Embed-812 n/a 62.0 g
Araldite 502 n/a 44.4 g
DDSA n/a 122.0 g
DMP-30 n/a 5.5 mL
Total n/a n/a
Caution: Unpolymerized embedding media are toxic. Handle them in a fume hood and wear personal protective equipment.
1%–3% formvar casting solution
Reagent Final concentration Quantity or volume
Formvar 15/95 resin 1%–3% (w/v) 1–3 g
Chloroform n/a 100 mL
Total n/a 100 mL
Laboratory supplies
Microcentrifuge tubes (0.2, 0.5, 1.5, and 2.0 mL) (e.g., Eppendorf, catalog number: 0030124332, 0030121023, 0030120086, and 0030120094, respectively)
Centrifuge tubes (15 and 50 mL) (e.g., Corning, catalog number: 430052 and 430290, respectively)
Petri dishes (30 and 100 mm) (e.g., Corning, catalog number: CLS430165 and CLS430167, respectively)
2 mL screwcap microtubes (Sarstedt AG & Co. KG, catalog number: 72.694.005)
0.22 μm syringe filter unit (Merck Millipore Ltd. catalog number: SLGPR33RS)
Aluminum foil (any supplier)
Pipette tips (0.1–20, 2–200, and 50–1,000 μL) (e.g., Eppendorf, catalog number: 0030000838, 0030000870, and 0030000919, respectively)
Cellulose capillary tubes (Leica Microsystems, catalog number: 16706869)
100 μm deep membrane carriers (Leica Microsystems, catalog number: 16707898)
Stainless steel surgical blades #23 (any supplier)
Surgical blade handles #3 (any supplier)
Plastic beakers (any supplier)
Plastic droppers (any supplier)
Amber glass bottles (any supplier)
Parafilm (Bemis, catalog number: PM996)
Fine tip needle (any supplier)
Glass slides (any supplier)
Slide storage boxes (any supplier)
Single-edge razor blades (any supplier)
Double-edge razor blades (any supplier)
Copper slot grids (Gilder Grids, catalog number: GS2X1-C3)
Filter papers (any supplier)
Cylindrical funnel (any supplier)
ACLAR® 33C film (Electron Microscopy Sciences, catalog number: 50425-10)
Kimwipes tissue (Kimberly-Clark, catalog number: 34155)
Grid storage box (any supplier)
Equipment
Fly anesthesia pad (e.g., Genesee Scientific, catalog number: 59-114)
Tabletop centrifuge (Beckman Coulter, model: Microfuge® 16)
Water purification system for ultrapure water (Merck Millipore, model: Milli-QTM Advantage A10TM)
Superfine vannas scissors (World Precision Instruments, catalog number: 501778)
Sharp forceps (Fine Science Tools, catalog number: 11251-30)
Forceps (Zhongjingkeyi Technology Co. Ltd., catalog number: EZ3-SA)
Pipettes (0.1–2.5, 2–20, 20–200, and 100–1,000 μL) (e.g., Eppendorf, catalog number: 3123000217, 3123000292, 3123000250, and 3123000268, respectively)
Ultrasonic cleaner (e.g., Shenzhen Sweep Technology Co., Ltd., model: SWP-DTP045)
Orbital shaker (e.g., Corning, catalog number: 6780-FP)
Analytical balance (e.g., Mettler-Toledo, catalog number: ME104E)
Stereoscope (Olympus Corporation, catalog number: SZ61)
High-pressure freezer (Leica Microsystems, catalog number: EM HPM100 or EM PACT2)
Automatic freeze-substitution device (Leica Microsystems, catalog number: EM AFS2)
Knifemaker (Leica Microsystems, catalog number: EM KMR3)
Ultramicrotome (Leica Microsystems, catalog number: EM UC7)
35° diamond knife (Diatome, catalog number: DU3530)
Magnetic stirrer (e.g., Heidolph, catalog number: MR3002)
Vortex mixer (e.g., Scientific Industries, model: Vortex-Genie2)
Tube revolver (e.g., Thermo Fisher Scientific, catalog number: 88881002)
Oven (any supplier)
Casting film device (Electron Microscopy Sciences, catalog number: 71305-01)
Grid staining matrix system (Electron Microscopy Sciences, catalog number: 71179-01), including a matrix body with handle and cover, a red staining vessel, and a blue staining vessel
FEI Tecnai F20 TEM equipped with a Gatan US4000 (895) CCD camera and controlled with FEI automated tomography software Xplore 3D
Software and datasets
TEM User Interface software is used in conjunction with TEM Imaging and Analysis controlling software and Digital Micrograph controlling software to acquire micrographs with the TEM
Xplore3D (TEM Tomography Version 3.0) is used for automated tilt series acquisitions
IMOD software package is used to build 3D reconstruction from the raw tilt series [22]
Amira ZIB edition 2016.16 is used for tracing microtubules, stitching consecutive sections, surface segmentation, and measurement
Procedure
Sample preparation using high pressuring and freeze-substitution
Sample dissection
Maintain all flies on standard medium at 23–25 °C.
Anesthetize flies with CO2 on a fly anesthesia pad and cut the fly′s head, wings, and legs off with superfine vannas scissors under a stereoscope.
Immediately immerse the remainder of the fly carcass into a big drop of phosphate buffer on a glass slide and remove the air bubbles around the haltere with the tip of sharp forceps.
Dissect the haltere in phosphate buffer and immediately transfer the haltere into a new drop of phosphate buffer on another slide.
High pressuring and freeze-substitution
Aspirate the sample into the cellulose capillary tube and use the back of the stainless-steel surgical blades (installed on the surgical blade handles when using) to press the cellulose capillary tube at both sides of the sample without damaging the sample. By doing so, a small compartment is created to trap the sample (Figure 1A).
Critical: Tissues with a hydrophobic surface do not have a strong affinity with the BSA aqueous solution. Therefore, the transparent, porous cellulose capillary tubes with an inner diameter of 200 μm can be used as special containers to help the samples (diameter lower than 200 μm) immerse into the cryoprotectant.
Figure 1. Sample preparation using high pressuring and freeze-substitution. A. A haltere is aspirated into the cellulose capillary tube. B. A sample capsule is transferred into the cavity of the membrane carrier filled up with cryoprotectant. C. A carrier assembly is formed by two membrane carriers with the sample in between. D. Side view of a Leica EM HPM100 high-pressure freezer. E. A carrier assembly is placed in a high-pressure freezer for cryo-fixation. F. Side view of a Leica EM AFS2 freeze-substitution device. G. A microtube containing freeze-substitution solution and samples is placed into an automatic freeze-substitution device for substitution. H. A Teflon-coated slide is wrapped with parafilm strips at both its sides. I. The samples are separated apart from the membrane carriers using forceps and a fine tip needle. J. After polymerization, the samples are flat embedded in the resin layer.
Cut along the cutting line to produce a small sample capsule (<1.4 mm long) to fit the membrane carrier.
Place a 100 μm deep membrane carrier onto a filter paper with the flat side down and add 20% BSA as cryoprotectant into the cavity of the carrier. Immediately transfer the sample capsule into the cavity and immerse it into the cryoprotectant (Figure 1B).
Place another carrier with the flat side down onto the first carrier and carefully press the top carrier down to close the carrier assembly (Figure 1C). The overflow of cryoprotectant is carefully soaked away using a wedge of absorbent paper.
Critical: Air bubbles will affect pressure transmission and compromise the freezing quality. Ensure that the membrane carrier is slightly overloaded with cryoprotectant to prevent any air inclusion inside the carrier.
Once the top carrier is in place, immediately transfer the carrier assembly to a high-pressure freezer like Leica EM HPM100 to freeze the sample (Figure 1D and E).
Note: The operating manual of this machine provides a complete, detailed freezing process and we will not describe it here. Be sure to read the operating manual carefully and be trained before use.
Pause point: The frozen samples can be stored in liquid nitrogen for several months.
Under liquid nitrogen, transfer the carriers into 2 mL screwcap microtubes (<10 carriers per microtube) containing 1 mL of liquid nitrogen–precooled freeze-substitution solution and immerse the microtubes into the liquid nitrogen.
Transfer these microtubes to the precooled (-90 °C) automatic freeze-substitution device (Figure 1F and G).
Critical: The freeze-substitution solution will expand during the process of freeze-substitution as the temperature will rise. In order to relieve pressure, be sure to loosen the microtube cap or poke a hole on the cap.
Perform freeze-substitution using the following program: incubate at -90 °C for 40 h, warm gradually from -90 °C to -30 °C at a rate of 5 °C per hour, incubate at -30 °C for 8 h, warm up to 0 °C at the rate of 5 °C per hour, and keep at 0 °C for 1–6 h.
Infiltration and flat embedding
Freshly prepare a mixture of embedding media and anhydrous acetone with the ratio of 1:3, 1:1, and 3:1 (vol/vol) at room temperature and mix until homogeneous.
When the substitution program comes to 0 °C, transfer the microtubes on ice.
Wash the samples in ice-cold anhydrous acetone three times for 3 min and in anhydrous acetone at room temperature three times for 3 min.
After washing, infiltrate the samples with 1:3, 1:1, and 3:1 embedding media/acetone mixture and infiltrate for 1 h at each step at room temperature.
Replace the mixture with pure embedding media and infiltrate for overnight.
Change the pure embedding media and infiltrate for another 5–8 h on a tube revolver at room temperature.
During infiltration, prepare Teflon-coated slides, which are made by dipping the glass slides into Teflon solution for seconds, allowing them to dry in the air, and cleaning with Kimwipe tissues for use.
Critical: Be sure to use the Teflon-coated slides as they are easier to break from the polymerized embedding media.
Wrap both sides of the Teflon-coated slide with a stack of two thin strips of parafilm and flatten the parafilm strips with the fingers (Figure 1H).
Add a big drop of pure embedding media onto the center of the Teflon-coated slide and transfer all the carriers and samples into the resin drop under a stereoscope.
With a firm clamp on the carriers by forceps, slowly scrape the rim of the carrier cavity and separate the samples from the cavity using a fine tip needle (Figure 1I).
Remove all the empty carriers and place another Teflon-coated slide onto the first slide with the parafilm strips as spacer.
Critical: The Teflon-coated slides can be replaced by ACLAR® 33C film. Note that the aclar film easily bends and causes the non-uniform thickness of the layer of the embedding media. Therefore, be sure to place the aclar film onto a glass slide to avoid bending.
Transfer the sandwiched slides in an oven to polymerize the embedding media at 60 °C for at least 48 h.
After polymerization, separate the sandwiched slides using a single-edge razor blade and store the slides in a slide storage box (Figure 1J).
Pause point: After polymerization, the samples are stable indefinitely under moisture-free conditions at room temperature.
Serial sectioning
Casting formvar-coated grids
Prepare 1%–3% formvar casting solution before use.
Wash copper slot grids with anhydrous acetone and transfer these grids to a new 100 mm Petri dish lined with filter paper. Let these grids air dry and then cover the dish with its lid.
Use a cylindrical funnel with a velocity controller or a casting film device to cast formvar.
Close the velocity controller and fill casting solution nearly to the top (Figure 2A).
Figure 2. Casting formvar-coated grids. A. A glass slide is placed into a cylindrical funnel almost filled with formvar casting solution. B. Four cutting lines on formvar film coating are made using a single-edge razor blade. C. The formvar film coating on the glass slide detaches from the glass slide and then floats onto the water surface. D. The grids are placed onto the formvar film with the light-colored side facing up. E. The formvar film along with grids adheres to the coverslip when the coverslip is plunged into the water.
Clean a glass slide with Kimwipe tissue and place it inside the cylindrical funnel.
Open the velocity controller and allow the formvar casting solution to drain with a steady flow.
Leave the glass slide in the cylindrical funnel for a few seconds and then air dry. Finally, the surface of the glass slide is coated with a thin layer of formvar film (Figure 2B).
Critical: For a selected cylindrical funnel and prepared formvar casting solution, the faster the drainage rate, the thicker the formvar film, and vice versa. Therefore, we recommend adjusting the drainage rate to control the thickness of the formvar film.
Fill up a big container with ultrapure water and clean the water surface by spreading out a Kimwipe tissue on the water surface and then dragging it across the water surface.
Use a single-edge razor blade to cut four lines parallel to the four edges of the slide on the formvar film coating (Figure 2B).
Blow a breath on the rectangle and slowly dip the slide into the ultrapure water. The formvar film coating on the rectangle comes off the glass slide and floats on the water surface (Figure 2C).
Critical: The formvar film shows a difference in color under an incandescent light when floating on the water surface. A purple, blue, or green color signifies that the formvar film is too thick and will affect the contrast of tilt-series images. Silver or grey color suggests that the formvar film is too thin and breaks easily while acquiring tilt series under a TEM. In order to meet the imaging requirements, we recommend using formvar film coating with light-gold or dark-gold color.
Use forceps to place the washed grids onto the formvar film with the light-colored side (rough side) facing up (Figure 2D).
Place a clean glass coverslip or a sheet of parafilm against the edge of the formvar film and plunge the coverslip or parafilm vertically into the water (Figure 2E). The formvar film along with grids is stuck to the surface of coverslip or parafilm.
Slowly lift the coverslip or parafilm out of the water and transfer it into a 100 mm Petri dish with grid-covered side facing up. Cover the dish with its lid and let the grids dry before use.
Poke a few holes around the outer rim of the grids and then scratch carefully along the outer rim using the tip of the forceps. Finally, pick up the formvar-coated grids with forceps and transfer the grids into a grid storage box.
Serial sectioning
Cut the sample out from the polymerized embedding media and mount the sample block onto a blank resin block using cyanoacrylic adhesive.
Note: Serial sectioning technique involves a great deal of skill. Users unfamiliar with this technique are advised to work with a skilled operator.
Under an ultramicrotome (Figure 3A and B), roughly trim the sample block to a pyramid frustum using a double-edge razor blade (Figure 3C).
Figure 3. Sample trimming. A and B. Side view of an ultramicrotome (A); the white box in (A) indicates the enlarged region shown in (B). C. The sample block is roughly trimmed to a pyramid frustum. D. The pyramid frustum is finely trimmed to a smaller one with parallel top and bottom edges on the block face surface.
Set the angle of the segment arc to 0°, clearance angle to 6°, and knife block to 0°.
Smooth the block face surface by repeated removal of 200 nm sections from the surface using a glass knife.
Finely trim the pyramid frustum to a smaller one using a double-edge razor blade (Figure 3D).
Critical: Straight section ribbons are ideal for pickup. Therefore, ensure that the top and bottom edges on the face surface are as parallel as possible because this is a must to produce a straight section ribbon.
Install a 35° diamond knife on knife block and manually move the knife block northward until the knife edge is very close to the block face surface.
Switch on the backlight of the ultramicrotome only.
Carefully move the diamond knife in east-west and north-south directions and move the sample block up and down until a band of reflected light appears on the block face surface (Figure 4A ).
Orientate the angle of the knife block until the top and bottom edges of the band of reflected light are parallel (Figure 4A).
Figure 4. Schematic drawing of the alignment process between the sample block and diamond knife. A. When the sample block is very close to the knife edge, a band of reflected light appears on the block face surface under the illumination of the backlight. Orientate the knife block (the knife is accordingly rotated in the direction indicated by the curved arrows) until the top and bottom edges of the band of reflected light are exactly parallel with each other. B. When the sample block is slightly higher than the knife edge, there is a gap between the bottom edge of the block face and the knife edge. Orientate the specimen holder (the sample block is accordingly rotated in the direction as indicated by the curved arrows) until the bottom edge of the block face surface is exactly parallel to the knife edge. C and D. Move the sample block to the positions so that a band of reflected light appears again and orientate the segment arc (the sample block is accordingly rotated in the direction as indicated by the curved arrows) until the band of reflected light remains the same width as the sample moves up and down.
Move the sample above the diamond knife and orientate the specimen holder until the bottom edge of the block face surface is parallel to the knife edge (Figure 4B).
Move the sample down until a band of reflected light appears on the block face surface again and orientate the segment arc until the width of the band of reflected light does not change when moving the sample up and down (Figures 4C and D).
Repeat the alignment steps until both the block face surface and its top and bottom edges are exactly parallel to the edge of the diamond knife.
Set cut start and cut end positions and switch on the top light.
Fill the knife reservoir with ultrapure water and adjust the water level until it becomes silvery.
Set feed at 300 nm and start cutting at a high cutting speed, e.g., 20 mm/s.
As soon as the first section is cut, reduce the speed to ~1 mm/s and remain at that speed for the entire cutting process. When the length of the section ribbon is suitable for the grid slot, stop cutting and use an eyelash tool to gently touch the knife edge to separate the section ribbon from the knife edge (Figure 5A).
Critical: As soon as the section ribbon is separated, immediately begin to cut the next ribbon. Otherwise, the first one or two sections of the next ribbon will be too thin or too thick.
Figure 5. Pickup of section ribbons. A. A section ribbon is separated from the knife edge. B. The section ribbons float on the water surface in the knife reservoir. C. A section ribbon is picked up onto the formvar-coated grid with the help of an eyelash tool. D. A section ribbon lays flat on a formvar-coated grid.
Arrange all the ribbons in the knife reservoir using an eyelash tool (Figure 5B).
Pick up the section ribbons onto formvar-coated grids (Figures 5C and D) and store the grids in a grid storage box. For pickup, clamp the grid bar with forceps, vertically dip the grid into the water with the dark-colored side (smooth side) facing to the section ribbon, and move the grid toward the ribbon or move the ribbon to the grid with the help of an eyelash tool. Once the section ribbon touches the formvar film at the center of the grid slot, slowly lift the grid and let the ribbon lay flat on the grid.
Pause point: Sections can be stored in the grid storage box for several months in dry conditions at room temperature.
Post-staining with heavy metals
Prepare 30%, 50%, and 70% aqueous methanol, 2% uranyl acetate, and 0.4% lead citrate before use.
Load the grids into the wells of the matrix body (one grid per well, up to 25 grids) of the grid-staining matrix system.
Place the matrix body into one staining vessel, add 70% aqueous methanol to completely cover all the grids for a few seconds, and replace it with 2% uranyl acetate to stain for ~10 min (Figure 6A).
Critical: At the beginning of each staining, be sure to move the matrix body back and forth several times inside the staining vessel to remove air bubbles wrapped within the wells. Otherwise, it will cause contamination of the sections.
Figure 6. Post-staining. A and B. Post-staining is performed using a grid staining matrix system. The red and blue staining vessel are used for uranyl acetate or lead citrate staining, respectively. C. Gold nanoparticles are added on both sides of the sections by immersing the grid into the drop of gold nanoparticle colloid.
Sequentially replace the staining solution with 70%, 50%, and 30% aqueous methanol for washing. Wash three times for each step, followed by washing with ultrapure water three times in the staining vessel and another three times in plastic beakers.
Transfer the matrix body into the other staining vessel and immediately add 0.4% lead citrate to stain for ~5 min (Figure 6B). Then, wash with ultrapure water three times in the staining vessel and another three times in plastic beakers.
Take the matrix body out, dry it with Kimwipe tissues, and carefully transfer the grids back into the grid storage box.
Critical: The application of post staining with soluble heavy metal–containing negative staining salts before imaging is indispensable for significantly improving the image contrast.
Pipette 100 μL of gold colloid into a 0.2 mL tube, vortex for a few seconds, centrifuge at ~295× g for 30 s at room temperature, and pipette the supernatant into a new 30 mm Petri dish.
Clamp the grid bar with forceps, dip the grid into the drop of supernatant for 10–30 s (Figure 6C), slowly lift the grid, carefully absorb the residual solution with a wedge of absorbent paper, and place the grid back into the grid storage box.
Critical: The gold nanoparticles serve as fiducial markers for subsequent image alignment. Be sure to check the density and distribution of gold nanoparticles in the area of interest under TEM before investing time in electron tomography. We recommend staining several times for 10 s each time until 40–100 gold particles are distributed on the surface of the area of interest.
Pause point: Sections can be stored in the grid storage box for several months in dry conditions at room temperature.
Acquisition of dual-axis tilt series
Check general conditions of TEM (Figure 7A). Here, we only describe the acquisition process using the FEI Tecnai F20 TEM equipped with Xplore3D.
Note: The tilt series in the protocol are acquired using a FEI Tecnai F20 or FEI Tecnai F30 TEM. Laboratories without this equipment can use existing TEMs equipped with automated acquisition software to achieve this purpose.
Critical: Microscope training is required before users are allowed to perform TEM. Please contact microscopists for training or assisted use.
Make sure that the vacuum level of the gun, column, and camera is reasonable and that the column valves are closed (yellow is closed, gray is open).
Fill up the Dewar flask with liquid nitrogen to cool down the Cold Trap.
Start the TEM User Interface, Digital Micrograph, and TEM Imaging and Analysis software if they are closed.
Make sure high tension is on and the value of the high tension is at 200 kV.
Make sure the FEG Control parameters are set correctly (e.g., extraction voltage is 3950 V and gun lens is 3).
Load the grid on the tomography single-tilt holder and insert the holder into the microscope.
Critical: Dual-axis tilt series are collected by tilting the object around two approximately orthogonal tilt axes (x- and y-axis, respectively) [22,23]. Be sure to load the grid on the holder with a constant orientation (Figures 7B, C, and D). Otherwise, data processing about stitching consecutive sections is going to be a problem.
Figure 7. The grid loaded on the tomographic holder with a constant orientation. A. Side view of a transmission electron microscope (TEM). B. The tip of a tomographic holder. C and D. The grid loaded on holder with a constant orientation. There are grid sides, grid bars, and sections that can be used as orientation indicators. To acquire first tilt series, for example, the grid is loaded on the holder with the dark-colored side (smooth side) facing up, the straight bar perpendicular to the long axis of the holder (holder tilt axis), and the top edges of sections facing forward (C). Rotate the grid 90° clockwise for acquisition of second tilt series (D).
Select a lower magnification (e.g., ~2,000×) and set spot size 1.
Click the Col. Valves Closed button to open the column. The beam should now be visible on the main screen. Adjust the intensity to spread the beam over the screen.
Scan across the grid to locate an area of interest and then center the beam.
Go to a higher magnification (e.g., 100,000×) and bring the area of interest to eucentric height.
Insert a selected condenser aperture and center it.
Correct condenser astigmatism to ensure the beam is round and expands concentrically.
Align gun tilt (optional), gun shift, beam tilt pp X/Y, beam shift, and rotation center.
Insert a selected objective aperture and center it.
Correct the objective astigmatism and focus the image.
Go back to a lower magnification (e.g., ~2,000×) and spread the beam out to fill the screen.
Center the area of interest and expose the sample to the beam for 2 min.
Critical: The plastic section will shrink in each dimension when viewed in electron microscope. Before data acquisition, be sure to bake the area of interest with an electron beam for inducing the rapid phase of the shrinkage so that the tilt series is recorded over the slow phase, which is tolerable for SS-ET.
Choose a working magnification (e.g., SA 29,000× resulting in a pixel size of ~1 nm).
Tilt to high angle to check what tilt range can be used without shielding the area of interest. A tilt-range of ±60° is needed for good tomography results.
Go back to zero tilt degree and use the Digital Micrograph to record images with 1 s of exposure time, binning 2 (an image has 2048 × 2048 pixels), and full image frame.
Adjust the beam intensity and defocusing until the image has good contrast and appearance. The optimal beam intensity and defocusing values are going to be used for acquiring the tilt series.
Start the Xplore 3D software; the Tomography Tasks panel comes out.
Critical: The application instructions of Xplore 3D provide a step-by-step guide for microscope users and the help file offers a detailed description of all parameter settings. Here, we just depict a typical procedure and parameters needed to acquire a good TEM tilt series at medium-high magnification.
In the Tomography Tasks panel, click the Holder Calibration and the Select View panel turn to Holder Calibration Data.
Click the button Load and load the selected hold calibration file specially created for plastic sample from the save directory into the system.
Go back to the Tomography Tasks panel, click the Acquire Tilt Series, and the Select View panel turns to Camera Acquisition Parameters.
Adjust the settings for Search, Focus, Exposure, and Tracking if necessary. For exposure mode, we recommend using full image size, binning 2, and 1 s of exposure time.
Press Apply to activate the changes and press the Proceed button to continue.
The Select View panel turns to Tilt Series Parameters. Set the parameters. Generally, Max. Negative Angle and Max. Positive angle are -60° and +60° and Start angle is -60°. Check the Continuous for the plastic section, which allows the object to be tilted from -60° to +60°. Stage relaxation time is 4–6 s, which is needed to stabilize the sample stage before focusing and exposure are performed. Choose MRC Series format for tilt series dataset and put the Series Name; note that the names of first and second tilt series are distinguished by adding the letter a or b to the same common name (e.g., “*a” for first tilt series and “*b” for second tilt series). Uncheck the Start At Eucentric Height and Start with AutoFocus since eucentric height and focus were done before. The 1° Tilt Step allows more image information to be obtained for tomographic reconstruction. Let the software do an autofocus every 4–6° at Low Tilt angle and every one degree at High Tilt (above 50–55°) for taking well-focused images. Set the Applied defocus value (e.g., -1 μm) to enhance the contrast of images. Check Track Before Acquisition to manually keep the feature of interest well centered throughout the whole tilt series.
Press Apply to activate the changes and press Proceed to automatically acquire tilt series.
Critical: The first few images at high tilts are normally of poor quality; be sure to manually center or focus the region of interest if necessary.
Wait until the acquisition of first tilt series is finished.
Go to the save directory and check the tilt series stored in a file with the suffix .mrc. Also, keep the log files (only a few kB in size) as they might help you in the future if data acquisition was not satisfactory.
Collect first tilt series for the other sections at the same working magnification.
After data acquisition, set the magnification to the lowest “M” setting, spread the beam to cover the screen, close the column valves, and pull the holder out of the microscope.
Rotate the grid by 90° clockwise and collect the second tilt series for each section at the same working magnification.
Build a dual-axis tomogram reconstruction with the Etomo program in the IMOD software package [22]. The IMOD software package and a detailed Etomo tutorial can be downloaded at the IMOD homepage. Therefore, we do not provide a comprehensive tutorial for reconstruction.
Tracing microtubules from tomographic reconstructions
Start the Amira ZIB edition 2016.16.
Click the Project button in the workroom toolbars to arrive at the Project workroom.
Load a reconstruction result with the suffix .rec into the system. Once the data object has been loaded, it is represented by a green icon in the Project View.
Click on the green data icon with the right mouse button and a popup menu appears containing all modules, which can be used to process this type of data. Choose the Slice module to display the data object in the 3D view window.
Soften the image object with a smoothing module or/and crop it using the Crop Editor module if needed.
Compute normalized cross-correlation for the image data. For computing, attach the Cylinder Correlation module to the image data, define the microtubules by setting the parameters in the Properties area, and finally press the Apply button.
After several hours of computation, two objects, i.e., “*CorrelationField.am” and “*OrientationField.am,” are produced.
Trace centerlines of microtubules. To trace, attach a Trace Correlation Lines module to the “*CorrelationField.am” object and, in the Properties area of Trace Correlation Lines module, select “*CorrelationField.am” object at the Data port and “*OrientationField.am” object in Orientation Field port to connect the two objects to the Trace Correlation Lines module.
Set all other parameters in the Properties area of Trace Correlation Lines module and finally press Apply button to proceed.
Just a few minutes later, a new object, “*CorrelationLines.am” for centerlines of microtubule, is produced and can be displayed with LineRaycast module.
Switch into the Filament workroom and edit the traced centerlines until they match exactly to the real microtubules.
Trace microtubules for the other tomographic reconstructions.
Stitching consecutive tomographic reconstructions to form a single larger volume
In the Amira ZIB edition 2016.16, switch to the Project workroom.
Right-click in the blank of the Project View area and select the Create Object. A dialog box then appears; enter and select the SerialSectionStack module in the drop-down menu. A green icon of SerialSectionStack appears in the Project view area.
Click Add Files in the Properties area of SerialSectionStack module, choose the tomographic reconstructions named “*.rec” and their corresponding microtubule objects named “*CorrelationLines.am,” and press the Open button to load these files.
Critical: After loading, ensure that the tomographic reconstructions are in the same order as their sectioning sequence and no changes are made to these reconstruction results and their corresponding microtubule objects. Otherwise, the process of stitching adjacent tomographic reconstructions cannot be performed.
Add the SerialSectionAligner module to SerialSectionStack and the 3D view window is separated into four panels. In the lower-right panel, each bar represents a tomographic reconstruction, and the blue and yellow bars represent the two working reconstructions, whose microtubules are displayed as blue or yellow dots in the upper and lower left panels and whose slices, together with these dots, are displayed in the upper-right panel.
Click the slide bars in the lower-right panel to choose the two working reconstructions.
Left-click twice in the upper-left panel and red solid circles appear on the upper- and lower-left panels.
Drag these red solid circles until the blue dots are well matched with the yellow dots.
Check Matching in the Properties area of SerialSectionAligner module.
Hold down the Ctrl key on the keyboard and left-click the matched blue and yellow dots, in the upper-right panel, to connect the two microtubule segments.
After connection, check the Alignment in the Properties area of SerialSectionAligner module and choose another two working reconstructions to complete the connection of microtubule segments in the same way mentioned above.
After all the microtubule segments are connected, press the Create button. A few minutes later, two Objects are produced, and their icons appear in the Project View area: one is an image object of stitched consecutive reconstructions and the other is a line object of connected microtubule segments.
Surface segmentation
Load the image object from the directory into the Amira ZIB edition 2016.16.
Go to the Segmentation workroom and a new green icon named “*labels.am” is created in the Project View area.
Set an appropriate magnification by pressing the zoom in and out buttons in the Zoom and Data Window region.
Draw the outlines of the same structure through all the slices using the brush. If the structure has little change among slices, draw the structure every few slices and click the Interpolate from the Selection menu bar to select the structure in between marked slices.
Note: The brush is one of the basic segmentation tools. Other segmentation tools can also be used for this job.
Select a Material in the material list and click the plus sign in the Selection region. The selected pixels in all slices are now assigned to the selected Material. Of course, more meaningful names or colors can be used to the material. The new Material can be added by pressing the New button or from the right-button menu.
Go to slices and assign the other structures into different Materials.
After drawing, add the Generate Surface module to “*labal.am” object in the Project workroom and press the Apply button. A dialog comes out; press Continue.
A few minutes later, a new icon named “*surf.am” is created and the segmentation result can be displayed with the Surface View module.
Add the Animate Ports to the Slice or other display modules and further adhere the Movie Maker module to Animate Ports.
Right-click in the blank of the Project View area to create the Camera-Orbit.
Adjust the setting of Animate Ports, Movie Maker, and Camera-Orbit to generate a movie of the segmentation results.
Measurement
In Amira ZIB edition 2016.16, left-click the Measuring Tool button at the top of the 3D view window and choose the 3D Length from the pull-down menu.
Note: There are several tools for measurement, including 2D Length, 2D Angle, 3D Angle, 3D Box, and 3D circle. Which one is selected depends on the structures that users want to measure.
Left-click one point on the object and then drag the mouse to another point in 3D space. The length of the line is shown on the 3D view window and a new icon named Measurement is created in the Project view area.
Measure all geometrical parameters of the fly MOs. These parameters can be used for further analysis.
Data analysis
In this protocol, SS-ET is used to resolve the 3D structures of fly MOs. The dual-axis tilt series are captured to reconstruct the tomogram (Figures 8A and B). Then, microtubules are traced and applied as fiducial markers to stitch the sections in z-axis (Figures 8C and D). Finally, tomograms of ten consecutive sections are combined into a single larger volume (Figure 8E). From the lateral view, the morphology of the modified cilium can be recognized (Figure 8F). In the modified cilium, the basal body is located at the base and the cilium extend from the basal body to the tubular body, which is nearly spherical. Above the distal end of the tubular body is the MO. Of note, the material loss induced by knife sectioning and distortions caused by electrons irradiation, while tolerable, are visible at the connection between sections. From the cross-sectional view of the MO, the smooth membrane, the microtubules arranged in two rows with electron-dense materials in between, and the high density of the regularly spaced membrane-microtubule connectors are all recognized (Figure 8G).
All structural measurements in 3D reconstruction were performed in Amira as described above.
Figure 8. Representative data using the protocol. A. Several individual images at different tilt angles are from the first tilt series (upper) and second tile series (lower). Scale bar: 0.5 μm. B. Tomogram reconstructed from dual-axis tilt series. Scale bar: 0.5 μm. C. Microtubules were traced from the tomographic reconstruction. Scale bar: 100 nm. Each microtubule was shown as a yellow rod. D. Microtubules were used as fiducial markers (upper) to stitch the sections (lower) in z-axis. Scale bar: 100 nm. E. Ten consecutive sections are combined into a single larger volume. Scale bar: 0.5 μm. F. Representative lateral view of a modified cilium generated from the single larger volume. Scale bar: 0.5 μm. G. Representative cross-sectional view of a mechanosensory organelle (MO) and its internal architectures. EDM, electron-dense materials. MMC, membrane-microtubule connector. Scale bar: 100 nm.
Validation of protocol
This protocol has been used and validated in the following published articles:
Song, X. et al. (2023). DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles. J Cell Biol 222(10): e202209116, DOI: 10.1083/jcb.202209116.
Sun, L. D. et al. (2021). Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia. J Cell Biol 220(1): e202004184, DOI: 10.1083/jcb.202004184.
Sun, L. D. et al. (2019). Ultrastructural organization of NompC in the mechanoreceptive organelle of Drosophila campaniform mechanoreceptors. Proc Natl Acad Sci USA 116(15): 7343–7352, DOI: 10.1073/pnas.1819371116.
General notes and troubleshooting
General notes
We suggest that scientists implementing the protocol described here should have extensive knowledge of TEM techniques. Also, scientists should already be familiar with manipulations of preparing and handling specimens and 3D tomographic image processing software.
This protocol, with minimal adaptive modifications, could also be applied to the studies on other sensory processes, like olfaction, gustation, vision, etc. Moreover, 3D structural analysis is helpful to understand non-sensory processes, such as neuronal cytoskeleton regulations, synaptic remodeling, membrane-bound vesicle dynamics, intracellular trafficking, etc.
This protocol is still a time-consuming approach for ultramicrotomy, microscopy, and data processing; therefore, the choice of the technology must be made based on the specific biological questions.
Troubleshooting (Table 1)
Table 1. Troubleshooting
Issue Possible reason Solution
Failure of aspirating the sample into the cellulose capillary tube Sample with hydrophobic cuticle easily floats on the surface of aqueous solution, which, in turn, leads to the failure. Press the sample to the bottom of the droplet with forceps and make sure there are no air bubbles around the sample.
Samples become black Osmium tetroxide in freeze-substitution solution forms osmium black at room temperature. Wash the samples several times with ice-cold anhydrous acetone to thoroughly remove the fixatives.
Section ribbon is not formed, or section ribbons easily break into short ribbons The trimmed sample block does not meet the requirements for serial sectioning. Retrim the sample block until the top and bottom edges of block face surface are parallel.
Improper alignment between block face surface and diamond knife. Repeat the alignment process to make the alignment perfect.
Diamond knife is contaminated with hydrophobic substances such as oil from hands. Thoroughly clean the knife with 70% alcohol.
Poor contrast after post-staining Different sources of heavy metals. Select the optimal staining time for 2% uranyl acetate and 0.4% lead citrate.
Contamination of sections Precipitates occur in the heavy metal solution. Filter or centrifuge the solution, then collect the filtrate or supernatant to use.
Acknowledgments
The authors thank members of the Cell Biology Facility and Electron Microscopy Facility of Tsinghua University for technical support. This work was supported by National Natural Sciences Foundation of China (32370730, 32070704 and 32350029).
Competing interests
The authors declare that they have no competing interests.
References
Gillespie, P. G. and Walker, R. G. (2001). Molecular basis of mechanosensory transduction. Nature 413(6852): 194–202. https://doi.org/10.1038/35093011
Lumpkin, E. A., Marshall, K. L. and Nelson, A. M. (2010). The cell biology of touch. J. Cell Biol. 191(2): 237–248. https://doi.org/10.1083/jcb.201006074
Singhania, A. and Grueber, W. B. (2014). Development of the embryonic and larval peripheral nervous system of Drosophila. WIREs Dev. Biol. 3(3): 193–210. https://doi.org/10.1002/wdev.135
Chalfie, M. (2009). Neurosensory mechanotransduction. Nat. Rev. Mol. Cell Biol. 10(1): 44–52. https://doi.org/10.1038/nrm2595
Cueva, J. G., Mulholland, A. and Goodman, M. B. (2007). Nanoscale organization of the MEC-4 DEG/ENaC sensory mechanotransduction channel in Caenorhabditis elegans touch receptor neurons. J. Neurosci. 27(51): 14089–14098. https://doi.org/10.1523/jneurosci.4179-07.2007
Effertz, T., Nadrowski, B., Piepenbrock, D., Albert, J. T. and Göpfert, M. C. (2012). Direct gating and mechanical integrity of Drosophila auditory transducers require TRPN1. Nat. Neurosci. 15(9): 1198–1200. https://doi.org/10.1038/nn.3175
Gillespie, P. G. and Müller, U. (2009). Mechanotransduction by hair cells: models, molecules, and mechanisms. Cell 139(1): 33–44. https://doi.org/10.1016/j.cell.2009.09.010
Keil, T. A. (1997). Functional morphology of insect mechanoreceptors. Microsc. Res. Tech. 39(6): 506–531. https://doi.org/10.1002/(sici)1097-0029(19971215)39:6<506::aid-jemt5>3.0.co;2-b
Liang, X., Madrid, J., Gärtner, R., Verbavatz, J. M., Schiklenk, C., Wilsch-Bräuninger, M., Bogdanova, A., Stenger, F., Voigt, A., Howard, J., et al. (2013). A NOMPC-dependent membrane-microtubule connector is a candidate for the gating spring in fly mechanoreceptors. Curr. Biol. 23(9): 755–763. https://doi.org/10.1016/j.cub.2013.03.065
Liang, X., Sun, L. and Liu, Z. (2017). Mechanosensory transduction in drosophila melanogaster. SpringerBriefs in Biochemistry and Molecular Biology: e1007/978–981–10–6526–2. https://doi.org/10.1007/978-981-10-6526-2
Sun, L., Gao, Y., He, J., Cui, L., Meissner, J., Verbavatz, J. M., Li, B., Feng, X. and Liang, X. (2019). Ultrastructural organization of NompC in the mechanoreceptive organelle of Drosophila campaniform mechanoreceptors. Proc. Natl. Acad. Sci. U.S.A. 116(15): 7343–7352. https://doi.org/10.1073/pnas.1819371116
Keil, T. A. (2012). Sensory cilia in arthropods. Arthropod Structure & Development 41(6): 515–534. https://doi.org/10.1016/j.asd.2012.07.001
Liang, X., Madrid, J. and Howard, J. (2014). The microtubule-based cytoskeleton is a component of a mechanical signaling pathway in fly campaniform receptors. Biophys J. 107(12): 2767-2774. https://doi.org/10.1016/j.bpj.2014.10.052
Thurm, U., Erler, G., Godde, J., Kastrup, H., Keil, T., Volker, W. and Vohwinkel, B. (1983). Cilia specialized for mechanoreception. J. Submicrosc. Cytol. Pathol. 15(1): 151-155. https://royalsocietypublishing.org/doi/10.1098/rstb.2019.0376
Zhang, W., Cheng, L. E., Kittelmann, M., Li, J., Petkovic, M., Cheng, T., Jin, P., Guo, Z., Göpfert, M. C., Jan, L. Y., et al. (2015). Ankyrin repeats convey force to gate the NOMPC mechanotransduction channel. Cell 162(6): 1391–1403. https://doi.org/10.1016/j.cell.2015.08.024
Song, X., Cui, L., Wu, M., Wang, S., Song, Y., Liu, Z., Xue, Z., Chen, W., Zhang, Y., Li, H., et al. (2023). DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles. J. Cell Biol. 222(10): e202209116. https://doi.org/10.1083/jcb.202209116
Sun, L., Cui, L., Liu, Z., Wang, Q., Xue, Z., Wu, M., Sun, T., Mao, D., Ni, J., Pastor-Pareja, J. C., et al. (2020). Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia. J. Cell Biol. 220(1): e202004184. https://doi.org/10.1083/jcb.202004184
Baumeister, W. (2002). Electron tomography: towards visualizing the molecular organization of the cytoplasm. Curr. Opin. Struct. Biol. 12(5): 679–684. https://doi.org/10.1016/s0959-440x(02)00378-0
Lučić, V., Förster, F. and Baumeister, W. (2005). Structural studies by electron tomography: From Cells to Molecules. Annu. Rev. Biochem. 74(1): 833–865. https://doi.org/10.1146/annurev.biochem.73.011303.074112
Soto, G. E., Young, S. J., Martone, M. E., Deerinck, T. J., Lamont, S., Carragher, B. O., Hama, K. and Ellisman, M. H. (1994). Serial section electron tomography: A method for three-dimensional reconstruction of large structures. NeuroImage 1(3): 230–243. https://doi.org/10.1006/nimg.1994.1008
Toyooka, K. and Kang, B. H. (2013). Reconstructing plant cells in 3D by serial section electron tomography. Methods Mol. Biol. : 159–170. https://doi.org/10.1007/978-1-62703-643-6_13
Mastronarde, D. N. (1997). Dual-axis tomography: An approach with alignment methods that preserve resolution. J. Struct. Biol. 120(3): 343–352. https://doi.org/10.1006/jsbi.1997.3919
Penczek, P., Marko, M., Buttle, K. and Frank, J. (1995). Double-tilt electron tomography. Ultramicroscopy 60(3): 393–410. https://doi.org/10.1016/0304-3991(95)00078-x
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4,941 | https://bio-protocol.org/en/bpdetail?id=4941&type=0 | # Bio-Protocol Content
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Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways
ZY Zilu Ye *
HW Hans H. Wandall
SD Sally Dabelsteen *
(*contributed equally to this work)
Published: Vol 14, Iss 4, Feb 20, 2024
DOI: 10.21769/BioProtoc.4941 Views: 1377
Reviewed by: Philipp WörsdörferHsih-Yin Tan Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Signaling Nov 2022
Abstract
Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models.
Key features
• 3D organotypic models with genetically engineered human cells.
• In-depth quantitative proteomics and phosphoproteomics in 2D cell culture.
• Careful handling of cell cultures is critical for the successful formation of theorganotypic cultures.
• For complete details on the use of this protocol, please refer to Ye et al. 2022.
Keywords: Organotypic model Cell signaling Phosphoproteomics Proteomics Gene editing Keratinocytes
Background
Signaling pathways are crucial regulators of cell growth and differentiation, playing significant roles in tissue formation and transformation; in many cases, they have been studied in simple 2D cell cultures or in animal studies. While many of the observations from murine models are expected to be applicable to human tissue, there are some inherent difficulties in translating these findings to a human context [1]. Consequently, it is important to use human cell and tissue systems to better understand and translate the functions of different signaling pathways into a human setting. We have previously integrated precise genetic engineering of the human N/TERT-1 and other keratinocyte cell lines to create a genetically traceable starting point to study individual gene products for the formation and transformation of fully differentiated human epidermal tissue [2–4]. The epidermis is a self-renewing, stratified tissue composed of proliferating basal cells and non-proliferating suprabasal cells [5]. Their properties are influenced by the underlying extracellular matrix (ECM) and dermal fibroblasts via intense bidirectional crosstalk mediated by secreted factors [6,7]. This creates the possibility to study both endogenous changes in cells and crosstalk between cell types in a controlled manner [2–4,8]. Therefore, in vitro human skin development is a relevant model to interrogate the various functions of signaling pathways, particularly when combined with proteomics and phosphoproteomics.
Quantitative liquid chromatography–mass spectrometry (LC–MS)-based proteomics and phosphoproteomics have emerged as the preferred methods to study cell signaling [9]. Isobaric labeling methods, like tandem mass tags (TMT), provide relative quantitation of identified proteins and phosphopeptides in multiple samples [10]. Combining the use of genetic engineering, proteomics, and phosphoproteomics with 3D organotypic human tissue models is an important tool in examining signaling pathways, as showed by our group [11]. In that study, we showed the potential different roles between Smad4-dependent and Smad4-independent TGF-β pathways and their role in diverse cellular behaviors such as human tissue homeostasis.
Taken together, these methods can be used as tools for characterizing signaling in human tissue formation and homeostasis. The described protocol details the creation of 3D organotypic cultures (protocol: organotypic tissue culture) that imitate in vivo tissue and its use to examine signaling pathways. Post incubation, the cultures are processed for histology or other analyses. In combination, parallel proteomics and phosphoproteomics analyses (protocol: Proteomics) of 2D cellular cultures, including cell lysis, protein digestion, phosphopeptide enrichment, and LC–MS analysis, allow for a detailed molecular investigation of the affected signaling pathways. The method provides novel insight into cell signaling, relevant for drug development and the understanding of disease pathology. The method can furthermore be used for drug testing and toxicology studies and minimizes the use of animal models.
Materials and reagents
Biological materials
For organotypic tissue culture
MRC-5 (ATCC, catalog number: CCL-171)
N/TERT-1 (James G. Rheinwald, Harvard Institute of Medicine)
For proteomics
Amine (NH2) functional microparticles, MagReSyn® Amine [ReSyn Biosciences (Pty) Ltd., South Africa]
Hydroxyl terminated beads, MagReSyn® Hydroxyl [ReSyn Biosciences (Pty) Ltd., South Africa]
Magnetic microparticles with chelated Ti4 + metal ions for highly specific phosphopeptide enrichment, MagReSyn® Ti-IMAC HP [ReSyn Biosciences (Pty) Ltd., South Africa]
Reagents
For organotypic tissue culture
Penicillin/streptomycin (Thermo Fisher, Gibco, catalog number: 151401)
DMEM (Thermo Fisher, Gibco, catalog number: 41966029)
Keratinocyte-SFM (Thermo Fisher, Gibco, catalog number: 17005034)
TrypLE (Thermo Fisher, Gibco, catalog number: 12605028)
Bovine pituitary extract (BPE) (Thermo Fisher, Gibco, catalog number: 13028014)
Fetal calf serum (FCS) (HyClone, catalog number: SH30071.03HI)
EGF (Thermo Fisher, Gibco, catalog number: PHG0314)
Insulin (Sigma-Aldrich, catalog number: 11070-73-8)
Hydrocortisone (Sigma-Aldrich, catalog number: H0888)
Triiodothyronine (T3) (Sigma-Aldrich, catalog number: T2752)
L-Glutamine (Thermo Fisher, Gibco, catalog number: A2916801)
Hams F12 (Thermo Fisher, Gibco, catalog number: 11765-054)
Rat tail collagen I (made in own lab [12])
Adenine (Sigma-Aldrich, catalog number: A8626)
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758)
Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D5652)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7979)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 655104)
Calcium chloride (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902)
Gentamycin sulphate (Sigma-Aldrich, catalog number: G1264)
Cholera enterotoxin (Sigma-Aldrich, catalog number: C8180)
10× minimum essential media (10× MEM) (Thermo Fisher, Gibco, catalog number: 11430-030)
Sodium bicarbonate (Thermo Fisher, Gibco, catalog number: 15750037)
Tissue-Tek® (SAKURA, catalog number: 4583)
Formalin solution neutral buffered, 10% (Sigma-Aldrich, catalog number: HT501128)
Sucrose (Sigma-Aldrich, catalog number: S0389)
Isopentane (Sigma-Aldrich, catalog number: PHR1661)
For proteomics
Lysis buffer, composed of 2% sodium dodecyl sulfate (SDS), 5 mM tris(2-carboxyethyl)phosphine (TCEP), 5.5 mM chloroacetamide (CAA), and 100 mM Tris pH 8.5; used for cell lysis
Acetonitrile (ACN), LC grade (SupelcoTM Analytical, catalog number: 75-05-8), used in various concentrations throughout the protocol, namely 70% for dilution of lysates, 95% for washing proteins, and 40% and 60% for elution on SepPak. ACN is also used for TMT-labeling
Ethanol (anhydrous) (Sigma-Aldrich, catalog number: 459836), used in 70% concentration for washing proteins after aggregation
50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich, catalog number: 09830), used for protein digestion and for resuspending TMT-labeled peptides
50% trifluoroacetic acid (TFA), HPLC (SupelcoTM Analytical, catalog number: TX1276), used for acidification of samples post digestion
1 M TEAB, pH 8 (Thermo ScientificTM, catalog number: 90114), used to achieve a final concentration of 100 mM in TMT-labeling
50% hydroxylamine (Thermo ScientificTM, catalog number: 90115), used to quench the TMT-labeling reaction
0.1% formic acid (v/v) in water (LC–MS grade) (Thermo ScientificTM, catalog number: 85170), used for resuspension of each TMT-labeled sample before pooling
Loading buffer, composed of 80% ACN, 5% TFA, and 1 M glycolic acid (Sigma-Aldrich, catalog number: 8.04104); used for resuspending peptides for phosphopeptide enrichment
1% ammonia (ammonia solution 25%) (Supelco, catalog number: 105428), used for elution of phosphopeptides
5 mM ABC (buffer A) and 100% ACN (buffer B), used in high pH reversed-phase peptide fractionation
PierceTM BCA Protein Assay kit (Thermo ScientificTM, catalog number: 23225)
TMTproTM 16plex label reagent set (Thermo ScientificTM, catalog number: A44520)
LysC, Lysyl endopeptidase (Fujifilm Wako, catalog number: 129-02541)
Trypsin, Sequencing grade modified trypsin (Promega, catalog number: V511X)
Sep-Pak C18 1 cc Vac Cartridge (Waters, catalog number: WAT054955)
Acquity CSH C18 1.7 μm, 1 mm × 150 mm column (Waters, catalog number: 186006935)
Solutions
Adenine (see Recipes)
Cholera enterotoxin (CT) (see Recipes)
Hydrocortisone (HC) (see Recipes)
Epidermal growth factor (EGF) (see Recipes)
Triiodothyronine (T3) (see Recipes)
Calcium chloride (CaCl2) (see Recipes)
Insulin (1,000× for 5 µg/mL) (see Recipes)
Sodium bicarbonate (71.2 mg/mL milli q water) (see Recipes)
Keratinocyte serum free medium (K-SFM) complete (see Recipes)
Organotypic culture medium (see Recipes)
DMEM 10% FBS (DMEM-10) (see Recipes)
Recipes
For organotypic tissue culture
Adenine
Prepare 2.4 mg/mL of adenine in HCl (this is a 100× stock):
Dissolve 243 mg of adenine in 100 mL of 0.05 M HCl.
Stir for approximately 1 h at room temperature (RT).
Filter sterilize with 0.2 μm filters.
Divide into 5 mL aliquots.
Store at -20 °C.
Cholera enterotoxin (CT)
Prepare 10 µM solution of cholera enterotoxin (this is 1,000× stock solution for 100 pM):
Add 1.18 mL of Milli-Q water to a 1 mg vial of cholera enterotoxin to prepare a 10-5 M solution.
Dilute this in 110 mL of PBS.
Filter sterilize with 0.2 µm filters.
Aliquot 2 mL per cryovial.
Store at -20 °C.
Note: MWCT = 84.000 Da
Hydrocortisone (HC)
Prepare 0.2 mg/mL hydrocortisone (this is a 500× stock for 0.4 µg/mL):
Dissolve a 25 mg vial in 5 mL of 100% ethanol. This solution is 5 mg/mL, which is 12.5× for 0.4 µg/mL. This can be stored at -20 °C indefinitely.
Add 2 mL of this concentrated stock to 48 mL of sterile PBS + 0.1% BSA.
Filter-sterilize with 0.2-µm filters.
Aliquot 2 mL into cryovials.
Store at -20 °C.
Epidermal growth factor (EGF)
Prepare 10 µg/mL of EGF (this is a 50,000× stock for 0.2 ng/mL):
Dissolve 100 µg of EGF in 10 mL of PBS + 0.1% BSA.
Filter sterilize with 0.2 µm filters.
Aliquot 1 mL into cryovials.
Store at -20 °C.
Triiodothyronine (T3)
Prepare 20 nM (2 × 10-8 M) of T3 [this is a 1,000× stock for 20 pM (2 × 10-11 M)]:
Measure 6.8 mg of T3 and dissolve in 7.5 mL of 0.02N NaOH.
Add 42.5 mL PBS (this is 100,000× for 20 pM T3).
Store at -20 °C in 10 mL aliquots.
To make the 1000× stock, dilute 0.4 mL of the 100,000× 1:100 into 39.6 mL of PBS.
Filter sterilize with 0.2 µm filters.
Divide into 2 mL aliquots.
Store at -20 °C.
Calcium chloride (CaCl2)
Prepare using calcium dihydrate CaCl2·2H2O (this is a 1,000× stock for 0.3 mM):
Dissolve 4.4 g of CaCl2·2H2O in 100 mL of Milli Q water.
Stir with magnetic bar to dissolve completely.
Filter sterilize with 0.2 µm filters.
Aliquot 2 mL per cryovial.
Store at -20 °C.
Insulin (1,000× for 5 µg/mL)
Use insulin from bovine pancreas.
Dissolve 100 mg of insulin in 20 mL of 0.005 M HCl in a 50 mL blue-capped tube.
Filter sterilize through a celluloseacetate, lowprotein binding 0.2 µM filter.
Aliquot 500 µL per tube.
Store at -20 °C.
Sodium bicarbonate (71.2 mg/mL milliQ water)
For organotypic cultures
Keratinocyte serum free medium (K-SFM) complete
Reagent Final concentration Quantity
Keratinocyte-SFM n/a 500 mL
CaCl2 (1,000×) 0.3 mM 500 µL
BPE (16.8 mg/mL) 25 µg/mL 750 µL
EGF (10 µg/mL) 0.2 ng/mL 10 µL
Total n/a 501.26 mL
Optional: Penicillin/Streptomycin can be added to K-SFM as well (100 U/mL).
Organotypic culture medium
Reagent Final concentration Quantity
DMEM n/a 375 mL
F12 n/a 125 mL
Pen/Strep 100 U/mL 5.14 mL
Adenine (2.4 mg/mL stock) 24 µg/mL 5.14 mL
HC (0.2 mg/mL stock) 0.4 µg/mL 1 mL
CT (10 µM stock) 100 pM 0.514 mL
T3 (1,000×) 20 pM 0.514 mL
Insulin (1,000×) 5 µg/mL 500 µL
FCS 0.3% 1.54 mL
Total 514.3 mL
DMEM 10% FBS (DMEM-10)
Reagent Final concentration Quantity
DMEM n/a 445 mL
FBS 10% 50 mL
Pen/Strep 100 U/mL 5 mL
Total 500 mL
Laboratory supplies
For organotypic tissue culture
Deep-well plates (Corning, catalog number: 355467)
Cell culture insert, PET membrane, 3.0 µm pore size (Corning, catalog number: 353092)
Airlift pads (cut in circles and autoclaved) (Whatman, GE Healthcare, catalog number: 10382461)
Cell culture Petri dishes or flasks from any brand (we use NUNC, Falcon, and Corning)
Equipment
For organotypic tissue culture
CO2 incubator at 37 °C (any brand can be used)
Centrifuge (any brand, for 15 mL tubes and a minimum of 100 g)
Freezers (-20 °C and -80 °C)
Equipment and software for proteomics
KingFisherTM Flex purification system (Thermo Scientific, catalog number: 5400610)
SpeedVac (Eppendor, model: VacufugeTM Concentrator)
Sonicator (Fisherbrand, model: 120 Sonic Dismembrator)
Spectrophotometer (Thermo Scientific, model: NanoDropTM 2000 C)
HPLC system [Thermo Scientific, model: UltiMateTM 3000 Standard (SD)]
Evosep ONE (Evosep)
Mass spectrometer (Thermo Scientific, model: Orbitrap ExplorisTM 480)
Proteome Discoverer software v2.4 (Thermo Scientific)
Chromeleon software v7.2 (Thermo Fisher Scientific)
Procedure
Part I: Organotypic tissue culture
Preparing cells for organotypic cultures (day 1–5)
Fibroblast cultures
Culture fibroblasts (we use MRC-5 cells) until you have an 80%–90% confluent T75 flask. You will need 1.8 million fibroblasts for one tray of organotypic cultures. For most fibroblasts, you will need to seed 0.8 million fibroblasts three days in advance (see Figure 1).
Figure 1. Fibroblasts suitable for organotypic collagen gels
Trypsinize cells for 5 min and neutralize with an equal volume of DMEM with 10% FCS. Count the cells (only viable cells) and spin down.
Resuspend fibroblasts at 8×105 per milliliter of DMEM-10.
Leave cells on ice while mixing the collagen gel.
Casting organotypic cultures
Note: Make sure you have enough fibroblasts ready at this point. You will need 1.8 million cells.
Acellular collagen gels
Note: All solutions must be ice cold or placed on ice.
For additional guidance watch Video 1.
Video 1. How to make an organotypic culture
Prepare a deep-well plate (6-well size) by adding one cell culture insert into each well. Use sterile forceps.
In a 50 mL tube on ice, mix the following components without making air bubbles:
650 µL of 10× MEM
70 µL of L-glutamine (100× stock of 200 mM)
10 µL of gentamicin sulphate (40 mg/mL)
725 µL of fetal calf serum (FCS)
725 µL of sodium bicarbonate (71.2 mg/mL)
5.5 mL of rat tail collagen (4 mg/mL in 0.05% acetic acid) (homemade, see [12])
850 µL of DMEM with 10% FCS
Add a few drops of 1 M NaOH to neutralize.
Carefully pipette 1 mL of the acellular gel mixture from the previous step into the culture insert and make sure it covers the whole bottom of the insert. Leave to polymerize in the incubator while preparing the fibroblasts and the cellular gel. The gel will be polymerized after 10–15 min.
Making of cellular gel
In a 50 mL tube on ice, mix the following components (avoid making air bubbles):
1.7 mL of 10× MEM
170 µL of L-glutamine (100× stock of 200 mM)
25 µL of gentamicin sulphate (40 mg/mL)
1.9 mL of FCS
525 µL of sodium bicarbonate (71.2 mg/mL)
14.4 mL of rat tail collagen (1 mg/ml in 0.05% acetic acid) (homemade)
2.1 mL of human fibroblasts (1.8 million cells) suspended in 10% calf serum DMEM
Mix gently and add a few drops of 1 M NaOH to neutralize the solution (the color should change to orange/light pink).
Quickly add 3 mL to the culture inserts on top of the acellular gel. Avoid air bubbles.
Place the tray in the incubator for approximately 30 min; the gels will be fully polymerized.
Fill DMEM 10% FCS in the well (18 mL) and 2 mL on top of the gel. Do not spray. See Figure 2 and Video 2.
Figure 2. Casted organotypic collagen gels, ready for media to be added
Video 2. How to fill media on organotypic cultures
Maturing collagen gels
Four to six days after the gels are contracted and appear macroscopically as a slightly shrunken, white disk, they are ready for keratinocytes.
Note: In our hands, this results in good organotypic cultures, possibly due to active deposition and reorganization of the ECM by fibroblasts.
Seeding keratinocytes on the casted collagen gels with fibroblasts (timing 2 h)
Trypsinize N/TERT-1 cells to be seeded (WT and validated knock-outs) and adjust concentration to 1 × 107 cells/mL (Figure 3).
Note: 3 × 105 cells need to be plated for each gel.
Aspirate medium from the bottom of each well in the deep-well plate.
Remove medium from the culture insert with a p1000 pipette.
Critical: Avoid keeping the pipette too close to the gel inside the insert, as you may aspirate the gel.
Add 30 µL of cell suspension (from step E1) in the center of the gel (Video 3).
Video 3. Adding keratinocytes to organotypic cultures
Add organotypic culture medium (see Recipes) at the bottom of the well in the deep-well plate until it reaches the bottom of the culture insert (∼9 mL).
Incubate at 37 °C for 20 min to let the cells adhere.
Add 2 mL of organotypic culture medium on top of the adhered cells and ∼16 mL of organotypic culture medium outside the insert and carefully place in the incubator. Do not spray as the keratinocytes will then detach.
Incubate for four days before lifting the gels.
Figure 3. Keratinocyte colonies. A. Colonies well-suited for organotypic collagen gels. B. Colonies not suited for organotypic cultures.
Air-liquid interface lifting of the keratinocyte. Timing: 30 min (Video 4)
Video 4. Placing airlift pads
On day 4 after seeding the keratinocytes, carefully aspirate medium from the culture inserts and from the plate wells.
Place the insert into a sterile 100 mm dish using sterile forceps.
Add two sterile airlift pads to the base of each well in the deep-well plate and place back the insert into the well over the pads.
Add approximately 9 mL of organotypic culture medium in the deep wells until the pads are completely wet.
Critical: The level of the medium should cover the pads and not be higher.
Adding inhibitors to organotypic cultures (Day 4–14)
For experiments using different inhibitors, culture medium is supplemented with inhibitors in the desired concentration when the inserts are raised to the air-liquid interface. Concentration of the inhibitor is replenished with subsequent medium changes. Note that for some inhibitors, concentration needs to be raised compared to the use on 2D cultured cells. Add the desired inhibitor directly to the media in the well. Replenish with every media change.
Maturing of organotypic cultures
Incubate the plate at 37 °C in a humidified incubator and change media in the deep wells every other day. Gels are ready to harvest in 10 days (Figure 4).
Figure 4. Organotypic culture ready to be fixed. Arrow points to the concavity in the contracted collagen gel where keratinocytes are growing.
Note: Organotypic cultures can now be fixed for histology or processed for western blot, RNAseq, or proteomics.
Formalin-fixed paraffin-embedded (FFPE) section processing
Cut the membranes from the insert and place the 3D cultures in a histology cassette in 10% formalin for >24 h followed by paraffin embedding.
Frozen sections
Note: The best morphology is obtained when the 3D cultures are incubated in 2 M sucrose (in dH2O) for >48 h before embedding in Tissue-Tek and freezing in isopentane on dry ice.
Cut the membranes from the insert and place the 3D cultures in a 2 M sucrose solution for >48 h.
Freeze in isopentane on dry ice and keep at -80 °C until sectioning.
Note: Sections cut at 4–6 μm give better staining results.
Part II: Proteomic analyses in 2D cell culture
Note: Steps K–P, Sample preparation for proteomics and phosphoproteomics and mass spectrometry analysis (2–3 days)
Cell lysis
Cultivate N/TERT-1 cells in 2D until they reach 80% confluence. Approximately 1 million cells are needed.
Lyse the cells by adding lysis buffer (500 μL) and boil the lysates for 10 min at 95 °C.
Further lyse by micro-tip probe sonication for 2 min, pulsing 1 s on and 1 second off at 60% amplitude.
Measure the protein concentrations using a BCA protein assay kit.
Protein digestion
Note: Digest protein extracts overnight using the KingFisher Flex robot following the protein aggregation capture (PAC) method [13], adapted for a 96-well plate format (Figure 5).
Dilute the lysates to a final concentration of 70% ACN.
Add magnetic hydroxyl terminated beads for protein aggregation using a 1:2 protein/bead ratio. The amine beads can be replaced by hydroxyl-terminated beads.
Wash proteins three times in 95% ACN and twice in 70% ethanol.
Digest in 300 μL of 50 mM ABC with LysC and trypsin at enzyme/protein ratios of 1:500 and 1:250, respectively, for 12 h at 37 °C.
Acidify samples with 50% TFA to a final concentration of 1% TFA.
Clean the peptide mixtures on SepPak C18 1 cc Vac cartridge and elute with 300 μL of 40% ACN followed by 300 μL of 60% ACN.
Evaporate the ACN in a SpeedVac.
Estimate the final peptide concentration by measuring absorbance at 280 nm on a NanoDrop 2000 C spectrophotometer.
Figure 5. Layout of the KingFisher plates for protein digestion
TMT labeling
Note: Label peptides from each sample with TMTpro 16-plex Label Reagent. After labeling, 10 µg of pooled peptides is reserved for proteome samples and the rest is used for phosphopeptide enrichment.
Add 1 M TEAB to each sample to reach a final concentration of 100 mM, followed by ACN to 50% final concentration.
Incubate the labeled peptides with TMT reagents for 1 h at RT.
Quench the reaction by adding 1% hydroxylamine (1:1 hydroxylamine/TMT) for 15 min at RT.
Pool the samples and evaporate the ACN by SpeedVac.
Phosphopeptide enrichment
Resuspend TMT-labeled peptides in 200 μL of loading buffer (Figure 6).
Figure 6. Layout of the KingFisher plates for phosphopeptide enrichment
Use Ti-IMAC beads in a 1:2 peptide/beads ratio and wash them in 500 μL of loading buffer for 5 min.
Incubate the beads with the peptides for 20 min.
Wash the beads successively with loading buffer, 80% ACN, 1% TFA, and finally 10% ACN and 0.2% TFA for 2 min each.
Elute the phosphopeptides in 200 μL of 1% ammonia for 10 min.
Repeat the enrichment process on the unbound remnant from the initial enrichment step with new elution buffers and beads.
Micro-flow high-pH reversed-phase peptide fractionation
Resuspend TMT-labeled peptides in 50 mM ABC.
Fractionate the peptides using a reversed-phase Acquity CSH C18 column on an UltiMate 3000 HPLC system with the Chromeleon software.
Follow the provided multi-step gradient.
Collect and concatenate to 12 fractions for phosphoproteome and 46 fractions for proteome, acidify with 10% formic acid, and dry with SpeedVac.
LC–MS/MS
Analyze all samples by LC–MS/MS using the Evosep One system with pre-programmed gradients.
MS settings:
Spray voltage to 1.8 kV
Heated capillary temperature at 275 °C
Funnel RF level at 40
Acquire data in profile mode with the full MS mass range set at 350–1,400 with an AGC target at 300%
Set full MS resolution at 60,000 at m/z 200 with a maximum injection time of 25 ms
Set the HCD fragment spectra resolution at 45,000 with an injection time of 86 ms and a Top10 method
Set AGC target value at 200% and the intensity threshold at 2E5
Isolation window set as at 0.8 m/z
Normalized collision energy at 32%
Data analysis
Data analysis (proteomics)
All raw data files are analyzed using the Proteome Discoverer 2.4 software (developed by Thermo Fisher Scientific). The human SwissProt FASTA database, with 20,355 entries as of March 2019, was used as the reference protein database.
Trypsin was selected as the digestion enzyme. Up to two missed cleavages were permitted. TMTpro is specified as a fixed modification on lysine and peptide N terminus, and methionine oxidation is specified as a variable modification. In addition, phosphorylation is set as a variable modification on serine, threonine, and tyrosine residues in phosphoproteome samples.
Validation of protocol
Organotypic tissue culture
The human skin organotypic model with N/TERT-1 cells (WT cells and genetic engineered cells), or any other keratinocyte cell type or cancer cell type we have used, is very robust. In our laboratory, we have made more than 400 organotypic cultures with WT cells and more than 4,000 organotypic cultures during the last decades. The outcome varies with +/- 1 cell layer counted on >100 organotypic wildtype cell cultures. Furthermore, the phenotype of the 3D organotypic models is highly similar to the normal human skin (see Ye et al. [11], figure 7 below).
Figure 7. Comparison of normal human skin with 3D organotypic skin cultures. Top row: staining of human skin with differentiation markers [involucrin (INV), filaggrin (Flg), loricrin (Lor), keratin 10 (K10), and keratin 1 (K1)] and the proliferation marker Ki67. Lower row: the same staining in N/TERT-1 WT organotypic cultures. Statistical analysis, columns to the right, using one-way ANOVA followed by Dunnett’s multiple comparison test. The organotypic cultures show the same distribution of these markers as the normal skin, but the basal lamina will be straight compared to the wavy nature of the normal human skin.
General notes and troubleshooting
General notes
The 3D organotypic culture with keratinocytes and fibroblasts is very robust and reproducible.
The media is cheaper than premade media bought from different companies, although it is a little time-consuming to make all the stock solutions the first time.
The fact that we use 6-well plates makes it easy to have enough material for many different assays and also have enough good tissue for immunohistochemistry.
Troubleshooting
Problem Potential solution 1 Potential solution 2
Collagen gels do not contract after point D
Good-quality collagen and fresh growing fibroblasts are pivotal for establishing good-quality organotypic cultures. Collagen gels may be left for up to seven days in the incubator before seeding keratinocytes, giving the fibroblasts more time to reorganize the ECM.
Fibroblasts should not have been cultured for more than two weeks and should be actively dividing. The passage number varies between fibroblast lines.
Keratinocytes do not form stratified layers
Keratinocytes should not have been cultured for more than three weeks and should be actively dividing. Do not shake the culture dishes during incubations, especially within the first 4 h after seeding the cells. Any passage number can be used of the N/TERT-1 cells as they are immortalized. Do not dry out the gel while lifting to the air-liquid interphase. Do not add too much media at this step as well (9 mL is usually enough; the media must not overflow into the inserts).
Avoid opening the incubator too often while growing the organotypic cultures. In our experience, organotypic cultures tend to fail more often in a very busy incubator (opened many times during the day).
The organotypic cultures are stable for up to three weeks after lifting.
Acknowledgments
The work was supported by the European Commission (GlycoSkin H2020-ERC), European Commission (Imgene H2O20), Lundbeck Foundation, The Danish Research Councils (Sapere Aude Research Leader grant to H.H.W.), Danish National Research Foundation (DNRF107), the Friis Foundation (H.H.W AND S.D), Danish Strategic Research Council, and the program of excellence from the University of Copenhagen (CDO2016). We thank Karin Uch Hansen, Birgit Poulsen, Karen Biré, and Louise Rosgaard Duus for the excellent technical assistance. We acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. We would like to thank James G. Rheinwald, Harvard Institute of Medicine, for providing the N/TERT-1 cells.
This work was described in Ye et al. [11] and Dabelsteen et al. [2].
Competing interests
The authors have no financial or non-financial competing interests concerning this manuscript.
Ethical considerations
The two human cell lines used in this study were either bought at ATCC or provided by James G. Rheinwald, Harvard Institute of Medicine where the use for academic purposes is approved.
References
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Dabelsteen, S., Pallesen, E. M. H., Marinova, I. N., Nielsen, M. I., Adamopoulou, M., Romer, T. B., Levann, A., Andersen, M. M., Ye, Z., Thein, D., et al. (2020). Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model. Dev. Cell. 54(5): 669-684 e667. https://doi.org/10.1016/j.devcel.2020.06.039.
Bagdonaite, I., Pallesen, E. M. H., Ye, Z. L., Vakhrushev, S. Y., Marinova, I. N., Nielsen, M. I., Kramer, S. H., Pedersen, S. F., Joshi, H. J., Bennett, E. P., et al. (2020). O-glycan initiation directs distinct biological pathways and controls epithelial differentiation. Embo. Reports 21(6). https://doi.org/10.15252/embr.201948885.
Nielsen, M. I., de Haan, N., Kightlinger, W., Ye, Z., Dabelsteen, S., Li, M., Jewett, M. C., Bagdonaite, I., Vakhrushev, S. Y. and Wandall, H. H. (2022). Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line. Nat. Commun. 13(1): 6257. https://doi.org/10.1038/s41467-022-33806-8.
Blanpain, C. and Fuchs, E. (2009). Epidermal homeostasis: a balancing act of stem cells in the skin. Nat Rev. Mol. Cell. Biol. 10(3): 207-217. https://doi.org/10.1038/nrm2636.
Briggaman, R. A. (1982). Epidermal-dermal interactions in adult skin. J. Invest. Dermatol. 79 Suppl 1: 21s-24s. https://doi.org/10.1111/1523-1747.ep12544628.
Smola, H., Stark, H. J., Thiekotter, G., Mirancea, N., Krieg, T. and Fusenig, N. E. (1998). Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture. Exp. Cell. Res. 239(2): 399-410. https://doi.org/10.1006/excr.1997.3910.
Aasted, M. K. M., Groen, A. C., Keane, J. T., Dabelsteen, S., Tan, E., Schnabel, J., Liu, F., Lewis, H. S., Theodoropulos, C., Posey, A. D., et al. (2023). Targeting Solid Cancers with a Cancer-Specific Monoclonal Antibody to Surface Expressed Aberrantly O-glycosylated Proteins. Mol. Cancer. Ther. 22(10): 1204-1214. https://doi.org/10.1158/1535-7163.MCT-23-0221.
Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P. and Mann, M. (2006). Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127(3): 635-648. https://doi.org/10.1016/j.cell.2006.09.026.
Li, J., Van Vranken, J. G., Vaites, L. P., Schweppe, D. K., Huttlin, E. L., Etienne, C., Nandhikonda, P., Viner, R., Robitaille, A. M. and Thompson, A. H. (2020). TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples. Nat. Methods. 17(4): 399-404. https://www.nature.com/articles/s41592-020-0781-4.
Ye, Z., Kilic, G., Dabelsteen, S., Marinova, I. N., Thøfner, J. F., Song, M., Rudjord-Levann, A. M., Bagdonaite, I., Vakhrushev, S. Y. and Brakebusch, C. H. (2022). Characterization of TGF-β signaling in a human organotypic skin model reveals that loss of TGF-βRII induces invasive tissue growth. Sci. Signal. 15(761): eabo2206. https://www.science.org/doi/10.1126/scisignal.abo2206
Rajan, N., Habermehl, J., Coté, M. F., Doillon, C. J. and Mantovani, D. (2006). Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc 1(6): 2753-2758. https://doi.org/10.1038/nprot.2006.430.
Batth, T. S., Tollenaere, M. A. X., Ruther, P., Gonzalez-Franquesa, A., Prabhakar, B. S., Bekker-Jensen, S., Deshmukh, A. S. and Olsen, J. V. (2019). Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics 18(5): 1027-1035. https://doi.org/10.1074/mcp.TIR118.001270.
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Correction Notice: Isolation and Culture of Murine Hepatic Stellate Cells
RM Rucha V. Modak
DZ Dietmar M. Zaiss
Published: Jan 20, 2024
DOI: 10.21769/BioProtoc.4942 Views: 314
Reviewed by: Gal HaimovichSara Johnson Anonymous reviewer(s)
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After official publication in Bio-protocol (https://bio-protocol.org/e3422), we realized that there is a typographical error in the Procedure, section C, point 1: where it reads “add 3.2 mL of OptiPrep to 4.8 mL of HBSS,” it should be corrected to “add 4.8 mL of OptiPrep to 3.2 mL of HBSS.”
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Analysis of DNA 5-methylcytosine Using Nanopore Sequencing
ZL Zhuowen Li
LZ Lihao Zheng
JZ Jixian Zhai
YL Yanping Long
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Abstract
DNA methylation is known to be a conserved repressive epigenetic modification ineukaryotic organisms, which involves the transfer of a methyl group to the C5position of cytosine by DNA methyltransferase. In plants, DNA methylation occursin CG and non-CG (including CHG and CHH, H = A, T, C) sequence contexts. It iswidespread in the genome and involved in various biological processes toregulate gene expression and genome stability. Nowadays, Nanopore sequencingenables the direct detection of DNA modification on native single-moleculelong-read DNA, overcoming the limitation of short-read bisulfite sequencing. Tofacilitate the Nanopore-based DNA methylation analysis in plants, in thisprotocol, we provide the guidance of the software DeepSignal-plant, which canaccurately call 5mC in all three contexts of CG, CHG, and CHH with highcorrelation with bisulfite sequencing in plants.
Keywords: Plant DNA methylation Nanopore sequencing DeepSignal-plant CG non-CG 5mC
Background
DNA methylation is one of the most important epigenetic modifications. It is involvedin the regulation of gene expression, gene imprinting, transposon silencing, andchromatin packaging in response to developmental and environmental stimulation [1].In mammals, DNA methylation mainly occurs in the CG context, and non-CG methylation(CHG and CHH) is only found in specific cell types such as brain cells andpluripotent cells [2]. In plants, however, in addition to the CG methylation, CHGand CHH methylations are also widespread throughout the genome and play importantroles in gene silencing [3]. There are various kinds of strategies for the detectionof DNA methylation. Among them, Illumina-based whole genome bisulfite sequencing(WGBS) can obtain global patterns at single-base resolution and thus serves as thegold standard for genome-wide DNA methylation analysis [4]. This method usesbisulfite to convert the cytosine into uracil; the latter turns into thymine afterPCR amplification, while the cytosine with 5mC can resist the conversion. Theirdifference can be identified by mapping to the reference genome after sequencing.However, WGBS has shortcomings like low coverage on repetitive regions due to theshort-read sequencing and false positives caused by incomplete conversion. Nanoporesequencing provides good solutions for all these problems. It allows the directdetection of methylation status on native single-molecule DNA without chemicaltreatment and PCR amplification. By studying the electric signal characters (called squiggle) produced when DNA goes through the Nanopore, the DNA sequence andassociated modification can be decoded [5]. The Nanopore long reads can cover largegenomic regions and enable the profiling of repetitive and complex regions as wellas the phasing haplotypes [6]. Currently, Nanopore sequencing can detect not only5mC but also other modifications like 5hmC, 4mC, and 6mA on DNA [7], and has beenapplied in bacteria [8,9], humans [10], and plants [11].
Many algorithms have been developed to decode the modification signal from Nanoporedata [7,12]. However, these methods failed to capture 5mC in the context of CHG andCHH with acceptable accuracies, which hinders their application in plant genomeresearch. To answer this requirement, DeepSignal-plant was developed. It uses deeplearning to recall 5mC in all contexts, having gained a high correlation with WGBSresults [13]. In this protocol, we introduce the data analysis process ofDeepSignal-plant for methylation study in plants.
Equipment
Linux version 3.10.0-862.el7.x86_64 (Red Hat 4.8.5-28) with 48 CPU (2*Intel Gold 6140, 18 cores, 2.3 Ghz) and GPU (2*Nvidia V100, 640 cores, 32 GB).
Software and datasets
Guppy (v4.0.11; https://timkahlke.github.io/LongRead_tutorials/BS_G.html) (Release date, June 18, 2020)
DeepSignal-plant (v0.1.5; https://github.com/PengNi/DeepSignal-plant) (Release date, March 31, 2022)
The pipeline for DeepSignal-plant depends on software listed as follows:
ont_fast5_api (v4.0.2; https://github.com/nanoporetech/ont_fast5_api) (Release date, March 22, 2017)
tombo (v1.5.1; https://github.com/nanoporetech/tombo) (Release date, February 20, 2020)
Conda (v23.3.1, https://docs.conda.io/en/latest/) (Release date, March 28, 2023)
h5ls tools we use to preview the FAST5 files should be installed automatically with conda.
Mamba (v1.4.2, https://mamba.readthedocs.io/en/latest/) (Release date, April 6, 2023)
The Integrative Genomics Viewer (IGV) (v2.6.1 https://software.broadinstitute.org/software/igv/) (Release date, July 26, 2019)
Python (3.7.12) (Release date, September 4, 2021)
Numpy (v1.20.3)
Pandas (1.3.4)
Click (8.1.3)
Seaborn (0.11.1)
Matplotlib (3.4.1)
hurry.filesize (0.9)
Input data
Data generated from Nanopore direct DNA sequencing in FAST5 format.
Reference genome in fasta format.
The annotation file of Arabidopsis in gff3 format.
Pre-trained model for plant 5mC calling.
Procedure
Case study
We will use a small sample data of Arabidopsis Nanopore sequencing for the case study. Figure 1 shows the overview of the workflow for genomic methylation analysis with Nanopore data.
Figure 1. DeepSignal-plant pipeline. This pipeline consists of nine steps. Steps 1 and 2 help us to get the nucleotide sequence in fastq format from FAST5 output files for Nanopore sequencers. Step 3 uses tombo to match the called sequences back to the electric current signal stored in FAST5 file, and Step 4 aligns the current signal to the reference genome. Then, in Steps 5 and 6, we use DeepSignal-plant to call the 5-methylcytosine and also calculate the frequency. Finally, in Steps 8 and 9, we calculate the bin methylation level and visualize the data with tools like IGV browser or python plot.
Preparation
Pipeline download
The pipeline we used in this protocol can be downloaded on the GitHub page ( https://github.com/Bio-protocol/DeepSignalplantPractise) with the command:
git clone https://github.com/Bio-protocol/DeepSignalplantPractise
Users can find the step-by-step commands in the folder “workflow,” which are also listed in the grey boxes in this protocol. The examples for intermediate and the final output are stored in “cache” and “output” folders, respectively. The “lib” folder contains python scripts used within the workflow. Users can prepare the input data in the “input” folder as the following guidance.
Data preparation
Nanopore data
The Nanopore sequence sample data “sample_data.tar.gz” we used here was modified from the sample data provided by DeepSignal-plant [13]. Users can download it from Google Drive with browser Edge or Chrome ( https://drive.google.com/drive/folders/1NZe6mQ5y1S8eaE-GwU124PvmONBoz5X7?usp=sharing) to a local computer and transfer it to the folder “DeepSignalplantPractise/input/Step1_Input.” The command below is used to decompress the file:
tar -zxvf sample_data.tar.gz
mv sample_data DeepSignalplantPractise/input/Step1_Input
In the “sample_data” folder, users will find four files ending in .fast5. These example files are in FAST5 format and were generated from Nanopore sequencing, containing the raw electric signal that we can call the base sequence and modification. Users can refer to https://hasindu2008.github.io/slow5specs/fast5_demystified.pdf for a detailed introduction of the FAST5 forma [14].
Reference genome and annotation
Here, we used TAIR10 as the reference genome for Arabidopsis. Users can download the genome file from the Ensemble Plants ( http://ftp.ensemblgenomes.org/pub/plants/), uncompressed for use:
#download reference genome
cd ./DeepSignalplantPractise/input/
mkdir reference
cd reference
wget -c http://ftp.ensemblgenomes.org/pub/plants/release-53/fasta/arabidopsis_thaliana/dna/Arabidopsis_thaliana.TAIR10.dna.toplevel.fa.gz
gunzip Arabidopsis_thaliana.TAIR10.dna.toplevel.fa.gz
Users can also download the annotation file of Arabidopsis in gff3 format from Ensemble Plants. The chromosomes coordinates are extracted from the gff3 file and written in bed format, which is used for bin methylation level calculation in step 8.
#download gff3 file
cd ./DeepSignalplantPractise/input/reference
wget -c http://ftp.ensemblgenomes.org/pub/plants/release-53/gff3/arabidopsis_thaliana/Arabidopsis_thaliana.TAIR10.53.gff3.gz
gunzip Arabidopsis_thaliana.TAIR10.53.gff3.gz
#extract the chromosomes coordinates
awk -F "\t" '{if($3=="chromosome") print($1"\t"$4-1"\t"$5)}' Arabidopsis_thaliana.TAIR10.53.gff3 > Tair10_genome.bed
Pre-trained model
Users can download the model provided by DeepSignal-plant on its GitHub page ( https://github.com/PengNi/DeepSignal-plant) and move it to the folder "DeepSignalplantPractise/input/model" for 5mC calling in Step5.
Environment preparation and software installation
Mamba
Mamba is recommended for the environment management of the tools we use. Conda should be installed before mamba. Users can find the right version for their system on the Conda product page ( https://www.anaconda.com/products/distribution). It can be installed with the commands below [use the 64-Bit (x86) Installer as the example]:
#install conda
wget -c https://repo.anaconda.com/archive/Anaconda3-2022.05-Linux-x86_64.sh
bash Anaconda3-2022.05-Linux-x86_64.sh
#install mambaconda install -c
conda-forge mamba
DeepSignal-plant
Users can set up the environment for DeepSignal-plant and install all its dependencies with Mamba. In this protocol, we use $PATHofDeepSignalPlant to indicate the path for DeepSignal-plant download and $MambaEnv to indicate the path of the Mamba environment. Users will need to replace these two variables manually with the path they use. For further information, users can find guidance on the DeepSignal-plant GitHub page ( https://github.com/PengNi/deepsignal-plant).
#create an environment
Mamba create -n deepsignalpenv python=3.7
#activate environment
mamba activate deepsignalpenv
#download DeepSignal-plant
cd $PATHofDeepSignalPlant
git clone https://github.com/PengNi/deepsignal-plant.git
#instal
lpip install deepsignal-plant
#re-install pytorch if needed
mamba install pytorch==1.11.0 cudatoolkit=10.2 -c pytorch
#install tombo
mamba install -c bioconda ont-tombo
After these steps, the DeepSignal-plant is installed in the Mamba environment to the path $MambaEnv/deepsignalpenv/. When using the tools in this environment, users should make sure it has been activated or set in the environment variable with “export PATH.”
Guppy
Guppy is used for Nanopore sequence basecalling. Users can download from the software download page in the Nanopore community ( https://community.nanoporetech.com/downloads, Figure 2). User registration is required. Users can find the version that matches their system and install it following the guidance it provides:
Figure 2. Guppy download page
IGV
The Integrative Genomics Viewer (IGV) is a widely used tool to visualize genomic data. Users can find the version suitable for their system and download it at https://software.broadinstitute.org/software/igv/download .
ont_fast5_api package
The ont_fast5_api_package is a tool for the FAST5 file operation. We will use the “multi_to_single_fast5” tool in this package in Section B, step 1. This package can be downloaded with the command:
#ont_fast5_api_package install
cd ./DeepSignalplantPractise/lib
git clone https://github.com/nanoporetech/ont_fast5_api.git
pip install ./ont_fast5_api
Practice
We provide the protocol from raw data preparation to visualization for Nanopore methylation analysis by Nanopore sequencing with nine steps in total. Users can find the commands and run them in the “DeepSignalplantPractise/workflow” folder.
Step 1. Convert the multi-read FAST5 into single-read form.
The FAST5 files generated by Nanopore sequencing are usually in multi-read FAST5 format, as are the sample data we provided, as shown in Figure 3 (1,000 reads per file in our samples). Users can use the h5ls tool provided by Conda to inspect the FAST5 files.
#preview all reads in FAST5 file
cd ./DeepSignalplantPractise/workflow
h5ls -r ../input/Step1_Input/sample_data/batch_0.fast5
Figure 3. Structure of the multi-read FAST5 file (previewed by h5ls, only the first four reads were shown)
Because Tombo only accepts single-read FAST5 format (one read per file), we use the “multi_to_single_fast5” tool from the ont_fast5_api package to convert multi-read FAST5 files into single-read form before using Guppy and Tombo. The command is shown below:
#01.multi_to_single_fast5.sh
mkdir ../cache/SINGLE_sample_data
multi_to_single_fast5 -i ../input/Step1_Input/sample_data -s ../cache/SINGLE_sample_data -t 30 --recursive
After conversion, you will find folders named with numbers containing multiple single-read FAST5 files in the output “../cache/SINGLE_sample_data” folder. The structure of single-read FAST5 is shown in Figure 4.
h5ls -r ../cache/SINGLE_sample_data/0/ffee8b3e-95ba-44e6-951c-5e7982d3b5f0.fast5
Figure 4. Structure of the single-read FAST5 file (previewed by h5ls)
Step 2. Basecall FAST5 files with Guppy.
This step is to basecall the single-read FAST5 file with Guppy. The task may require a huge amount of calculating resources and is recommended to be run with GPU for faster processing. Users can find details for Guppy usage in the Guppy protocol available in the Nanopore community ( https://community.nanoporetech.com/docs/prepare/library_prep_protocols/Guppy-protocol/v/gpb_2003_v1_revae_14dec2018) (registration needed). If users cannot access Guppy, they can download the basecalled fastq files we prepared from Google Drive ( https://drive.google.com/drive/folders/1pk4vecjdC48gslbeXGNKforUb0jxRPpz?usp=sharing) and decompress it under the “cache” folder for the downstream analysis. The basic use of the parameters is listed as follows:
-i input folder
-s output folder
-c basecalling model, select based on the kit
--recursive search for FAST5 in the sub-folder of input
--qscore_filtering enable filtering of reads into pass/fail folders. The default q-score for the filter is 7
--device "cuda:all:100%" Use all GPUs in this system without memory limit
The commands used for basecalling are listed below:
#02.basecall.sh
guppy_basecaller \
-i ../cache/SINGLE_sample_data \
-s ../cache/fastq \
-c dna_r9.4.1_450bps_hac_prom.cfg \
--recursive \
--disable_pings \
--qscore_filtering \
--device "cuda:all:100%"
The basecalling model set by “-c” can be selected based on the flowcell and kit for sequencing. Users can use the command below to see the corresponding model (Figure 5):
Figure 5. Corresponding basecalling model for flowcell and kit (part)
This step produces two folders, “fail” and “pass,” and two files named “sequencing_summary.txt” and “sequencing_telemetry.js.” The fastq files in the “pass” folder containing the base sequence information and the file “sequencing_summary.txt” containing the basecalling information for each read are used for Tombo preprocess in the next step.
Step 3. Add the basecalled sequence back to FAST5 with Tombo preprocess.
Tombo provides useful tools for raw Nanopore signal processing and can be used for modification identification as well [15]. In this step, we use Tombo to allocate the basecalled fastq sequence back to the raw signal. Here are the commands.
#03.tombo_preprocess.sh
#environment setting, replace $MambaEnv/deepsignalpenv with your actual path
export PATH=$MambaEnv/deepsignalpenv/bin:$PATH
# Tombo preprocess
# “*fastq” represents all the files with names end with “fastq” in the “pass” directory.
cat ../cache/fastq/pass/*fastq > ../cache/fastq/pass.fastq
tombo preprocess annotate_raw_with_fastqs \
--fast5-basedir ../cache/SINGLE_sample_data \
--fastq-filenames ../cache/fastq/pass.fastq \
--sequencing-summary-filenames ../cache/fastq/sequencing_summary.txt \
--overwrite \
--processes 30
After this operation, the basecalled information is added back to the corresponding FAST5 file under the “Basecall_1D_000” group in the “Analyses” section (Figure 6). When users need to rerun this step, they can use the parameter “--overwrite” to overwrite the previously written information and files. If the command is rerun without the parameter “--overwrite,” there will be multiple groups of basecalled folder added under the “Analyses” group, named “Basecall_1D_001,” “Basecall_1D_002,” and so on.
h5ls -r ../cache/SINGLE_sample_data/0/ffee8b3e-95ba-44e6-951c-5e7982d3b5f0.fast5
Figure 6. File structure of fast5 after preprocess (previewed by h5ls). The red box highlights the new generated files at step 3.
Step 4. Map the raw signal to reference genome with Tombo resquiggle.
This step aims to map the raw electric signal to the reference genome. Here are the commands:
#04.tombo_resquiggle.sh
#environment setting, replace $MambaEnv/deepsignalpenv with your actual path
export PATH=$MambaEnv/deepsignalpenv/bin:$PATH
# resquiggler
tombo resquiggle \
../cache/SINGLE_sample_data \
../input/reference/Arabidopsis_thaliana.TAIR10.dna.toplevel.fa \
--processes 30 \
--corrected-group RawGenomeCorrected_000 \
--basecall-group Basecall_1D_000 \
--overwrite \
--ignore-read-locks
The parameter “--basecall-group” uses the group name “Basecall_1D_000” generated in the previous step in the FAST5 file (Figure 6) to define which basecalled sequence is to be used. After this step, the sequences with the signal assignment are written back into the FAST5 file, generating a group named “RawGenomeCorrected_000” (set by “--corrected-group,” Figure 7).
h5ls -r ../cache/SINGLE_sample_data/0/ffee8b3e-95ba-44e6-951c-5e7982d3b5f0.fast5
Figure 7. Structure of the FAST5 file after resquiggle (previewed by h5ls). The red box highlights the new generated files at step 4.
With the shell command “ll -a,” users can find a hidden index file (“.SINGLE_sample_data.RawGenomeCorrected_000.tombo.index”) accompanying “SINGLE_sample_data” folder (Figure 8). The index file is generated for faster later steps. In addition, users can use “--overwrite” to overwrite files when rerunning this step. Details for resquiggle can be found in https://nanoporetech.github.io/tombo/resquiggle.html?highlight=corrected%20group .
Figure 8. Hidden index file generated after resquiggle
Step 5. Call methylation of reads with DeepSignal-plant call_mods.
This step uses the “call_mods” command in DeepSignal-plant to call the modification from input resquiggled FAST5 files. All three contexts of 5mC can be called. To accelerate this step, GPU is recommended. The parameter “CUDA_VISIBLE_DEVICES” is used to set the mode for GPU use (CPU only: =0; use one: =0; use two: =0,1 and so on). The model file ending in .ckpt is required for 5mC calling, and users can download it from the GitHub page of DeepSignal-plant ( https://github.com/PengNi/DeepSignal-plant). Here are the commands:
#05.deepplant-met-mod.sh
#environment setting, replace $MambaEnv/deepsignalpenv with your actual path
export PATH=$MambaEnv/deepsignalpenv/bin:$PATH
#call 5mC
CUDA_VISIBLE_DEVICES=0,1 deepsignal_plant call_mods \
--input_path ../cache/SINGLE_sample_data \
--model_path ../input/model/model.dp2.CNN.arabnrice2-1_120m_R9.4plus_tem.bn13_sn16.both_bilstm.epoch6.ckpt \
--result_file ../cache/fast5s.C.call_mods.tsv \
--corrected_group RawGenomeCorrected_000 \
--reference_path ../input/reference/Arabidopsis_thaliana.TAIR10.dna.toplevel.fa \
--motifs C --nproc 30 --nproc_gpu 2
The output file lists the methylation status of individual C with the genomic position and the name of the read it locates (Figure 9). When the unmethylated probability (column 7) is higher than the methylated probability (column 8), the site is considered as unmethylated and labeled as “0” in column 9, and vice versa.
Figure 9. Output of DeepSignal-plant call_mods
Step 6. Calculate methylation frequency with DeepSignal-plant call_freq.
This step calculates the methylation frequency of each C in the reference genome based on the file “fast5s.C.call_mods.tsv” generated in the previous step and outputs a modification frequency file for all three contexts together. For each site, the ratio of the read counts with methylated cytosine to total read counts is defined as methylation frequency or site methylation level [16]. GPU is not required for this step. We use the parameter “--bed” to set the output format as bedMethyl, which can be dealt with bed format–related tools (Figure 10). The parameter “--sort” is used to sort the results according to the genomic location for further processing.
#06.deepplant-met-freq.sh
#environment setting, replace $MambaEnv/deepsignalpenv with your actual path
export PATH=$MambaEnv/deepsignalpenv/bin:$PATH
#calculate frequency
deepsignal_plant call_freq \
--input_path ../cache/fast5s.C.call_mods.tsv \
--result_file ../cache/fast5s.C.call_mods.freq.bed \
--sort --bed
Figure 10. Output of DeepSignal-plant call_freq
Step 7. Split the result into CG, CHG, and CHH context.
This step uses the script “split_freq_file_by_5mC_motif.py” provided by DeepSignal-plant to split the methylation frequency according to the three different contexts CG, CHG, and CHH. The python script was downloaded and stored in the “scripts” folder when we installed the DeepSignal-plant (see 2.2 DeepSignal-plant). Users can also find it on the GitHub page of DeepSignal-plant ( https://github.com/PengNi/deepsignal-plant/tree/master/scripts). The input file is fast5s.C.call_mods.freq.bed generated in the previous step. The reference genome file is also required. The output is methylation frequency files for three individual contexts (Figure 11). Here are the commands:
#07.split_context.sh
#replace $PATHofDeepSignalPlant with your actual path
python $PATHofDeepSignalPlant/scripts/split_freq_file_by_5mC_motif.py \
--freqfile ../cache/fast5s.C.call_mods.freq.bed \
--ref ../input/reference/Arabidopsis_thaliana.TAIR10.dna.toplevel.fa
Figure 11. Three methylation frequency files generated for CG, CHG, and CHH in bedMethyl format
Step 8. Calculate the weighed methylation level in the bin.
Up to now, we have gained the single-site DNA methylation level for all three contexts. However, for downstream analysis and visualization, we usually have to calculate the methylation level in bins rather than in sites. To calculate the weighed methylation of each bin [16], we define the sites with more than three read counts (coverage > 3) as the valid site, and bins with more than three valid cytosine sites as the valid bin. The methylation level for the invalid bin was recorded as NaN [17]. In valid bins, the weighed methylation level is calculated as follows:
In this formula, M represents the methylated read counts of the valid site i, and C represents its coverage. The python script “met_level_bin.py” for the methylation level calculation we provided here is also used in our previously published Pore-C data [18].
We used the small sample data in the previous steps for time-saving practices. However, its coverage is insufficient for bin-weighed methylation level calculation and visualization. For this reason, we use the methylation frequency files generated from the Pore-C data (Project: PRJCA006702, Run Accession CRR327363, Pore-C Rep2 for Col-0) [18] in the following analysis, with binsize = 100 bp for IGV, and binsize = 100,000 bp for python plotting. Users can find the preprocessed data of Pore-C in https://drive.google.com/drive/folders/14xw6gvQz_gjUi6p86NrSHZq59YABlzZO?usp=sharing .
Users can run the python script (met_level_bin.py) in the DeepSignalplantPractise folder downloaded from our GitHub page (refer to Section 3. Download scripts) and get the bin weighed methylation level (binsize = 100,000 bp) as follows:
#08.met_level_bin.sh
python ../lib/met_level_bin.py \
--region_bed ../input/reference/Tair10_genome.bed \
--met_bed ../input/Step8_Input/Rep2_fast5s.C.call_mods.CG.frequency.bed \
--prefix Rep2_fast5s.C.call_mods.CG \
--binsize 100000 \
--outdir ../output
The parameter “--region_bed” uses a bed file to define the genome coordinates for calculation. Here, we use the file “reference/Tair10_genome.bed” generated in data preparation (refer to Section 1.2 Reference genome preparation), which contains all chromosome coordinates of the Arabidopsis to set the input region. The parameter “--met_bed” uses each context’s methylation frequency file as the input. The parameter “--prefix” is to set the prefix of the output file. This step outputs a “bedGraph” file with the prefix “Rep2_fast5s.C.call_mods.CG.”
Step 9. Visualize the methylation level by IGV and python plotting.
The “bedGraph” files created in the previous step contain four columns: chromosome name, start, end, and the bin weighed methylation level (Figure 12). Users can find a detailed explanation of the format “bedGraph” on the help page in UCSC Genome Browser https://genome.ucsc.edu/goldenpath/help/bedgraph.html). This format file can be explored by genome browsers as IGV with different zoom levels (Figure 13). Users can find more information on IGV usage at https://software.broadinstitute.org/software/igv/UserGuide.
Figure 12. Bedgraph format
Figure 13. Methylation level shown in IGV browser in different zoom levels. A. All chromosomes except Pt and Mt. B. Chromosome 4. C. Chr4:9,112,570-9,202,278.
Users can plot the “bedgraph” file by python scripts in the DeepSignalplantPractise folder (refer to Section 3. Download scripts) (Figure 14). Here, we use chromosome 4 as an example. The bedGraph files with binsize=100,000 bp are used as input:
#09.chrom_met_visulization.sh
python ../lib/chrom_met_visulization.py \
--cg_bedg ../output/Rep2_fast5s.C.call_mods.CG_binsize100000.bedgraph \
--chg_bedg ../output/Rep2_fast5s.C.call_mods.CHG_binsize100000.bedgraph \
--chh_bedg ../output/Rep2_fast5s.C.call_mods.CHH_binsize100000.bedgraph \
--region_bed ../input/reference/Tair10_genome.bed \
--chrom 4 --outdir ../output
Figure 14. Methylation distribution in chromosome 4 plotted by python
Discussion
DeepSignal-plant has been applied to methylation detection in Arabidopsis, rice, and black mustard with high accuracy and sensitivity [13]. Compared with other existing analysis tools for Nanopore data in plants, DeepSignal-plant can achieve 5mC in all three contexts of CG, CHG, and CHH and shows high correlations with bisulfite sequencing [17]. Furthermore, DeepSignal-plant can profile the methylation of more cytosines, especially in the repetitive regions. It also provides the tools for new model training when applied to the genomes of other plants [13].
There are some points that users should pay attention to. First, although DeepSignal-plant works well with models trained across species, the CHH model in Arabidopsis shows relatively poor performance, which might be due to the low CHH methylation level in Arabidopsis. It is suggested that users should use a dataset containing enough valid sites for model training. Second, DeepSignal-plant is based on deep learning, indicating a high requirement for computational resources. There are two steps in which we recommend using GPU with the Arabidopsis data. When applied to large plant genomes, the memory and processing time required will increase drastically. If non-CG methylation analysis is not required, methods that use fewer resources like Nanopolis [10] may be a better choice. What′s more, sequence depth does not significantly impact the performance of the DeepSignal-plant. It can obtain acceptable results (Pearson correlation with bisulfite sequencing: > 0.95 in CG context, > 0.90 in CHG context, >0.8 in CHH context in Arabidopsis and rice) at low coverages (20×), and the increase of coverage does not significantly improve the output. This feature serves as a cost-saving option for experimental design.
Acknowledgments
This work was supported by the National Key R&D Program of China Grant (2019YFA0903903); an NSFC to J.Z. (31871234); National Natural Science Foundation of China (32300479); the Shenzhen Sci-Tech Fund (KYTDPT20181011104005); the Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes (2019KSYS006) and the Stable Support Plan Program of Shenzhen Natural Science Fund Grant (20200925153345004). We thank the members of the Zhai lab-Dr. Weipeng Mo, Hong Zhang, and Dr. Yiming Yu for proofreading and comments.
The main pipeline for Nanopore methylation calling (Steps 1–7) is based on the guidance provided by DeepSignal-plant ( https://github.com/PengNi/deepsignal-plant [13], and the part for bin calculation and visualization (Steps 8–9) is based on our recent work [18].
Competing interests
The author declares no conflict of interest.
References
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Supplementary information
Data and code availability: All data and code have been deposited to GitHub: https://github.com/Bio-protocol/DeepSignalplantPractise
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Utilisation of Methylome Data to Identify Stably Unmethylated Regions in Plant Genomes
JE Judith I. M. Eglitis-Sexton
LM Leroy M. Mangila
HA Haylie L. Andrews
LH Lee T. Hickey
PC Peter A. Crisp
Published: Feb 20, 2024
DOI: 10.21769/BioProtoc.4944 Views: 132
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Abstract
DNA methylation is a key chromatin modification that provides a mechanism for epigenetic inheritance. However, DNA methylation profiles can also be used to annotate or filter plant genomes by partitioning a genome into methylated and unmethylated regions (UMRs). UMRs comprise only a very small fraction of moderate to large plant genomes, yet these regions are known to be highly enriched in functionally significant genomic sequences, including genes and cis-regulatory elements. Therefore, methods to efficiently and accurately identify UMRs in plant genomes are useful for genome annotation and functional genomics and potentially for crop improvement. In this protocol, we provide a reproducible vignette to identify UMRs in the maize methylome, starting from raw fastq files obtained by whole-genome bisulfite sequencing. This method determines the average methylation per 100 bp tile of the genome and classifies tiles as methylated and unmethylated. To support training and learning, this step-by-step guide uses a small data subset corresponding to a 20 Mb region of the maize genome so that this analysis could be completed on a standard desktop computer with minimal computational resources.
Keywords: DNA methylation Epigenetics Unmethylated regions Plants Whole-genome bisulfite sequencing Methylome Bioinformatics
Background
Groundbreaking research into epigenetics has opened up possibilities for its application to human diseases, modern agriculture, synthetic biology, and studies of evolution. Covalent attachment of a methyl group to the 5′ carbon of cytosine in DNA (5-methylcytosine) is generally known as DNA methylation, and 5-methylcytosine is sometimes referred to as the fifth base [1]. DNA methylation can provide a mechanism for epigenetic inheritance of phenotypes and, accordingly, is often referred to as an epigenetic modification. DNA methylation plays critical roles in transposon silencing, genome stability and organisation, heterochromatin formation, gene regulation, development, and imprinting [2]. More recently, we have demonstrated that there is great utility in identifying regions of the genome that lack DNA methylation, referred to as unmethylated regions (UMRs) [3]. In this protocol, we provide a step-by-step guide to identify UMRs from DNA methylation sequencing data.
There are a variety of technologies that can be used to detect and quantify the levels of DNA methylation at a particular locus, and many of these methods can also be scaled to genome-wide profiling of the entire methylome [4,5]. Technologies include bisulfite based, digestion based, or affinity based. Whole-genome techniques include whole-genome bisulfite sequencing (WGBS), enzymatic methyl-seq (EM-seq), reduced representation bisulfite sequencing (RRBS-seq), which uses bisulfite coupled with enzymatic digestions, and methyl-DNA immunoprecipitation (MeDIP), which attracts methylation through antibody enrichment [6–9]. In addition, over the last few years, nanopore sequencing has emerged as another alternative, allowing direct detection of DNA and RNA methylation on long reads [10]. Importantly, the ability to identify methylation at single-base-pair resolution has allowed specific understanding of how methylation—or the lack thereof—influences specific regions of the genome, including regulatory regions that may be important for transcriptional changes and phenotypic variation for traits of interest. In this protocol, we analyse WGBS sequence data; we routinely apply the same analysis pipeline to EM-seq data, to which it is equally applicable. Together, these methyl-seq whole-genome sequencing approaches (WGBS and EM-seq) are the current gold standard for DNA methylation profiling, as they provide a whole-genome analysis of regions that are methylated and the types of methylation present [11,12].
The key step in WGBS involves treating genomic DNA with sodium bisulfite, which converts unmethylated cytosines to uracils by deamination (which are converted to thiamine following PCR amplification), while 5’-methylcytosines remain unaffected [8]. After bisulfite treatment is complete, converted DNA can be prepared for sequencing, commonly on the Illumina platforms, allowing inference of methylation at single-base resolution by comparison to a reference genome. Due to the cytosine conversion step in WGBS (and in EM-seq), a key requirement in the bioinformatic analysis is to use bisulfite aware mapping software to identify the T-converted unmethylated cytosines, which will appear as polymorphisms compared to a reference genome. Commonly used mapping software includes BSMAP, BWA-meth, and Bismark [13,14].
In plants and animals, DNA methylation occurs in different sequence contexts including CG, CHG, and CHH (where H is A, T, or C). Studies have found that the type of methylation can correspond to different functions; for example, CG-only methylation has been correlated with actively transcribed gene bodies, while transcriptionally silenced regions are associated with high levels of methylation in all contexts (CHH, CHG, and CG) [15]. The output of this analysis pipeline includes files for visualisation of each type of methylation. Recently, the research community has demonstrated that regions that lack DNA methylation in all contexts are of particular significance [3,15–23]. Partitioning genomic regions into different categories of methylation types allows us to identify unmethylated regions (UMRs). This approach can be very useful for annotating a genome [24] because UMRs tend to align with regions containing functional genes and also cis-regulatory elements (CREs) [3,25]. CREs are non-coding elements and include enhancers, promoters, and silencers, which can influence gene expression. These have the potential to be important targets for selection and breeding and also as targets for genetic engineering. Researchers have yielded promising results from genetic modification of CREs for tailoring gene expression without causing developmental problems [26,27]. For example, improvements in yield in rice [28,29], maize [30], and tomato [31–33] have been produced by gene editing non-coding cis-regulatory promoter alleles. However, efficiently identifying functional regions of regulatory importance is challenging [3]; identification of UMRs can narrow the search for genetic targets. As genomic sequencing and tools for genome annotation improve, our ability to understand the functionality of the genome is made increasingly powerful when combining knowledge of transcription binding sites, conservation of sequences through evolution, epigenomic markers for transcriptional activity, and other emerging technologies.
In the case study outlined below, we have provided a subset of raw WGBS reads extracted from SRX5532987, a published study of DNA methylation in maize leaf tissue [3]. This subset of reads aligns to a small 20 Mb region of maize chromosome 1; we provide this subset of the genome as a reference sequence for mapping. These files enable processing this example data with minimal computational resources and could be completed on a basic laptop computer with the appropriate software installed. The pipeline was originally optimised for maize, but it is generally applicable to other species without modification [34]; however, users could consider modifying the thresholds in the UMR-calling step if applied to a plant genome that has particularly unusual levels or distribution of DNA methylation. The key steps of the workflow are outlined in Figure 1.
Overview of the workflow to identify unmethylated regions (UMRs) in a plant genome. The bioinformatic workflow presented in the protocol includes six major steps: 1) read trimming, 2) read mapping, 3) filtering, 4) extraction and quantification of methylation levels per cytosine, 5) data visualisation, and lastly 6) summarisation and identification of UMRs. The output file formats are indicated above each step and the software tools and their purpose are summarised.
Software and datasets
The required software, references, and websites for download are provided below:
trim_galore! [16], v0.6.4_dev ( https://github.com/FelixKrueger/TrimGalore)
cutadapt [20], v1.8.1 ( https://cutadapt.readthedocs.io/en/stable/)
fastQC [3], v0.11.5 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
BSMAP [45], v2.74 ( https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-10-232 )
samtools [9], v1.16 ( https://github.com/samtools/samtools)
bamtools [4], v2.4.0 ( https://github.com/pezmaster31/bamtools/)
Java [1], v1.8.0_45 ( https://www.oracle.com/java/technologies/javase/8u45-relnotes.html )
Picard, v2.9.0 ( https://broadinstitute.github.io/picard/)
bamUtil [15], v1.0.13 ( https://genome.sph.umich.edu/wiki/BamUtil)
Python [39], v2.7.5 (https://www.python.org/)
bedGraphToBigWig (UCSC; download from http://hgdownload.soe.ucsc.edu/admin/exe/ )
perl [40], v5.26.2 (https://www.perl.org/)
IGV [32], v2.5.3 ( https://software.broadinstitute.org/software/igv/)
R, v4.1 (https://www.r-project.org/ )
Each of the above software was run in a terminal application on a server running the software Linux but could also be run on any personal machine running Linux or macOS.
Input data
To demonstrate the identification of UMRs from WGBS data, we have provided a small subset of reads (3,396 reads) for analysis in paired fastq files (“B73_chr1_subset_reads_1.fastq” and “B73_chr1_subset_reads_2.fastq”). These reads were extracted from SRR8738272 [7] SRA PRJNA527657, WGBS from a maize B73 seedling, V1 stage, leaf shoot. These reads map to a section of the maize V4 genome between 80 and 100 kb on chromosome 1. We have provided a fasta reference sequence for this portion of the maize genome for mapping the reads (“maize_chr1_reference.fa”). This minimal example will run with minimal hardware requirements and should only take a few seconds per step.
Users interested in analysing their own data should first perform a quality check before proceeding with this pipeline. For example, FastQC can be used to perform basic checks of sequence data quality. Additionally, when analysing bisulfite data, it is critical to check the conversion efficiency. This is not performed in this example for simplicity; however, it should be performed on every dataset. This can be done either by using a spike-in unmethylated DNA sequence such as lambda gDNA and then mapping reads to this reference sequence or, in plants, mapping to the unmethylated chloroplast genome; good conversion rates should preferably be >99%. In this example, the WGBS data is paired end data; however, this pipeline can equally be run on single-end data; paired end data is not a requirement for UMR analysis.
All steps in this pipeline are run in the same folder that contains the provided input data and other required files. The output files are written to the same folder.
Link to the input data and scripts:
Repository: https://github.com/Bio-protocol/unmethylated-regions_UMR-extractor-WGBS/tree/master
Input data located in `/input`
Other required scripts in `/lib`
Procedure
Case study
Trim the reads.
In the first step, we trim the reads for quality and remove any contaminating adapter sequences. This step requires the software packages trim_galore!, cutadapt, and fastQC. Once these software and the fastq reads are loaded, the code laid out below can be run in the same folder that contains the fastq files. The parameter “phred33” instructs cutadapt to use ASCII+33 quality scores as Phred scores for quality trimming. The parameter “clip” removes 20 base pairs from the 5’ end of both reads one and two; this is required for some library preparation methods, for example if using the ACCEL-NGS Methyl-Seq DNA Library Kit (SWIFT Biosciences). The parameter “o” indicates that the output will be placed in the current directory, and the paired end fastq files are provided following the “paired” argument. Please note that the length of adapters is also dependent on the library preparation methods.
```
trim_galore \
--phred33 \
--clip_R1 20 --clip_R2 20 \
-o ./ \
--paired B73_chr1_subset_reads_1.fastq B73_chr1_subset_reads_2.fastq
```
For each fastq file, two new files are produced: 1) a trimming report and 2) a new fastq with trimmed reads as shown below.
```
B73_chr1_subset_reads_1.fastq_trimming_report.txt
B73_chr1_subset_reads_1_val_1.fq
B73_chr1_subset_reads_2.fastq_trimming_report.txt
B73_chr1_subset_reads_2_val_2.fq
```
Align the reads to the genome reference.
In this step, we use BSMAP v2.74 to align the reads from the fastq file with the supplied maize genome reference file “maize_chr1_reference.fa.” The input parameter “-v 5” allows up to five mismatches, “-r 0” reports only unique mapping pairs, and “-q 20” performs quality trimming to q20. The output file generated is in SAM format.
```
bsmap \
-a B73_chr1_subset_reads_1_val_1.fq \
-b B73_chr1_subset_reads_2_val_2.fq \
-d maize_chr1_reference.fa \
-o mapped.sam \
-v 5 \
-r 0 \
-q 20
```
Below is an example of the standard error report that should be generated from the code above; these reads should have a paired mapping rate of approximately 97%.
```
BSMAP v2.74
Start at: Tue Jun 14 22:44:54 2022
Input reference file: maize_chr1_reference.fa (format: FASTA)
Load in 1 db seqs, total size 20000 bp. 0 secs passed
total_kmers: 43046721
Create seed table. 1 secs passed
max number of mismatches: 5 max gap size: 0
kmer cut-off ratio: 5e-07
max multi-hits: 100 max Ns: 5 seed size: 16 index interval: 4
quality cutoff: 20 base quality char: '!'
min fragment size:28 max fragemt size:500
start from read #1 end at read #4294967295
additional alignment: T in reads => C in reference
mapping strand (read_1): ++,-+
mapping strand (read_2): +-,--
Pair-end alignment(8 threads)
Input read file #1: B73_chr1_subset_reads_1_val_1.fq (format: FASTQ)
Input read file #2: B73_chr1_subset_reads_2_val_2.fq (format: FASTQ)
Output file: mapped.sam (format: SAM)
Thread #2: 3395 read pairs finished. 1 secs passed
Total number of aligned reads:
pairs: 3277 (97%)
single a: 21 (0.62%)
single b: 16 (0.47%)
Done.
Finished at Tue Jun 14 22:44:55 2022
Total time consumed: 1 secs
```
Fix and sort the mapped reads.
This step requires samtools (v1.3) to perform fixing and sorting of the BSMAP output provided by the previous step. First, we convert the files to BAM format and then name-sort them. The fixmate option of samtools can then be used to ensure that mates have the pair’s coordinates and insert sizes (to ensure compliance with downstream software) and mappings are again sorted and also indexed. Indexing is not strictly required but can be performed so the mapping file could be visualised in IGV.
```
samtools view -bS mapped.sam > mapped.bam
samtools sort -n mapped.bam -o mapped_nameSrt.bam
samtools fixmate mapped_nameSrt.bam mapped_nameSrt_fixed.bam
samtools sort mapped_nameSrt_fixed.bam -o mapped_sorted.bam
samtools index mapped_sorted.bam
rm mapped.sam mapped_nameSrt.bam mapped_nameSrt_fixed.bam mapped.bam
```
The output files generated are:
```
mapped_sorted.bam
mapped_sorted.bam.bai
```
To see some summary statistics about the mapping file, we can use amtools stats.
```
samtools stats mapped_sorted.bam | grep ^SN | cut -f 2-
```
Some select metrics from samtools stats are shown below.
```
raw total sequences: 6591
reads mapped: 6591
reads mapped and paired: 6566 # paired-end technology bit set + both mates mapped
reads unmapped: 0
reads duplicated: 0 # PCR or optical duplicate bit set
reads QC failed: 0
average length: 104
maximum length: 106
average quality: 36.6
insert size average: 156.3
```
Filter the mapping file.
This step removes improperly paired reads (for example, pairs that map to different chromosomes), read duplicates, and any overlapping portion of read pairs. Removing duplicate reads and trimming overlaps is important because these represent redundant information originating from the same DNA molecule; retaining duplicates or overlaps can lead to biassed data.
This step requires bamtools; we use bamtools filter to remove any improperly paired or unmapped reads. The second part of this step also requires an installation of Java to run picard for the removal of duplicate reads.
```
bamtools filter \
-isMapped true \
-isPaired true \
-isProperPair true \
-in mapped_sorted.bam \
-out mapped_sorted_pairs.bam
```
Now, we remove duplicate reads using picard. This is an essential step in any DNA methylation analysis; however, in this example there are no duplicate reads, so the output file should contain all the input mappings.
```
java -jar /path/to/picard.jar MarkDuplicates \
I=mapped_sorted_pairs.bam \
O=mapped_sorted_MarkDup_pairs.bam \
METRICS_FILE=mapped_MarkDupMetrics.txt \
ASSUME_SORTED=true \
CREATE_INDEX=False \
REMOVE_DUPLICATES=true
```
Finally, we trim any overlapping portion of paired reads so that overlapping regions are only counted once in the analysis. Clipping is required; otherwise, cytosines in the overlapping regions are counted twice. These cytosines represent the same biological information, measured in technical replication, so should only be counted once.
```
bam clipOverlap \
--in mapped_sorted_MarkDup_pairs.bam \
--out mapped_sorted_MarkDup_pairs_clipOverlap.bam \
--stats
```
Below is an example of the standard error report from the clipOverlap step.
```
Overlap Statistics:
Number of overlapping pairs: 2922
Average # Reference Bases Overlapped: 61.3809
Variance of Reference Bases overlapped: 727.063
Number of times orientation causes additional clipping: 176
Number of times the forward strand was clipped: 1420
Number of times the reverse strand was clipped: 1502
Completed ClipOverlap Successfully.
```
Extract cytosine methylation levels.
The mapping files must now be analysed to determine the level of methylation at each cytosine. A script methratio.py is provided with the BSMAP software to extract methylation data; this requires the installation of python and BSMAP. In addition, samtools is required, which is also provided with BSMAP; importantly, this script requires an older version of samtools (< v1.1.18). The parameter “s” is used to direct methratio.py to the correct version of samtools, which can be found in the installation folder of BSMAP. The code displayed first directs python to the location of methratio.py; the parameter “o” is the output summary text file. The option “d” is used to indicate the reference sequence file (FASTA format); in this case, “maize_chr1_reference.fa,” the 20 Mb subset of maize chromosome 1, which is provided. The option “u” processes only unique mappings and pairs, while “z” reports the loci with zero methylation ratio; the parameter “r” is used to remove duplicates.
```
python2 ~/software/bsmap-2.74/methratio.py \
-o methratio.txt \
-d maize_chr1_reference.fa \
-u -z \
-s ~/software/bsmap-2.74/samtools \
-r mapped_sorted_MarkDup_pairs_clipOverlap.bam
```
* “~/software/”should be replaced with the user’s path to the location of the BSMAP software installation.
An example standard error report is provided below.
```
total 5701 valid mappings, 7121 covered cytosines, average coverage: 11.16 fold.
```
An example of the output text file called “methratio.txt” is provided below.
```
chr pos strand context ratio eff_CT_count C_count CT_count rev_G_count rev_GA_count CI_lower CI_upper
maize_chr1_reference 8 + ATCAT 0.000 1.00 0 1 0 0 0.000 0.793
maize_chr1_reference 15 + TTCAC 0.000 2.00 0 2 1 1 0.000 0.658
maize_chr1_reference 17 + CACAA 0.000 2.00 0 2 1 1 0.000 0.658
maize_chr1_reference 22 + AACCA 0.000 3.00 0 3 3 3 0.000 0.562
maize_chr1_reference 23 + ACCAC 0.000 3.00 0 3 3 3 0.000 0.562
```
Parse the output of BSMAP.
The output of methratio.py provides the sequence context of each cytosine with the two adjacent nucleotides on either side of the cytosine (column 4 “context”). Here, we use a custom awk function to convert this output file into a new file with the general methylation context of each cytosine (CG, CHG, or CHH) and parse coordinates to zero-based format "BED" type for bedtools. Subsequently, awk is used to convert to bedgraph format (columns: chromosome, start, stop, and ratio) for downstream tools.
```
# awk function to parse the output of bsmap methratio.py script
awk_make_bed='BEGIN {OFS = FS} (NR>1){
if(($3=="-" && $4~/^.CG../ ) || ($3=="+" && $4~/^..CG./))
print $1, $2-1, $2, $3, "CG", $5, $6, $7, $8, $9, $10, $11, $12;
else if(($3=="-" && $4~/^C[AGT]G../ ) || ($3=="+" && $4~/^..C[ACT]G/))
print $1, $2-1, $2, $3, "CHG", $5, $6, $7, $8, $9, $10, $11, $12;
else if(($3=="-" && $4~/^[AGT][AGT]G../ ) || ($3=="+" && $4~/^..C[ACT][ACT]/))
print $1, $2-1, $2, $3, "CHH", $5, $6, $7, $8, $9, $10, $11, $12;
else
print $1, $2-1, $2, $3, "CNN", $5, $6, $7, $8, $9, $10, $11, $12
}
'
# run the awk function
awk -F$"\\t" "$awk_make_bed" \
"methratio.txt" > "BSMAP_out.txt"
# output file:
maize_chr1_reference 7 8 + CHH 0.000 1.00 0 1 0 0 0.000 0.793
maize_chr1_reference 14 15 + CHH 0.000 2.00 0 2 1 1 0.000 0.658
maize_chr1_reference 16 17 + CHH 0.000 2.00 0 2 1 1 0.000 0.658
maize_chr1_reference 21 22 + CHH 0.000 3.00 0 3 3 3 0.000 0.562
maize_chr1_reference 22 23 + CHH 0.000 3.00 0 3 3 3 0.000 0.562
```
Generate bigWig files for viewing in IGV.
Now we can further process the output files into a format compatible with IGV for inspecting the data. Use bedGraphToBigWig to make a bigWig file for IGV. The awk function filters by required columns and gives us the average percentage of methylation. We are then able to split this into three files based on methylation context (CG, CHH, CHG).
```
# awk function to filter to only the required columns and to calculate the average percent methylation
awk_make_bedGraph='BEGIN {OFS = FS} (NR>1){
print $1, $2, $3, $8/$9*100, $5
}
'
# awk function to split the bedgraph into three files by methylation context
awk_make_bedGraph_context='BEGIN {OFS = FS} (NR>1){
print $1, $2, $3, $4 > "BSMAP_out_"$5".bedGraph"
}
'
# run the two above functions
awk -F$"\\t" "$awk_make_bedGraph" \
"BSMAP_out.txt" | \
awk -F$"\\t" -v ID=$ID "$awk_make_bedGraph_context" -
# Example for CG context (columns: chromosome, start, end, percent-methylation)
maize_chr1_reference 24 25 100
maize_chr1_reference 25 26 75
maize_chr1_reference 95 96 85.7143
maize_chr1_reference 96 97 64.7059
maize_chr1_reference 125 126 60
# Make bigWigs files for IGV per context
bedGraphToBigWig BSMAP_out_CG.bedGraph maize_chr1_reference.chrom.sizes BSMAP_out_CG.bigWig
bedGraphToBigWig BSMAP_out_CHG.bedGraph maize_chr1_reference.chrom.sizes BSMAP_out_CHG.bigWig
bedGraphToBigWig BSMAP_out_CHH.bedGraph maize_chr1_reference.chrom.sizes BSMAP_out_CHH.bigWig
# remove intermediate files
rm -rv BSMAP_out*.bedGraph
```
Summarise methylation levels into 100 bp tiles.
The output of the previous step is a text file per context with the average methylation level of each individual cytosine. DNA methylation can either be analysed at the single-cytosine level or at a regional level. Single-cytosine analysis can be useful for investigating rates of epimutation; however, it is generally agreed that methylation over a contiguous region is of most biological relevance because this can affect chromatin conformation, chromatin accessibility, and gene expression. A powerful and simple (and computationally cheap) way to determine region-level methylation is to divide the genome into small equal-sized tiles (also sometimes called windows or bins). We have found that 100 bp tiles are an optimal compromise between resolution and computational efficiency. We note that there are many different software tools that provide alternative (often more complex) algorithms for defining regional methylation and partitioning the genome into different methylation states, for example DSS [10], methylkit [2] or MethylScore [14]. Here, we provide a simple perl script met_context_window.pl to parse and summarise the BSMAP output into the average methylation per context per 100 bp tile. The argument “100” sets the tile size; users can also parse the data into different sized tiles by changing the last argument.
```
# summarise into 100bp tiles
perl met_context_window.pl BSMAP_out.txt 100
# output (columns: chromosome, start, end, sites, “Cs”, “C+Ts”, percent-methylation)
maize_chr1_reference 0 100 4 23 31 0.741935483870968
maize_chr1_reference 100 200 6 52 70 0.742857142857143
maize_chr1_reference 200 300 6 70 95 0.736842105263158
maize_chr1_reference 300 400 2 27 34 0.794117647058823
maize_chr1_reference 400 500 6 94 114 0.824561403508772
```
Identify unmethylated regions.
The final step requires classifying each 100 bp tile into one of six methylation categories. This step requires R and the R package tidyverse. The methylation categories include “missing data” (including “no data” and “no sites”), “RdDM,” “heterochromatin,” “CG-only,” “unmethylated,” or “intermediate”. In this analysis, we are primarily interested in using this classification to identify the UMRs; however, users might also be interested in other types of methylation, which could be extracted from this same analysis strategy. We also suggest removing organelles from the data before proceeding with this step; however, in this example data, the organelle genomes have already been removed.
This analysis is performed using a custom R script that we have provided: Call-umrs.R. Regions are classified according to the following hierarchy: tiles are classified as missing data if they have less than two cytosines in the relevant context or if there is less than the specified coverage threshold of reads (e.g., 3–5× coverage); RdDM if CHH methylation is greater than 15%; heterochromatin if CG and CHG methylation is 40% or greater; CG-only if CG methylation is greater than 40%; unmethylated if CG, CHG, and CHH are less than 10%; and intermediate if methylation is 10% or greater but less than 40%. Note that the levels of CHH methylation are hard coded in this script, while the level of CG and CHG are specified when calling the script. We have found these levels to be appropriate for a range of species; however, they could be adjusted if a genome has a different or unusual distribution, for example if CHH methylation is known to be higher.
This script also requires a genome reference cytosine tile file that provides the number of cytosines that occur in each context for each tile. We have provided this file, maize_chr1_reference_100 bp_tiles.bed, for the analysis of the 20 Mb region in this example. Users will need to create this file to analyse a different plant genome; some reference genome files are linked in the git repository https://github.com/Bio-protocol/unmethylated-regions_UMR-extractor-WGBS/tree/master. The format of the file is shown below (columns: chromosome, start, end, #CG sites, #CHG sites, #CHH sites):
```
maize_chr1_reference 0 100 4 6 29
maize_chr1_reference 100 200 6 7 25
maize_chr1_reference 200 300 6 4 36
maize_chr1_reference 300 400 2 10 28
maize_chr1_reference 400 500 6 2 31
```
When calling the R script, the following arguments are required in this order; suggested default settings are indicated in the brackets:
The reference genome cytosine tile file (“maize_chr1_reference_100 bp_tiles_sites_counts.txt”).
Minimum coverage (suggestion 3× or 5×).
Minimum number of sites (suggestion 2).
Minimum percent to be considered methylated (suggestion 40%).
Maximum percent to be considered unmethylated (suggestion 10%).
```
R -f Call-umrs.r \
--args maize_chr1_reference_100bp_tiles_sites_counts.txt \
3 \
2 \
0.4 \
0.1
```
An example of the output bed file is below (columns: chromosome, start, stop, methylation category).
```
maize_chr1_reference 2900 3000 Unmethylated
maize_chr1_reference 3000 3100 Unmethylated
maize_chr1_reference 3100 3200 Unmethylated
maize_chr1_reference 3200 3300 Unmethylated
maize_chr1_reference 12500 12600 Unmethylated
```
The output of the R script is a bed file UMTs.bed that lists the coordinates of all the tiles that were categorised as unmethylated. In addition, a file called mC_domains_cov_3_sites_2_MR_0.4_UMR_0.1_tiles_with_data.bed is also produced by this script and provides a list of all regions that had sufficient data for UMR testing. The UMR file can now be sorted, and then adjacent tiles are merged to yield the final unmethylated regions.
```
sort -k1,1 -k2,2n UMTs.bed > UMTs_sorted.bed
bedtools merge -i UMTs_sorted.bed > all_UMRs.bed
awk '($3-$2) >= 299' all_UMRs.bed > UMRs.bed
```
The entire final output bed files are shown below (columns: chromosome, start, stop). The “all_UMRs.bed” file contains 6 UMRs; however, we highly recommend that the UMRs are further filtered to only retain UMRs that are 300 bp or longer in size. Some unmethylated regions of 300 bp or less could be functionally important. However, we have found that the majority of these small unmethylated regions (and there are a lot) lack evidence of functionality; for example, 99.5% did not have accessible chromatin [7]. However, further research is needed to develop methods to identify the potentially small minority of functionally important small (<300 bp) unmethylated regions.
```
# all_UMRs.bed
maize_chr1_reference 2900 3300
maize_chr1_reference 12500 12800
maize_chr1_reference 12900 13600
maize_chr1_reference 13800 13900
maize_chr1_reference 14000 14800
maize_chr1_reference 15200 15300
# UMRs.bed
maize_chr1_reference29003300
maize_chr1_reference1250012800
maize_chr1_reference1290013600
maize_chr1_reference1400014800
```
Results interpretation
The key output from this analysis workflow is the bed file with the coordinates of the UMRs, “UMRs.bed.” The bed file can be used for a number of downstream analyses. The total number, size distribution, and genomic location of UMRs can be analysed. Most diploid genomes analysed to date have approximately 100 Mb of UMRs in total across the whole genome, so it would be expected that the total number of UMRs should be around this number (or greater for polyploids). If no UMRs are detected, this would be surprising and would suggest that an error has occurred; users are advised to step backwards in the protocol to identify which step and output file may be empty or incomplete. The output file of this example can also be visualised using IGV. In Figure 2 below, we show an IGV screenshot of this UMR bed file (track labelled “UMRs”) along with the bigWig files that display the per-cytosine methylation (track labelled “methylation”). In the methylation track, we can see the three different coloured bars, which represent CG, CHG, and CHH methylation in each region of the 20 kb fragment. This has been aligned to the genome annotation for maize, so that we can see where genes are in relation to the methylation. The one gene within this 20 kb region is Zm0001d027232, which corresponds to an mRNA-hypothetical protein; as we can see, there is reduced methylation present where the gene is located. The analysis has identified five UMRs in this gene region and a sixth present downstream of the gene. This singular downstream UMR may represent a regulatory region containing cis-regulatory elements that could influence expression of the nearby gene, either as enhancers or silencers. Importantly, as highlighted in the figure, there are also other gaps in the methylation data upstream of the gene; however, rather than being unmethylated regions, these regions lack data as can be seen by the gaps in the “tiles with data” track.
Figure 2. Example output of the unmethylated region (UMR)-calling pipeline. The output is viewed in IGV for a 20 kb section of the maize genome on chromosome one surrounding the gene Zm00001d027232. Bars in the “methylation” track (bigwig files) represent percent methylation (0%–100%) for each cytosine in the CG (blue), CHG (green), and CHG (orange) context. UMRs are marked by the large red rectangles (UMRs.bed file); there are several UMRs overlapping the gene locus and one UMR located 9 kb downstream of the gene. Tiles with sufficient data (coverage and minimum number of cytosines) are marked by the small grey rectangles; the dashed boxes mark examples of gaps in the methylation data that are not UMRs because these regions are instead missing data.
Acknowledgments
We wish to acknowledge the University of Queensland's Research Computing Centre (RCC) for its support in this research. PAC was supported by an ARC Discovery Early Career Researcher Award (DE200101748). This protocol was adapted from our previous work [7].
Competing interests
The authors declare no competing interests.
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Supplementary information
Data and code availability: All data and code have been deposited to GitHub: https://github.com/Bio-protocol/unmethylated-regions_UMR-extractor-WGBS/tree/master
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4,945 | https://bio-protocol.org/en/bpdetail?id=4945&type=0 | # Bio-Protocol Content
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Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique
FB Federico Berdun
MV Matías Valiñas
Gabriela Pagnussat
EZ Eduardo Zabaleta
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4945 Views: 659
Reviewed by: Noelia ForesiEmilia KrypotouIsmail Tahmaz
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Original Research Article:
The authors used this protocol in Molecular Microbiology Feb 2021
Abstract
Diatoms serve as a source for a variety of compounds with particularbiotechnological interest. Therefore, redirecting the flow to a specific pathwayrequires the elucidation of the gene’s specific function. The mostcommonly used method in diatoms is biolistic transformation, which is a veryexpensive and time-consuming method. The use of episomes that are maintained asclosed circles at a copy number equivalent to native chromosomes has become auseful genetic system for protein expression that avoids multiple insertions,position-specific effects on expression, and potential knockout of non-targetedgenes. These episomes can be introduced from bacteria into diatoms viaconjugation. Here, we describe a detailed protocol for gene expression thatincludes 1) the gateway cloning strategy and 2) the conjugation protocol for themobilization of plasmids from bacteria to diatoms.
Keywords: Conjugation Diatom Bacteria Mobilization plasmid Destination vector Phaeodactylum tricornutum
Background
Diatoms are unicellular, predominantly photosynthetic, free-living microorganisms that play a central role in trophic webs as primary producers [1]. Diatoms represent the most abundant group within the phytoplankton community, contributing to 40% of global CO2 fixation in the oceans. These organisms are found in freshwater bodies, making them one of the most ecologically successful microalgae worldwide [2,3]. One physiological aspect that explains its ecological success is the energetic-metabolic coupling between mitochondria and chloroplasts [4,5].
From a biotechnological point of view, these organisms produce a variety of compounds of interest such as silica frustule, used as filtering and abrasive materials, and terpenes like fucoxanthin, lupeol, and betulin with antitumoral and antioxidant properties [6]. Furthermore, these organisms are a source of traditional biofuels including methane, through the anaerobic digestion of algae biomass, and biodiesel derived from oil. These organisms show an interesting lipid profile, rich in very long– chain polyunsaturated fatty acids (ω-3 and ω-6), which are of great interest to the industry, especially because they are not usual in other organisms like chlorophytes and flowering plants [7].
The advancement of genetic tools and increasing knowledge on metabolic pathways have facilitated the development of strategies to enhance the productivity of diatoms byredirecting the metabolic flow towards desired products [8]. Thus, diatom genetic manipulation is essential for the elucidation of specific gene functions. Biolistic transformation methods are standard for many diatom species; however, they are time-consuming and require high-yield plasmid DNA preparations and access to expensive specific equipment and reagents (e.g., gene gun). On the other hand, episodes provide a reliable, consistent, and predictable platform for protein expression by avoiding the complications of random chromosomal integration including multiple insertions, position-specific effects on expression, and potential knockout of non-targeted genes. They can be efficiently transferred from bacteria into the diatom via the conjugation method developed by Karas et al. [9] and improved by Diner et al. [10]. Here, we thoroughly describe the procedure previously published [11] and frequently employed in our lab for cloning genes of interest and the subsequent mobilization of vector constructions from bacteria into diatoms by conjugation.
Materials and reagents
Biological materials
Pipette tips: 1–2 µL, 2–200 μL, and 100–1,000 μL (Deltalab, catalog number: 200070 and 301-09)
90 mm diameter Petri dishes (Deltalab, catalog number: 200209)
1.5 mL microcentrifuge tubes (Deltalab, catalog number: 200400P)
50 mL conical centrifuge tubes (Deltalab, catalog number: 42993)
0.22 μm nylon membrane filters (e.g., GVS, catalog number: FJ13BNPNY002AD01)
Reagents
Phaeodactylum tricornutum liquid cultures [e.g., Culture collection of algae, University of Texas, Austin (Utex), catalog number: 646]
PCR kit
dNTPs (Promega, catalog number: U123A)
Platinum Pfx (Invitrogen, catalog number: 11708-013)
10× Pfx amplification buffer (Invitrogen, catalog number: 52806)
MgCl2 25 mM (Promega, catalog number: A351H)
Taq Pegasus (Productos Bio-Logicos, catalog number: EA01M)
GoTaq green buffer 5× (Promega, catalog number: M791A)
Destination plasmid pFcpB (Addgene, catalog number: 90098)
pTA-Mob (Addgene, catalog number: 149662)
Escherichia coli DH10B (Thermo Fisher, catalog number: Eco 113)
E. coli pTA-Mob liquid cultures (homemade)
pENTR/D-TOPO Vector kits (Thermo Fisher Scientific, catalog number: K240020SP)
LR Clonase Enzyme mix (Thermo Fisher Scientific, catalog number: 11791019)
Agar medium 2% (Britannia, catalog number: B0101406)
Luria Broth (LB) medium (homemade)
Gentamicin (Gn) stock solution 25 mg/mL (1:1,000) (Merck, catalog number: G3632)
Kanamycin (Kn) stock solution 50 mg/mL (1:1,000) (Merck, catalog number: BP861)
Ampicillin (Amp) stock solution 100 mg/mL (1:1,000) (Merck, catalog number: A9518)
Zeocin (Zeo) stock commercial solution (Invitrogen, catalog number: R25001); use a concentration of 7.5 µL commercial solution stock in 10 mL of BG11 medium
Amphotericin (Amph) stock solution B 2.5 mg/ mL (1:1,000) (Richet S.A laboratory, https://www.richet.com.ar/en/list?t=n&i=13); use a concentration of 2.5 µg/ mL
BG11 medium (homemade)
Selection plate (0.5× BG11, 1% agar + antibiotics (Amp + Kn + Amph + Gn + Zeo)
Conjugation plate (0.5× BG11 + 0.9% agar + 5% LB + antibiotics (Amp + Amph + Gn)
Tryptone (BD, catalog number: 211705)
Yeast extract (Oxoid, catalog number: LP0021)
NaCl (J.T. Baker, catalog number: 3624-19)
Na2Mg EDTA (Sigma-Aldrich, catalog number: 14402-88-1)
Ferric citrate (Merck, catalog number: 3522-50-7)
CaCl2·2H2O (Merck, catalog number: 10035-04-8)
MgSO4·7H2O (Cicarelli, catalog number: 1054214)
K2HPO4 (Cicarelli, catalog number: 1015214)
H3BO3 (Cicarelli, catalog number: 771214)
MnCl2·4H2O (Merck, catalog number: 13446-34-9)
ZnSO4·7H2O (Merck, catalog number: 7446-20-0)
CuSO4·5H2O (Merck, catalog number: 1.02790.1000.1026)
CoCl2·6H2O (Merck, catalog number, 7791-13-1)
Na2MoO4·2H2O (Merck, catalog number: 10102-40-6)
NaCO3 (Merck, catalog number: 497-19-8)
NaNO3 (Merck, catalog number: 7631-99-4)
Solutions
LB medium (1 L)
BG11 medium (1 L):
Stock I (1 L)
Stock II (1 L)
Stock III (1 L)
Stock V - microelements (1 L)
Carbonate supplement (50 mL)
Nitrate supplement (50 mL)
Recipes
LB medium (1 L)
10 g of tryptone
5 g of yeast extract
10 g of NaCl
1 L of dH2O
BG11 medium (1 L)
Stock I (1 L):
0.1 g of Na2Mg EDTA
0.553 g of ferric citrate
3.6 g of CaCl2·2H2O
Filter sterilize into a sterile bottle or autoclave.
Stock II (1 L):
7.5 g of MgSO4·7H2O
Filter sterilize into a sterile bottle or autoclave.
Stock III (1 L):
3.05 g of K2HPO4
Filter sterilize into a sterile bottle or autoclave.
Stock V - microelements (1 L):
2.86 g of H3BO3
1.81 g of MnCl2·4H2O
0.222 g of ZnSO4·7H2O
0.074 g of CuSO4·5H2O
0.05 g of CoCl2·6H2O
0.4451 g of Na2MoO4·2H2O
Filter sterilize into a sterile bottle or autoclave.
Carbonate supplement (50 mL)
1 g of NaCO3
Filter sterilize into a sterile bottle.
Nitrate supplement (50 mL)
15 g of NaNO3
Filter sterilize into a sterile bottle.
For basic BG11 (1 L), combine the following stock solutions:
Add 10 mL of stock I, II, and III.
Add 1 mL of stock V and carbonate supplement.
Add 5 mL of nitrate supplement.
Add sterilized dH2O to complete to 1 L.
Equipment
Pipettes
Neubauer counting chamber
Laminar air flow equipment
Centrifuge with 50 mL tube capacity
Erlenmeyer
Room or chamber at 37 ºC
Room or chamber at 18 ºC with light 100-200 PAR
Shaker (Vicking, model: Shaker Pro)
Spectrophotometer (Gene Quant, model: 1300)
Binocular light microscope
Applied Biosystems Veriti Thermocycler
Procedure
Preparation of a donor bacterial strain (E. coli pTA-Mob)
The donor bacterial strain should contain a plasmid that allows the formation of the conjugative pili between the bacterium and the diatom. This plasmid, called pTA-Mob (Gnr) [12], is a large plasmid of 52.7 kb. Transform competent E. coli cells (e.g., DH10B) with pTA-Mob plasmid using the appropriate form (chemical/electro competents). Mix DH10B competent cells with ~50 ng of pTA-Mob plasmid and gently flick the tube several times. Incubate the cells on ice for 2 min. Transfer the tube from ice to a 42 °C water bath and heat shock for exactly 45 s. After treatment, add 700 µL of LB medium and incubate at 37 °C for 1 h. Plate on LB solid medium (LB medium + 10 g/L agar) containing gentamicin and incubate at 37 ºC overnight. Pick a resistant colony and check the presence of plasmid by colony PCR using a specific primer, e.g., Gentamycin_fw: TTAGGTGGCGGTACTTGGGT and Promoter_Rv: GTTGACATAAGCCTGTTCGGT (expected PCR product of 752 bp), and/or by digestion with restriction enzymes, e.g., EcoRI (expected digested fragments: 29 kb, 11,7 kb, 8,8 kb, 6,84 kb). Once corroborated, prepare ultracompetent cells from DH10B pTA-Mob strain (Gn r) following the Inoue method for preparation of competent cells (Figure 1A) [13].
Preparation of constructs to mobilize from bacteria into diatoms
Amplify the sequence of interest with a high-fidelity polymerase (Pfx) using a forward primer containing a CACC sequence in the 5′ end by PCR and clone it into a pENTRTM TOPO® entry vector. Once the construct is obtained, check it by PCR using a primer from your gene of interest and Universal M13 primers (M13_fw: GTAAAACGACGGCCAG, M13_rv: CAGGAAACAGCTATGAC), and digestion by restriction enzymes. Finally, corroborate the construct by sequencing.
The pENTR vector has a recombination site (attL1/attL2) that allows recombination with other plasmids containing attR1/attR2 sequences (destination plasmids). The recombination reaction is facilitated by the use of Gateway LR Clonase Enzyme mix. The pFcpB vector is used as a destination plasmid to express proteins in diatoms. Note that this vector contains a light-induced promoter. Check the destination plasmid construct by PCR (using Taq polymerase), using a primer from your gene of interest and PfcpB primer (pFcpB_fw: TTCACGGTTGCCAGAAGTCAAGTCG, pFcpB_rv: TCGAGGTAGCTCAGAATTCACCAC), and digestion by restriction enzymes (Figure 1B).
Figure 1. Simplified diagram of the conjugation protocol. A. Transform DH10B competent cells with ~50 ng of pTA-Mob plasmid, plate on LB solid medium containing gentamicin, and incubate at 37 ºC overnight. Pick a resistant colony and check the presence of plasmid by colony PCR or/and digestion with restriction enzymes. B. Amplify the sequence of interest (SOI) using a forward primer containing a CACC to clone it into a pENTR™ TOPO® entry vector. Transform competent cells and check the entry clone by PCR and digestion with restriction enzymes. With the entry clone corroborated, make a Gateway LR Clonase Enzyme Mix reaction to obtain a destination plasmid. Once the destination plasmid is corroborated by sequencing, transform the pTA-Mob competent cells. C. Centrifuge fresh diatoms (1 × 108 cells) and E. coli (OD = 0.8–1.2) cultures and resuspend in 500 µL of the corresponding culture medium. Then, combine and plate in conjugation plates. After 20 days, exconjugant diatom colonies should appear.
Transformation of DH10B pTA-Mob (Gnr) strain with a destination plasmid
Transform the E. coli pTA-Mob strain with ~50 ng of destination plasmid and plate on LB solid medium with both antibiotics (Gn for pTA-Mob plasmid and Amp for pFcpB) to select both plasmids (three days before the conjugation step).
Incubate overnight at 37 °C.
Pick a colony and inoculate 5 mL of fresh LB medium with antibiotics (Gn + Amp).
Grow overnight at 37 °C with shaking.
In the afternoon of the day before the conjugation, inoculate 50 mL of fresh LB medium (with antibiotics Amp + Gn) using a 1/100 dilution of the overnight culture.
Let the culture grow overnight at 20 °C and 190 rpm instead of 3 h at 37 °C as described in Karas et al. (2015) [9]. This modification results in significantly higher conjugation efficiency, presumably because the lower temperature favors the expression of recombinant proteins. In both cases, the E. coli pTA-Mob strain with the destination plasmid at the time of harvest must have an OD between 0.8 and 1.2.
Growth of diatom (Phaeodactylum tricornutum) cells
The general growth conditions for diatoms used in this protocol include long-day photoperiod (16:8 h light/dark), 100 μmol photons m-2·s-1 (100 μE m-2·s-1) light intensity, 18 °C constant temperature, and 120 rpm constant agitation.
Pick a diatom colony grown on solid BG11 and inoculate 50 mL of liquid BG11 containing ampicillin and amphotericin.
After approximately seven days, this culture will be in exponential state (1 × 106 cells/mL). Determine the exact cell number with the Neubauer counting chamber using a binocular light microscope and take a volume of culture to obtain a final concentration of 1 × 104 cells/mL in 50 mL of liquid BG11 in an Erlenmeyer flask. Then, follow the culture until it reaches the stationary phase of the growth curve (approximately 1 × 106 cells/mL). The final cell yield in each culture may vary depending on the medium and growth conditions. In our conditions, it takes between one and two weeks. A higher cell number results in a higher number of exconjugant colonies.
Cell harvest and control plates
Take the entire volume of the Erlenmeyer flask for both bacteria and diatoms and centrifuge them at room temperature (always work under the laminar flow). Centrifuge at low speed (2,000 × g) for 5 min. Do not use a brake in order to ensure cell viability.
Resuspend the pellets in 500 µL of the corresponding culture medium: BG11 for diatoms and LB for E. coli.
Take 50 µL of diatoms and plate:
Plate without Zeo to check growth (positive control).
Plate with Zeo to corroborate wild-type diatom antibiotic susceptibility (negative control).
Incubate these plates at 18 °C under 100 μmol photons m-2·s-1 light intensity.
Mix diatoms and DH10BpTA-Mob bacteria (conjugation)
In a 1.5 mL Eppendorf tube, combine 200 µL of diatoms with 200 µL of E. coli DH10B pTA-Mob bacteria strain containing the construct of interest. Spread the mixture on conjugation plates (Figure 1C).
Incubate for 90 min in darkness at 30 °C.
Incubate at 18 °C under 100 μmol photons m-2·s -1 light intensity for 48 h. During this time, the conjugation is carried out.
Collect all the cells grown on the plate by adding 200 µL of BG11 medium and then take the entire volume and plate it on a selection plate containing Zeo.
Incubate at 18 °C under 100 μmol photons m-2·s -1 light intensity. After 20 days, exconjugant colonies resistant to Zeo should appear.
Check the presence of the plasmid in the exconjugant diatom cells by colony PCR.
To perform colony PCR, proceed as a standard colony PCR. Take a colony with a toothpick and introduce it into a PCR tube with 20 μL of PCR solution mix containing primers (the best combination will be one of the particular contrast and a second PfcpB primer, as suggested in section B).
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Cainzos, M., Marchetti, F., Popovich, D., Leonardi, P, Pagnussat, G and Zabaleta E. (2021) Gamma Carbonic Anhydrases are subunits of the Mitochondrial Complex I of diatoms. Mol. Microbiology 116(1), pp. 109–125
General notes and troubleshooting
The efficiency of conjugation strongly depends on the length of the construct, which is in turn determined by the length of the sequence of interest cloned into the destination vector. The highest number of exconjugants is obtained with small plasmids.
Acknowledgments
This work was financially supported by ANPCyT grant PICT 18 0652.
Competing interests
The authors declare no conflicts of interest.
References
Sommer, U., Stibor, H., Katechakis, A., Sommer, F. and Hansen, T. (2002). Pelagic food web configurations at different levels of nutrient richness and their implications for the ratio fish production:primary production. Sustainable Increase of Marine Harvesting: Fundamental Mechanisms and New Concepts : 11–20.
Falciatore, A. and Mock, T. (Eds.). (2022). The Molecular Life of Diatoms. Springer International Publishing.
Wilhelm, C., Büchel, C., Fisahn, J., Goss, R., Jakob, T., LaRoche, J., Lavaud, J., Lohr, M., Riebesell, U., Stehfest, K., et al. (2006). The Regulation of Carbon and Nutrient Assimilation in Diatoms is Significantly Different from Green Algae. Protist 157(2): 91–124.
Allen, A. E., LaRoche, J., Maheswari, U., Lommer, M., Schauer, N., Lopez, P. J., Finazzi, G., Fernie, A. R. and Bowler, C. (2008). Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc. Natl. Acad. Sci. U.S.A. 105(30): 10438–10443.
Murik, O., Tirichine, L., Prihoda, J., Thomas, Y., Araújo, W. L., Allen, A. E., Fernie, A. R. and Bowler, C. (2018). Downregulation of mitochondrial alternative oxidase affects chloroplast function, redox status and stress response in a marine diatom. New Phytol. 221(3): 1303–1316.
Butler, T., Kapoore, R. V. and Vaidyanathan, S. (2020). Phaeodactylum tricornutum: A Diatom Cell Factory. Trends Biotechnol. 38(6): 606–622.
Zulu, N. N., Zienkiewicz, K., Vollheyde, K. and Feussner, I. (2018). Current trends to comprehend lipid metabolism in diatoms. Prog. Lipid Res. 70: 1–16.
Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G. and Tredici, M. R. (2008). Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low‐cost photobioreactor. Biotechnol. Bioeng. 102(1): 100–112.
Karas, B. J., Diner, R. E., Lefebvre, S. C., McQuaid, J., Phillips, A. P., Noddings, C. M., Brunson, J. K., Valas, R. E., Deerinck, T. J., Jablanovic, J., et al. (2015). Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 6(1): e1038/ncomms7925.
Diner, R. E., Bielinski, V. A., Dupont, C. L., Allen, A. E. and Weyman, P. D. (2016). Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA Delivery Methods. Front. Bioeng. Biotechnol. 4: e00065.
Cainzos, M., Marchetti, F., Popovich, C., Leonardi, P., Pagnussat, G. and Zabaleta, E. (2021). Gamma carbonic anhydrases are subunits of the mitochondrial complex I of diatoms. Mol. Microbiol. 116(1): 109–125.
Strand, T. A., Lale, R., Degnes, K. F., Lando, M. and Valla, S. (2014). A New and Improved Host-Independent Plasmid System for RK2-Based Conjugal Transfer. PLoS One 9(3): e90372.
Sambrook, J. and Russell, D. W. (2006). The Inoue Method for Preparation and Transformation of CompetentE. Coli: “Ultra-Competent” Cells. Cold Spring Harb. Protoc. 2006(1): 10–1101.
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© 2024 CONICET: Consejo Nacional de Investigaciones Científicas y Técnicas; This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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4,946 | https://bio-protocol.org/en/bpdetail?id=4946&type=0 | # Bio-Protocol Content
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Analysis of Cleavage Activity of Dengue Virus Protease by Co-transfections
LG Lekha Gandhi
MV Musturi Venkataramana §
(§ Technical contact)
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4946 Views: 408
Reviewed by: Luis Alberto Sánchez VargasRan ChenVaibhav B. ShahDay-Yu Chao
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Original Research Article:
The authors used this protocol in iScience 2023
Abstract
The genome of the dengue virus codes for a single polypeptide that yields three structural and seven non-structural (NS) proteins upon post-translational modifications. Among them, NS protein-3 (NS3) possesses protease activity, involved in the processing of the self-polypeptide and in the cleavage of host proteins. Identification and analysis of such host proteins as substrates of this protease facilitate the development of specific drugs. In vitro cleavage analysis has been applied, which requires homogeneously purified components. However, the expression and purification of both S3 and erythroid differentiation regulatory factor 1 (EDRF1) are difficult and unsuccessful on many occasions. EDRF1 was identified as an interacting protein of dengue virus protease (NS3). The amino acid sequence analysis indicates the presence of NS3 cleavage sites in this protein. As EDRF1 is a high-molecular-weight (~138 kDa) protein, it is difficult to express and purify the complete protein. In this protocol, we clone the domain of the EDRF1 protein (C-terminal end) containing the cleavage site and the NS3 into two different eukaryotic expression vectors containing different tags. These recombinant vectors are co-transfected into mammalian cells. The cell lysate is subjected to SDS-PAGE followed by western blotting with anti-tag antibodies. Data suggest the disappearance of the EDRF1 band in the lane co-transfected along with NS3 protease but present in the lane transfected with only EDRF1, suggesting EDRF1 as a novel substrate of NS3 protease. This protocol is useful in identifying the substrates of viral-encoded proteases using ex vivo conditions. Further, this protocol can be used to screen anti-protease molecules.
Key features
• This protocol requires the cloning of protease and substrate into two different eukaryotic expression vectors with different tags.
• Involves the transfection and co-transfection of both the above recombinant vectors individually and together.
• Involves western blotting of the same PVDF membrane containing total proteins of the cell lysate with two different antibodies.
• Does not require purified proteins for the analysis of cleavage of any suspected substrate by the protease.
Graphical overview
Keywords: Dengue virus Protease Co-transfection Flavivirus Anti-protease molecules Western blotting
Background
Viral encoded proteins mitigate cellular activities in order to bring the host under their control. In this direction, proteases of viral origin cleave the host proteins and cause irreparable damage to the host cells, hence being prime drug targets. Poliovirus-encoded 2A protease [1] cleaves the eukaryotic initiation factor 4G (eIF4G) and stalls cellular mRNA translation. Proteases of hepatitis C virus (HCV) [2] and dengue virus [3] are known to target the mitochondrial homeostasis, which leads to mitochondrial dysfunction and failure of the immune response. The main proteases 3CLpro and the papain-like protease (PLPro) of SARS CoV2 [4] possess protease activity, and the substrates need to be characterized. For this purpose, in silico techniques are useful, but experimental evidence is needed to confirm the analysis. In vitro pull-down assays or immunoprecipitation (IP) assays are being used to identify the novel substrates of viral-encoded proteases. 2D gel electrophoresis followed by MALDI-TOFF is also being used to this purpose but often yields false positives or negatives.
Dengue virus infections are hyperendemic in more than 130 countries across the globe, causing millions of infections and thousands of deaths annually. There is no vaccine or specific drugs developed to date, in spite of several attempts. One of the reasons for this is the failure to identify suitable drug/vaccine targets. The genome of this virus encodes three structural and seven non-structural (NS) proteins, along with two untranslated regions, one at each end. Among NS proteins that play significant roles during viral replication, NS3, alone or along with NS2B, possesses a crucial role and is the prime drug target for developing antivirals. NS3 is a 70 kDa protein that contains a 180 amino acid N-terminal trypsin-like serine protease domain followed by a C-terminal helicase domain [5]. This protein is a multifunctional serine protease, forming the catalytic triad with amino acids histidine (H-51), aspartate (D-75), and serine (S-135). NS3 is also known to regulate several host proteins to induce and maintain pathogenesis. Along with cleavage of the self-polypeptide to yield functional proteins, this protease is also known to cleave the host cellular proteins FAM134B (endoplasmic receptor), Ikα/β (cellular factors), nucleoporins (Nups), MITA, MFN1, and MFN2, thereby enhancing viral replication and affecting host metabolism [6-10]. However, there is still a lack of knowledge regarding the full list of host proteins that NS3 cleaves and its biological effects, particularly with regard to pathogenicity. In vitro cleavage experiments are useful to characterize such substrates but require purified proteins. In addition, the in vitro conditions sometimes fail to mimic the natural conditions and lead to erroneous results. NS2B–NS3 interactions have been targeted for drug development [11]. Therefore, this protocol is expected to be useful in the quick screening of anti-protease molecules against NS2B–NS3 interactions. The present protocol, an ex vivo study, describes the co-transfection followed by western blotting in order to evaluate EDRF1 as a novel substrate of dengue virus protease. Furthermore, this protocol is expected to be useful in the identification of any such substrates using NS3 of any of the four serotypes of dengue virus infections.
Materials and reagents
All materials and reagents listed below can be acquired from other suppliers. In our lab, we use molecular and cell culture grade for all reagents. High-quality products are recommended for the cell culture experiments.
Biological materials
Cell lines: human embryonic kidney 293 cells (HEK 293), obtained from National Centre for Cell Science (NCCS), Pune, India
Reagents
Anti-GFP (D5.1) rabbit mAB (monoclonal antibody) (Cell Signalling Technology, catalog number: 2956)
Anti-Myc tag mouse monoclonal antibody (Proteintech, catalog number: 60003-2-Ig)
Rabbit anti-mouse-IgG HRP conjugated antibody (GeNei, catalog number: 1140580011730)
Mouse anti-rabbit-IgG HRP conjugated antibody (Santa Cruz Biotechnology, catalog number: sc-2357)
Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number: 11995-065)
Fetal bovine serum (FBS) (Gibco, catalog number: 10270106)
Antibiotic (penicillin and streptomycin) (Gibco, catalog number: 15240062)
Trypsin-EDTA (1×) solution (HiMedia, catalog number: TCL007)
Lipofectamine 2000 (Invitrogen, catalog number: 11668-027)
Radio Immunoprecipitation Assay (RIPA) buffer (Sigma Aldrich, catalog number: R0278)
Femto LUCENT™ PLUS-HRP (G-Biosciences, catalog number: 786-003)
Protease inhibitor cocktail (Protease Arrest™) 100× (G-Biosciences, catalog number: 786-331)
Bradford reagent (Bio-Rad, catalog number: 5000201)
Immobilon®-P PVDF membrane (Merck Millipore, catalog number: IPVH00010)
Trypan blue (Sigma, catalog number: T6146)
OptiMEM (Gibco, catalog number: 31985062)
Ponceau S stain (Sigma, catalog number: P3504)
Tris base (HiMedia, catalog number: TC072)
Glycine (Merck, catalog number: 1.94907.0521)
Sodium dodecyl sulphate (SDS) (SR Lifesciences, catalog number: 14374)
NaCl (Merck, catalog number: 1.93206.0521)
KCl (HiMedia, catalog number: 7447-40-7)
NaH2PO4 (Merck, catalog number: MB024)
KH2PO4 (HiMedia, catalog number: PCT0009)
BSA (HiMedia, catalog number: GRM3151)
Skimmed milk powder (HiMedia, catalog number: GRM1254)
Tween-20 (SR Life Sciences, catalog number: 28599)
Absolute ethanol (Analytic CS, catalog number: 64-17-5)
Phosphate buffer saline (PBS) (Gibco, catalog number: 10010023)
GeneJet Plasmid MidiPrep kit (Thermo Scientific, catalog number: K0481)
Solutions
10× PBS (see Recipes)
PBST (1× PBS and Tween-20) (see Recipes)
Blocking buffer (see Recipes)
70% ethanol (see Recipes)
10× transfer buffer (see Recipes)
Stripping buffer (see Recipes)
Recipes
10× PBS
Reagent Final concentration Quantity
NaCl 1.37 M 80 g
KCl 27 mM 2 g
NaH2PO4
KH2PO4
100 mM
18 mM
14.4 g
2.4 g
H2O - Up to 1 L
Note: Adjust pH to 7.4. Stock can be stored at 25 °C up to one month.
Note: From this 10× stock, 500 mL of 1× PBS solution can be prepared: mix 50 mL of 10× PBS with 450 mL of H2O (can be stored at 25 °C for a week). Use double-distilled water (ddH 2O) (autoclaved) for preparing 1× PBS solution.
PBST
Reagent Final concentration Quantity
10× PBS
Tween-20
H2O
1×
0.1%
n/a
50 mL
500 μL
450 mL
Note: Use ddH2O.
Blocking buffer
Reagent Final concentration Quantity
BSA/skimmed milk powder
PBST
2% or 7%
1×
0.2 or 0.7 g
10 mL
Note: Prepare fresh when required. 7% blocking buffer solution is required for the blocking the PVDF membrane.
Optional: 2% blocking buffer solution is required for preparing antibody dilutions.
70% ethanol
Reagent Final concentration Quantity
Ethanol
H2O
70%
n/a
350 mL
50 mL
Note: Prepare fresh when required.
10× transfer buffer
Reagent Final concentration Quantity
Tris 25 mM 30.2 g
Glycine 192 mM 144 g
H2O - up to 1 L
Note: From this 10× stock, 1× transfer buffer can be prepared: dissolve 100 mL of 10× transfer buffer in 200 mL of methanol and make up the volume with water up to 1 L. Store at 4 °C; this can be reused for two weeks.
Stripping buffer
Reagent Final concentration Quantity
Glycine 199 mM 1.5 g
SDS 0.1% 0.1 g
Tween-20 1% 1 mL
H2O up to 100 mL
Note: Adjust to pH 2.2.
Laboratory supplies
Polypropylene conical-bottom centrifuge tubes, 15 and 50 mL Falcon (Tarsons, catalog number: 546021 and 546041) (sterile)
T-25 cell culture flasks (Tarsons, catalog number: 950040)
60 mm cell culture dishes (Tarsons, catalog number: 960020)
Pipettes (1000, 200, and 20 μL) (Eppendorf, catalog numbers: 3123000063, 3123000055, 3123000098) and tips (Tarsons, catalog numbers: 523104, 523101, 523109)
Hand gloves (Kimberlay Gloves, catalog number: KC500-S)
1.5 mL Eppendorf tubes (Tarsons, catalog number: 500010) and CRYOCHILL™ Vial 2D Coded (Tarsons, catalog number: 883192)
Parafilm 4" × 125' (SR Lifesciences, catalog number: 000814)
Aluminum foil
Discard box (any supplier)
Spray bottles (any supplier)
Cell scrapper (Tarsons, catalog number: 960051)
Equipment
-80 °C deep freezer (Eppendorf, catalog number: F570)
Microcentrifuge (Eppendorf, catalog number: 5424R)
CO2 incubator (Eppendorf, catalog number: Galaxy 48R)
Inverted brightfield microscope (Lawrence and Mayo, catalog number: TC5400)
Chemidoc image analyzer (Bio-Rad, catalog number: 12003028)
Electroblot transfer unit (BioNova)
SDS-PAGE unit (Bio-Rad, model: Mini-PROTEAN Tetra Cell, catalog number: 1658038)
Hemocytometer (Neubeur Blood Counting Chamber)
Laminar hood (Laminar Flow Systems)
Animal cell culture facility
Autoclave (KETAN, catalog number: PA21)
Water bath (GeNei, catalog number: 107931GB)
Vortex mixer (Tarsons, catalog number: 3002)
Software and datasets
Image Lab software (Bio-Rad) (v6.1.0, 2020)
Zen software for Carl Zeiss fluorescence microscope (Zen 2.3, blue edition, v2.3.69.1000)
Procedure
Culturing and maintenance of cells
Take a 2.0 mL cryovial of HEK 293 cells from -80 °C or liquid nitrogen.
Caution: Use cryogenic hand gloves for taking the vial from liquid nitrogen or -80 °C.
Thaw the cryovial in a water bath for 2–3 min at 37 °C.
Add 1 mL of serum-free DMEM to the thawed vial.
Collect the cells and transfer in a 15 mL Eppendorf tube.
Centrifuge the cells at 800× g for 3 min.
Replace the medium with 1 mL of fresh complete DMEM [containing heat-inactivated 10% FBS and 1% antibiotic (v/v) (penicillin and streptomycin)].
Count ~8 × 105 cells using a hemocytometer and seed in T-25 flask.
Incubate the cells at 37 °C in a humidified incubator for 12–16 h with 5% CO2.
The next day, observe the cells under the inverted brightfield microscope for their proper adherence (Figure 1 ).
Figure 1. Representative image showing HEK cell confluency (70%–80%) and adherence analyzed under brightfield inverted microscope
Replace the medium with 3 mL of fresh complete DMEM (containing 10% FBS and 1% antibiotics).
Cell splitting, counting, and seeding
From the above cultured T-25 flask, discard the medium.
Add 1 mL of Trypsin-EDTA solution (1×) and incubate for 3 min at 37 °C in a CO2 incubator.
Observe the detached cells under the brightfield inverted microscope.
Once the cells are detached, add 1 mL of complete DMEM to inactivate the trypsin and collect cells into a fresh 15 mL Falcon tube.
Optional: Add 1 mL of serum-free DMEM to collect the leftover cells from T-25 flasks.
Harvest cells by centrifugation at 500× g for 3 min.
Wash the cell pellet with 1 mL of serum-free DMEM medium (or mix 500 μL of DMEM and 500 μL of 1× PBS in 1:1 ratio). Resuspend the cells gently by pipetting up and down.
Centrifuge again at 500× g for 3 min.
Discard the medium, add 1 mL of fresh complete DMEM medium, and suspend gently.
Prepare two or three T-25 flasks with 3 mL of complete DMEM medium and add 200 μL of the above resuspended cell suspension (in ratios 1:10 or 1:15) to the flasks.
Gently shake the flasks (back and forth) and allow cells to grow in a humidified CO2 incubator at 37 °C.
To perform counting of the cells, follow the above steps B1a–g.
Take 1 mL of cell suspension and count using a hemocytometer with trypan blue.
In a fresh 1.5 mL Eppendorf tube, add 100 μL of trypan blue dye and 100 μL of cell suspension. Mix gently with a pipette.
Place the coverslip on the hemocytometer and add 10 μL of trypan blue cell suspension under the coverslip using a pipette. Allow the cells to spread evenly on the counting chamber.
Count the number of viable cells from the four big squares of the hemocytometer using the formula:
No. of cells/mL = (No. of cells in 4 big squares/4) × 104 × dilution factor*
*Dilution factor = 2 (100 μL of trypan blue dye: 100 μL of cell suspension)
104 = Conversion factor to 1 mL
For example:
No. of cells in 4 big squares: 1000
No of cells/mL = (1000/4) x 104 × 2 i.e., 5 × 106 cells/mL
To seed 1 × 106 cells/mL, add 200 μL of cell suspension in 800 μL of complete DMEM.
Seed 1 × 106 cells per dish (60 mm dishes) and allow to grow to attain ~80% confluency at 37 °C in a CO2 incubator for 12–14 h (see Troubleshooting 4).
Co-transfection
Observe the cells seeded in 60 mm dishes in the above step to confirm confluency (70%–80%).
Use the fresh plasmids (pcDNA3.1 c-myc, pEGFP-N1 vectors, and the recombinant vectors, i.e., pcDNA3.1 c-myc EDRF1 and pEGFP-N1 NS2BNS3pro) for transfection (Please see supplementary Figures S1A and B for the recombinant construct development).
Note: Isolate the plasmids using MidiPrep two days before the transfection experiment. Prepare 20–30 μL of aliquots in 0.5 mL tubes and store at -20 °C.
Take 1.5 mL Eppendorf tubes, add 1 μg of pcDNA3.1 c-myc vector + 1 μg of pEGFP-N1 vector (tube 1), 1 μg of pcDNA3.1 c-myc EDRF1 (tube 2), 1 μg of pcDNA3.1 c-myc vector (tube 3), 1 μg of pcDNA3.1 c-myc EDRF1 + 1 μg of pEGFP-N1 NS2BNS3pro (tube 4), 1 μg of pEGFP-N1 vector (tube 5), and 1 μg of pEGFP-N1-NS2BNS3pro (tube 6) in OptiMEM (Mix-1) (see Troubleshooting 5).
Optional: Any other vector containing two different tags can also be used for transfection (FLAG or HA tag vectors).
Incubate the OptiMEM-diluted plasmids for 2–3 min at room temperature.
Take separate 1.5 mL tubes and add 2 μL and 4 μL of the Lipofectamine 2000 in OptiMEM (Mix-2).
Incubate the OptiMEM-diluted Lipofectamine for 2–3 min at room temperature.
See Table 1 for an example.
Table 1. Preparation of transfection and co-transfection reaction mixtures using Lipofectamine 2000
Mix-1 Mix-2
S. No Transfection/ Co-transfection Quantity of plasmids Volume of OptiMEM (μL) Volume of Lipofectamine (μL) Volume of OptiMEM (μL)
1. pcDNA3.1 c-myc vector + pEGFP-N1 vector 2 μL (1 μg) + 2 μL (1 μg) 46 4 46
2. pcDNA3.1 c-myc EDRF1 2 μL (1 μg) 48 2 48
3. pcDNA3.1 c-myc vector 2 μL (1 μg) 48 2 48
4. pcDNA3.1 c-myc EDRF1 + pEGFP-N1 NS2BNS3pro 2 μL (1 μg) + 2 μL (1 μg) 46 4 46
5. pEGFP-N1 vector 2 μL (1 μg) 48 2 48
6. pEGFP-N1-NS2BNS3pro 2 μL (1 μg) 48 2 48
Note: Each plasmid (1 μg) with lipofectamine (2 μL) is in 1:2 ratio. Plasmid to Lipofectamine can also be used in ratios 1:3, 1:4, and 1:5. In the co-transfected condition, the total plasmid concentration is 2 μg (1 μg of each plasmid), so 4 μL of Lipofectamine is used.
Add Mix-1 to Mix-2 and incubate for 20 min to form a plasmid–Lipofectamine complex.
Add the complex by gentle pipetting onto the cells drop by drop and further incubate the cells at 37 °C in a CO2 incubator for 5–6 h.
Replace the media with fresh complete DMEM and allow the cells to grow for 48 h.
After 48 h of transfection, remove the media from the plates and add 1 mL of 1× PBS.
Scrap the cells with a cell scrapper and collect the cells by pipetting into a fresh 1.5 mL Eppendorf tube.
Centrifuge at 800× g for 5 min.
Wash the cells with 1 mL of 1× PBS and centrifuge at 800× g for 5 min.
Repeat the wash step twice if pelleted cells still contain traces of DMEM medium.
Add 200 μL of RIPA buffer and 20 μL of 1× Protease inhibitor cocktail to the cell pellet and resuspend gently.
Vortex the suspension thrice at 5 min intervals and keep on ice for 30 min.
Centrifuge the cell suspension at 20,000× g for 15 min.
Collect the supernatant into a fresh Eppendorf tube as whole-cell lysate.
Quantify the total protein of the lysates by Bradford reagent.
Resolve 60 μg of the quantified protein on 10% SDS PAGE.
Optional: Before proceeding with the cell harvesting step C8, GFP expression can be analyzed using a fluorescence microscope.
Note: GFP expression was analyzed to confirm the transfection.
Western blotting
Transfer the above resolved proteins onto PVDF membrane at 80 V current for 3 h (or 50 V overnight) using a western blot transfer unit in 1× transfer buffer.
Stain the PVDF membrane with Ponceau S stain.
Prepare 50 mL of fresh Ponceau S stain solution.
Add 4–5 mL of Ponceau S stain onto the PVDF membrane and immerse the membrane completely.
Incubate the membrane for 5 min with slow agitation on the rocker.
Remove the Ponceau S stain and collect it in an Eppendorf tube for reuse (at least twice).
Add ddH2O water to wash out the excess stain and, as the bands start appearing immediately, record the image.
Wash the membrane with 1× PBST until the Ponceau is removed completely.
Note: Ponceau stain must be removed as it may hinder the blocking step.
Prepare 7% blocking buffer and block the membrane for 2 h at room temperature under gentle shaking.
Note: Blocking step needs to be optimized for each antibody. 7% Blocking buffer is used to avoid non-specificity.
Rinse the membrane once with 1× PBST and add the primary antibody solution [anti-Myc Tag (1.4 μg/mL, 1:1000) in 1× PBST or 2% blocking buffer solution].
Note: 2% Blocking buffer is used for diluting the primary antibodies.
Incubate the membrane at room temperature for 2 h or at 4 °C overnight with gentle rocking.
Note: Incubation time and dilution of antibody need to be optimized based on the non-specific bands.
Wash the membrane with 1× PBST three times for 10 min each on a rocker with mild agitation.
Add secondary antibody (anti-mouse IgG HRP conjugated; 1:10,000) diluted in 1× PBST.
Incubate the membrane for 2 h at room temperature.
Wash the membrane with 1× PBST three times for 15 min each with high agitation.
Develop the washed membrane with western blot Femto LUCENT™ PLUS-HRP substrate solution and capture the image using Chemidoc Image System (Bio-Rad) (see Troubleshooting 6).
Stripping and re-probing the PVDF membrane
Stripping is performed to re-probe the membrane with the different antibodies. All the steps for stripping can be carried out at room temperature.
From the above step D9, use the membrane for stripping.
Wash the membrane with ddH2O two times for 5 min each on the rocker with medium agitation.
Repeat the wash step two times with 1× PBST for 5 min each on the rocker with medium agitation.
Add ~10 mL of stripping buffer and incubate the membrane for 30 min on the rocker with medium agitation at room temperature.
Discard the stripping buffer and wash the membrane with ddH 2O for 5 min on a rocker.
Wash the membrane with 1× PBS two times for 5 min each on the rocker.
Repeat the wash steps with 1× PBST two times for 10 min each on the rocker.
The membrane is ready for the re-probing with different antibodies.
For re-probing with the different antibodies, begin with the blocking steps as mentioned in the above western blotting steps (D4–D9).
Note: For re-probing, in our experiment, we have used anti-GFP antibody (1:1,000) (primary antibody) and anti-rabbit HRP conjugated antibody (0.04 μg/mL, 1:10,000) (secondary antibody).
Data analysis
In this protocol, we analyzed EDRF1 cleavage by co-transfection followed by western blotting. The western blot data was obtained using anti-Myc tag antibody for pCDNA 3.1 c-myc EDRF1 and GFP-tag antibody for pEGFPN1 NS2BNS3pro expressions. In pcDNA3.1 c-myc vector containing EDRF1 as an insert, the EDRF1 band was detected as intact showing the presence of expressed EDRF1 alone (Figure 2, lane 2). As expected, no expression was observed in pcDNA3.1 c-myc vector alone. Importantly, the expressed EDRF1 completely disappeared in the presence of protease in co-transfected pCDNA3.1 c-myc EDRF1 and pEGFP-N1 NS2BNS3pro conditions, suggesting EDRF1 as a substrate of protease (Figure 2, lane 4). pEGFP-N1 vector alone and pEGFP-N1 NS2BNS3pro lysates were loaded as controls (Figure 2, lanes 11 and 12). To confirm the expression of NS2BNS3pro in the above experiment, we have checked the expression of protease using anti-GFP antibody (pEGFPN1-NS2BNS3pro) after stripping the same membrane. It was observed that, in co-transfected vectors and pEGFP-N1 vector alone, GFP was expressed. In pcDNA3.1 c-myc EDRF1 and pEGFP-N1 NS2BNS3pro co-transfected cell lysates, the protease was detected, thus confirming the expression of NS2BNS3pro in the co-transfected conditions (Figure 2, lane 10). pEGFP-N1 NS2BNS3pro was also found to be expressed alone, which is a control (Table 2). We expect that this analysis will be useful in identifying the novel substrates of viral encoded proteases, as this protocol is simple, easy, and quick to perform. Further, this protocol can be used to evaluate anti-protease molecules.
Table 2. Tabulated summary of the data
Myc-Tag antibody GFP-Tag antibody
S. No Plasmid Transfected Myc-tag fusion protein GFP-tag fusion protein
1. pcDNA 3.1 c-myc vector + pEGFP-N1 vector
No bands will be detected as the Myc tag is 1.2 kDa
(co-transfected empty vectors)
GFP protein will be detected due to the presence of pEGFP-N1 vector ~27 kDa
(co-transfected empty vectors)
2. pCDNA 3.1 c-myc EDRF1 ~ 50 kDa pcDNA3.1c-myc EDRF1 detected No band detected
3. pcDNA 3.1 c-myc vector No band detected No band detected
4. pcDNA 3.1 c-myc EDRF1 + pEGFP-N1 NS2BNS3pro
No band detected due to cleavage of EDRF1 in presence of protease
(co-transfected)
~56 kDa pEGFP-N1 NS2BNS3pro band detected showing the presence of GFP tag protease (co-transfected)
5. pEGFP-N1vector No band detected ~ 27 kDa GFP band detected
6. pEGFP-N1 NS2BNS3pro No band detected ~56 kDa pEGFP-N1 NS2BNS3pro band detected
Figure 2. Western blotting analysis of co-transfected lysates. A. Ponceau image after electroblot transfer. The lysate loaded is indicated on each lane. B. and C. Simulated images of western blotting probed with anti-Myc and anti-GFP antibodies.
Validation of protocol
This protocol was described in our published article in iScience (2023), https://doi.org/10.1016/j.isci.2023.107024. We have repeated the protocol and found that it is easy to execute, and the outcome is consistent. In vitro pulldown assay followed by mass spectrometry identification/western blotting confirmed the interaction between EDRF1 and protease. In vitro sequence analysis suggested the existence of a total of five protease cleavage sites in EDRF1. Further, superimposed models of EDRF1 and protease indicated the location of the cleavage site within the catalytic triad of protease. The above observations support the outcome of the protocol described.
General notes and troubleshooting
General notes
This protocol requires either the two differently expressing vectors with different tags or proteins specific for analyzing the expression, if using the same tag for the co-transfected plasmids. The protocol is more convenient to be performed with two different tagged vectors.
Troubleshooting
Potential problem Possible cause Corrective measures
1. Cell line contamination and cross contamination
Presence of bacterial/fungal/mycoplasma.
Cell line obtained from other labs.
Improper handling during cell culture maintenance of two cell lines together.
Preparation of cryovial with contaminated cryomedium.
Proper use of sterile equipment and reagents in cell culture will allow maintenance of the aseptic environment and minimize contamination.
Collect the cells from the certified cell repositories (NCCS, Pune, India, or ATTC).
Culture and split the cells one at a time or perform the splitting of cells on alternate days.
Prepare the cryomedium fresh, if possible. Use cell culture grade DMSO or glycerol or commercially available cryomedium. Check the expiry date of the FBS, media, and antibiotics used for culturing the cells.
2. Cell culture media color changes
Highly confluent dishes.
CO2 levels are low.
Bacterial contamination.
Thaw a new cryovial or split the cells as needed.
Set the CO2 levels to 5% and maintain humidity.
Add antibiotics and wash the cells with 1× PBS (cell culture grade).
Discard the media and sterilize the laminar hood cabinets and CO2 incubator.
3. Cells did not attach after being obtained from cryogenic stage
Cryomedium shows toxicity.
Too many apoptotic cells.
Less cell numbers during cryofreezing.
Higher number of continuous passages.
Use 5%–7% DMSO in cryomedium.
Use 90% FBS during cryofreezing.
Prepare cryovials with 80% confluent freshly split cells.
4. Cell growth slows
Less or inaccurate supplemental cell culture components.
Cell culture conditions (temperature, humidity, and CO2 levels).
Protein of interest not expressing.
Begin with fresh cryovials.
Growth media with 10%–12% FBS will be good for attaining a good confluency.
Keep the incubator at 37 °C with 5% CO2 and water tray.
Grow the cells without antibiotics.
5. Co-transfection not working
Transfection reagents may not be suitable.
Cell lines may not be efficient for co-transfection.
Co-transfecting plasmids not expressing together.
Check the quantity and quality of plasmids. Use high quality plasmid isolation kits (Thermo MidiPrep or High Pure Links Kits)
Lipofectamine 2000 works best for most cell lines. Lipofectamine 3000 can also be used.
Check the transfection efficiency before proceeding with the co-transfection experiments.
Perform and analyze the transfection of the individual plasmids that need to be co-transfected.
6. Western blot of the co-transfected plasmids
Antibody not detecting the proteins in co-transfecting plasmids.
No signal in the blot.
High background.
Optimize the transfection timing for the co-transfected plasmids. Forty-eight hours of co-transfection give conclusive results.
Use individual plasmids as controls to confirm the transfection.
Optimize the antibody dilution (1:500 to 1:3,000).
Use specific tagged antibodies or protein-specific antibodies.
Use species-specific secondary antibodies (anti-mouse or anti-rabbit HRP conjugated).
Optimize the primary and secondary antibody incubation times and washing steps.
Optimize the blocking buffer (5%–7% BSA or skimmed milk).
Use 1× PBST and up to 0.3% Tween-20.
Acknowledgments
STARS-MoE-IISc (STARS/APR2019/BS/584/FS) for the financial support and Indian Council of Medical Research (ICMR) for providing fellowship to L.G.
Competing interests
The authors declare no competing interests.
References
Castelló, A., Álvarez, E. and Carrasco, L. (2011). The Multifaceted Poliovirus 2A Protease: Regulation of Gene Expression by Picornavirus Proteases. J. Biomed. Biotechnol. 2011: 1–23.
Li, X. D., Sun, L., Seth, R. B., Pineda, G. and Chen, Z. J. (2005). Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc. Natl. Acad. Sci. U.S.A. 102(49): 17717–17722.
Gandhi, L., Maisnam, D., Rathore, D., Chauhan, P., Bonagiri, A. and Venkataramana, M. (2023). Differential localization of dengue virus protease affects cell homeostasis and triggers to thrombocytopenia. iScience 26(7): 107024.
Moustaqil, M., Ollivier, E., Chiu, H. P., Van Tol, S., Rudolffi-Soto, P., Stevens, C., Bhumkar, A., Hunter, D. J. B., Freiberg, A. N., Jacques, D., et al. (2021). SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species. Emerg. Microbes. Infect.10(1): 178–195.
Gandikota, C., Mohammed, F., Gandhi, L., Maisnam, D., Mattam, U., Rathore, D., Chatterjee, A., Mallick, K., Billoria, A., Prasad, V. S. V., et al. (2020). Mitochondrial Import of Dengue Virus NS3 Protease and Cleavage of GrpEL1, a Cochaperone of Mitochondrial Hsp70. J. Virol. 94(17): e01178–20.
Lennemann, N. J. and Coyne, C. B. (2017). Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B. Autophagy 13(2): 322–332.
Lin, J. C., Lin, S. C., Chen, W. Y., Yen, Y. T., Lai, C. W., Tao, M. H., Lin, Y. L., Miaw, S. C. and Wu-Hsieh, B. A. (2014). Dengue Viral Protease Interaction with NF-κB Inhibitor α/β Results in Endothelial Cell Apoptosis and Hemorrhage Development. J. Immunol. 193(3): 1258–1267.
De Jesús-González, L. A., Cervantes-Salazar, M., Reyes-Ruiz, J. M., Osuna-Ramos, J. F., Farfán-Morales, C. N., Palacios-Rápalo, S. N., Pérez-Olais, J. H., Cordero-Rivera, C. D., Hurtado-Monzón, A. M., Ruíz-Jiménez, F., et al. (2020). The Nuclear Pore Complex: A Target for NS3 Protease of Dengue and Zika Viruses. Viruses 12(6): 583.
Yu, C. Y., Chang, T. H., Liang, J. J., Chiang, R. L., Lee, Y. L., Liao, C. L. and Lin, Y. L. (2012). Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity. PLoS Pathog. 8(6): e1002780.
Yu, C. Y., Liang, J. J., Li, J. K., Lee, Y. L., Chang, B. L., Su, C. I., Huang, W. J., Lai, M. M. C. and Lin, Y. L. (2015). Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins. PLoS Pathog. 11(12): e1005350.
Goethals, O., Kaptein, S. J. F., Kesteleyn, B., Bonfanti, J. F., Van Wesenbeeck, L., Bardiot, D., Verschoor, E. J., Verstrepen, B. E., Fagrouch, Z., Putnak, J. R., et al. (2023). Blocking NS3–NS4B interaction inhibits dengue virus in non-human primates. Nature 615(7953): 678–686.
Supplementary information
The following supporting information can be downloaded here
Figure S1A: Cloning and expression of DENV protease ex vivo.
Figure S1B: Cloning and expression of EDRF1 ex vivo.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Category
Microbiology > Microbe-host interactions > Virus
Cell Biology > Cell-based analysis > Enzymatic assay
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Peer-reviewed
Monitoring Intestinal Organoid–Derived Monolayer Barrier Functions with Electric Cell–Substrate Impedance Sensing (ECIS)
SO Sarah Ouahoud
FG Francesca P. Giugliano
VM Vanesa Muncan
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4947 Views: 601
Reviewed by: Philipp WörsdörferValeria Fernandez Vallone Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Scientific Reports Oct 2023
Abstract
The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids.
Key features
• We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers.
• The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.
Keywords: Intestinal organoids ECIS Impedance TEER Intestinal barrier Wounding
Background
The intestinal epithelium is represented as a cellular monolayer that separates the luminal content from the rest of the body [1]. Apart from enabling digestion and absorption of food, it serves as a crucial barrier for warding off potentially pathogenic microbes [1]. Damage and impairment of the gut barrier are observed through the course of various intestinal diseases such as necrotizing enterocolitis and inflammatory bowel disease [2,3].
A major boost to the in vitro modeling of the intestinal epithelial barrier came with the emergence of organoid technology. Organoids are self-organizing, three-dimensional (3D) structures that are grown in vitro from stem cells [4]. They recapitulate many structural and functional aspects of their parent organ more accurately than the commonly used two-dimensional (2D) cell lines [4]. Therefore, organoids have become a frequently used model among researchers. Since it remains challenging to study epithelial barrier formation in 3D structures, several studies cultured the intestinal organoids as a monolayer in a Transwell system [1].
The Transwell system is the most commonly used in vitro model for intestinal barrier research [5,6]. It consists of a porous cell culture insert that can be placed in a traditional cell culture well plate [5]. The gut epithelial cells are grown on the permeable membrane of the insert to create a cell monolayer with a luminal and basolateral compartment. Evaluation of barrier formation is assessed by measuring the transepithelial electrical resistance (TEER) with electrodes placed on either side of the membrane [5,6]. While measuring TEER with the typical handheld chopstick electrode epithelial voltohmmeter (EVOM) device remains the gold standard for assessing the integrity of barrier models, this technique requires removal of the cultures from the incubator to test each well individually. Such approach might disrupt the cell layers and, in addition, variations in electrode positioning could hinder reproducibility of measurement because of non-uniform electric field created by the chopstick electrodes [6].
An alternative to EVOM is electrical cell–substrate impedance sensing (ECIS). ECIS uses the same principle as the EVOM, except that the electrodes are integrated into the bottom of the well on which the cells are grown [6]. The presence of the electrodes in the wells allows cells to attach and proliferate directly on top of the electrodes, resulting in more localized and sensitive impedance measurements of the cell barrier. The ECIS enables impedance measurements at a broad scale of electrical frequencies, ranging from 62.5 Hz to 64 kHz. In addition, depending on the electrode placement and size, the ECIS can be used to determine additional properties of the epithelial cell layer such as cell attachment, migration, and proliferation [6]. Studying cell attachment, proliferation, and spreading yields insights into fundamental cellular behaviors, enhancing our understanding of basic biological processes in health and disease. Apart from monitoring cell barrier formation, the cells can be subjected to (lethal) electrical currents to inflict cellular damage [7]. The ECIS is therefore a potentially interesting cell-based system to study gut barrier formation and to screen for drugs capable of resolving cell damage and achieving higher mucosal healing rates. Here, we show that the ECIS technology can be successfully applied on gut organoid 2D monolayers.
Materials and reagents
Biological materials
Established 3D intestinal organoid lines isolated from either fetal (gestational age 18–22 weeks) or adult tissue (see general note 1)
Reagents
Advanced DMEM/F12 (Gibco, catalog number: 12634-028)
GlutaMAX 100× (Gibco, catalog number: 35050-038)
HEPES 1 M (Gibco, catalog number: 15630-056)
Penicillin-streptomycin (Pen/strep) 10,000 U/mL (Gibco, catalog number: 15140-122)
Glacial acetic acid (VWR, catalog number: 1.00056.2500)
L-cysteine (Sigma-Aldrich, catalog number: C7352)
Collagen type I, rat tail, 5 mg/mL (IBIDI, catalog number: 50201)
TrypLE express (1×), phenol red (Gibco, catalog number: 12605036)
Y27632 (ROCK-inhibitor) (Sigma-Aldrich, catalog number: Y0503)
Human IntestiCult organoid growth medium (includes 50 mL of IntestiCultTM OGM human basal medium and 50 mL of organoid supplement) (Stemcell Technologies, catalog number: 06010)
Human IntestiCult organoid differentiation medium (includes 50 mL of IntestiCultTM ODM human basal medium and 50 mL of organoid supplement) (Stemcell Technologies, catalog number: 100-0214)
Solutions
Ad-DF+++ (see Recipes)
Human IntestiCultTM organoid growth medium (see Recipes)
Human IntestiCultTM organoid differentiation medium (see Recipes)
100 mM L-cysteine (see Recipes)
0.1% acetic acid solution (see Recipes)
Collagen/0.1% acetic acid solution (see Recipes)
Recipes
Media preparations
Ad-DF+++
Supplement 500 mL of advanced DMEM/F12 with 5 mL of Pen/strep, 5 mL of HEPES, and 5 mL of GlutaMAX.
Human IntestiCultTM organoid growth medium
Combine 50 mL of IntestiCultTM OGM human basal medium with 50 mL of organoid supplement and 1 mL of Pen/strep.
Human IntestiCultTM organoid differentiation medium
Combine 50 mL of IntestiCultTM ODM human basal medium with 50 mL of organoid supplement and 1 mL of Pen/strep.
Coating solutions
100 mM L-cysteine
Dissolve 12.12 mg of L-cysteine in 1 mL of Milli-Q water. Pass the solution through a 0.2 µm syringe sterilization filter. The solution can be kept in the fridge for up to two weeks. Dilute the stock solution 10× in sterile Milli-Q water before the start of the experiment.
0.1% acetic acid solution
Add 25 µL of acetic acid to 25 mL of Milli-Q water. Pass the solution through a 0.2 µm syringe sterilization filter.
Collagen/0.1% acetic acid solution
Wells should be coated with 10 µg collagen/cm2. Each well has a growth area of 0.8 cm2. For 17 wells (16 wells + 1 extra), add 27.2 µL of collagen type I to 5,072.8 µL of 0.1% acetic acid solution.
Laboratory supplies
Applied BioPhysics ECIS 8W1E array (Ibidi, catalog number: 72001)
Applied BioPhysics ECIS 8W10E array (Ibidi, catalog number: 72010)
15 mL tubes (Falcon, catalog number: 352096)
0.2 µm syringe sterilization filters (Sarstedt, catalog number: 83.1826.001)
15 mL tubes (Falcon, catalog number: 352096)
200 µL pipette tips (Sapphire, catalog number: 775355)
1,000 µL pipette tips (Sapphire, catalog number: 777355)
Equipment
ECIS® Z-Theta instrument (Applied Biophysics, catalog number: 71617)
16-well array module housed in incubator including 8W test array (Applied Biophysics, catalog number: 71612)
Cell culture equipment (centrifuge, incubator, pipets, sterile hood, etc.)
Software and datasets
ECIS Zθ software (Applied Biophysics)
(Optional) Excel
(Optional) GraphPad Prism
Procedure
Conduct all cell and array manipulations within a sterile environment using a laminar flow cabinet.
Preparation of the 16-well station
Place the ECIS 16-well station in an incubator at 37 °C at least one day before the experiment to prewarm the array holder (Figure 1A).
Add water to the incubator's water reservoir to prevent the wells from drying out.
Preparation of the arrays
The choice of array depends significantly on the specific scientific question and the type of cells involved. Choices for array selection include the 8W1E array for wound healing studies due to enhanced sensitivity to cell motion–induced resistance fluctuations. Multielectrode arrays, such as the 8W10E, average signals over several electrodes and are more suitable for studying cell proliferation and barrier formation by minimizing bias from uneven cell distribution.
Prepare the 100 mM L-cysteine stock solution and dilute the stock 10 times in sterile Milli-Q water. For two arrays, 3,500 µL of 10 mM L-cysteine solution is needed. Therefore, add 350 µL of 100 mM L-cysteine solution to 3,150 µL of sterile Milli-Q water. See general note 2.
Add 200 µL of the 10 mM L-cysteine solution to each well.
Incubate the arrays for 30–45 min at room temperature (RT).
Wash the wells two times with 300 µL of sterile Milli-Q water.
Add 300 µL of the Collagen/0.1% acetic acid solution to each well.
Incubate arrays for 60 min at RT.
Wash the wells two times with 300 µL of sterile Milli-Q water.
Add 400 µL of Ad-DF+++ to each well.
Calibration of the station and start of baseline measurements
Turn on the computer and the ECIS Zθ machine (Figure 1B). See general note 3.
Figure 1. Electrical cell–substrate impedance sensing (ECIS) setup. A. The 16-well station is placed in the incubator at 37 °C. B. The ECIS® Z-Theta instrument, including laptop, is placed in close proximity to the incubator holding the 16-well station. C. The 8W test array is used to calibrate the ECIS. Running the test array should provide measurements with values (close-up picture) mentioned on the array. D. The 8W ECIS arrays hold eight wells, with the 8W1E having one electrode per well and the 8W10E (close-up picture) having 10 electrodes.
Open the ECIS software.
Place the test array in the station, click Setup, and check the connection (Figure 1C and Figure 2A). If the connection is good, the wells are given a green color; the ones with a bad connection are shown in red. Reinsert the array and click Setup until a good connection is achieved for all wells (Figure 2B).
Select the RC test array in the array type pop-up screen.
Run the check for the test array (Figure 2C).
If the RC test is OK and correct values are shown, as mentioned on the test array, you can continue using the ECIS for your experiment (Figure 1C and 2D). Contact the manufacturer if the values are incorrect.
Place the array(s) with the medium in the 16-well station and perform a check. If electrodes have been properly stabilized with the L-cysteine, the 8W1E and the 8W10E should give values of approximately 5 nF and 50 nF, respectively. See general note 4.
Select array type.
Choose the Multiple Frequency/Time (MFT) or another mode (Figure 2C). See general note 5.
Figure 2. Electrical cell–substrate impedance sensing (ECIS) software. A. Upon opening the ECIS software, click Setup (black arrow) in the right window to measure the impedance of the device and detect an open circuit. B. Arrays/wells that have been properly connected are indicated in green in the left-bottom window of the screen, whereas arrays or wells with an open circuit are shown in red. C. Readjust the placement of the array and select Setup (black arrow) again to confirm correct placement of the array. By selecting check (button underneath Setup), measurements of the test array will be made using the default frequency of 4,000 Hz. If electrodes were not properly stabilized after the L-cysteine treatment, an additional stabilization step can be performed with the ECIS by selecting stabilize (green arrow). The same right pane holds options for the acquisition mode (outlined with a black square), the Wound/Electroporate Setup function (outlined with a red square), and the Start (red arrow) button. D. Measurements performed by selecting Check, e.g., results from the test array, are shown on a pop-up screen. E. Data acquisition is halted by selecting Pause (black arrow) and can be resumed by clicking on Resume (red arrow). F. After selecting Wound/Electroporate Setup, additional settings emerge in the bottom-right pane. Select Wound (black arrow), adjust the wound settings, and click Activate (red arrow) to perform the wounding. Wounding can be postponed to a later time point by selecting Delay Until Hour (outlined with a black square).
Click on Start and enter the file name to start the run (Figure 2C).
Measure the baseline for at least 30 min. Continue with harvesting the cells during this step.
Preparation of cells
Remove the culture medium from the organoids and replace it with 0.5 mL of cold Ad-DF+++ for each well in a 24-well plate or 1 mL for each well in a 12-well plate.
Pipette several times up and down with a p1000 pipette to disrupt the Matrigel and collect the Ad-DF+++ with the organoids in a 15 mL tube.
Centrifuge at 200× g for 5 min at 4 °C to remove the Matrigel.
Take off the supernatant and add 1–3 mL of TrypLE, depending on pellet size, to the 15 mL tube. Resuspend the cells in the TrypLE and incubate the 15 mL tube for 5–7 min in a water bath at 37 °C. See general note 6.
Resuspend the cells vigorously for 15–20× with a p1000 pipette to generate a single-cell suspension. Adding a 200 µL tip on top of the 1,000 µL tip greatly improves the mechanical disruption of the organoids. No cell clumps or organoid structures should be visible with the naked eye. See general note 7.
Inactivate the TrypLE with 10 mL of Ad-DF+++.
Spin the cells at 340× g for 5 min at 4 °C. Remove the supernatant.
Resuspend the cells in organoid growth medium (OGM) supplemented with ROCK-inhibitor (1:1,000) and perform a cell count.
Prepare the single-cell suspension in the range of 150,000–300,000 cells per well. Adjust the volume with OGM supplemented with ROCK-inhibitor.
Pause the baseline measurements and take the array out of the incubator (Figure 2E).
Remove the media in the array.
Add 400 µL of the homogenized cell suspension to each well. Do not forget to include an empty control.
Put the array back in the 16-well station and resume the measurements (Figure 2E). See general note 8.
Experiment run and measurements
The frequency-selection ECIS experiments rely on the research goal and the characteristics of the cells under investigation. In resistance analysis, the 4,000 Hz frequency is used as the default setting, as this frequency is regularly used due to its relevance in capturing subtle changes associated with cell behavior and barrier function, particularly in the context of tight junction formation and integrity. However, the optimal frequency may vary, depending on the cell type and the specific experimental conditions. The frequency scan performed with the ECIS software on fetal and adult intestinal organoids showed that capacitance measurements should be analyzed at 64 kHz, whereas resistance should be analyzed at 500 Hz (Figure 3A, 3B). The capacitance and resistance data shown in this protocol paper are therefore measurements performed at a frequency of 64 kHz and 500 Hz, respectively.
Compared to cell lines, much higher organoid cells numbers are needed to reach confluence shortly after seeding. As an example, approximately 50,000 CaCo2 cells are needed to obtain a monolayer within 24 h. ECIS capacitance measurement, which indicates electrode coverage, shows that if you seed two different organoid cell numbers per well, 150,000 or 300,000, no monolayers are formed within the first 30 h after seeding (Figure 4A–4C). After approximately 60 h, a monolayer has formed in the wells with 300,000 cells per well, and an additional 30 h is needed before a monolayer has formed in the wells with 150,000 cells per well (Figure 4D–4E). ECIS resistance measurements (500 Hz) correlate inversely with the capacitance measurements, showing that the transepithelial resistance is increasing while the monolayer is forming (Figure 4F–4J). Approximately 300,000 cells per well are needed for human intestinal organoids to reach confluence within three days, but this could differ slightly per donor depending in its growing rate. Perform a pilot experiment to determine the optimal cell number that fits your research question.
Figure 3. Optimal alternating current (AC) frequencies for analysis of intestinal organoid monolayers. The frequency displaying the greatest difference between cell-free (empty) and cell-covered conditions is deemed ideal for subsequent analysis. A. Capacitance data from fetal organoids grown on organoid growth medium (OGM), displayed as cell-to-cell-free ratios, exhibits a maximum response at 64 kHz (dotted line). B. However, 500 Hz (dotted line) or 1 kHz seem to be the optimal frequencies for analysis of resistance data.
Figure 4. Barrier formation in 2D intestinal organoid–derived monolayers. Intestinal organoid cells from the same fetal tissue culture were seeded in two different ECIS 8W10E arrays (indicated in the graph as round or triangle shaped). A. As cells proliferate and cover the electrode surfaces, electrical current is impeded. The capacitance measured during cell proliferation is inversely correlated with surface coverage until a complete monolayer has formed. B–E. The capacitance measured at 64 kHz (indicative of electrode coverage) indicates that a full monolayer was achieved after 60 and 90 h when 150,000 (n = 12) or 300,000 (n = 3) cells were seeded, respectively. F. A significant increase in resistance is observed as the monolayers form. G–I. Resistance continues to increase and reaches a plateau at 4,000 Ω when measured at 500 Hz (indicating intact cell barrier). Bars represent mean ± SD. P < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****) as determined with an unpaired t-test.
Changing the medium during an experiment
Click on Pause to halt data acquisition. The experimental clock will continue to run.
Take the array out of the holder and change the medium under a laminar flow bench. Change the medium to medium without ROCK-inhibitor 2–3 days after seeding and continue to refresh medium every 2–4 days. See general note 9.
Return the array to the holder and click Check to check if arrays were placed back correctly.
Wait for 5–10 min before resuming the measurements, as temperature changes affect the measurements.
Click Resume to restart data acquisition. The measurement software will include a time mark in the data set. See general note 10.
Differentiation of cells
If the research question requires the cells to reflect the mature intestine and the monolayer to contain all major cell types present within tissues, an additional differentiation step might be performed [8]. By switching the culture medium from OGM to organoid differentiation medium (ODM) after the gut monolayer has been formed, an additional increase in the transepithelial resistance is observed (Figure 5). Gut cells cultured in ODM contain physiologically relevant properties of differentiated cells, such as nutrient absorption and expression of digestive enzymes and tight junction proteins.
Figure 5. Organoid cell differentiation results in increased resistance values. Intestinal organoid cells from adult tissue were seeded into 8W10E ECIS arrays and cultured in IntestiCult organoid growth medium (OGM). After 118 h, when the capacitance plateau phase was reached but resistance values were still increasing (indicated by the dotted line), cells were further cultured in either OGM (n = 5) or organoid differentiation medium (ODM) (n = 3). A. Capacitance measurements showed significant changes between cells cultured in OGM or ODM. B) Resistance measurements, on the other hand, revealed that resistance continued to increase in cells cultured in ODM. C–F. The capacitance data of 118 h and 200 h indicate that a slight but significant change occurred in how the cells cover the electrodes, suggesting that the increase in resistance can be attributed to the formation of a tighter barrier by differentiated cells. Bars represent mean ± SD. P < 0.01 (**) as determined with an unpaired t-test.
Wound-healing assay
Damage to the intestinal epithelial barrier is observed in a number of gut diseases. Once a stable monolayer has formed, the ECIS can be used to inflict cellular damage by subjecting the cells to a lethal electrical current. The electrical current specifically kills the cells on top of the small gold electrodes, which subsequently die and detach from the electrode, creating a wound that is healed by neighboring cells that have not been submitted to the current [7]. This function allows for the screening of drugs that might provide significant clinical benefit by improved or faster wound healing.
Determining the appropriate settings for the specific cell type is crucial for electrical wounding. If the wounding duration is too brief, it may lead to inadequate cell removal, while excessively long or harsh wounding can potentially damage the electrodes. For the wound-healing assays, 8W10E confluent intestinal epithelial monolayers derived from organoids were subjected to different currents. While applying a current of 4,000 μA and a frequency of 50,000 Hz for 30 s is capable of inducing enough cell damage for the cells to break their tight junction, the capacitance data shows that the current is not strong enough to kill all the cells present on the electrode. However, successful wounding on organoid-derived monolayers is achieved after applying a current of 5,000 μA and a frequency of 50,000 Hz for 60 s (Figure 6).
Figure 6. Wounding of 2D organoid monolayers. Monolayers formed by adult organoids were grown in organoid growth medium (OGM). A. Applying a current of 4,000 μA and a frequency of 50,000 Hz for 30 s was insufficient to eliminate all the cells present on the electrodes of an 8W10E array, whereas almost no cells were present when a current of 5,000 μA and a frequency of 50,000 Hz was used for 60 s. Images are representative pictures of the conditions. B–C. Capacitance and resistance measurements also demonstrated that successful wounding was only achieved when a current of 5,000 μA and 50,000 Hz was applied for 60 s, as the capacitance values did not drop to the levels observed in the empty control when a current of 4,000 μA and 50,000 Hz for 30 s was used. Dotted lines indicate time of wounding. Bars represent mean ± SD of two replicates.
Select the wells you want to subject to wounding.
Click the Wound/Electroporate Setup box (Figure 2C).
Select Wound (Figure 2F).
Adjust the baseline settings for the wound time (s), wound current (μA), and frequency (Hz) (Figure 2F). See general note 11.
You can delay the wounding to another time by selecting the Delay until hour (Figure 2F).
Click Activate to perform the wounding.
Data analysis
Navigate to File.
Click on Export data.
Choose All data or Selected wells/time.
Select either To Excel or Graph data. Use the exported data in Excel for statistical analysis and to generate graphs.
Validation of protocol
The protocol described in this paper has undergone a meticulous optimization process through a series of experiments to ensure its effectiveness. For this protocol paper, several smaller experiments were conducted to illustrate crucial findings that guarantee a successful experiment. These smaller experiments aimed to highlight key insights and refine procedural details. Multiple replicates were carried out for each experimental condition, with careful consideration given to the distribution of replicates across two arrays. This approach not only validates the protocol's robustness but also ensures its adaptability and reliability across various conditions.
This protocol has been validated and used in the following research article: ten Hove et al. [9].
General notes and troubleshooting
General notes
This protocol might potentially be applied, with or without minor adjustments, for human organoids derived from various tissue types.
Stabilizing the ECIS arrays with L-cysteine is crucial for improving electrode performance, promoting enhanced cell attachment, and sustaining a biocompatible environment. This approach guarantees that impedance measurements derived from ECIS arrays yield reliable data.
A detailed protocol on how to use the ECIS has been published by Szulcek et al. [10] and Anwer et al. [11].
If some of the wells have not reached acceptable stabilization, an alternative way to clean the electrodes can be performed using the ECIS electrical stabilization method. If the ECIS electrical stabilization method is used, you have to wait 30 min before running baseline measurements. More information about this function can be found in the online manual of the manufacturer.
The Multiple Frequency Time (MFT) program automatically measures selected wells across various AC frequencies and is recommended for various cell-related assays with compound effects observed over periods longer than 15 min. The Single Frequency Time (SFT) mode is suitable for rapid data acquisition at a single frequency.
If an incubation step of 7 min and subsequent mechanical disruption proves insufficient to obtain a single-cell suspension, the incubation step can be prolonged by an additional 1–3 min. However, do not leave the organoids for longer than a total of 10 min in TrypLE, as this affects the viability of the cells. Resuspend again after the second incubation step.
It is crucial to obtain a homogenous single-cell suspension, as cell clumps tend to grow as organoids after seeding. This might influence the resistance and capacitance measurements.
You can start the measurements directly after seeding the cells if you are interested in proliferation and barrier formation. However, if you are interested in the wounding function of the ECIS, you can put the arrays in the incubator and transfer them to the 16-well station to perform the wounding when a stable monolayer, as determined through microscopic assessment, has formed.
Be careful to not damage the monolayer while removing and adding the medium. Tilt the upper side of the plate slightly up and to the left (or right) so that the medium will gather into one corner of the well. Remove the medium by placing the tip in the corner where the medium has gathered. Add 400 µL of medium per well by gently pipetting the medium down the wall of the well.
Marks for pausing (green) and wounding (red) actions are shown as vertical dashed lines at the time points on which they were performed.
Wounding settings might differ between different array types. A pilot experiment to determine the optimal wounding conditions is therefore needed if another array type is used.
Experimental conditions might influence the susceptibility of the organoids to the wounding settings. A pilot experiment to determine the optimal wounding conditions might, therefore, be needed if cells are, for example, grown on ODM or exposed to a compound that affects cell viability.
Troubleshooting
Problem 1: Large variation in measurements between wells and/or abnormal high background signals during baseline measurements.
Possible cause: Electrodes have not been properly stabilized.
Solution: Always ensure that electrodes have undergone adequate stabilization before initiating baseline measurements. An extra stabilization step may be conducted using the ECIS electrical stabilization method. Alternatively, you may proceed with cell seeding, as cells also possess the capability to clean the electrodes. However, this process requires several hours, and measurements taken during this period may exhibit unusual patterns.
Problem 2: Large variation in measurements between wells.
Possible cause: Organoids are not properly converted to a single-cell suspension and grow as clumps on the electrodes.
Solution: Carefully check your single-cell suspension for the presence of organoid structures. Continue disrupting the cells if organoid structures can still be observed with the naked eye. Alternatively, include more replicates per condition to account for well variability.
Problem 3: Cells grow as cell clumps on the array.
Possible cause: Organoids are not properly converted to a single-cell suspension.
Solution: Carefully check your single-cell suspension for the presence of organoid structures. Continue disrupting the cells if organoid structures can still be observed with the naked eye.
Problem 4: Cells do not survive after seeding.
Possible cause: Cells were kept too long in TrypLE or were cultured in medium without ROCK-inhibitor.
Solution: Do not leave the cells for longer than 10 min in TrypLE and do not forget to add ROCK-inhibitor to the medium.
Competing interests
The authors declare that there is no conflict of interest.
References
Roodsant, T., Navis, M., Aknouch, I., Renes, I. B., van Elburg, R. M., Pajkrt, D., Wolthers, K. C., Schultsz, C., van der Ark, K. C. H., Sridhar, A., et al. (2020). A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine. Front. Cell. Infect. Microbiol. 10: e00272.
Halpern, M. D. and Denning, P. W. (2015). The role of intestinal epithelial barrier function in the development of NEC. Tissue Barriers 3(1–2): e1000707.
Oshima, T. and Miwa, H. (2016). Gastrointestinal mucosal barrier function and diseases. J. Gastroenterol. 51(8): 768–778.
Rossi, G., Manfrin, A. and Lutolf, M. P. (2018). Progress and potential in organoid research. Nat. Rev. Genet. 19(11): 671–687.
Vancamelbeke, M. and Vermeire, S. (2017). The intestinal barrier: a fundamental role in health and disease. Exp. Rev. Gastroenterol. Hepatol. 11(9): 821–834.
Yeste, J., Illa, X., Alvarez, M. and Villa, R. (2018). Engineering and monitoring cellular barrier models. J. Biol. Eng. 12(1): 18.
Gu, A., Kho, D., Johnson, R., Graham, E. and O’Carroll, S. (2018). In Vitro Wounding Models Using the Electric Cell-Substrate Impedance Sensing (ECIS)-Zθ Technology. Biosensors 8(4): 90.
Hanyu, H., Sugimoto, S. and Sato, T. (2023). Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures. Methods Mol. Biol. 2650: 141–153.
ten Hove, A. S., Mallesh, S., Zafeiropoulou, K., de Kleer, J. W. M., van Hamersveld, P. H. P., Welting, O., Hakvoort, T. B. M., Wehner, S., Seppen, J., de Jonge, W. J., et al. (2023). Sympathetic activity regulates epithelial proliferation and wound healing via adrenergic receptor α2A. Sci. Rep. 13(1): 17990.
Szulcek, R., Bogaard, H. J. and van Nieuw Amerongen, G. P. (2014). Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility. J. Vis. Exp. (85): e51300.
Anwer, S. and Szászi, K. (2020). Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS). Bio Protoc. 10(16): e3729.
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4,948 | https://bio-protocol.org/en/bpdetail?id=4948&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Simple Rescue of Opaque Tissue Previously Cleared by iDISCO
HM Haylee Mesa *
JM Jonathan Meade *
PG Paula Gajewski-Kurdziel
RB Randy D. Blakely
QZ Qi Zhang
(*contributed equally to this work)
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4948 Views: 826
Reviewed by: Hong LianOneil Girish Bhalala Anonymous reviewer(s)
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Abstract
Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time.
Key features
• This simple protocol rescues previously cleared tissue that has turned opaque.
• The method does not cause detectable loss of immunofluorescence from previously stained samples.
Graphical overview
Keywords: Tissue clearing Rescue iDISCO Dehydration Delipidation Reflective index rematching Light-sheet imaging
Background
Tissue clearing paired with light-sheet microscopy rejuvenated the century-old histochemistry field [1]. Clearing can render organs or even whole animals transparent [2], while the light-sheet microscopy can image fluorescent labels deep inside tissues or bodies [3]. Available clearing techniques generally fall into two categories based on the solvents used, specifically whether using hydrophilic or hydrophobic (including hydrogel-based methods) solutions [4]. Despite technical differences, the underlying principles between the two are the same, i.e., substituting molecules that diffract lights (e.g., lipids, pigments, and calcium phosphate) with other molecules that match the refractive index (RI) of the imaging media [2]. The transparency of cleared tissues allows fluorescence imaging of biomolecules deep inside the tissue (i.e., intrinsically expressed fluorescent proteins or after labeling with fluorescently tagged antibodies), which is further facilitated by light-sheet microscopy that illuminates the sample using an adjustable thin sheet of UV light [3]. Notably, fluorophore-tagged antibodies are often used to boost the detection of endogenous fluorescent proteins. iDISCO, a hydrophobic clearing method integrated with immunohistochemistry [5], has become very popular and given rise to multiple derivatives including uDISCO [6] and vDISCO [7]. Conventionally, cleared samples are stored in RI-matching media, often for months, for repeated imaging (please see https://idisco.info for more information). However, during storage, iDISCO-cleared specimens can turn cloudy and some even become opaque, which makes them unsuitable for imaging accurately by light-sheet microscopy. We suspected that such changes in tissue opacity with storage arose from residual lipids, a loss of RI matching deep inside the tissue, or the rehydration of samples by moisture in the air or dissolved in the storage solvent. To counteract those changes, we developed a simple rescue protocol based on immunofluorescence-compatible iDISCO. The approach is comprised by three steps—dehydration, delipidation, and RI rematching. The protocol we describe can successfully re-clear entire, murky mouse brains that had been previously cleared by iDISCO. We imaged the re-cleared brains by light-sheet microscopy, observing little or no loss of immunofluorescence as compared to imaging prior to storage, allowing the samples to be stored and studied far beyond the time of initial scanning.
Materials and reagents
Biological materials
Murky tissues previously cleared by iDISCO and possibly other hydrophobic clearing methods
Reagents
Methanol (Sigma-Aldrich, catalog number: 179337)
Dichloromethane (Sigma-Aldrich, catalog number: 270997)
Dibenzyl ether (Sigma-Aldrich, catalog number: 33630)
Solutions
Dichloromethane and methanol mix (see Recipes)
Recipes
Dichloromethane and methanol mix (per sample)
Reagent Final concentration (vol%) Volume
Dichloromethane 66% 3.3 mL
Methanol 34% 1.7 mL
Total n/a 5.0 mL
The mix should be made freshly and kept at room temperature before use.
Laboratory supplies
Organ holding forceps (e.g., Fine Science Tools, catalog number: 11031-15)
Screw-cap glass vials (e.g., Millipore Sigma, catalog number: 27151)
Nitrile gloves (any brand)
Kimwipes and paper towels (any brand)
Equipment
Fume hood (e.g., Hamilton Lab, model: SafeAire II)
Orbital shaker (e.g., Stovall Life Science, Belly Dancer, model: BDRAA115S)
Light-sheet microscope (e.g., Miltenyi Biotec, model: UltraMicroscope II Blaze)
Computer workstation equipped with, at least, Intel Xeon CPU, 128 G RAM, GPU with 16 GB memory, 2 TB storage, and USB3.0 port.
Software and datasets
Cloud-based lab management software (e.g., Labguru.com) is recommended to track and record the rescue procedure
Microscope system control and image acquisition software (e.g., ImSpector 7.5.3, free)
Image processing software (e.g., ImageJ1.53, free)
Atlas-matching and immunofluorescence quantification software (e.g., NeuroInfo, licensed by MBF Bioscience)
Procedure
Dehydration
Note: Please see note 2 and 3 about waste disposal and light avoidance.
Transfer the murky tissue sample (Figure 1) to a 5 mL screw-cap glass vial filled with 100% methanol and seal tightly.
Figure 1. Two previously cleared but murky brain samples over the course of rescue. Brain hemisphere (IDT3WT) and whole brain (S3) previously cleared by iDISCO but that turned murky during months of storage. Samples in A) and B) become totally opaque after dehydration; in C) they remain totally opaque after delipidation, with transparency restored with a light-yellow tinge, D) after refractive index (RI) matching.
Place the vial on an orbital shaker (e.g., Belly Dancer) and shake at 70 rpm at room temperature for 1 h.
Discard methanol and repeat steps A1 and A2 three more times.
Delipidation
Transfer tissue sample (Figure 1B) to a 5 mL screw-cap glass vial filled with 66% dichloromethane + 34% methanol and seal tightly.
CAUTION: Wear double nitrile gloves and work in a ventilation hood when handling dichloromethane. Wipe the exterior of the vials with paper towels.
Place the vial on an orbital shaker and shake at 70 rpm at room temperature for 48 h.
Transfer tissue sample to a 5 mL screw-cap glass vial filled with 100% dichloromethane and seal tightly.
CAUTION: One can always keep the sample in the same vial and change the solution instead (see note 4).
Place the vial on an orbital shaker and shake at 70 rpm at room temperature for 1 h.
RI rematching
Transfer tissue sample (Figure 1C) to a 5 mL screw-cap glass vial filled with 100% dibenzyl ether and seal tightly.
CAUTION: Wear double nitrile gloves and work in a ventilation hood when handling dibenzyl ether. Wipe the exterior of the vials with paper towels.
Place the vial on an orbital shaker and shake at 70 rpm at room temperature for 48 h.
Storage
Transfer tissue sample (Figure 1D) to a 5 mL screw-cap glass vial filled with fresh 100% dibenzyl ether and seal tightly.
CAUTION: Ensure no air bubbles are visible in the storage vials, which can be done by slight overfill of the glass vial and closing the cap very slowly.
Store the sample at room temperature and refresh dibenzyl ether once a month.
Data analysis
Tissue samples are imaged by light-sheet microscopy (e.g., Blaze) to localize immunofluorescence. Compiling of Z-stack and stitching of tiles is carried out using image processing software (e.g., Imaris). Atlas-matching and immunofluorescence measuring are achieved by image analysis programs (e.g., NeuroInfo). Figure 2A shows fluorescence light-sheet images of mouse brain before and after rescue. Figure 2B summarizes the fluorescence changes in different brain regions before and after rescue.
Figure 2. Rescued mouse brain. A. Sample images before and after the rescue. Overlay images are the composite of CF568, 640, and 770 signals. CF568 images show an apparent haze in midbrain and thalamus (before), which was cleared after rescue. Scale bar, 2 mm. B. Average fluorescence for all three immunofluorescent labels (arbitrary unit, a.u.; calculated from 15–30 randomly selected regions of interest within those brain regions). The increases are likely due to significantly lowered background (i.e., much less diffraction) after the rescue.
Validation of protocol
We have applied this rescue protocol to three different batches of murky brain tissues previously cleared by iDISCO and two more batches of murky brain tissues similarly cleared by a different researcher from another lab. In total, we tested this protocol using eight murky brain samples and successfully re-cleared all of them while preserving immunofluorescent signal.
General notes and troubleshooting
Unless specified, all steps are carried out at room temperature.
Waste disposal. As with initial iDISCO clearing, users should follow institutional requirements for waste disposal as established by local Environmental Health and Safety officers. All reagents used in this protocol should be disposed of in appropriate liquid chemical waste containers.
Light avoidance. If the tissue samples to be rescued have immunofluorescence labels, caution is needed to reduce loss of fluorescence. Options include always using aluminum foil to cover the glass vials containing tissue samples and operating under reduced light.
Solution change. Although the protocol above describes solution change as involving a transfer of samples to new vials containing the needed solutions, one can also discard the solution with retention of the tissue in the original vials, followed by adding new solutions to the original vial. It is important that containers (i.e., glass vials) holding samples are completely filled with solutions.
This rescue protocol is tested for the rescue of iDISCO-cleared samples. In rescued mouse brains, we used the following primary antibodies: one is the combination of guinea pig anti-GFAP antibody, chicken anti-Tuj-1 antibody, and rabbit anti-SREBP2 antibody; the other is rabbit anti-cFos alone. Accordingly, the secondary antibodies are highly cross-absorbed CF770-conjugated goat anti-guinea-pig IgG, CF640-conjugated goat anti-chicken IgG, and CF-568-conjugated goat anti-rabbit IgG (all from Biotium).
The timeline of this protocol is adjustable depending on the size of the samples.
Like iDISCO, the survival of different epitopes as well as corresponding antibodies during the rescue needs to be evaluated on a case-by-case basis. Since the rescue procedure and solutions used are largely the same to those of iDISCO, it is likely that antibodies suitable for iDISCO should be compatible with this rescue procedure.
Because this protocol is based on the hydrophobic iDISCO method, it may also work for samples prepared by other hydrophobic clearing methods but not for samples cleared by hydrophilic methods.
Acknowledgments
H.M., J.M., and Q.Z. were supported by NIH award GM147912, a Florida Department of Health award 21A04, and a pilot award from the Stiles-Nicholson Brain Institute. P.G.-K. and R.B. were supported by NIH Award MH094527.
Competing interests
The authors declare no competing interests.
Ethical considerations
Animal use and related procedures were approved by the Florida Atlantic University Institutional Animal Care and Use Committee (IACUC Protocol #A21-06). Mice were housed in pairs. A total of eight male and female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) between the age of 6 and 30 months were used for collecting brain tissues for this study.
References
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Ueda, H. R., Ertürk, A., Chung, K., Gradinaru, V., Chédotal, A., Tomancak, P. and Keller, P. J. (2020). Tissue clearing and its applications in neuroscience. Nat. Rev. Neurosci. 21(2): 61–79. https://doi.org/10.1038/s41583-019-0250-1
Stelzer, E. H. K., Strobl, F., Chang, B. J., Preusser, F., Preibisch, S., McDole, K. and Fiolka, R. (2021). Light sheet fluorescence microscopy. Light sheet fluorescence microscopy. Nat. Rev. Methods Primers 1(1): e1038/s43586–021–00077–4. https://doi.org/10.1038/s43586-021-00077-4
Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T. and Ueda, H. R. (2016). Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling. Annu. Rev. Cell Dev. Biol. 32(1): 713–741. https://doi.org/10.1146/annurev-cellbio-111315-125001
Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P. and Tessier-Lavigne, M. (2014). iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159(4): 896–910. https://doi.org/10.1016/j.cell.2014.10.010
Pan, C., Cai, R., Quacquarelli, F. P., Ghasemigharagoz, A., Lourbopoulos, A., Matryba, P., Plesnila, N., Dichgans, M., Hellal, F., Ertürk, A., et al. (2016). Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13(10): 859–867. https://doi.org/10.1038/nmeth.3964
Cai, R., Pan, C., Ghasemigharagoz, A., Todorov, M. I., Förstera, B., Zhao, S., Bhatia, H. S., Parra-Damas, A., Mrowka, L., Theodorou, D., et al. (2018). Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull–meninges connections. Nat. Neurosci. 22(2): 317–327. https://doi.org/10.1038/s41593-018-0301-3
Article Information
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Neuroscience > Neuroanatomy and circuitry > Fluorescence imaging
Cell Biology > Cell imaging > Fixed-tissue imaging
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4,949 | https://bio-protocol.org/en/bpdetail?id=4949&type=0 | # Bio-Protocol Content
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Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed
AS Arnold Sipos
KK Kwang-Jin Kim
JA Juan R. Alvarez
EC Edward D. Crandall
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4949 Views: 930
Reviewed by: Olga KopachJohn P Phelan Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Autophagy Reports Mar 2023
Abstract
Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer’s disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models.
Key features
• Allows real-time qualitative imaging of autophagic flux at single-cell level.
• Primary cells and cell lines can also be utilized with this technique.
• Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.
Keywords: Autophagic flux DAPRed Live cell imaging Confocal microscopy A549 cells
Background
Autophagy is a highly conserved cellular process that removes unwanted or damaged cellular components [1,2]. Although first described as a mechanism to protect cells against starvation [3], it was later understood that autophagy has an important role in non-starving cells to maintain cellular homeostasis [4,5]. Currently, autophagy is recognized as being involved in organ development [6,7] and in coping with infections [8], environmental stressors [9], and aging [10]. It is also known that extreme cellular stress or insufficient autophagic response leads to the deterioration of cellular functions, possibly leading to cell death [11–14].
There are a number of different tools available to study autophagy [15], including electron microscopy and measurement of Atg8-family proteins [e.g., microtubule-associated protein 1A/1B-light chain 3 (LC3)] by various techniques including western blot, flow cytometry, immunofluorescent staining, or fluorescent microscopy, providing an assessment of autophagic activity. Autophagic proteins exhibit tissue- and cell-specific expression and different turnover rates. In addition, some of these approaches require cell fixation, permeabilization, or transfection of exogeneous fluorescent proteins. Since these procedures can alter the physiological expression of autophagy markers, they are not entirely suitable for the accurate assessment of autophagic flux (the rate of autophagosome formation over time, an indicator of autophagic activity) per se.
Live cell imaging is a valuable tool for studying biological processes. When combined with suitable fluorescent marker(s), it allows the visualization and quantification of complex biological processes, such as autophagy, in real time, even at single-cell resolution. Recently, a novel fluorescent dye, DAPRed, was developed for autophagy studies [16]. DAPRed is a small fluorescent molecule that is incorporated into the autophagosome membrane during the double membrane formation [16]. DAPRed allows the real-time visualization of autophagosomes (i.e., formation and subcellular tracking of autophagosomes), enabling autophagic flux measurement in living cells without the need for complex molecular techniques such as cloning or transduction. When DAPRed is combined with adequate imaging techniques, such as confocal or super resolution microscopy, it allows the study of autophagy kinetics and the interactions with other cellular processes (e.g., lysosomal degradation [17]). DAPRed has already been used in semiquantitative studies to address the activation of autophagy [18–20] or autophagic flux measurement [21]. Here, we describe a procedure for the measurement of autophagic flux in single live cells using DAPRed. This procedure involves cell stimulation with the positive control Rapamycin to induce autophagy and marking the plasma membrane with a fluorescently tagged tomato lectin for high spatial precision for single-cell DAPRed fluorescence detection. Autophagy is a dynamic biological process in which autophagosomes are generated and consumed upon merging with lysosomes. Autophagic activity is best described by autophagic flux, which is the rate of autophagosome generation over time. In this protocol, we explain the importance of inhibiting autophagosome–lysosome fusion and the conditions for correctly determining autophagic flux.
Materials and reagents
Polycarbonate Transwell® (tissue culture treated, permeable support of 1.13 cm2 or 12 mm diameter), 12-well plate (Costar, catalog number: 3401)
Centrifuge tubes, 15 mL (VWR, catalog number: 89039-670)
Centrifuge tubes, 50 mL (VWR, catalog number: 89079-494)
N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) hemisodium salt (HEPES) solution (Sigma-Aldrich, catalog number: H0887)
Dulbecco's modified Eagle's medium/nutrient mixture Ham's F-12 medium (DME/F-12) (Sigma-Aldrich, catalog number: 6421) including 15 mM HEPES and sodium bicarbonate, without L-glutamine
Fetal bovine serum (HyClone, catalog number: SH30071.03)
Bovine serum albumin (Jackson ImmunoResearch Laboratories, catalog number: 001-000-162)
L-Glutamine (Sigma-Aldrich, catalog number: G7513)
Nonessential amino acid solution (Sigma-Aldrich, catalog number: M7145)
Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P4333)
Primocin (VWR, catalog number: MSPP-ANTPM1)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418)
Microscope cover glass, 22 mm × 22 mm, No. 1. (Denville Scientific Inc., catalog number: M1100-01)
DAPRed (Dojindo Molecular Technologies, catalog number: D677-10)
Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628)
Rapamycin (Selleck Chemical, catalog number: s1039)
Dylight 488-conjugated tomato lectin (Vector Laboratories, catalog number: DL-1174-1)
Hoechst 33342 (Sigma-Aldrich, catalog number: H6024)
Human lung adenocarcinoma cell line A549 (ATCC, catalog number: CCL-185)
Solutions
A549 cell culture medium (MDS) (see Recipes)
Krebs-Ringer solution (see Recipes)
Recipes
A549 cell culture medium (MDS)
DME/F-12 medium supplemented with 10% fetal bovine serum, 1 mM nonessential amino acid solution, 100 U/mL primocin, 10 mM HEPES, 1.25 mg/mL bovine serum albumin, 2 mM L-glutamine, and 1 U penicillin-streptomycin.
Krebs-Ringer solution
136 mM NaCl, 4.7 mM KCl, 1 mM NaH2PO4, 1 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, 1 mM D-glucose. Adjust pH to 7.4.
Equipment
FormaTM Series II Water-Jacketed CO2 incubator (Thermo Scientific, model: 3110)
Centrifuge (Eppendorf, model: 5424)
Confocal microscope (Leica Microsystems, model: Leica STELLARIS 8)
Temperature-controlled chamber: Ludin chamber type 1 (Life Imaging Services, Switzerland)
Software and datasets
Leica LAS 3D Process and Quantify Packages (Leica Microsystems)
ImageJ (National Institutes of Health)
Microsoft Excel
Procedure
Plate A549 cells onto Transwell filters at 100,000 cells/1.13 cm2 and culture until reaching 80% confluence (typically until culture day 4).
Note: Cell culturing parameters, including culturing substrate, plating density, level of confluency, and time to reach desired confluency should be adjusted individually to different cell/tissue types.
Freshly prepare chloroquine [water soluble; vehicle: culture fluid (MDS) (see Recipes)], an inhibitor of autophagosome fusion with lysosome (stock solution: 40 mM), and add to the culture fluid to reach 40 μM final concentration on both apical and basolateral sides for 1 h before imaging; the control should receive culture fluid alone.
Note: Live cell imaging is carried out with and without chloroquine with different sets of cells.
Add DAPRed [0.1 μM; lipid soluble; vehicle: 0.1% (v/v) DMSO] (1 μL) to the culture fluid in the apical compartment for 30 min before imaging.
To label cell plasma membranes, remove culture fluid and apply Dylight 488-conjugated tomato lectin (5 μg/mL: 5 μL from stock of 1 mg/mL) directly onto the apical surface of the cells just prior to imaging.
Note: Although Dylight 488 proved to be resistant to photobleaching in our hands, it is advisable to avoid unnecessary light exposure.
Carefully cut out the Transwell filter with the cell monolayer of interest grown on it using a scalpel along the rim. Mount the Transwell filter with monolayer on a coverslip and add Krebs-Ringer solution (~1–2 mL), as a bathing solution, to the coverslip chamber.
Live cell imaging:
Perform confocal imaging at 63× magnification, 12 bit, and 1,024 × 1,024 resolution with an SP8 confocal microscope system.
In xyz series, measure intracellular fluorescence intensity (DAPRed: 561/570–600 nm, Dylight 488-conjugated tomato lectin: 488/490–530 nm) stack by stack over the entire volume of a single, live A549 cell.
Aim to zoom in sufficiently to get to the level of ~150–200 nm/pixel, which is the resolution limit of current confocal microscopy approaches, and to include as many cells as possible.
Using the signal of Dylight 488-conjugated tomato lectin, set the upper limit for xyz series. For a typical A549 cell, this is 60 × 60 × 20 μm for x, y, and z dimensions, respectively.
Move the objective in the opposite direction to find the lower limit for xyz series (i.e., where the fluorescence signal is lost) and set the sectioning interval between 0.35 and 0.5 μm. This seems to be the most practical interval to produce detailed 3D image quality of autophagosomes in live A549 cells while avoiding phototoxicity and photobleaching caused by more aggressive laser scanning.
Data analysis
Live cell imaging approach: measure DAPRed fluorescence intensity in a single live A549 cell in a stack-by-stack mode and integrate over the entire volume of the cell.
Open image file in ImageJ.
Separate fluorescence channels (DAPRed and Dylight 488-conjugated tomato lectin) in ImageJ software (ImageJ: image/color/split channels).
Use the plasma membrane marker Dylight 488-conjugated tomato lectin to delineate the area of cytoplasm in which fluorescence of DAPRed needs to be measured.
Have the two fluorescence channels open next to each other in two separate windows (see step 1b above).
Synchronize windows (ImageJ: analyze/tools/synchronize windows).
Click on the DAPRed channel and delineate the border of the cell to be measured [create an ROI (Region of Interest)], which is guided by the synchronized cursor of plasma membrane marker.
Figure 1. Representative image showing plasma membrane–guided ROI selection. Plasma membrane markers (e.g., Dylight 488-conjugated tomato lectin, left panel) can help precisely delineate the cytosolic border of individual cells (e.g., A549 cells). Plasma membrane–guided ROIs (yellow highlighted areas) were drawn, and measurement was conducted in the DAPRed channel (right panel).
Measure DAPRed fluorescence in the ROI in the yellow highlighted area (Figure 1) and export the measured DAPRed fluorescence intensity to Microsoft Excel for subsequent calculations.
Calculation of autophagic flux:
(1) Øcontrol = Fcontrol (with chloroquine) - Fcontrol (without chloroquine)
(2) Øexposure = (Fexposure (with chloroquine) - Fexposure (without chloroquine)) - Øcontrol
Ø: autophagic flux
F: DAPRed fluorescence intensity
Representative data
We recently published an article using this method to assess the kinetics of autophagy in nanoparticle-exposed A549 cells [17]. We also successfully applied this method for autophagy flux detection in rat alveolar epithelial cell monolayers. An in-depth characterization of DAPRed, including an assessment of its photobleaching, was provided in collaboration with the manufacturer [16]. In addition, here we provide a representative dataset in which Rapamycin, an autophagy inducer, was used to stimulate autophagy (Figure 2) and a video showing typical DAPRed labeling (Video 1).
Figure 2. Autophagic flux measurement using DAPRed in single live A549 cells. A. Representative images showing time-dependent activation of autophagic marker DAPRed (red) in A549 cells upon 50 nM Rapamycin exposure. Rapamycin activates autophagy by repressing the activity of the mammalian target of Rapamycin complex 1 (mTORC1). Exposure times are shown above images. Images were captured at a single focal plane representing one cross-section only of the cells. For quantifications (see panel B), DAPRed fluorescence was combined from multiple focal planes covering the entire volume of a single cell. The plasma membrane was labeled by Dylight 405-conjugated tomato lectin to differentiate extracellular from intracellular space and fluorescent signal arising from adjacent cells (see Figure 1). Scale bars, 25 µm. B. Rapamycin-induced (50 nM) autophagic flux. Quantification of autophagic flux measured via DAPRed method was carried out using the formula described in Data analysis. Each time point represents a different set of A549 cells. Cells at each condition were imaged for a short (< 30 min) period of time. n = 7–8.
Video 1. Representative video showing 3D rendering of autophagosomes in a single, live A549 cell (see Video 1). A549 cells were exposed to 50 nM Rapamycin in the apical culture media for 24 h. Cells were treated with 40 μM chloroquine in the apical culture fluid for 1 h prior to imaging. Autophagosomes were labeled by DAPRed (0.1 μM, 37 °C, 5% CO2 for 30 min), while nuclei were marked with Hoechst 33342 (50 μg/mL, 37 °C, 5% CO2 for 30 min). Plasma membrane was labeled with Dylight 488-conjugated tomato lectin (5 μg/mL, labeling is instantaneous, no incubation needed). Autophagosomes distribute over the entire cytoplasm with noticeable enrichment around the perinuclear cytosolic area.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Sipos, A., Kim, K. J., Sioutas, C. and Crandall, E. D. (2023). Kinetics of autophagic activity in nanoparticle-exposed lung adenocarcinoma (A549) cells. Autophagy Rep. 2(1): e2186568.
Data in this research article (Figures 1–6) were generated using the methods described in this current Bio-protocol paper.
General notes and troubleshooting
Detector sensitivity should be adjusted in a way that avoids oversaturated DAPRed signal. For this, under maximal autophagic activity (e.g., in our case, 3–4 h of Rapamycin stimulation), set the detector gain to a level where no oversaturated pixels are present. If needed, increase signal detection from 8 to 12 bit.
A 1,024 × 1,024 resolution is optimal for single-cell analysis. Lower resolution, although it allows faster scanning with less phototoxicity, does not provide the needed spatial resolution. By choosing resolutions higher than 1,024 × 1,024, it may be possible to gain more detailed spatial information. However, increasing the resolution beyond 1,024 × 1,024 was not practical in our experiments, since it also increased the pixel dwell time, predisposing the cells and intracellular organelles to phototoxicity.
DAPRed showed minimal to no photobleaching under optimized, moderate (laser intensity < 10% power) imaging conditions [16].
Photobleaching can be a potential source of artifacts when (typically older) confocal and fluorescent microscopes are used. However, recent developments in detector systems, especially with the Leica Hybrid detectors we utilized for this work, exhibit drastically decreased photobleaching due to their improved sensitivity and increased scanning speed. In our setup, a 561 nm laser was used at 2.5% full power. In addition, pixel dwell time was 0.6 µs while imaging time at a certain region was < 5–8 min. Finally, imaging was performed in a sequential fashion, meaning that quantitative imaging of the DAPRed signal is collected first, followed by qualitative imaging of the plasma membrane marker Dylight 488-conjugated tomato lectin.
Laser power was kept constant (488 nm: 2%, 561 nm: 2.5% full power) during the entire experiment to be able to compare different datasets.
Acknowledgments
This work was supported in part by the Will Rogers Motion Picture Pioneers Foundation, Whittier Foundation and Hastings Foundation. E.D.C. is Hastings Professor in the Keck School of Medicine. This protocol is based on Sipos et al. [17].
Competing interests
Authors declare no conflict of interest.
References
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Doherty, J. and Baehrecke, E. H. (2018). Life, death and autophagy. Nat. Cell Biol. 20(10): 1110–1117.
Green, D. R. and Levine, B. (2014). To Be or Not to Be? How Selective Autophagy and Cell Death Govern Cell Fate. Cell 157(1): 65–75.
Klionsky, D. J., Abdel-Aziz, A. K., Abdelfatah, S., Abdellatif, M., Abdoli, A., Abel, S., Abeliovich, H., Abildgaard, M. H., Abudu, Y. P., Acevedo-Arozena, A., et al. (2021). Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 17(1): 1–382.
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Sipos, A., Kim, K. J., Sioutas, C. and Crandall, E. D. (2023). Kinetics of autophagic activity in nanoparticle-exposed lung adenocarcinoma (A549) cells. Autophagy Rep. 2(1): e2186568.
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Article Information
Copyright
© 2024 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-based analysis > Autophagic activity
Cell Biology > Cell imaging > Confocal microscopy
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Immunofluorescent Staining Assay of 3D Cell Culture of Colonoids Isolated from Mice Colon
TM Trisha Mehrotra
XS Xiaodi Shi
DM Didier Merlin
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4950 Views: 941
Reviewed by: Samantha Hallerabhijnya kanugovi Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Cell Death & Disease Sep 2020
Abstract
Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules—both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody–marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients’ colons, monitoring the biomarkers, and customizing their treatments.
Key features
• Analysis of molecular markers of cell behavior.
Protocol to visualize proteins in 3D cell culture.
• This protocol requires colonoids isolated from mice colon grown on matrigel support.
• Protocol requires at least eight days to complete.
Graphical overview
Keywords: Inflammatory bowel disease (IBD) Colonoids 3D cell culture Matrigel Immunofluorescent staining
Background
Inflammatory bowel disease (IBD) represents a chronic intestinal inflammation with unknown etiology, which has been linked to genetic and environmental factors. Dysregulation of epithelial-mucosal homeostasis is a key feature of IBD pathogenesis. Epithelial-mucosal lining acts as a defensive barrier against harmful luminal microenvironment. In the past, to understand the progression of IBD, immunofluorescent staining was used to assess the key markers of cell differentiation and proliferation. Staining has been used mostly either for fixed intestinal tissues (as an in vivo model) or for traditional 2D cultures of immortal cell lines (as an in vitro model), which lack the spatial 3D orientation of the cells. There are other staining techniques such as in situ hybridization, immunohistochemistry, and Alcian blue staining utilizing colonoids [1]. Here, we describe the use of immunofluorescent staining of colonoids to mimic the intestinal physiological and pathophysiological relevance. This is an assay that shows the expression of a protein as well as its subcellular location in a realistic way, providing significant benefits to translational research by screening potential therapeutic drugs and their effects on cell differentiation and proliferation for IBD patients.
Materials and reagents
Biological materials
4–6-weeks-old C57BL/6Jmice (Jackson Laboratories, strain number: 000664)
Reagents
Pipette tip: 1,000 µL, 20–200 µL, 1–10 µL (Thermo Fisher Scientific, catalog numbers: P1126, P1179, and P1060, respectively)
Matrigel phenol-red free (Fisher Scientific, catalog number: CB-40234C)
Gibco DMEM/F-12 (Thermo Fisher Scientific, catalog number: 11320033)
Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122)
L-Glutamine solution (Sigma-Aldrich, catalog number: G7513-100ML)
Fetal bovine serum (FBS), heat inactivated (Corning, catalog number: 35-011-CV)
GlutaMAX supplement (Thermo Fisher Scientific, catalog number: 35050079)
HEPES buffer solution (Sigma-Aldrich, catalog number: 83264-100ML-F)
N-2 supplement (100×) (Thermo Fisher Scientific, catalog number: 17502048)
B27 supplement (50×), serum free (Thermo Fisher Scientific, catalog number: 17504044)
Epidermal growth factor (EGF) (Sigma-Aldrich, catalog number: E1257-0.1MG)
ROCK inhibitor (Sigma-Aldrich, catalog number: 688001-500UG)
1× phosphate buffered saline (PBS) (without calcium and magnesium) (Fisher Scientific, catalog number: MT21040CM)
Paraformaldehyde 16% (Thermo Fisher Scientific, catalog number: 043368-9M)
FocusClear solution (CelExplorer, catalog number: FC-101)
Triton X-100 solution (Fisher Scientific, catalog number: P185111)
Tween 20 solution (Fisher Scientific, catalog number: P185113)
Sodium azide (Sigma-Aldrich, catalog number: S2002-5G)
Vector Laboratories goat serum (Cole-Parmer, catalog number: S-1000)
ProLong Gold Antifade Mountant with DNA blue fluorescence stain DAPI (Thermo Fisher Scientific, catalog number: P36931)
Ted Pella Inc clear nail polish (Fisher Scientific, catalog number: NC 1849418)
Solutions
Minigut medium (see Recipes)
4% paraformaldehyde solution (see Recipes)
Immunofluorescence (IF) buffer (see Recipes)
Permeabilization solution (see Recipes)
Blocking buffer (see Recipes)
Dilution buffer (see Recipes)
Recipes
Minigut medium
Conditioned medium (1 volume of L-cell medium and 1 volume of DMEM F12 containing 1:100 vol/vol penicillin-streptomycin, 1:100 vol/vol glutamine, and 20% vol/vol FBS) [2]
GlutaMAX 1:100 vol/vol
10 mM HEPES buffer
Penicillin-streptomycin 1:100 vol/vol
N-2 supplement 1:100 vol/vol
B27 supplement 1:50 vol/vol
EGF 50 ng/mL (final concentration)
ROCK inhibitor 10 µM (final concentration)
4% paraformaldehyde solution
1:4 vol/vol 16% paraformaldehyde and 1× PBS
Immunofluorescence (IF) buffer
Triton X-100 0.2% (vol/vol), Tween 20 0.05% (vol/vol), 1× PBS 99.75% (vol/vol)
Permeabilization solution
Triton X-100 2% (vol/vol) and 1× PBS 98% (vol/vol)
Blocking buffer
Goat serum 10% (vol/vol), Triton X-100 2% (vol/vol), sodium azide 0.02% (wt/vol), 1× PBS 88% (vol/vol)
Dilution buffer
Triton X-100 0.25% (vol/vol), goat serum 1% (vol/vol), sodium azide 0.02% (wt/vol), 1× PBS 98.75% (vol/vol)
Equipment
Costar 24-well clear TC-treated multiple well plates individually wrapped, sterile (Corning, catalog number: 3524)
Pipetman—PK1000, PK200, PK20 (Gilson, catalog numbers: F144059M, F144058M, and F144056M, respectively)
Lab-Tek chamber slide 8-well glass slide (Nunc, catalog number: 154941)
Laminar flow hood (LabGard Class II type A2 Biological Safety cabinet) (Nuaire, model: NU-540)
3110 CO2 water jacketed cell incubator (Thermo Fischer Scientific, model: 4120)
Digital water bath (VWR, catalog number: 89501-460)
Sorvall ST 16R centrifuge (Fisher Scientific, catalog number: 75-004-240)
All-in-One fluorescence microscope (Keyence BZ-X series, model: BZ-X810) (Figure 1)
Rectangular ice pan (ice tray/bucket) (Fisher Scientific, catalog number: 07-210-107)
Micro cover glass (VWR, catalog number: 48393252)
Figure 1. Keyance microscope. A. Keyence microscope with software showing the immunofluorescent staining of a colonoid. B. Keyance microscope showing the holder to be used for 8-well Lab-Tek chamber slide.
Software and datasets
BZ-X analyzer (https://www.keyence.com/bz-x810)
Procedure
As described by O’Rourke et al. [2], use 5–6-week-old wild-type mice to isolate colonoids. Briefly, the colon of the mice was cut into small pieces and strained to remove tissues. Filtrate was shaken vigorously with 1× PBS-EDTA and was continuously checked for crypts enrichment under a basic microscope. Once optimum crypts are observed, the supernatant is centrifuged at 112× g (rcf) at 10 °C. The pellet is mixed with matrigel. Mix isolated colonoids with matrigel (10 µM) in a laminar flow hood. Mix the suspension by pipetting using a PK200 without creating bubbles.
Add 50 µL of matrigel and colonoid mix (~10 colonoids/50 µL matrigel) in each well of a prewarmed (at 37 °C) 24-wells plate in the laminar flow hood and keep it in a cell incubator set at 37 °C with 5% CO2 and 90% humidity for 5 min for polymerization.
After polymerization, overlay each well with 1 mL of prewarmed Minigut medium using a PK100 in the laminar flow hood. Dispense the medium along the wall of the well.
Replace the medium of the wells after 18–24 h and on day 3 with 1 mL of prewarmed Minigut medium using a PK1000 in the laminar flow hood. Use PK1000 to aspirate the medium along the wall of the well without disturbing the matrigel drop.
On day 5, aspirate the medium using a PK1000 in the laminar flow hood and use prewarmed 1× PBS (without calcium and magnesium) as one-time wash to remove the medium.
Place the 24-well plate on an ice pan in the laminar flow hood and add 1 mL of ice-cold 1× PBS (without calcium and magnesium) using a PK1000. Gently break the matrigel drop by repeated pipetting using a PK200 avoiding bubbling.
Collect the 1× PBS containing the dissolved matrigel with colonoids in a 50 mL centrifuge tube and centrifuge at 28× g (rcf) for 5 min at 4 °C.
Aspirate out 1× PBS with dissolved matrigel using a PK1000 as quick as possible, keeping the centrifuge tube in ice in the ice pan.
Resuspend the colonoid pellet in 400 µL of matrigel and mix it by pipetting with 200 µL Pipetman. Add 50 µL of the suspension to prewarmed 8-well Lab-Tek chamber slide and place it straight (not upside down) in the cell incubator for 5 min. After polymerization, add 500 µL of prewarmed Minigut medium to each well of the Lab-Tek chamber slide and place it in the cell incubator (Figure 2).
Figure 2. 8-well Lab Tek chamber slide. A. 50 μL of matrigel drop with colonoid suspension. B. Minigut medium overlaying the polymerized matrigel drop with colonoid in cell incubator.
Replace the medium of the wells on days 1 and 3 with 1 mL of prewarmed Minigut medium using a PK1000 in the laminar flow hood. Use a 1 mL Pipetman to aspirate the medium along the wall of the well without disturbing the matrigel drop.
On day 4 or day 5, colonoids will show branching (Figure 3), which is the optimum state to start immunofluorescent staining. Take out the Lab-Tek chamber slide from the cell incubator. Use a PK1000 to aspirate the medium along the wall of the well without disturbing the matrigel drop. Wash each well (once only) with 500 µL of prewarmed 1× PBS using a PK1000.
Figure 3. Day 2 (no branching), Day 4 (branching starts appearing), Day 6 (branching is not very clear in multilayer colonoid), and Day 8 (branching is not visible in very dense colonoid) of wild-type mice culture on matrigel in 8-well Lab-Tek chamber plate. Magnification is 20×.
After aspirating 1× PBS, fix the colonoids using 4% paraformaldehyde solution at room temperature for 2 h.
Wash two times with 500 µL (per well) of prewarmed 1× PBS using a PK1000 (5 min each wash, no shaking).
Add 200 µL of FocusClear solution to each well using a PK1000 and incubate for two nights at room temperature on a working laboratory bench.
Wash two times with 200 µL (per well) of IF buffer using a PK200 (5 min each wash, no shaking).
Incubate with 200 µL of permeabilization solution in each well for three nights at room temperature.
Aspirate out the permeabilization solution using a P200 and wash three times with IF buffer (200 µL per well) at room temperature for 5 min (each wash) without shaking.
Add 200 µL of blocking buffer to each well and incubate for 4 h at room temperature.
Aspirate out the blocking buffer using a P200.
Use a P200 to add 150 µL (per well) of primary antibody diluted in dilution buffer and incubate overnight at room temperature.
Aspirate out the primary antibody using a P200 and wash three times with IF buffer (200 µL per well) at room temperature for 5 min (each wash) without shaking.
Add 150 µL (per well) of secondary antibody (against the primary antibody, as per the manufacturer’s instruction) conjugated with, for example, green fluorescence (GFP). Dilute the secondary antibody (as per the manufacturer’s instruction) in dilution buffer and incubate for two nights at room temperature in the dark.
Aspirate out the secondary antibody using a P200 and wash three times with IF buffer (200 µL per well) at room temperature for 5 min (each wash) without shaking.
Add 150 µL (per well) of fluorescence-counterstaining antibody (for example, if secondary antibody is conjugated with green fluorescence, then red fluorescence conjugated Phalloidin, which stains actin filaments, should be used) diluted in dilution buffer for one night at room temperature.
Aspirate out the counterstaining antibody using a P200 and wash three times with IF buffer at room temperature (200 µL per well) for 5 min (each wash) without shaking.
Add 100 µL of ready-to-use ProLong Gold Antifade Mountant with DNA blue fluorescence stain DAPI to each well for one night at room temperature.
As per manufacturer’s instructions, a tool is provided which separates and removes the upper chamber from the silicone gasket of the Lab-Tek slide.
Coverslip the Lab-Tek slide without upper chamber with a micro cover glass and seal with white-colored nail polish.
Store in the dark at room temperature.
The mounted and cover-slipped slide should be used for taking images at the earliest (within 2–3 days of staining) and can be stored at room temperature for 4–5 days only under dark conditions.
Representative data: For example, we demonstrate in Figure 4 that 4-day-old colonoids isolated from wild-type mice were stained with anti-PCNA (proliferating cell nuclear antigen, a proliferation marker) conjugated with green fluorescence. TRITC-conjugated phalloidin and DAPI (blue fluorescence) were used to counterstain actin filaments with red fluorescence and nuclei with blue fluorescence, respectively, using 40× magnification. We also captured the image in brightfield using 40× magnification.
Figure 4. Representative result of immunofluorescence staining of 4-day-old organoid of wild-type mice, cultured and fixed on matrigel in 8-well Lab-Tek chamber plate and probed with anti-PCNA (proliferation marker). FITC as green fluorescence was used as secondary antibody. Images are the Z-sections at 40× magnification. DAPI was used to counterstain nuclei with blue fluorescence and red fluorescence is for actin-phalloidin. Branching of colonoid shows proliferating cells as indicated by white arrows.
Data analysis
We used a Keyence microscope (BZ-X810 model) and the BZ-X series software to capture images of the immunofluorescent-stained colonoid in brightfield and under green, red, and blue channel. With the help of software, an overlay image of all three channels can be created, which will depict the expression of the protein of interest as well as its location due to actin and nuclear counterstaining.
Validation of protocol
We have demonstrated in Walter et al. [3] that 4-day-old colonoids were stained with anti-SEPP1 conjugated with green fluorescence (Figure 5C-ii). TRITC-conjugated phalloidin and DAPI were used to counterstain actin filaments with red fluorescence and nuclei with blue fluorescence, respectively, using 40× magnification.
General notes and troubleshooting
The protocol was used on freshly isolated colonoids from mice colon. However, it should work with any colonoid culture as long as they are grown on matrigel and are healthy and less branched. See in Figure 4 the difference in branching of colonoids on Day 4 and Day 6.
The 50 µL of colonoid and matrigel suspension in prewarmed 8-well Lab-Tek chamber slide should not be incubated in the cell for more than 5 min. This is the optimum duration for the polymerization of matrigel as well as avoiding nutrient deprivation of colonoids.
While breaking the matrigel-containing colonoid, temperature should be maintained icy cold. Furthermore, frothing and bubbling due to repeated pipetting should be avoided as much as possible.
The entire procedure takes approximately eight days to finish. Therefore, there is a higher chance of losing the polymerized matrigel-containing colonoid during the procedure. Therefore, one slide (meaning all 8 wells) should be used for one condition. This means that, if we are comparing wild-type mice with transgenic mice, one slide should be seeded with wild-type mice colonoids and another slide should be seeded with transgenic mice colonoids. This will give a sufficient number of ‘ns’ for data analysis.
Mounted and cover-slipped slides should be processed for data analysis as soon as possible. Due to matrigel, slides cannot be stored at 4 °C, and fluorescence intensity fades out slowly at room temperature.
Handling of paraformaldehyde should be done in the fume hood while wearing PPE and safety goggles.
Troubleshooting
Matrigel polymerizes at 37 °C and becomes liquid at 4 °C. Therefore, while passaging the colonoids, icy conditions are important to be maintained; during the staining procedure, prewarmed solutions should be used at room temperature.
After seeding colonoids on 8-well Lab-Tek chamber slides, they should be observed daily. Branching of colonoids can start as early as on Day 3 and as late as Day 5 or 6 depending on mice strain or if it is a transgenic mouse. Multilayered colonoids with several branching should be avoided for data analysis purposes, as it will not result into sharp pictures.
If primary antibody staining is not visible, then consider decreasing its dilution.
Acknowledgments
This protocol was adapted from Walter et al. [3].
Competing interests
The authors declare no conflicts of interest within this work.
Ethical considerations
Georgia State University’s Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee (IBC) have approved the experiments described in the protocol.
References
Wilson, S. S., Mayo, M., Melim, T., Knight, H., Patnaude, L., Wu, X., Phillips, L., Westmoreland, S., Dunstan, R., Fiebiger, E., et al. (2021). Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids. Front. Immunol. 11: e547102. https://doi.org/10.3389/fimmu.2020.547102
O’Rourke, K., Ackerman, S., Dow, L. and Lowe, S. (2016). Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells. Bio Protoc 6(4): e1733. https://doi.org/10.21769/bioprotoc.1733
Walter, L., Canup, B., Pujada, A., Bui, T. A., Arbasi, B., Laroui, H., Merlin, D. and Garg, P. (2020). Matrix metalloproteinase 9 (MMP9) limits reactive oxygen species (ROS) accumulation and DNA damage in colitis-associated cancer. Cell Death Dis. 11(9): 767. https://doi.org/10.1038/s41419-020-02959-z
Article Information
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Cell Biology > Cell staining > Protein
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4,951 | https://bio-protocol.org/en/bpdetail?id=4951&type=0 | # Bio-Protocol Content
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Bacterial Pathogen-mediated Suppression of Host Trafficking to Lysosomes: Fluorescence Microscopy-based DQ-Red BSA Analysis
MM Mădălina Mocăniță
KM Kailey Martz
VD Vanessa M. D'Costa
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4951 Views: 395
Reviewed by: Lucy XieXin Xu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Cell Reports Sep 2015
Abstract
Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen–mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention.
Key features
• Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model.
• Live microscopy–based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification.
• Assay can be adapted to a broad array of pathogens and diverse host cell types.
• Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.
Keywords: Bacterial pathogenesis Intracellular pathogen DQ-BSA Lysosome Intracellular trafficking Host-pathogen interactions Salmonella enterica
Background
Bacterial pathogens employ diverse strategies to manipulate host cellular function to establish infection [1]. Among the many approaches, modulation of host cells can occur at the level of immune signalling and immune cell function [1,2], cellular immune responses such as lysosomal and autophagy function [3–6], organelle function [7,8], or cytoskeletal dynamics [1,9,10]. The lysosomal pathway represents a fundamental signalling hub and cellular immune response pathway in eukaryotic cells [11,12]. Lysosomes are membrane-bound organelles responsible for the degradation of proteins, lipids, nucleic acids, and carbohydrates [11,12]. These vesicles exhibit a characteristically low pH that promotes the activation of over 60 different hydrolases, ultimately contributing to cellular homeostasis and clearance of intracellular pathogens [11,12]. Defects in lysosome function can lead to one of more than 50 different disorders, collectively defined as lysosome storage diseases, with implications in neurodegeneration and aging [12–15].
Given that lysosomes function as a primary organelle responsible for the clearance of intracellular bacterial pathogens, they represent an important target for pathogen manipulation. Intracellular bacteria have evolved diverse mechanisms to manipulate host function to evade lysosomal degradation [2,10]. Lysosomal evasion can occur through either direct or indirect means. An example of an indirect mechanism of evasion is the manipulation of host trafficking to acquire membrane from other organelles. For example, the pathogen Legionella pneumophila uses a cooperative approach involving a series of secreted virulence proteins to acquire membrane from the endoplasmic reticulum [16]. This acquisition of membrane disguises the pathogen-containing vacuole as the host compartment, ultimately evading detection by the lysosomal pathway [17]. Many pathogens have evolved more direct means of targeting lysosomal function [10,18]. For example, the foodborne pathogen Vibrio parahaemolyticus uses the secreted virulence protein VepA to target the lysosomal V-ATPase and induce lysosome rupture [5].
Effective characterization of pathogen-mediated suppression of host lysosome function involves identifying the mechanism of host evasion and ultimately establishing the mechanism of action of the virulence factor responsible. This requires the development of robust cell biological assays for the study of these phenotypes. Among the most commonly proposed direct mechanisms of evasion is the suppression of trafficking of host lysosomes [10, 18]. Here, we describe a host–pathogen infection assay that can be used to investigate this phenotype [19]. This fluorescence-based microscopy assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA). This highly labelled BSA derivative exists as a self-quenched, non-fluorescent form (Figure 1A) and, upon treatment of host cells, is internalised by endocytosis. Upon trafficking to functional lysosomes, the acquisition of lysosomal proteases results in BSA cleavage, resulting in unquenching and bright-red fluorescence [20]. DQ-Red BSA is a substrate for a broad array of lysosome hydrolases and, therefore, is capable of detecting global lysosome function [21].
Figure 1. DQ-Red BSA assay for bacterial pathogen–mediated suppression of host trafficking. A. DQ-Red BSA dye mechanism. The fluorophores within the DQ-Red BSA dye exist in a quenched form and therefore do not generate intrinsic fluorescence. When host cells are treated with the dye, the compound is internalised by general endocytic mechanisms, and is trafficked to intermediates of the endocytic pathway. When dye-containing vesicles are trafficked to lysosomes, active lysosomal proteases cleave the BSA backbone, resulting in unquenching of the fluorophores and the generation of bright-red fluorescence. B. Host–pathogen infection assay to assess suppression of host lysosomal trafficking. Host cells are seeded in a microscopy chamber slide and infected with the bacterial pathogen of interest expressing green fluorescent protein (GFP). At the timepoint of interest, cells are subjected to a pulse-chase with DQ-Red BSA. Infected host cells are imaged by fluorescence microscopy, and suppression of host lysosome trafficking is quantified by imaging software.
The following summarises a method to quantitatively assess bacterial pathogen–mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model (Figure 1B). S. enterica is a Gram-negative pathogen that is among the leading causes of foodborne gastroenteritis worldwide [22]. As an intracellular vacuolar pathogen, S. Typhimurium evades targeting for death by lysosomes to establish a replicative niche within host cells [10]. S. Typhimurium was shown to suppress host trafficking to lysosomes (Figure 2) using this fluorescence microscopy–based imaging assay [19], which allows the visualization of trafficking to lysosomes in individual live-infected host cells alongside neighbouring non-infected host cell counterparts. This method was also used to identify the secreted virulence factor responsible for the phenotype, as well as domains within the bacterial protein that contribute to the phenotype [19]. The methods outlined highlight important considerations for adapting this method for the study of other host cell types and other bacterial pathogens, as well as for modifying the assay setup to tailor to the experimental question of interest.
Figure 2. Salmonella virulence factor–mediated suppression of host trafficking to lysosomes. Salmonella Typhimurium manipulates the host cytoskeleton to direct its uptake into host epithelial cells using early-secreted virulence proteins. Upon uptake, Salmonella-containing vacuoles (SCVs) begin to undergo interactions with early intermediates of the endocytic pathway. As a mechanism of evasion, Salmonella produces late-secreted virulence proteins to suppress trafficking to lysosomes and promote intracellular survival and replication.
Materials and reagents
Biological materials
HeLa (ATCC, catalog number: CCL-2)
Salmonella enterica serovar Typhimurium SL1344 harbouring pFPV25.1 [23]
Note: The pathogen of interest must be expressing a fluorescent protein that is not associated with red wavelengths (e.g., GFP) for detection purposes. This may be in the form of a shuttle plasmid such as pFPV25.1 (expressing GFP) or a strain harbouring a chromosomal insertion. Should this not be previously established for the pathogen of interest, broad host spectrum shuttle plasmids exist that might be worthy of investigation [24].
Reagents
Dulbecco modified Eagle medium (DMEM), 4.5 g/L glucose, with L-glutamine, sodium pyruvate, and phenol red) (Wisent, catalog number: 319-005-CL)
Fetal bovine serum (FBS) (Wisent, catalog number: 080-450)
LB Miller broth (BioShop, catalog number: LBL407)
Agar, bacteriological grade (BioShop, catalog number: AGR001)
Streptomycin sulfate (BioShop, catalog number: STP101)
Carbenicillin sodium salt (BioShop, catalog number: CAR544)
Phosphate-buffered saline with calcium and magnesium (PBS+/+) (Wisent, catalog number: 311-011-CL)
Gentamicin sulfate (BioShop, catalog number: GTA401)
DQ-Red BSA (Thermo Fisher, catalog number: D12051)
Phosphate-buffered saline without calcium and magnesium (PBS-/-) (Wisent, catalog number: 311-425-CL)
Roswell Park Memorial Institute 1640 (RPMI), L-glutamine & HEPES, no sodium bicarbonate (Wisent, catalog number: 350-025-CL)
Solutions
Streptomycin stock solution (see Recipes)
Carbenicillin stock solution (see Recipes)
LB-agar solution (for LB-agar plates) (see Recipes)
DQ-Red BSA solution (see Recipes)
Recipes
Streptomycin stock solution (1 mL)
Note: Dissolve powder in water and vortex well until powder dissolves. In a sterile area such as a Bunsen burner or Class II biosafety cabinet, filter sterilise the stock into a sterile tube.
Reagent Final concentration Quantity or Volume
Streptomycin sulfate 50 mg/mL 50 mg
Double-distilled water (ddH2O) n/a to 1 mL
Carbenicillin stock solution (1 mL)
Note: Dissolve powder in water and vortex well until powder dissolves. In a sterile area such as a Bunsen burner or Class II biosafety cabinet, filter sterilise stock into a sterile tube.
Reagent Final concentration Quantity or Volume
Carbenicillin sodium salt 100 mg/mL 100 mg
Double-distilled water (ddH2O) n/a to 1 mL
LB-agar solution (500 mL)
Note: Dissolve powder in water and mix well using a stir bar until powder dissolves as much as possible. Autoclave using standard settings for liquid laboratory media. Where supplementation with antibiotics is applicable, when the autoclaved media is cooled such that it is warm to the touch, add the appropriate concentration of antibiotic stock in a sterile area. After stirring on a stir plate, use a sterile area to pour a layer of agar-supplemented media in each Petri dish. Allow to solidify at room temperature and store solidified plates at 4 °C.
Reagent Final concentration Quantity or Volume
LB Miller broth (powder) 25 g/L 12.5 g
Agar 15 g/L 7.5 g
Double-distilled water (ddH2O) n/a to 500 mL
DQ-Red BSA stock solution (1 mL)
Note: Dissolve powder in PBS-/- and mix well by vortexing. If needed, sonicate to assist with solubilizing the reagent. Once solubilised, the reagent can be stored at 4 °C for several weeks. Note that DQ-Red BSA is light-sensitive. Store in a sample tube that is either covered in foil or opaque and, wherever possible, handle in the dark.
Reagent Final concentration Quantity or Volume
DQ-Red BSA 2 mg/mL 1 mg
PBS-/- n/a to 500 μL
Laboratory supplies
Glass-bottom microscopy chambers (ibidi, catalog number: 80827-90)
Centrifuge tubes, polypropylene, sterile (Corning, catalog number: C352098)
Serological pipettes, sterile (Sarstedt, catalog number: 86.1253.001)
Petri dishes for bacteriology, sterile (Sarstedt, catalog number: 82.1473.001)
Round-bottom culture tubes, sterile (Corning, catalog number: C352059)
Syringe, sterile (BD, catalog number: B309659)
Syringe filter, sterile (Millipore Sigma, catalog number: SLGP033RS)
Magnetic stir bar (Fisherbrand, catalog number: 800371113)
Equipment
CO2 incubator (Nuaire, model: NU-425-400)
Bacterial incubator (New Brunswick, model: Innova 42)
Quorum spinning disk fluorescence microscope (Leica, model: DMIRE2, Hamamatsu, model: CMOS FL-400)
Bunsen burner (EISCO, model: CH0095B) or Class II biosafety cabinet (Nuaire, model: NU-602-600)
Analytical balance (Mettler Toledo, model: ML304T)
Stir plate (Thermo Scientific, model: SP131525)
Autoclave
Vortex (VWR, model: G-560)
Microcentrifuge (Eppendorf, model: 5425)
Software and datasets
Fiji (ImageJ, v1.54f 29, June 2023) (free analysis software)
Procedure
Bacterial pathogen infection
The following method is used for the study of S. Typhimurium infection of host epithelial cells (Figure 1B). The protocol is adaptable for infection with diverse intracellular pathogens and a broad array of cell types. Highlighted within the protocol are important considerations for tailoring this assay to study different host cell types, pathogens, and applications. Information is also provided regarding commonly encountered challenges and suggested troubleshooting strategies.
Seed host cells in glass-bottom microscopy chambers. For comparison studies, such as identifying virulence factor(s) associated with the phenotype or domains within the virulence factor responsible for function (additional information in Table 1), one well is needed per strain. For analysis of S. Typhimurium infection of HeLa cells, seed μ-Slide 8-well glass-bottom chambers with HeLa cells at 6 × 104 cells/well using DMEM supplemented with 10% FBS as a culture medium.
Note: For comparison studies with isogenic knockout strains, the use of a complementation strain is required to rule out the possibility of polar effects. Complementation plasmids for these purposes have been established for diverse intracellular pathogens.
Table 1. Applications of DQ-Red BSA assay for the study of bacterial pathogenesis
Assay purpose Bacterial strains required Additional information and considerations
Validation of suppression of host lysosomal trafficking Wildtype
• Kinetics of trafficking suppression may vary by strain and host cell line; if the timepoint of interest has not been established, a time-course analysis may be needed
• Seeding: one well per timepoint of interest
Virulence factor identification Wildtype, isogenic knockout(s), complementation strain(s)
• If needed, assay can be scaled down to a smaller format to be amenable for larger scale screen of knockouts
• Once a candidate virulence factor is identified, the experiment must be repeated in comparison to a complementation strain
• Complementation: all strains must harbour the same complementation base plasmid, either with or without the virulence factor gene of interest
Virulence factor domain mapping Wildtype, isogenic knockout, complementation strains harbouring wildtype and truncation mutants
• All strains must harbour the same complementation base plasmid, either with or without the virulence factor gene of interest
• For virulence factors with limited information, in silico tools such as Phyre2 [26] may provide valuable insight to guide hypotheses
• If the virulence factor has known or predicted catalytic sites or functional residues, point mutations can also be assessed in parallel
Grow host cells in a CO2 incubator until the optimal confluency for infection of the pathogen of interest. For S. Typhimurium infection of epithelial cells, incubate overnight until host cells are at a confluency of 70%–80%.
Concurrently, prepare the pathogen inoculum so that it is ready for infection when the host cells are at the optimal level of confluency for infection. For S. Typhimurium SL1344 harbouring pFPV25.1, this requires setting up an overnight culture on the day of seeding host cells. Inoculate a sterile bacterial culture tube with 2 mL of LB supplemented with 50 μg/mL streptomycin (for selection of the strain) and 100 μg/mL carbenicillin (for selection of pFPV25.1) with several single colonies grown on LB-agar supplemented with 50 μg/mL streptomycin and 100 μg/mL carbenicillin. Incubate for 16 h at 37 °C and 250 rpm.
On the day of infection, prepare the pathogen inoculum as required prior to infection. For S. Typhimurium SL1344 infection of epithelial cells, in a sterile 125 mL flask containing 10 mL of LB, subculture from the overnight culture (1:33) [25] and incubate for 3 h at 37 °C and 250 rpm. Prepare inocula by pelleting 1 mL of the culture at 10,000× g for 2 min, resuspending the pellet in PBS+/+ to 1 mL, and diluting in PBS+/+ to the appropriate multiplicity of infection. For S. Typhimurium SL1344, 1:100 is suggested.
Note: The goal of this experimental setup should be to have host cells infected with the intracellular pathogen such that, at the time of microscopy analysis, primary infected cell(s) are in the same field of view with neighbouring non-infected host cells. The multiplicity of infection and host cell confluency can be adjusted during the optimization process to achieve this.
Aspirate culture medium from host cells and wash with 200 μL of PBS+/+. Apply bacterial inoculum as previously established for the pathogen of interest. A volume of 200 μL is suggested. For S. Typhimurium infection of epithelial cells, incubate infected host cells in CO2 incubator at 37 °C for 10 min.
After the appropriate amount of time previously established for the pathogen, aspirate the bacterial inoculum from the host cells and wash three times with PBS+/+. Apply growth medium appropriate for the host cell type of interest and incubate in CO2 incubator.
Note: Prewarm all culture media used in this experiment to 37 °C. This step is important to allow completion of bacterial uptake such that the pathogen-containing vacuole has completed fission from the host plasma membrane. Proceeding to Step A7 too early could result in bacterial killing and therefore little or no infected host cells.
At the appropriate timepoint for the pathogen, change the culture media to growth medium supplemented with antibiotic for the selection of intracellular bacteria. For S. Typhimurium SL1344 infection of epithelial cells, change the media at 30 min post-infection (p.i.) to DMEM supplemented with 10% FBS and 100 μg/mL gentamicin [19]. At 2 h p.i., change the media to DMEM supplemented with 10% FBS and 10 μg/mL gentamicin for maintenance purposes.
Note: Selection for intracellular bacteria is essential to ensure that the phenotype of interest is not due to factors produced by extracellular bacteria. The antibiotic of interest is chosen based on previous knowledge about antibiotic internalization into host cells. For example, gentamicin is commonly used because of its limited penetration into a wide range of host cells, allowing selection for intracellular bacteria [27]. The sensitivity profile of the pathogen strain of interest, as well as previously established acceptable antibiotic concentration ranges for host cell treatment, will affect the concentration selection. With respect to pathogen sensitivity, this information has been previously established in the literature for many intracellular pathogens. If unknown for the pathogen of interest, a minimum inhibitory concentration assay can be performed.
At the timepoint of infection where the suppression of host trafficking to lysosomes occurs, change media to the culture medium used at the end of Step A7 supplemented with DQ-Red BSA. For S. Typhimurium infection of HeLa cells, perform this step at 8 h p.i. using DMEM supplemented with 10% FBS, 10 μg/mL gentamicin, and 0.25 mg/mL DQ-Red BSA [19].
Note: The infection timepoint associated with the phenotype will most often be determined based on previous literature for the pathogen. Should the pathogen be poorly understood in this context, a series of timepoints can be investigated to determine this. The concentration of DQ-Red BSA for treatment will vary depending on the cell line used and its ability to uptake dye. For example, for phagocytic RAW 264.7 cells, a concentration of 10 μg/mL may be used [19]. This is often previously established in the literature but may be optimised experimentally if needed.
Incubate in CO2 incubator for 1 h. This treatment, with culture medium supplemented with dye, is called the pulse step.
Note: The pulse period can be adjusted if 1 h proves to be ineffective for the cell type of interest.
Wash infected host cells with PBS+/+ and change media to the culture medium used at the end of Step A7. Incubate in CO2 incubator for 4 h. This incubation in the absence of dye to allow for trafficking to lysosomes is called the chase step.
Note: This chase period will allow the DQ-Red BSA to traffic to lysosomes for detection. The time period associated with this step may vary depending on the cell line. Should the pathogen of interest function to block trafficking to lysosomes, DQ-Red BSA trafficking should occur at a reduced level.
Wash infected host cells with PBS+/+ and change medium to HEPES-buffered RPMI 1640 or an equivalent culture medium with HEPES buffer, supplemented with 10% FBS and the antibiotic used for intracellular selection (10 μg/mL gentamicin for S. Typhimurium). The infected sample is ready for live imaging by fluorescence microscopy.
Note: The HEPES-buffered media is used to allow live-cell imaging on a routine microscope. Should the microscope of interest be outfitted with a CO2-infused system, a culture medium with sodium bicarbonate may be used.
Fluorescence microscopy imaging and image quantification
On a fluorescence microscope with a 40× or 63× objective, examine each chamber for primary infected cells in the channel associated with the fluorescent protein expressed. For S. Typhimurium SL1344 expressing pFPV25.1, use the green channel. For each infected host cell identified, examine the field of view for an adjacent non-infected cell. Image each field of view, capturing the channel to image the bacterially expressed fluorescent protein and DQ-Red BSA (Figure 3). Host cells can also be captured in the differential interference contrast (DIC) channel to assist with identifying cell boundaries.
Figure 3. DQ-Red BSA assay: Salmonella-infected epithelial cells. HeLa cells were infected with wildtype Salmonella enterica serovar Typhimurium SL1344 harbouring the GFP-expressing plasmid pFPV25.1 (green) for 8 h. Cells were subjected to a pulse-chase with DQ-Red BSA (red) and subsequently imaged live. The scale bar represents 10 μm.
Continue imaging as described in Step B1. For each strain of interest, it is ideal to capture images of at least 100 infected cells with adjacent non-infected counterparts.
Note: If the microscope is outfitted with a platform capable of automated imaging, capture as many fields of view as required to fulfil the criteria described above.
Analysis can be performed using the commonly available software Fiji (ImageJ). Open imaging file of interest in the software (Figure 4A).
Note: An equivalent analysis can be performed using other microscopy analysis software, such as Volocity.
For each primary infected cell, assess fluorescence intensity per area in the DQ-Red BSA channel:
Open the image of interest. To assist with assessment, use the Composite setting to allow all channels to be visualised as an overlay.
Open the Channels Tool (Image → Color → Channels Tool) to allow for the turning on and off of individual channels.
To select which measurements will be performed, go to Analyze → Set Measurements. Select Area and Mean Value.
Open ROI Manager by selecting Analyze → Tools → ROI Manager.
As the DQ-Red BSA channel will be measured, use the slider at the bottom of the image to select the corresponding channel.
Select the Freehand Tool (Figure 4B).
Figure 4. Fiji analysis of DQ-Red BSA assay: Salmonella-infected epithelial cells. (A) Cells should be imaged such that each field of view contains an infected cell of interest and a neighbouring non-infected cell. Visualization in the Composite mode allows for the viewing of both bacterial (green) and DQ-Red BSA (red) channels simultaneously. (B) DQ-Red BSA quantification is performed in Fiji. The Freehand Tool is used to outline both infected and non-infected cells as regions of interest (ROIs) prior to measurement using ROI Manager.
Draw a region of interest corresponding to the cell boundary of the infected host cell.
In ROI Manager, add the newly selected ROI by clicking Add.
Draw a region of interest corresponding to the cell boundary of the neighbouring non-infected host cell.
In ROI Manager, add the newly selected ROI by clicking Add.
To take measurements, in ROI Manager, select Measure. A Results window will appear with the corresponding measurements for the two ROIs. These values can be copied and pasted into Excel for subsequent analysis.
Repeat steps B4g–k for all images of interest.
Note: Should the host cell type demonstrate abnormal cells that are characteristically smaller and/or larger than normal cells, a size threshold range can be predetermined such that abnormal cells are excluded from analysis. Also, if needed, a background correction can be performed.
Data analysis
For each primary infected cell and its neighbouring non-infected host cell counterpart, individually calculate the associated intensity/µm2 of the DQ-Red BSA channel by dividing the total intensity measured for the ROI (mean value) by the area of the ROI (area).
For each primary infected cell and its neighbouring non-infected host cell counterpart, calculate the intensity/µm2 of the infected cell as a percentage of the non-infected comparison. This can be accomplished by dividing the intensity/µm2 of the infected cell by the intensity/µm2 of the non-infected cell and multiplying by a factor of 100.
Repeat steps 1 and 2 for each primary infected cell and neighbouring non-infected cell set. A total of 100 data points is ideal.
For each strain or condition of interest, calculate the mean of the 100 data points. For comparison studies of wild type with isogenic knockout strains and complementation strain(s), mean DQ-Red BSA signal intensity/µm2, presented as a percentage of control (non-infected cells), can be compared by bar graph.
Note: Should the data indicate a statistically significant difference with a new pathogen, or should a virulence factor be implicated, a supplementary analysis is needed to rule out the possibility of a defect in dye internalization. This can be performed as a separate independent assay or in combination with DQ-Red BSA using a secondary dye (e.g., dextran Alexa Fluor 647) [19]. Also, it would be valuable to consider validating that DQ-Red BSA dye uptake in non-infected cells is equivalent to that of uninfected cells. To assess this, a comparison of uninfected conditions can be performed for subsequent analysis.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
D’Costa et al. [19]. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7. Cell Reports (Figure 1, panels A–D; Figure 3, panels C–D; Figure 5, panel B; Supplemental Figure 1, panels A–C; Supplemental Figure 2).
General notes and troubleshooting
Troubleshooting
Problem 1: Poor infection efficiency.
Possible cause: Suboptimal host cell conditions.
Solution: Host cell confluency can affect bacterial uptake. If this is suspected, a confluency assessment can be performed by seeding at several host cell densities.
Problem 2: Poor infection efficiency.
Possible cause: Suboptimal bacterial inoculum.
Solution: A colony-forming unit analysis of the inoculum can be performed to confirm that the bacterial growth conditions resulted in viable bacteria. If applicable, it may also be possible that virulence factor production to induce uptake was suboptimal. As virulence factor production in pathogens such as Salmonella can be growth phase–dependent, a growth curve analysis can be performed to assess this.
Problem 3: No intracellular bacteria.
Possible cause: Ineffective intracellular protection conditions.
Solution: If intracellular protection is performed before pathogen uptake into intact pathogen-containing vacuoles is complete, the antibiotic is accessible to the bacteria, which will subsequently be targeted for death. The time period prior to antibiotic treatment can be increased if needed.
Problem 4: Extracellular bacteria observed.
Possible cause: Ineffective intracellular protection conditions.
Solution: Perform a minimum inhibitory concentration assay with the strain of interest to confirm its sensitivity to the antibiotic of interest. This may indicate that a slight adjustment in the drug concentrations used in the assay may be needed. If the strain is resistant to the antibiotic of preference (e.g., gentamicin), an alternate drug class may be considered (e.g., polymyxins).
Problem 5: DQ-Red BSA signal is too low.
Possible cause: Low sensitivity microscopy system.
Solution: Increase light source intensity or exposure length.
Problem 6: DQ-Red BSA signal is too low.
Possible cause: Suboptimal dye uptake.
Solution: The pulse treatment conditions may need optimization, depending on the intrinsic uptake properties of the host cell line. If needed, consider increasing the dye concentration or uptake period.
Acknowledgments
This protocol was adapted from a previously published study [19]. We thank members of the D’Costa Lab for helpful discussions. This work was funded by Operating Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (PJ4-175369 and PJT-178191) to V.M.D.
Competing interests
The authors have no conflicts of interest or competing interests to declare.
Ethical considerations
There are no ethical considerations relevant to this paper.
References
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Tuli, A. and Sharma, M. (2019). How to do business with lysosomes: Salmonella leads the way. Curr. Opin. Microbiol. 47: 1–7.
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An Optimized P. berghei Liver Stage–HepG2 Infection Model for Simultaneous Quantitative Bioimaging of Host and Parasite Nascent Proteomes
JM James L. McLellan
AG Andreu Garcia-Vilanova
KH Kirsten K. Hanson
Published: Vol 14, Iss 5, Mar 5, 2024
DOI: 10.21769/BioProtoc.4952 Views: 424
Reviewed by: Clara Morral MartinezRaniki Kumari Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in mSphere Nov 2023
Abstract
The Plasmodium parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the P. berghei-HepG2 infection model, followed by automated confocal image acquisition and computational separation of P. berghei vs. H. sapiens nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis.
Key features
• Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in Plasmodium berghei liver-stage parasites in actively translating hepatoma cells.
• This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells.
• The P. berghei–infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification.
Graphical overview
Keywords: Plasmodium berghei Liver stage Translation Automated confocal feedback microscopy HepG2 cell culture Quantitative bioimaging Protein synthesis Antimalarial drug discovery
Background
Plasmodium parasites, the causative agents of malaria, have a complex lifecycle spanning both mosquito and human hosts. The first obligate step for parasite development in humans occurs in the liver following the introduction of motile parasites forms, called sporozoites, via an anopheline mosquito bite. Here, the liver-stage parasites, or exoerythrocytic forms (EEFs), undergo an extensive growth and replication process entirely within a parasitophorous vacuole in the cytoplasm of a hepatocyte; a single sporozoite will result in the formation of thousands of hepatic merozoites, which initiate the blood stage of infection [1]. While the continuous asexual replication cycles of blood-stage parasites can give rise to the signs and symptoms of malaria, the liver stage is asymptomatic and a key target for both vaccination strategies and chemoprophylaxis. The most widely used model species for studying the Plasmodium liver stage is P. berghei, a rodent malaria parasite that is capable of invading and growing in a variety of cell lines, including human hepatoma cell lines, during its liver-stage development [2,3]. Several immortalized cell lines are in regular use today, including HepG2 (human hepatoma), Huh-7 (human hepatoma), and HeLa (human cervical adenocarcinoma). Hepatocytes are highly specialized, polarized epithelial cells making up the liver parenchyma, and HepG2 are unique amongst the cell lines used for P. berghei liver-stage infections in their substantial maintenance of hepatic polarity, thus providing the closest in vitro cellular environment to that of the native host hepatocytes within a mammalian liver [4,5]. Hepatocytes in the liver are arranged in chords within hexagonal lobules in an architecture where each hepatocyte has direct basal surface contact with blood flowing through the fenestrated sinusoidal endothelium, while its multiple apical surfaces form the lumina into which bile is secreted at lateral points of contact with other hepatocytes [4,5]. The regenerative capacity of the liver relies on the ability of quiescent hepatocytes to reenter the cell cycle and divide, and the organizing principle that keeps the architecture of the chords intact relies on precise alignment of the daughter cells along the sinusoid [6]. These structural and functional particularities of the native hepatocyte environment have consequences for the in vitro growth of polarized HepG2 cells, which have a reputation for forming 3-dimensional layers or clumps in the absence of the liver architecture, which can hinder quantitative bioimaging in the P. berghei–HepG2 infection model. Careful attention to HepG2 culture and passage conditions can minimize this, allowing quantitative bioimaging of both host and parasite characteristics like translational output, which we present here.
Core cellular processes are somewhat difficult to study in Plasmodium liver stages. Protocols for routine isolation of EEFs have not been reported, so signals from the parasite must be separated from those of both infected and uninfected hepatocytes in the culture. While quantitative translation assays exist for Plasmodium blood-stage parasites [7–9], to our knowledge this is the first protocol for direct quantification of liver-stage translation in single cells and populations in the P. berghei–HepG2 infection model [10,11]. This assay relies on the use of the modified puromycin analogue, o-propargyl puromycin (OPP) [12], to label the HepG2 and P. berghei nascent proteomes during a 30 min window, followed by fixation, click chemistry–based attachment of a fluorophore to the OPP-labeled nascent polypeptides, and immunolabeling of the parasites, followed by confocal imaging, batch image segmentation and feature extraction to computationally separate HepG2 and parasite signals, and data analysis.
Materials and reagents
Biological materials
Plasmodium berghei ANKA (676 m1cl1)–infected Anopheles stephensi mosquitos (University of Georgia SporoCore)
HepG2 human hepatoma cell line (ATCC STR profiling-verified)
Reagents
DMEM (Gibco, catalog number: 10313-021)
Pen Strep (Gibco, catalog number: 15140-122)
Heat inactivated fetal bovine serum (FBS) (Gibco, catalog number: 16140-071)
GlutaMAX (Gibco, catalog number: 35050-061)
TrypLE Express (Gibco, catalog number: 12605-028)
Dulbecco’s PBS (DPBS) (Sigma, catalog number: D8537)
PBS (Sigma, catalog number: P3813)
Triton X-100 (Sigma, catalog number: T9284)
Paraformaldehyde (PFA) solution, 4% in PBS (Thermo Scientific, catalog number: 30525-89-4)
Penicillin Streptomycin Neomycin (PSN) (100×) (Gibco, catalog number:15640-055)
Kanamycin sulfate (Corning, catalog number: 30-006-CF)
Amphotericin B (Gibco, catalog number: 15290-026)
Gentamycin (Gibco, catalog number: 15750-060)
DMSO (Sigma, catalog number: D2650)
Anisomycin (EMD Millipore-Sigma, catalog number: 176880)
O-propargyl-puromycin (OPP) (Invitrogen, catalog number: 10459)
O-propargyl-puromycin (OPP) [Vector labs (formerly Click Chemistry Tools), catalog number: CCT-1407-5]
Click-iT® Plus Alexa Fluor® 555 picolyl azide toolkit (Invitrogen, catalog number: C10642)
Click-&-Go® Plus 555 Imaging kit (Vector labs, catalog number: CCT-1317)
Bovine serum albumin (BSA) (Fisher Bioreagents, catalog number: 9048-46-8)
Mouse anti-PbHsp70 primary antibody [13]
Donkey anti-mouse Alexa Fluor® 488 (Invitrogen, catalog number: A21202)
Hoechst 33342 (10 mg/mL) (Thermo Scientific, catalog number: 62249)
70% ethanol
Laboratory supplies
25 cm2 cell culture flask (Corning, catalog number: 430168)
75 cm2 cell culture flask (Corning, catalog number: 430720U)
Cell strainer, 40 μm nylon (Corning, catalog number: 431750)
Cell strainer, 100 µm nylon (Corning, catalog number: 431752)
0.22 µm filters (Millex, catalog number: SLGV004SL)
0.20 µm filters (Corning, catalog number: 431229)
1 mL syringes (BD, catalog number: 309659)
10 mL syringes (BD, catalog number: 302995)
1 mL syringes with hypodermic needle (BD, catalog number: 309626)
50 mL conical tubes (Corning, catalog number: 430828)
24-well cell culture plate (Corning, catalog number: 3524)
12 mm round coverslips, 1.5 thickness (Azer, catalog number: ES0117520)
Fluoromount-G (SouthernBiotech, catalog number: 0100-01)
96-well µClear cell culture plate (Greiner bio-one, catalog number: 655098)
96-well round bottom plate (Corning, catalog number: 3799)
5 mL serological pipettes (Corning, catalog number: 4487)
10 mL serological pipettes (Corning, catalog number: 4488)
25 mL serological pipettes (Corning, catalog number: 4489)
Reagent reservoirs (Eppendorf, catalog number: 022265806)
Live insect forceps (FST, catalog number: 26029-10)
Dumont #5/45 coverslip forceps (FST, catalog number: 11251-33)
6-well plate (Corning, catalog number: 351146)
Neubauer chamber (Brand GMBH, catalog number: 717805)
Pestle (Fisherbrand, catalog number: 12-141-363)
Multichannel racked pipette tips, 1,200 µL (Rainin, catalog number: 17002921)
Multichannel racked pipette tips, 300 µL (Rainin, catalog number: 30389255)
P1000 barrier pipette tips (Thermo Scientific, catalog number: 2079)
Microscope slides 75 × 25 × 1 mm (VWR, catalog number: 16004-368)
1.7 mL snap top tubes (Axygen, catalog number: MCT-175-A)
Kimwipes
Paper towels
Ice bucket
Solutions
Complete DMEM (cDMEM) (see Recipes)
Infection DMEM (iDMEM) (see Recipes)
DMSO control treatment (see Recipes)
Anisomycin control treatment (see Recipes)
0.5% Triton-X (see Recipes)
Blocking solution (see Recipes)
Click-iT® Plus Master Mix (see Recipes)
1° (primary) antibody solution (see Recipes)
2° (secondary) antibody solution (see Recipes)
Recipes
Complete DMEM (cDMEM)
Reagent Final concentration Quantity or Volume
DMEM 88% (v/v) 440 mL
FBS
Pen Strep
GlutaMAX
10% (v/v)
1% (v/v)
1% (v/v)
50 mL
5 mL
5 mL
Total n/a 500 mL
Infection DMEM (iDMEM)
Reagent Final concentration Quantity or Volume
cDMEM 97.5% (v/v) 97.5 mL
PSN
Kanamycin sulfate
Amphotericin B
Gentamycin
1% (v/v)
1% (v/v)
0.33% (v/v)
0.1% (v/v)
1 mL
1 mL
334 µL
100 µL
Total n/a 100 mL
DMSO control treatment
Reagent Final concentration Quantity or Volume
iDMEM n/a 1,998 µL
DMSO 0.1% (v/v) 2 µL
Total n/a 2,000 µL
Anisomycin control treatment
Reagent Final concentration Quantity or Volume
iDMEM n/a 1,998 µL
10 mM stock of anisomycin 0.1% (v/v) 2 µL
Total n/a 2,000 µL
0.5% Triton-X
Reagent Final concentration Quantity or Volume
PBS n/a 4,750 µL
10% Triton X-100 0.5% (v/v) 250 µL
Total (optional) n/a 5 mL
Blocking solution (2% BSA in PBS)
Reagent Final concentration Quantity or Volume
BSA 2% (w/v) 1 g
PBS n/a 50 mL
Total n/a 50 mL
Click iT® Plus Master Mix
Note: All reagents should be prepared and stored in accordance with manufacturer’s specifications. Components should be brought to room temperature before making master mix. Each master mix should be prepared fresh and used within 15 min. Each component of the master mix should be added in the order listed. The CuSO4 and copper protectant mix should be combined separately before addition to the master mix; these components are stored separately to allow you to change the concentration of copper catalyst in the reaction master mix by adjusting the ratio of CuSO4 to copper protectant buffer. We have found that a 1:4 ratio of copper–copper protectant provides nearly identical signal compared to those of higher ratios. The total volume reported here is designed for a 96-well plate (32 wells at 27 µL each) with ample dead volume.
Reagent Final concentration Quantity or Volume
Molecular grade H2O 78.3% (v/v) 783 µL
10× Click-iT® reaction buffer
500 µM Alexa Fluor® PCA solution
CuSO4-copper protectant mix
1×x Click-iT® buffer additive
8.7% (v/v)
1% (v/v)
2% (v/v)
10% (v/v)
87 µL
10 µL
20 µL
100 µL
Total (optional) n/a 1,000 µL
1° antibody solution
Note: Anti-PbHSP70 working dilution will depend on antibody concentration; the dilution given is for an unpurified batch produced from the 2E6 hybridoma in the Hanson lab. Antibody solutions are filtered using 0.20–0.22 µm filters before use.
Reagent Final concentration Quantity or Volume
Blocking solution n/a 1,592 µL
Mouse anti-PbHSP70 1:200 (v/v) 8 µL
Total (optional) n/a 1,600 µL
2° antibody solution
Note: Antibody solutions are filtered using 0.20–0.22 µm filters before use.
Reagent Final concentration Quantity or Volume
Blocking solution n/a 1595.2 µL
Donkey anti-mouse Alexa Fluor® 488
Hoechst 33342 (10mg/mL)
1:500 (v/v)
1:1,000 (v/v)
3.2 µL
1.6 µL
Total (optional) n/a 1,600 µL
Equipment
SP8 confocal microscope (Leica, model: TCS SP8)
Dissecting stereoscope (Leica, model: S4E)
Inverted phase contrast microscope (Zeiss, model: Invertoskop 40 C)
Inverted fluorescence microscope (Leica, model: DM IL LED)
Fluorescence light source (Leica, model: EL6000)
Water bath (Thermo, model: 280 series)
CO2 incubator (Thermo, model: Napco Series 8000 WJ)
Biological safety cabinet (LABGARD, model: Class II, Type A2)
Centrifuge with swinging bucket rotor (Thermo, model: Sorval ST 40R, TX-750 rotor)
Titanium liquid removal chambers for swinging bucket rotor centrifuge (custom fabrication by Autotiv)
Small bench centrifuge (Eppendorf, model: 5415D)
Multichannel pipette 300 µL LTS (Rainin, model: E4 XLS)
Multichannel pipette 1200 µL LTS (Rainin, model: E4 XLS)
Software and datasets
LAS AF 3 (Leica)
MatrixScreener (HCS A Developer Full with CAM Leica)
CellProfiler (v2.1.1 rev6c2d896, 7/25/2014), used for image segmentation and feature extraction
CellProfiler (v2.0.11710, 01/24/2014), used for online image segmentation and parasite ID during automated confocal feedback microscopy (ACFM)
KNIME version 4.7.7 and earlier v4 releases (01/20/2022), used for data analysis
Procedure
HepG2 cell culture optimized for imaging
Note: All steps involving live HepG2 cells are performed in a biosafety cabinet.
Prepare cDMEM (Recipe 1).
Ensure cDMEM, DPBS, and TrypLE are at room temperature (RT).
Remove the T75 cell culture flask containing HepG2 cells from the 5% CO2, 37 ºC incubator, decant medium from the flask, and gently wash cell monolayer two times with 10 mL of DPBS.
Critical: These DPBS washes are important for the removal of residual cDMEM, which can hinder the breakdown of cell–cell junctions in the TrypLE digestion step.
Decant the second DPBS wash and add 7.5 mL of room-temperature TrypLE. Return flask to the 5% CO2, 37 ºC incubator for 10 min.
Critical: Do not disturb the flask during incubation and do not attempt to disrupt the HepG2 monolayer by tapping the flask. It is far easier to disrupt the cell-substrate attachment than to break down the cell–cell junctions, and the latter is crucial for maintaining the cells in a monolayer.
Remove the flask and add 7.5 mL of cDMEM to the TrypLE using a serological pipette, repeatedly washing the cell monolayer using the shearing forces of the dispensed liquid to dislodge the attached cells and break up any connected clumps or groups of cells.
After all cells have been removed from the plastic substrate, aspirate the cell suspension using a serological pipette and dispense through a sterile Ø40 µm filter into a 50 mL conical tube.
Critical: Filtering removes any large clumps or sheets of cells that could interfere with monolayer formation in continuous culture or when seeding cells for infection.
Centrifuge the filtered cell suspension at 315× g (1,200 rpm) for 5 min.
Discard supernatant and resuspend the pellet with 900 µL of cDMEM using p1000 with a filtered tip (>30 up-down cycles).
Add 9.1 mL of cDMEM to resuspended cells and invert several times to thoroughly mix cell suspension.
Prepare a 1:5 dilution of cell suspension by removing 10 µL of cell suspension and adding it to 40 µL of cDMEM in a snap top tube.
Place the bulk cell suspension at 4 °C while counting the cell suspension dilution.
Dispense 10 µL of the 1:5 dilution of the cell suspension into a Neubauer chamber and count all four outer quadrants (see illustration). Using the average of these four quadrants, calculate the concentration of cells in suspension using the formula shown in figure 1.
Figure 1. Counting chamber example formula. Illustration of counting chamber showing four quadrants used for counting with an explanation for calculating the cells/µL and the total number of cells in suspension.
Prepare cell suspensions at the desired seeding concentration for continued propagation of cells (see Table 1).
Table 1. Schedule for HepG2 cell passage. We always seed a backup flask during each cell passage in case of delayed mosquito arrival, contamination of the primary flask during passage, etc. We maintain each backup flask in the incubator through two passages of the main flask. We use HepG2 cells in assays from p2 through p15. M = million.
Monday Tuesday Wednesday Thursday Friday
T-75 primary flask Seed 2.4 m cells for Thursday split Seed 1.8 m cells for Monday split
T-25 backup flask 0.8 m cells 0.6 m cells
With the remaining cell suspension, proceed to section B.
Seeding HepG2 cells for infection
Note: This protocol details the quantification of liver-stage translation and small molecule–induced translation inhibition in P. berghei–infected HepG2 cells in a 96-well plate format, but this can also be performed in a coverslip format. See general note 3 for details.
Calculate the number of cells required for experiments and prepare an appropriately diluted seeding solution in cDMEM. To seed a 24-well plate with 150,000 HepG2 per well in a volume of 500 µL [(24 wells + 1 well dead volume) × 150,000 cells] = 3,750,000 cells to be suspended in (25 wells × 500 µL) = 12.5 mL of seeding suspension.
Note: HepG2 cells should never be a limiting reagent. Since sporozoite yield cannot be fully predicted in advance of the dissection, we always seed more HepG2 wells than we predict we will need.
Invert tube containing the cell suspension several times to ensure it is properly mixed.
Dispense 500 µL of cell suspension to each well of the 24-well plate.
Carefully redistribute cells within each well by tracing the figure ∞ 10–13 times, rotate the plate 180°, and trace the figure ∞ an additional 10–13 times.
Critical: An uneven cell distribution can affect invasion success and hinder imaging.
Note: Inspect the cell distribution of several wells using a microscope at a low magnification. You can get a good idea of the cell distribution by adjusting the focal plane and moving through the well contents in the Z-axis. If the cells are not evenly distributed upon visual inspection, repeat figure ∞ steps again.
Return plates to the incubator for 20–24 h before the anticipated time of infection the next day.
P. berghei sporozoite infection of HepG2 cells
Note: Up until the point of HepG2 infection, it is crucial to keep sporozoites on ice whenever a solution is not being directly handled. As before, all steps involving live HepG2 cells are performed in a biosafety cabinet. For more information regarding mosquito dissection and sporozoite isolation, see general note 1.
Visually inspect the cells seeded the previous day to ensure that they are adherent and growing normally and that no wells are contaminated.
Prepare iDMEM (Recipe 2).
Collect the mosquitos that are to be dissected into a 50 mL conical tube, cold-anesthetize them for 5 min at -20 °C, and then remove and place the tube of mosquitos horizontally on ice in a benchtop ice bucket.
Critical: Prolonged exposure to -20 °C or exposure to lower temperatures can affect sporozoite viability.
Using insect forceps, carefully transfer ~50 mosquitos into a Ø100 µm cell strainer. Using a 6-well plate as a wash station, immerse them into a well containing 70% ethanol (5–6 s), blot excess ethanol on a paper towel, then rinse in HBSS by immersion into two successive wells.
Note: This washing procedure helps to reduce contamination by the mosquito-surface microbiome.
Transfer the mosquitos into a large drop of DMEM on a glass microscope slide and immobilize a mosquito by gently pressing down on the thorax with the blunt side of a hypodermic needle using the non-dominant hand. Using another needle in the dominant hand, gently pull the mosquito head (with salivary glands attached) away from the thorax and cleave the salivary glands from the head with the sharp edge of the needle.
Note: If the salivary glands do not separate with the head, applying gentle pressure to the thorax will allow gland removal from the opening of the thorax. See general note 1 for additional resources and instructional guides for mosquito dissection and anatomy.
Transfer each set of glands to a 1.6 mL snap top tube containing 100 µL of DMEM, which is kept on ice.
Note: Cold anesthetized mosquitos and salivary glands should be kept on ice throughout the dissection procedure, and total time spent dissecting should be minimized to ensure sporozoite viability.
Release sporozoites by grinding the isolated salivary glands for 15–30 s using a sterile pestle and wash the walls of the tube and the pestle with additional DMEM, keeping the total volume of DMEM below 1 mL.
Note: The mosquito salivary glands must be disrupted to release sporozoites into the media. If this step is not done properly, the sporozoites will remain in the salivary glands and will be removed by filtering in the next step. For additional resources and alternative methods for releasing sporozoites from the salivary gland see general note 1.
Pass sporozoite material through a Ø100 µm cell strainer into a 50 mL conical tube. Using a micropipette, very gently recover any remaining medium from the bottom of the strainer and add it to the collection.
Centrifuge the sporozoite solution at 100× g for 30 s to ensure that all liquid is collected and transfer it into a 1.6 mL snap top tube.
Prepare a 1:5 counting dilution, mixing the sporozoite solution vigorously by manually flicking the tube; then, remove 10 µL of sporozoite solution and add it to 40 µL of DMEM. Mix this solution by vigorous pipetting using the same tip that transferred the sporozoites. Then, transfer 10 µL of this solution into a Neubauer chamber, place the chamber into a humidified, foil-covered dish, and allow the sporozoites to settle for 8 min; then, count the sporozoites as in Figure 1.
Note: If the sporozoite count in a single 4 × 4 counting grid exceeds 100 sporozoites, it is preferable to further dilute the counting dilution and then repeat the steps here and recount (e.g., adding 40 µL of medium to the remaining 40 µL of counting solution would yield a 1:10 dilution).
Based on the total number of sporozoites recovered, calculate the number of wells that can be infected with 100,000 sporozoites per well.
Dilute concentrated sporozoites into iDMEM to infect each well using a total volume of 300 µL. Assuming a recovery of 1 million sporozoites in a total volume of 800 µL, 10 wells could be infected. To reach a final volume of 3 mL, 800 µL containing 1 × 106 sporozoites would be added to 2.2 mL of iDMEM.
Remove the HepG2 cell plate from the incubator, carefully aspirate the cDMEM from each well, and replace with 300 µL of the sporozoite suspension prepared in the previous step.
Centrifuge plate at 2,000× g (3,000 rpm) for 5 min and return the plate to the 5% CO2, 37 °C incubator for 2 h.
Note: Record the exact time when the plates are returned to the incubator, as this will correspond to t0, and future steps will take place at a set number of hours post-infection (hpi).
At 2 hpi, remove the plate from the incubator, extract the iDMEM from each well using a p1000 filter-tipped micropipette, and add 1,000 µL of room-temperature DPBS.
Remove dPBS from each well and replace it with 200 µL of TrypLE. Return to the 5% CO2, 37 °C incubator for 10 min.
Transfer plate from incubator to sterile hood and add 200 µL of fresh iDMEM to each well containing TrypLE.
Using a filter-tipped P1000 with volume set to 300 µL, repeatedly pipette up and down across the entire bottom of the well, using fluid force to dislodge the infected HepG2 (iHepG2) from the substrate into a single-cell suspension. Then, transfer the entire 400 µL into a 50 mL collection tube and immediately add 200 µL of iDMEM to the well.
Once all wells have been processed, inspect the plate well by well to ensure that all iHepG2 from the monolayer were successfully harvested.
Critical: Once exposed to the sporozoite solution, HepG2 are even stickier than normal, and an inexperienced investigator may find that many cells remain in each well. If so, repeat well washing steps to detach cells by fluid force with a micropipette and add to the collection tube.
Using a serological pipette, transfer the suspended iHepG2 through a Ø40 µm strainer into a 50 mL conical tube to remove any large cell clumps and mosquito debris.
Centrifuge at 315× g (1,200 RPM) for 5 min.
Discard supernatant and resuspend the pellet with 900 µL of iDMEM using a p1000 with a filtered tip (at least 50 up and down cycles, as infected HepG2 are harder to separate compared with uninfected cells).
Add iDMEM to a final volume equal to 1 mL × the number of wells trypsinized; so, with 10 wells harvested, add 9.1 mL.
Remove a small sample of infected cell suspension for counting and place the bulk cell suspension at 4 °C while counting.
Dispense 10 µL of the undiluted infected cell suspension into a Neubauer chamber and count infected cells as previously described (Figure 1).
Based on the number of iHepG2 cells recovered, calculate the number of wells and plates needed for reseeding and prepare iHepG2 cell suspension for seeding. Each well of a 96-well plate will be seeded with 25,000 iHepG2 in a total volume of 200 µL per well. Typical iHepG2 recovery is ~200,000 cells per well of a 24-well plate, so the concentration will be 200 cells/µL. For this example, we will seed one 96-well plate for imaging. Due to steric hindrance from the microscope objective used, we work only in the inner 32 wells of each plate, and for each plate to be seeded with an electronic 8-channel pipette, a dead volume corresponding to one well is added. For this, (33 wells × 25,000 cells) = 825,000 iHepG2 are required in a total volume of 6.6 mL; 825,000 iHepG2/200 = 4.13 mL of iHepG2 suspension is needed, which will be added to 2.35 mL of iDMEM.
Transfer the iHepG2 seeding suspension into a sterile reagent reservoir and use a serological pipette to thoroughly mix the solution by up and down pipetting 3–5 times.
Using a p1200 8-channel electronic pipette, dispense 200 µL of iHepG2 suspension into the desired number of wells.
Let cells settle at RT for 10 min shielded from the light and then place plates in 5% CO2, 37 °C incubator.
Compound treatments, OPP labeling, and fixation
Note: We developed this protocol to quantify the activity of translation inhibitors, which is compared to DMSO controls (test compound vehicle) and known active controls such as anisomycin. The protocol steps listed below are a detailed demonstration of one assay modality—the acute pretreatment format, where test compounds and controls are applied to iHepG2 from 24–28 hpi; during the last 30 min of the treatment period, OPP is added to each well and the nascent proteome is labeled in the continued presence of treatment compounds. This assay can be modified in several ways; see general note 2.
Prepare test compound treatments, DMSO control treatments, and anisomycin control treatment, transferring them to a 96-well round-bottom plate matching the desired assay plate layout, if not already in this format. Do this in sufficient time to ensure that solutions are at RT at 24 hpi.
Critical: Regardless of compound concentration being tested, the concentration of the DMSO solvent must be identical between treatments, positive controls, and DMSO controls. DMSO concentration in every well should be 0.1% (see Recipes 3–4).
Note: We include four DMSO control wells and one anisomycin active control well on each plate to ensure robust normalization during later analysis.
At 24 hpi, carefully aspirate all iDMEM from each well using a p300 multichannel pipette and replace it with 200 µL of treatment-containing medium from the compound plate prepared in step D1. Then return assay plate to 5% CO2, 37 °C incubator.
At 26.5 hpi, prepare 250 µL of a 20× (400 µM) OPP solution in iDMEM, transfer 30 µL to wells A1–A8 of a round-bottom 96-well plate, and prewarm it in the 5% CO2, 37 °C incubator.
Critical: OPP labeling needs to occur at 37 °C for efficient and effective labeling. Ensure that samples and OPP labeling media are properly warmed to 37 °C before labeling.
Note: We have used two different sources of OPP (see reagents list) and detected no difference in labeling.
At 27 hpi, remove 105 µL of treatment media from each well using a multichannel pipette, leaving 95 µL in each well.
Return the treatment plate with reduced treatment volumes to the incubator for 30 min.
Note: Reducing the medium volume and returning samples to the incubator for 30 min in advance of OPP addition helps to ensure the temperature of the samples is 37 °C when OPP is added.
At 27.5 hpi, transfer the sample plate and the plate containing OPP-labeling solution to the biosafety cabinet. Using a multichannel pipette, dispense 5 µL of 20× OPP solution to each well.
Note: This step should be performed quickly to prevent everything from cooling of the medium.
Gently swirl the plate 5–6 times, quickly return the plate to the incubator, and start a 30 min timer.
After 30 min (at ~28 hpi), remove the plate from the incubator, take off the lid, and place the plate face-down in a swinging bucket rotor liquid removal chamber. Remove media by centrifuging plate at 79× g for 15 s.
Critical: Swinging bucket rotor liquid removal chambers need to be manufactured in weight-matched pairs and, in the case of handling a single plate, the centrifuge should be balanced by adding the equivalent volume (here, 3.2 mL) of water to the other liquid removal chamber and topping it with a matching empty 96-well plate.
Fix cells by dispensing 100 µL of 4% PFA in PBS that has been prewarmed to RT; allow samples to fix for 15 min at RT.
After fixation, flick out the PFA and wash the samples with 200 µL of PBS three times.
Seal plates in parafilm and store at 4 °C overnight.
Note: It is also possible to proceed directly to permeabilization and fluorophore addition by click chemistry. We have successfully performed click reactions on samples that have been stored for several days at 4 °C without reducing the fluorescence signal intensity or fidelity of the click reaction, but samples should be processed in a timely manner to avoid sample degradation. If plates are being stored at 4 °C for several days, the addition of 0.05% sodium azide in PBS helps to reduce bacterial contamination.
Permeabilization and click-chemistry fluorophore addition
Remove plate from 4 °C and allow it to warm to RT.
To permeabilize, flick out PBS and replace with 100 µL of 0.5% triton X-100 in PBS (Recipe 5) for 20 min at RT.
Note: Triton solution should be made fresh from 10% stock each time.
Flick out permeabilization solution and wash cells three times with 100 µL of PBS.
Incubate cells in 100 µL of blocking solution (Recipe 6) while click reaction master mix is prepared (minimum 5 min at RT).
Make click reaction master mix (MM) (Invitrogen click-iT® Alexa Fluor® 555 picolyl azide) according to manufacturer’s protocol. To label 32 wells with a minimum of 27 µL per well with ample dead volume, you need to prepare 1,000 µL (detailed in Recipe 7).
Critical: All components should be brought to room temperature before making MM.
Critical: When preparing the master mix, combine each component in the order listed in Recipe 7, per manufacturer’s recommendation.
Note: We have also successfully used Vector Labs Click-&-Go® Plus 555 Imaging Kit for click reactions with no detectable difference in the fluorescence labeling.
Flick out blocking solution and dispense 27 µL of click reaction to each well. Allow reaction to proceed shielded from light for 30 min at RT.
Flick out click reaction MM and wash the plate with blocking solution three times.
Using a fluorescence microscope, visually inspect each well of the clicked sample to ensure that DMSO controls are brightly labeled before proceeding to DNA or immunofluorescence labeling.
Prior to immunolabeling, block samples by incubating in blocking solution for 1 h at RT.
After blocking, flick out blocking solution, replace with 1° antibody solution (Recipe 8), and incubate at RT for 4 h shielded from light.
Remove 1° antibody solution and wash sample three times with blocking solution.
Flick out blocking solution and replace with 2° antibody and Hoechst solution (Recipe 9).
Incubate samples in 2° antibody solution for 1 h at RT shielded from light.
Remove 2° antibody solution and wash samples three times with PBS.
Proceed to image acquisition steps or seal plates with parafilm and store at 4 °C until imaging.
Note: Plates are imaged in PBS.
Image acquisition
Note: We use automated confocal feedback microscopy (ACFM) on a computer-aided microscopy-licensed Leica SP8 confocal running MatrixScreener directly interfacing with an early version of Cell Profiler (v 2.0.11710) via plugins (https://github.com/VolkerH/MatrixScreenerCellprofiler/wiki) [14]. These coordinate task control between CellProfiler and MatrixScreener to allow real-time online image segmentation of low-resolution monolayer images to identify potential parasites in the sample based on fluorescence and size properties. Coordinates for the center of each low-resolution EEF object identified are then passed from CellProfiler to MatrixScreener, which triggers a high-magnification autofocus Z-stack for each EEF object with X and Y position set by the initial center coordinates, followed by high magnification, high resolution multichannel image acquisition. This process has been described in detail in [15]. Different implementations of feedback microscopy are possible, and the process allows for collection of large, unbiased single-parasite image sets; however, many laboratories will not have access to such a setup but can still implement OPP-imaging of host and parasite nascent proteomes through conventional user-controlled image acquisition. So, we present here the critical steps in the process agnostic to the software and hardware used to control the image acquisition process. Regardless of automated or manual image acquisition, the user should be (or consult with) an experienced microscopist, capable of determining optimal image acquisition settings for their samples and system. We run ACFM imaging experiment on mounted glass coverslips, optical plastic-bottom 96- and 384-well plates, and a variety of glass-bottom imaging dishes; choices of substrate for the cell monolayer, mounting medium, etc. will all influence the choice of objective and are best determined by an experienced microscopist familiar with the options available to the user. Here, we will present an optical plastic-bottom 96-well plate sample containing test compounds, a DMSO vehicle control, and an anisomycin-treated control, which will have both HepG2 and P. berghei translation inhibited.
After securing the plate to the stage, use a 63× water objective to inspect the DMSO control samples, searching for the brightest OPP-A555 fluorescence, and manually image a few parasites to determine appropriate settings for laser power and PMT gain so that no pixels are saturated. Repeat this process to determine optimal settings for the DNA image and the EEF marker (P. berghei HSP70) image.
Note: To assess the sensitivity and specificity of OPP-A555 labeling, a control labeling experiment can be performed as described in Figure S1 of McLellan et al., 2023 for our imaging setup. The key controls needed are 1) a well that was not treated with OPP but was click-labeled and immunostained and 2) a well that was OPP-treated and click-labeled but with omission of the Alexafluor555 picolyl azide from the reaction mix.
With these settings saved, move to the anisomycin control well and verify if the EEF marker image allows easy visual identification of the parasite in images acquired with the settings saved in 1). If not, make slight adjustments to the EEF marker image acquisition settings until this is achieved and save the settings.
A naming scheme for image files must be decided prior to the beginning of image acquisition so that images can be unambiguously assigned to a particular well downstream in the data analysis process. We suggest consulting the REMBI framework [16] to identify a suitable image naming scheme that facilitates metadata capture. For manual acquisition of data in multiple wells of a 96-well plate, we would recommend Experiment#_96wpANcoordinate_YYYYMMDDimaged_Parasite#, e.g., E15_C7_20230803_P12, where the experiment number can be matched to full experimental details in a lab notebook, the well ID can be used to match the images to the treatment received in a plat map, the date of imaging ensures that appropriate control images can be easily identified without needing to resort to image-encoded metadata inspection, and assigning each of the imaged parasites an integer starting from 1 allows each image set from the well to be easily distinguished.
Images must be acquired in an unbiased manner. If manually acquiring images, do so without visualizing anything but the parasite marker, and acquire images of parasites systemically according to predefined rules that mimic a computational process, e.g., from a starting point in the upper-left corner of each well, move rightward in the sample and acquire images of the first five parasites encountered. Then, move down in the sample such that the last parasite imaged is well outside the field of view and repeat the process moving to the left. Continue until ~30 image sets are acquired.
When a parasite is encountered, use an automated focusing algorithm to determine the Z plane, if possible, or manually focus each parasite to maximize parasite area (without visual inspection of anything except the parasite marker) and then sequentially acquire each channel using the saved settings.
Iterate this process for each well on the plate.
Critical: All images that are to be directly compared should be collected from a single imaging session and have identical handling from sections A–C described above. Both biological and technical variables can contribute to experiment-to-experiment variation, and this necessitates data normalization in downstream steps. It is absolutely essential that robust DMSO control image sets are collected in each and every imaging session.
Image segmentation and feature extraction
Note: We perform batch image segmentation and feature extraction in CellProfiler. Resources for learning to use this software are available at cellprofiler.org. For each step in the analysis (termed modules in CellProfiler), there are numerous options and parameter setting adjustments that can be optimized for your needs and specific samples. Batch processing means that an identical setting throughout the Cell Profiler analysis pipeline must be used to process all samples from the same infection, which are handled identically and in parallel at every step in the process. Due to technical and biological variation between experiments, image intensities may vary between replicates and necessitate small tweaks to settings between experiments; however, the identical pipeline should always be used. For each image set analyzed in CellProfiler, a corresponding tiled image is generated, metadata-linked to the corresponding data by filename, and saved, along with all the features extracted and the specifically configured pipeline. For additional information on quantitative bioimaging please see A biologist’s guide to planning and performing quantitating bioimaging experiments [17].
Load all image sets from a single experiment into CellProfiler for batch analysis. Each image set is comprised of three images from different fluorescence channels: 1) parasite and HepG2 DNA, 2) PbHSP70-marked parasite, and 3) the OPP-A555 labeled nascent proteome of the parasite and host.
Using metadata fields encoded in the file names (described in image acquisition), define each image channel in CellProfiler.
Using the well position–encoded metadata, filter images to include only DMSO parasites, which are used to establish the settings of each analysis step of the pipeline.
First, use the Smooth module to reduce digital noise using a Gaussian filter. The typical artifact diameter setting will be dependent upon your image resolution and feature space, but for much of our ACFM imaging we use an artifact diameter of 2.
With the RescaleIntensity module, perform an intensity rescaling of the EEF image and the DNA image to improve contrast and further reduce noise for segmentation. We typically define intensity minima and maxima based on several DMSO image sets and then rescale pixels to the full intensity range. Image rescaling is needed for segmentation but not used in generating fluorescence intensity metrics.
Using the IdentifyPrimaryObjects module, identify the EEF object. We use global thresholding strategy with a two class Otsu method. We define the lower and upper bounds of the EEF object area (in pixels) based on the known size range of parasites at the given timepoint to reduce creation of artifacts. Further, we exclude any EEF objects that are touching the border of the image.
Use the ExpandOrShrinkObjects module to generate EEF objects both expanded and shrunken by several pixels.
Note: Often, EEFs are in close proximity to the HepG2 nucleus, where the most intense OPP signal in the HepG2 is usually found; these shrunken and expanded EEF objects are used in subsequent masking steps to eliminate the pixels at the host–parasite interface for accurate quantification of fluorescence from parasite vs. HepG2 nascent proteomes. See Figure 2 for example.
Figure 2. Tiled image example generated in CellProfiler. Tiled images provide a visual overview of the raw images, objects identified, and masking. They can be very helpful in understanding how to optimize batch segmentation parameters for a given dataset. Each tiled image is metadata-linked to the corresponding data, and image tiles for data points of interest in quality control or exploratory data analysis are routinely inspected via an interactive KNIME workflow ( https://hub.knime.com/-/spaces/-/~TZCrKvv3sbJwM_xP/current-state/). The representative parasite in this tiled image is from a 48 hours post-infection (hpi) timepoint. The shrunken exoerythrocytic form (EEF) outline indicated by the orange arrow is inside of the expanded EEF outline indicated by the blue arrow. OPP, o-propargyl puromycin.
Use the MaskImage module to apply an inverse shrunken EEF object mask to both DNA and OPP-A555 images for analysis of parasite-specific fluorescence.
Critical: You need to create a unique and unambiguous name for every object and masked image generated in the CellProfiler modules, as these objects and masked images are used for downstream measurements of object area and image intensity.
In another MaskImage module, use the expanded EEF object to mask the DNA and OPP-A555 images for analysis of HepG2 features.
Apply IdentifyPrimaryObjects modules to the masked images to further define any other features of interest, such as EEF DNA objects and HepG2 DNA objects.
Using a “Tile” module, construct a tiled image (Figure 2) containing raw images, merged channels, outlines of objects from key segmentation steps, masked images, etc. Then, with the SaveImages module, define folder location where the tiles will be written and define the tiled image output name using the metadata-encoded input file names. Tiled images (Figure 2) provide a single point of reference for quality control (QC) and ground truth determination of the image contents, which we routinely examine using an interactive KNIME workflow (described in section H); an example workflow is available to download on the KNIME hub ( https://hub.knime.com/-/spaces/-/~TZCrKvv3sbJwM_xP/current-state/ ).
Use MeasureObjectSizeShape, MeasureImageAreaOccupied, and MeasureImageIntensity modules to extract features for objects and masked images that were defined in upstream modules.
Finally, use ExportToSpreadsheet module to define the output file location, naming scheme, and which features are to be exported to spreadsheets.
Note: All measurements pertaining to objects will be written into separate CSVs and the image measurements will be written to the image CSV.
Critical: If you are manually selecting which feature measurements to include, be sure to include all necessary metadata to join object features to the image CSV during data analysis steps in section H.
When the pipeline is ready to run, save a copy to disk and click Analyze images.
Data analysis
Note: We use KNIME for all data cleaning and exploratory analyses. Our ACFM workflow generates images of anything identified as meeting the fluorescence and size properties that define EEF object in the low resolution, low magnification images, but not every image from which data is extracted will meet our criteria for inclusion in downstream data analyses. To do so, the image must contain a single, centered, well-focused parasite in the HepG2 monolayer. Since we strive to meaningfully quantify single parasite variation in protein synthesis, we have iteratively optimized our QC filters (Figure 3) to meet our objective. Some images will fail QC based on the ground truth of what the image contains, while others will be filtered when segmentation failures are identified. In our analyses, each row contains data from a single ACFM image. Each image is thus passed through QC filters via the extracted data, and the entire row will be tagged and removed from the main dataset by the first filter where a QC criterion is not met. When all data have been analyzed, the filtered images then comprise a separate dataset, which is explored via an interactive dashboard to identify any systematic issues and ensure data integrity. (See the Validation section for link to our QC and exploratory data analysis workflow on the KNIME hub.) Once we have our final dataset comprised of all images that passed QC, we also perform exploratory analyses and visualizations in KNIME and prepare data for any statistical tests or visualizations in other platforms.
Figure 3. Image data quality control steps with example images. Quality control filters designed to identify and remove confounding image data are consistently applied across the entire dataset prior to data normalization or analysis. Example images are representative of image artifacts and image segmentation artifacts from a 28 hours post-infection (hpi) dataset. Several types of artifacts would be recognized and removed by more than one filter. ACFM, automated confocal feedback microscopy; EEF, exoerythrocytic form.
The key features extracted in CellProfiler are the fluorescence intensity values of OPP-A555 (corresponding to the 30 min nascent proteome) in both the parasite and the in-image HepG2 cells. In our 96-well plate, we have four wells of DMSO controls. Calculate the OPP-A555 mean fluorescence intensity (MFI) of all DMSO-treated EEFs parasites from the four wells. This value is set to 100.
Similarly calculate the OPP-A555 mean fluorescence intensity (MFI) of all DMSO-treated in-image HepG2 from the four wells. This value is also set to 100.
Normalize the data for each EEF and an in-image HepG2 as a percentage of the relevant DMSO control OPP MFI; e.g., if the control OPP MFI is 0.27, which is set to 100, then a well with an OPP MFI of 0.11 would be normalized to [(0.11/0.27) × 100] = 40.7
Note: In KNIME, we normalize data using the Normalize Plates (POC) node.
Use the normalized data to compare, for instance, the ability of the anisomycin control to inhibit both P. berghei liver stage and HepG2 translation in three independent experiments.
For analyses or visualizations outside of KNIME, use a CSV writer node to write datasets to disk.
Validation of protocol
This protocol has been used and validated in the following research articles:
McLellan et al., 2023. Single-cell quantitative bioimaging of Plasmodium berghei liver stage translation. mSphere. DOI: https://doi.org/10.1128/msphere.00544-23 (entire paper).
McLellan & Hanson, 2023. Translation inhibition efficacy does not determine the Plasmodium berghei liver stage antiplasmodial efficacy of protein synthesis inhibitors. BioRxiv. DOI: https://doi.org/10.1101/2023.12.07.570699 (Figures 1–3, 6C, S1–4, S6)
KNIME exploratory data analysis and QC workflows:
KNIME hub QC and exploratory data analysis interactive workflow corresponding to Figure 1 of [11]: https://hub.knime.com/-/spaces/-/~TZCrKvv3sbJwM_xP/most-recent/
KNIME hub exploratory data analysis interactive workflow with data used to generate Figure 2–3 and associated supplementary figures of [11]: https://hub.knime.com/-/spaces/-/~EcnvMwYtqylu2reV/current-state/
General notes and troubleshooting
General notes
The preferred methods for dissecting infected mosquitos and isolating sporozoites from the salivary glands tend to vary from laboratory to laboratory, and detailed protocols for several of these approaches have been previously published.
Mosquito dissection:
A detailed Bio-protocol from the Kyle group [18] outlines the isolation of salivary glands from Anopheles dirus in section E, steps 1–8. This also includes a video demonstrating the dissection steps in video 2.
Another Bio-protocol [19] provides a step-by-step tutorial on salivary gland removal from non-infected mosquitos with detailed diagrams.
For detailed videos of mosquito dissection and salivary gland removal, please see the Journal of Visual Experiments [20] and the Wellcome Trust film unit [21]. These contain accurate depictions of what isolated salivary glands look like under a dissection microscope.
Sporozoite isolation from salivary glands:
A protocol for sporozoite isolation and counting from the Sinnis laboratory website [22] details their use of a Dounce homogenizer to disrupt the salivary glands and isolate the sporozoites by centrifugation.
Another previous Bio-protocol [23] outlines their use of a Teflon homogenizer and filtering steps to isolate sporozoites.
A protocol for the dissection-independent isolation of sporozoites from whole-mosquito lysates using a gradient centrifugation [24] highlights an alternative approach.
Example images of sporozoites visualized with transmitted light microscopy are available in Figure 5A of [25] and Figure 1A [26].
While we detail a 4 h compound pretreatment format here, the OPP assay is very flexible. We have also extensively run the assay in competition (co-OPP) mode, where a compound of interest is added at the same time as OPP. Known direct translation inhibitors show similar potency and efficacy in co-OPP and acute pretreatment assays [10], and we would recommend co-OPP mode to test whether an uncharacterized compound of interest likely functions as a Plasmodium translation inhibitor. We have used a 30 min period of OPP labeling without compound treatment to visualize translation throughout P. berghei liver-stage development, from sporozoites to hepatic merozoites. As parasite size and shape change dramatically during development, parameters for image acquisition, segmentation, feature extraction, and QC will need to be adjusted accordingly for quantification. We have also demonstrated specific OPP-A555 labeling in P. falciparum blood-stage parasites, from ring stages to schizonts, and others have used OPP labeling to quantify P. falciparum asexual blood-stage translation using flow cytometry [27]. We only use a 30 min period of OPP labeling for quantifying P. berghei translation, but we have been able to visualize the P. berghei nascent proteome with OPP treatments periods as short as 5 min.
In addition to the 96-well plate format described in the main protocol, we also quantify translation indirectly infecting HepG2 monolayers cultured on glass coverslips in a 24-well plate. The protocol is identical with the following exceptions:
Section B, Step 1: 75,000 HepG2 cells are seeded in 500 µL per well onto a glass coverslip. After dispensing cell seeding suspension to each well, use the pipette tip to gently press the coverslip to the bottom of the dish to avoid cells attaching to the bottom of the coverslip.
Section C, Step 10: Coverslips are infected with a maximum of 50,000 sporozoites/well, but the minimum number can be adjusted depending on imaging modality and sporozoite availability.
Section C, Step 14: Instead of detaching and reseeding infected cells, remove the plate from the incubator, aspirate the sporozoite suspension media, and replace with 1 mL of fresh iDMEM. Then, return to incubator and move on to step D.
Section D, several steps: The volumes during treatment and OPP labeling are increased to account for larger well volumes. Compound pretreatments are performed using 500 µL for each well and OPP labeling is performed in 300 µL.
Section E, several steps: Use 500 µL for permeabilization and wash steps. To perform a click reaction on a coverslip (CS), remove the CS from 2% BBS using coverslip forceps, gently blot the edge on a paper towel to wick away BBS solution, place inverted CS on a piece of parafilm (cells facing up), and dispense 30 µL of click reaction MM. We have found that using a tinfoil-covered slide-box containing a water-saturated Kimwipe works well to prevent evaporation of the MM during the click reaction. Return the CS to the 24-well plate for PBS washes. Antibody labeling steps are performed in the same way. After labeling is completed, mount coverslips onto a glass slide, cell side down, into a drop of Fluoromount-G and allow them to cure overnight before imaging.
HepG2 cells are available from ATCC (item number HB-8065) and other national cell banks. If you source HepG2 cells from ATCC or start from any HepG2 cell stock in which the cells grow largely in clumps, you will likely need to perform several cell passages in a 12.5 cm2 dish/flask (or smaller, if necessary), removing the clumped cells as described in Procedure section A, until enough cells are recovered to scale up the culture.
Troubleshooting
Problem 1: Low iHepG recovery during trypsinization.
Possible cause: Failure to dislodge all cells from the plastic.
Solution: As noted in section C steps 17–18, add 200 µL of iDMEM to each well immediately after collecting the cell suspension and carefully inspect each well on a phase contrast microscope to make sure that no cells remain attached to the substrate. This can be done multiple times if necessary. iHepG2 (exposed to sporozoite solution) are noticeably more difficult to detach from the plastic substrate than uninfected HepG2.
Acknowledgments
The iHepG2 recovery and reseeding protocol was first developed for work supported by the Bill and Melinda Gates Foundation (OPP1141284 to KKH). P. berghei liver-stage protein synthesis quantification assay development was supported by the National Institutes of Health (R21AI149275 to KKH). JLM was supported by a South Texas Center for Emerging Infectious Diseases fellowship. The protocol was adapted from the publication McLellan et al. [10] and preprint McLellan and Hanson [11].
Competing interests
The authors declare no competing interests.
References
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Computational Analysis of Maize Enhancer Regulatory Elements Using ATAC-STARR-seq
AM Alexandre P. Marand
Published: Mar 5, 2024
DOI: 10.21769/BioProtoc.4953 Views: 156
Reviewed by: G. Alex MasonPrashanth N Suravajhala Anonymous reviewer(s)
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Abstract
The blueprints for development, response to the environment, and cellularfunction are largely the manifestation of distinct gene expression programscontrolled by the spatiotemporal activity of cis-regulatory elements. Althoughbiochemical methods for identifying accessible chromatin—a hallmark ofactive cis-regulatory elements—have been developed, approaches capable ofmeasuring and quantifying cis-regulatory activity are only beginning to berealized. Massively parallel reporter assays coupled to chromatin accessibilityprofiling present a high-throughput solution for testing thetranscription-activating capacity of millions of putatively regulatory DNAsequences in parallel. However, clear computational pipelines for analyzingthese high-throughput sequencing-based reporter assays are lacking. In thisprotocol, I layout and rationalize a computational framework for the processingand analysis of the transposase accessible chromatin profiling followed byself-transcribed active regulatory region sequencing (ATAC-STARR-seq) data froma recent study in Zea mays. The approach described herein canbe adapted to other sequencing-based reporter assays and is largely modelorganism–agnostic with appropriate input substitutions.
Keywords: STARR-seq ATAC-seq ATAC-STARR-seq Regulatory activity cis-regulatory elements Accessible chromatin Transcriptional regulation Enhancers
Background
Eukaryotic cells exhibit remarkable functional and morphological diversity despite containing a generally invariant copy of the same genomic sequence. Cellular heterogeneity arises in part due to the activities of cis-regulatory elements (CREs), short DNA-binding motifs recognized by sequence-specific transcription factors (TFs). CREs are often found in clusters termed cis-regulatory modules (CRMs) that dictate highly dynamic spatiotemporal patterns of gene expression via the cooperative activities of DNA-bound TFs (Schmitz et al., 2022). For proper activation of transcription, the cell strictly regulates CRM activity by controlling TF access of CRM sequences through nucleosome dynamics. Genome-wide approaches, such as the assay for transposase accessible chromatin sequencing (ATAC-seq), have been developed to profile accessible chromatin regions (ACRs) (Buenrostro et al., 2013; Minnoye et al., 2021). In general, CRMs that localize to accessible chromatin reflect active regulatory elements (Marand et al., 2017; Schmitz et al., 2022). Thus, activation and silencing of gene expression is effectively controlled by the relative chromatin accessibility of cognate CRMs.
CREs can be classified into distinct functional groups based on their regulatory effect on transcription, including enhancers, silencers, promoters, and insulators (Schmitz et al., 2022). Of these, enhancers are of particular interest due to their transcription-activating properties that function independently of location and orientation of their target genes, in contrast to the stereotypical locations of promoters surrounding gene transcription start sites (TSSs) (Marand et al., 2017; Schmitz et al., 2022). While analysis of chromatin accessibility in distinct tissues and cell types has been central to the identification of CRMs (Marand et al., 2021), chromatin profiling techniques are largely qualitative and lack the ability to quantitatively estimate regulatory activity. To overcome these challenges, massively parallel reporter assays (MPRAs) have been developed to quantify the transcription-activating properties of diverse sequences (Melnikov et al., 2012; Arnold et al., 2013). In particular, self-transcribing active regulatory region sequencing (STARR-seq) demonstrates the greatest potential for broad application by eliminating the need for homogenous cell lines available only in mammalian models typical of other MPRA methods (Arnold et al., 2013; Ricci et al., 2019; Sun et al., 2019; Jores et al., 2020). Although STARR-seq was originally designed to profile the entire genome for regulatory activity, recent implementations have successfully utilized ATAC-seq libraries as input (ATAC-STARR-seq), reducing the search space to potential regulatory regions and offsetting sequencing costs and library complexity requirements (Figure 1). Despite its promise as a powerful approach towards understanding cis-regulatory activity, computational analysis of ATAC-STARR-seq data remains challenging, particularly due to a lack of dedicated software and computational pipelines.
Here, I present a computational pipeline for analysis of ATAC-STARR-seq data generated in Zea mays L., cultivar B73 (Ricci et al., 2019). After processing and evaluation of data quality, I demonstrate how ATAC-STARR-seq data analysis allows for the interrogation of new biological questions. The pipeline can be run entirely from the code below or through freely available bash, perl, and R scripts hosted at https://github.com/Bio-protocol/Maize_ATAC_STARR_seq.
Figure 1. Schematic of assay for transposase accessible chromatin profiling followed by self-transcribed active regulatory region sequencing (ATAC-STARR-seq). ATAC-STARR-seq begins by first generating an ATAC-seq library. The ATAC fragments are then cloned into a reporter assay and transformed into maize protoplasts. Transformed protoplasts are then split into two pools: the first for sequencing the input fragments (ATAC-seq DNA) and the second for purifying transcribed (mRNA) ATAC-seq fragments that facilitate their own transcription from the reporter construct. Raw sequenced reads for the ATAC-seq input and mRNA output are processed and aligned to the maize reference genome and compared to provide estimates of cis-regulatory activity.
Equipment
This pipeline assumes that a user has knowledge of shell commands and is comfortable working on a Linux-based operating system.
Computational requirements
The following procedure can be run on any Linux-like system. However, this protocol and publicly available code is written for executing commands via a high-performance computing (HPC) cluster managed by a SLURM scheduler. Still, the code presented here can be readily converted to TORQUE or other HPC systems. The pipeline assumes a working Perl interpreter version 5.30.0 or greater and R version 3.6.2 or greater.
Software
The following analytical procedure makes use of several standard computational tools that are assumed to be available in the user’s shell environment:
BWA MEM (Li and Durbin, 2009) v0.7.17; http://bio-bwa.sourceforge.net/bwa.shtml
SAMtools (Li et al., 2009) v1.14; http://www.htslib.org
BEDtools (Quinlan and Hall, 2010) v2.27.1; https://bedtools.readthedocs.io/en/latest/
SRA-toolkit (Leinonen et al., 2011) v2.11.1; https://github.com/ncbi/sra-tools
fastp (Chen et al., 2018) v0.20.0; https://github.com/OpenGene/fastp
pigz v2.4; https://zlib.net/pigz/
MACS2 (Liu, 2014) v2.2.7.1; https://pypi.org/project/MACS2/
UCSC binaries (Kent et al., 2010) v1.04.0; http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/
tabix (Li, 2011) v0.2.6; http://www.htslib.org/doc/tabix.html
IGV (Thorvaldsdottir et al., 2013) v2.11.1; https://software.broadinstitute.org/software/igv
MEME (Grant et al., 2011) v5.4.1; https://meme-suite.org/meme/index.html
CrossMap (Zhao et al., 2014) v0.5.1; http://crossmap.sourceforge.net/
DeepTools (Ramirez et al., 2014) v3.5.1; https://deeptools.readthedocs.io/en/develop/index.html
Input data
The starting input for this computational pipeline uses paired-end sequencing data from an ATAC-STARR-seq experiment performed on maize protoplasts (Ricci et al., 2019). The ATAC-STARR-seq experiment consisted of a DNA input (ATAC-seq library) and a mRNA readout (self-transcribed regulatory regions) to identify genomic regions exhibiting transcription-activating regulatory activity.
Transfected ATAC-seq DNA-input FASTQ
Transcribed ATAC-seq mRNA FASTQ
Procedure
An overview of the ATAC-STARR-seq pipeline is presented in Figure 2.
Figure 2. Computational workflow for the assay for transposase accessible chromatin profiling followed by self-transcribed active regulatory region sequencing (ATAC-STARR-seq) data. Raw sequence ATAC-STARR-seq data are first acquired from NCBI GEO, processed, and aligned to the reference genome with BWA MEM, filtered via SAMtools, reformatted as fragments with BEDtools, and compared via BEDtools and custom R scripts to provide estimates of enhancer activity for downstream analysis.
Download and prepare the requisite data and reference genome sequence
Raw mRNA ATAC-STARR-seq data generated from transfection of Zea mays leaf ATAC-seq fragments in Z. mays protoplasts, and the accompanying ATAC-seq input fragments, are publicly available on NCBI GEO. ATAC-STARR-seq mRNA and DNA input can be downloaded with fasterq-dump available from the SRA-toolkit package:
# set variables and download FASTQ files
mkdir FASTQ_files
cd FASTQ_files
fasterq-dump -o B73_maize_DNA_input.fastq SRR10964904
fasterq-dump -o B73_maize_mRNA_output.fastq SRR10964905
To save disk space, we will compress the FASTQ files with pigz. By default, pigz uses all available processors or eight if the number of available processors is unknown. Alternatively, users can use the unix tool, gzip, to compress the STARR mRNA and ATAC input DNA FASTQ files.
# compress fastq files
pigz *.fastq
# NOT RUN
# Tip: gzip can be used as an alternative to pigz (parallel gzip)
# gzip *.fastq
Download the B73 reference genome sequence and gene annotation. The original article mapped raw reads to version 4 of the B73 maize reference genome. In this case study, I map and analyze maize ATAC-STARR-seq data to version 5 of the B73 maize reference genome to showcase how updated reference genomes and read mapping strategies enable informative reanalysis of publicly available data sets (Hufford et al., 2021).
# download reference data
cd ../
mkdir Genome_Reference
cd Genome_Reference
wget https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/Zm-B73-REFERENCE-NAM-5.0.fa.gz
wget https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3.gz
To map data to the reference genome, we first need to decompress the FASTA file. Constructing reference genome indices is a prerequisite for BWA alignment and allows for faster post-alignment processing of BAM/SAM/BED formatted files with command line tools such as SAMtools and BEDtools.
# create indices for reference genome FASTA
gunzip Zm-B73-REFERENCE-NAM-5.0.fa.gz
samtools faidx Zm-B73-REFERENCE-NAM-5.0.fa
bwa index Zm-B73-REFERENCE-NAM-5.0.fa
Trim adapters and remove low quality reads
Illumina platforms may produce reads with adapter sequences on the 3’ ends if the DNA insert is shorter than the number of cycles. Additionally, the fidelity of sequencing by synthesis deteriorates with each additional cycle due to phasing, the desynchronization of cycles that results from unremoved terminator caps ultimately leading to greater uncertainty of base calls in later cycles. Removing adapter contamination and low-quality bases increases the total number of alignable reads, particularly important when analyzing a relatively lower sequence complexity experiment, such as ATAC-STARR-seq. We will use fastp to remove sequencing adapters and low-quality reads for the mRNA output and DNA input. A script to perform read trimming can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step01_trim_raw_reads.sh
# set variables
cd ../
fastqdir=$PWD/FASTQ_files
dna=B73_maize_DNA_input
rna=B73_maize_mRNA_output
threads=16
# trim and filter DNA input reads
fastp -j $dna.json -h $dna.html -w $threads \
-i $fastqdir/${dna}_1.fastq.gz -I $fastqdir/${dna}_2.fastq.gz \
-o $fastqdir/${dna}_1.trim.fastq.gz -O $fastqdir/${dna}_2.trim.fastq.gz
# trim and filter mRNA output reads
fastp -j $rna.json -h $rna.html -w $threads \
-i $fastqdir/${rna}_1.fastq.gz -I $fastqdir/${rna}_2.fastq.gz \
-o $fastqdir/${rna}_1.trim.fastq.gz -O $fastqdir/${rna}_2.trim.fastq.gzbS - | samtools sort - > $outdir/B73_maize_mRNA_output.raw.bam
# tidy up log files
mkdir fastp_log_files
mv *.json *.html fastp_log_files
Align and process sequenced reads
After trimming and quality filtering reads, we align the input DNA and output mRNA reads to the maize B73 version 5 reference genome. To speed up the alignment and downstream processing, we are using 24 CPUs (-t 24) to align the input and output reads. However, users should modify this value to reflect the number of available cores on their system. Additionally, we mark split hits as secondary alignments (-M) to be filtered out downstream, as the maize genome is highly repetitive. The output of BWA MEM is piped to SAMtools view for compression (SAM to BAM) and sorted by alignment coordinate prior to further processing to minimize the footprint on disk. A script to perform these steps can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step02_align_STARR_data.sh
cd ../
mkdir BAM_files
outdir=$PWD/BAM_files
refdir=$PWD/Genome_Reference
fastqdir=$PWD/FASTQ_files
# align DNA input and pipe to samtools for SAM to BAM conversion
bwa mem -M -t 24 $refdir/Zm-B73-REFERENCE-NAM-5.0.fa $fastqdir/B73_maize_DNA_input_1.trim.fastq.gz $fastqdir/B73_maize_DNA_input_2.trim.fastq.gz | samtools view -bS - | samtools sort - > $outdir/B73_maize_DNA_input.raw.bam
# align RNA output and pipe to samtools for SAM to BAM conversion
bwa mem -M -t 24 $refdir/Zm-B73-REFERENCE-NAM-5.0.fa $fastqdir/B73_maize_mRNA_output_1.trim.fastq.gz $fastqdir/B73_maize_mRNA_output_2.trim.fastq.gz | samtools view -bS - | samtools sort - > $outdir/B73_maize_mRNA_output.raw.bam
To ensure that only high-quality alignments remain, here we remove non-properly paired reads (-f 3), secondary hits (-F 256), alignments with low mapping quality (-q 10), and multiple mapped reads (grep -v -E -e '\bXA:Z:’) using a combination of SAMtools and unix commands. The header, which contains information on the reference genome and the read mapping parameters, is retained in the output by setting the -h flag in the SAMtools view command.
# filter DNA input alignments
samtools view -h -q 10 -f 3 -F 256 $outdir/B73_maize_DNA_input.raw.bam
| grep -v -E -e '\bXA:Z:' | samtools view -bSh - > $outdir/B73_maize_DNA_input.mq10.pp.unique.bam
# filter mRNA output alignments
samtools view -h -q 10 -f 3 -F 256 $outdir/B73_maize_ mRNA_output.raw.bam
| grep -v -E -e '\bXA:Z:' | samtools view -bSh - > $outdir/B73_maize_mRNA_output.mq10.pp.unique.bam
A major difference between analysis of ATAC-seq and STARR-seq data is how assay information is captured by sequencing. For paired-end ATAC-seq, since Tn5 inserts sequencing adapters adjacent to its bound genomic location, chromatin accessibility is encoded as the 5’ ends of sequenced reads. In contrast, STARR-seq produces mRNA transcripts from fragments that are capable of activating their own transcription; thus, the entire STARR-seq mRNA and DNA fragment is informative for analysis. The following commands extract the coordinates of sequenced fragments by leveraging the CIGAR strings in BAM paired-end alignments for DNA input and mRNA output. A script to extract fragments can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step03_extract_fragments.sh
# create output directory
mkdir BED_files
# variables
outdir=$PWD/BAM_files
beddir=$PWD/BED_files
dna=B73_maize_DNA_input
rna=B73_maize_mRNA_output
# extract DNA input fragments (ignore the warnings from bedtools with respect to “missing” mate pairs, these reflect one of the pairs that had its mate filtered in prior steps)
echo " extracting fragments from STARR DNA input ..."
samtools sort -n $outdir/$dna.mq10.pp.unique.bam \
| bedtools bamtobed -bedpe -i - \
| sort -k1,1 -k2,2n - \
| cut -f1,2,6 - > $beddir/$dna.fragments.bed
# extract mRNA output fragments
echo " extracting fragments from STARR mRNA output ..."
samtools sort -n $outdir/$rna.mq10.pp.unique.bam \
| bedtools bamtobed -bedpe -i - \
| sort -k1,1 -k2,2n - \
| cut -f1,2,6 - > $beddir/$rna.fragments.bed
Identify regions with enriched activity over background
To identify regions of the genome with the capacity to activate transcription, we assess enrichment of mRNA reads relative to the input ATAC-seq fragments using MACS2. As MACS2 is a general peak caller, we need to adjust the default settings to tailor the analysis specifically for ATAC-STARR-seq data. Since this experiment did not use unique molecular identifiers, and the number of transcripts from fragment is a direct reflection of its regulatory activity, duplicate mRNA fragments are retained (--keep-dup all). To directly use coverages determined by the input fragment coordinates, we turn off the default fragment shifting model (--nomodel) and set the input type to BEDPE (-f BEDPE). Additionally, we reduce the maximum gap size between candidate peaks to allow for the identification of fine-mapped regulatory elements within a broader regulatory region (--max-gap 50) by setting the minimum peak size to 300 (--min-length 300). Finally, we use the background coverage rates in place of the local bias, which aids in peak detection (--nolambda). A script to perform peak calling can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step04_call_peaks.sh
# prepare output directory and input files
mkdir Peak_data
# generate input files
uniq $beddir/$rna.fragments.bed > $beddir/$rna.fragments.uniq.bed
uniq $beddir/$dna.fragments.bed > $beddir/$dna.fragments.uniq.bed
cat $beddir/$rna.fragments.uniq.bed $beddir/$dna.fragments.uniq.bed | sort -k1,1 -k2,2n - > $beddir/ALL.fragments.uniq.bed
# find regulatory regions
echo " calling regulatory regions without duplicate removal ..."
macs2 callpeak -t $beddir/$rna.fragments.bed \
-c $beddir/ALL.fragments.uniq.bed \
--keep-dup all \
--max-gap 50 \
--min-length 300 \
--nolambda \
--nomodel \
-f BEDPE \
-g 1.6e9 \
--bdg \
-n STARR_wdups
# find regulatory regions
echo " calling regulatory regions with duplicate removal ..."
macs2 callpeak -t $beddir/$rna.fragments.uniq.bed \
-c $beddir/$dna.fragments.uniq.bed \
--keep-dup all \
--max-gap 50 \
--min-length 300 \
--nolambda \
--nomodel \
-f BEDPE \
-g 1.6e9 \
--bdg \
-n STARR_nodups
# find regulatory regions using all unique fragments
echo " calling regulatory regions by aggregating all unique fragments ..."
macs2 callpeak -t $beddir/ALL.fragments.uniq.bed \
--keep-dup all \
--max-gap 50 \
--min-length 300 \
--nolambda \
--nomodel \
-f BEDPE \
-g 1.69e9 \
--bdg \
-n STARR_ALL
# clean-up output
mv STARR_* Peak_data
# merge peaks
cd Peak_data
cat STARR_wdups_peaks.narrowPeak STARR_nodups_peaks.narrowPeak STARR_ALL_peaks.narrowPeak | sort -k1,1 -k2,2n - | bedtools merge -i - > STARR_merged_peaks.bed
To estimate regulatory activity at fine scale, first we need to create a list of unique intervals based on mRNA and DNA fragments. Next, we count the intersection of mRNA and DNA fragments for each unique interval. Finally, we remove all the temporary files to reduce the footprint on disk. A script to estimate enhancer activity can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step05_estimate_enhancer_activity.sh
# variables
beddir=$PWD/BED_files
dna=B73_maize_DNA_input
rna=B73_maize_mRNA_output
ref=./Genome_Reference/Zm-B73-REFERENCE-NAM-5.0.fa.fai
# sort the reference
sort -k1,1 -k2,2n $ref > $ref.sorted
# merge RNA/DNA
cat $beddir/$rna.fragments.uniq.bed $beddir/$dna.fragments.uniq.bed \
| sort -k1,1 -k2,2n - \
| bedtools genomecov -i - -bga -g $ref.sorted \
| sort -k1,1 -k2,2n - \
| cut -f1-3 - > $beddir/Unique_genomic_intervals.bed
# count fragments
bedtools intersect -a $beddir/Unique_genomic_intervals.bed \
-b $beddir/$rna.fragments.bed \
-c -sorted -g $ref.sorted > $beddir/$rna.activity.raw.bed
bedtools intersect -a $beddir/$rna.activity.raw.bed \
-b $beddir/$dna.fragments.bed \
-c -sorted -g $ref.sorted > $beddir/B73_maize_mRNA_DNA.activity.raw.bed
# clean up temporary files
rm $beddir/Unique_genomic_intervals.bed
rm $beddir/$rna.activity.raw.bed
We and others define enhancer activity as the enrichment of mRNA transcripts that are produced by DNA fragments in the assay in terms of log2(mRNA/DNA) at unique fragment intervals. Prior to taking the log2 ratio of mRNA to DNA, we normalize both the input and output to per million to account for differences in sequencing depth and complexity. A pseudocount of one is added to any interval with at least one RNA or DNA fragment. The following code can also be run from the command line using >Rscript Estimate_Enhancer_Activity.R with the following script: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/bin/Estimate_Enhancer_Activity.R
# open an interactive R session to estimate enhancer activity
cd $beddir
R
# load data
a <- read.table("B73_maize_mRNA_DNA.activity.raw.bed")
# reformat
rownames(a) <- paste(a$V1,a$V2,a$V3,sep="_")
a[,1:3] <- NULL
a <- as.matrix(a)
colnames(a) <- c("mRNA", "DNA")
a <- a[rowSums(a)!=0,]
a <- a + 1
# normalize
a <- a %*% diag(x=1e6/colSums(a))
colnames(a) <- c("mRNA", "DNA")
a <- as.data.frame(a)
# estimate enhancer activity
a$enhancer_activity <- log2(a$mRNA/a$DNA)
# reformat output
rownames(a) <- gsub("scaf_","scaf", rownames(a))
df <- data.frame(do.call(rbind, strsplit(rownames(a), "_")))
df$X1 <- gsub("scaf", "scaf_", as.character(df$X1))
mrna <- df
dna <- df
df$X4 <- a$enhancer_activity
mrna$X4 <- a$mRNA
dna$X4 <- a$DNA
# cap negative activity at 0
df$X4 <- ifelse(df$X4 < 0, 0, df$X4)
# save enhancer activity BEDGRAPH file to disk
write.table(df, file="B73_maize.enhancer_activity.bdg",quote=F, row.names=F, col.names=F, sep="\t")
write.table(mrna, file="B73_maize.mRNA.bdg",quote=F, row.names=F, col.names=F, sep="\t")
write.table(dna, file="B73_maize.DNA.bdg",quote=F, row.names=F, col.names=F, sep="\t")
# exit interactive mode
q()
To visualize enhancer activity and the normalized mRNA and DNA fragments at any given locus, per million coverage values in bedGraph format from the previous step can be converted into bigwig files (using bedGraphToBigWig from UCSC Utils) for facile visualization using the Integrated Genomics Viewer (IGV) or JBrowse instances.
bedGraphToBigWig
B73_maize.enhancer_activity.bdg ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0.fa.fai.sorted B73_maize.enhancer_activity.bw
bedGraphToBigWig
B73_maize.mRNA.bdg ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0.fa.fai.sorted B73_maize.mRNA.bw
bedGraphToBigWig
B73_maize.DNA.bdg ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0.fa.fai.sorted B73_maize.DNA.bw
To view the enhancer activity, mRNA, and DNA fragment bigwig files, download and install IGV ( https://software.broadinstitute.org/software/igv/download) on your local machine.
Unpack, bgzip, and index the gene product annotation. Then, load all bigwig, narrowPeak, and genome annotation files using File > Load from File… in IGV. An example of an IGV screenshot is shown in Figure 3.
# change directory
cd ../Genome_Reference
# unzip
gunzip Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3.gz
# sort by coordinate and remove whole chromosome intervals
sort -k1,1 -k4,4n Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3 | grep -v assembly - > Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.sorted.gff3
# compress with bgzip
bgzip Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.sorted.gff3
# index with tabix
tabix -p gff Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.sorted.gff3.gz
Figure 3. Visualization of the assay for transposase accessible chromatin profiling followed by self-transcribed active regulatory region sequencing (ATAC-STARR-seq) data in Zea mays. Normalized (reads per million) coverages of the DNA ATAC-seq input (blue), self-transcribed mRNA fragments (pink), and enhancer activity [purple; log2(mRNA/DNA)] of a 23 kb window. Regulatory regions are shown as black bars, while the grey loops reflect predicted enhancer-gene links.
Create a list of control regions
To assess enrichment of STARR regulatory regions determined by MACS2 relative to random control regions, we first need to identify regions of the genome that can be uniquely mapped given the sequencing output and read lengths. Although there are numerous methods for estimating mappability, I illustrate a simple approach using synthetic reads tailored to the sequencing parameters of the present experiment. First, we generate the same number of random read pairs with the same sequencing length (36 nucleotides) for mRNA and DNA input using the wgsim tool that is supplied to SAMtools. The synthetic reads are then remapped and uniformly processed as the original STARR-seq sequencing experiments to identify regions that are uniquely mappable. By constraining randomized control region selection to uniquely mappable genomic intervals, we ensure that downstream comparisons will not be biased by mappability and repeat composition. A script to construct control regions can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step06_create_control_regions.sh
# move into the “Genome_Reference” directory
cd ./Genome_Reference
# estimate read counts
mRNA_counts=$(samtools view -c ./BAM_files/B73_maize_mRNA_output.raw.bam)
DNA_counts=$(samtools view -c ./BAM_files/B73_maize_DNA_input.raw.bam)
# generate simulated reads matching the mRNA output using wgsim from the SAMtools package
wgsim -1 36 -2 36 -d 300 -N $mRNA_counts Zm-B73-REFERENCE-NAM-5.0.fa simulated_STARR_mRNA_r1.fq simualted_STARR_mRNA_r2.fq
# generate simulated reads matching the DNA input
wgsim -1 36 -2 36 -d 300 -N $DNA_counts Zm-B73-REFERENCE-NAM-5.0.fa simulated_STARR_DNA_r1.fq simualted_STARR_DNA_r2.fq
# compress synthetic fastq files
pigz *.fq
Remap the synthetic reads using the same pipeline as for the original STARR-seq data.
# variables
outdir=$PWD/BAM_files
refdir=$PWD/Genome_Reference
ref=$refdir/Zm-B73-REFERENCE-NAM-5.0.fa.fai.sorted
fastqdir=$refdir
# align synthetic mRNA output and pipe to samtools for SAM to BAM conversion
bwa mem -M -t 24 $refdir/Zm-B73-REFERENCE-NAM-5.0.fa \
$fastqdir/simulated_STARR_mRNA_r1.fq.gz \
$fastqdir/simualted_STARR_mRNA_r2.fq.gz \
| samtools view -bS - \
| samtools sort - > $outdir/simulated_STARR_mRNA.raw.bam
# align synthetic DNA input and pipe to samtools for SAM to BAM conversion
bwa mem -M -t 24 $refdir/Zm-B73-REFERENCE-NAM-5.0.fa \
$fastqdir/simulated_STARR_DNA_r1.fq.gz \
$fastqdir/simulated_STARR_DNA_r2.fq.gz \
| samtools view -bS - \
| samtools sort - > $outdir/simulated_STARR_DNA.raw.bam
Process and filter reads using the original STARR-seq pipeline.
# filter synthetic mRNA alignments
echo " filtering synthetic STARR mRNA alignments ..."
samtools view -h -q 10 -f 3 $outdir/simulated_STARR_mRNA.raw.bam \
| grep -v -E -e '\bXA:Z:' \
| samtools view -bSh - > $outdir/simulated_STARR_mRNA.mq10.pp.unique.bam
# filter synthetic DNA alignments
echo " filtering STARR DNA alignments ..."
samtools view -h -q 10 -f 3 $outdir/simulated_STARR_DNA.raw.bam \
| grep -v -E -e '\bXA:Z:' \
| samtools view -bSh - > $outdir/simulated_STARR_DNA.mq10.pp.unique.bam
Identify uniquely mappable regions.
# extract mRNA output fragments
echo " extracting fragments from simulated STARR mRNA output ..."
samtools sort -n $outdir/simulated_STARR_mRNA.mq10.pp.unique.bam \
| bedtools bamtobed -bedpe -i - \
| sort -k1,1 -k2,2n - \
| cut -f1,2,6 - > $refdir/simulated_STARR_mRNA.fragments.bed
# extract DNA input fragments
echo " extracting fragments from simulated STARR DNA input ..."
samtools sort -n $outdir/simulated_STARR_DNA.mq10.pp.unique.bam \
| bedtools bamtobed -bedpe -i - \
| sort -k1,1 -k2,2n - \
| cut -f1,2,6 - > $refdir/simulated_STARR_DNA.fragments.bed
# merge all fragments (sorting by coordinate at this step may take a while)
cat $refdir/simulated_STARR_mRNA.fragments.bed $refdir/simulated_STARR_DNA.fragments.bed \
| sort -k1,1 -k2,2n \
| bedtools merge -i - > $refdir/mappable_genomic_regions.bed
Construct control regions using only mappable regions and excluding putative regulatory regions output by MACS2.
# create controls
peaks=$PWD/Peak_data/STARR_merged_peaks.bed
bedtools shuffle -i $peaks \
-g $ref \
-incl $refdir/mappable_genomic_regions.bed \
-excl $peaks \
| sort -k1,1 -k2,2n - > $PWD/Peak_data/STARR_CONTROL.bed
Compare enhancer activity
Determine enhancer activity for predicted enhancers output by MACS2 as well as the negative control regions.
# create directory to contain analysis
cd ../
mkdir 01_Peak_Analysis
cd 01_Peak_Analysis
# map maximum enhancer activity to putative regulatory regions (wdups)
bedtools map -a ../Peak_data/STARR_merged_peaks.bed -b ../BED_files/B73_maize.enhancer_activity.bdg -o max -c 4 > STARR_merged_peaks.enhancer_activity.bed
# map maximum enhancer activity to control
bedtools map -a ../Peak_data/STARR_CONTROL.bed -b ../BED_files/B73_maize.enhancer_activity.bdg -o max -c 4 > STARR_CONTROL.enhancer_activity.bed
To remove regulatory regions with enhancer activity similar to background, we filter STARR regulatory regions using an empirical false discovery rate (eFDR) based on the matched control regions. A user-specified eFDR threshold identifies the enhancer activity value in the control set that removes 1-eFDR percent of control regions. In this example, we set the FDR to a widely used rate of 0.05. The following code performs and plots eFDR filtering and enhancer activity distributions and can be run from the command line using >Rscript eFDR_Filter_STARR_Peaks.R. Filtering STARR peaks based on eFDR thresholds derived from the control regions is visualized in Figure 4A. An R script of the following code can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/bin/eFDR_Filter_STARR_Peaks.R
# start an interactive R session
> R
# load libraries
library(scales)
# load data
starr <- read.table("STARR_merged_peaks.enhancer_activity.bed")
con <- read.table(”STARR_CONTROL.enhancer_activity.bed")
# set missing to 0
starr$V4[starr$V4=='.'] <- 0
con$V4[con$V4=='.'] <- 0
# convert to numeric
starr$V4 <- as.numeric(as.character(starr$V4))
con$V4 <- as.numeric(as.character(con$V4))
# get empirical thresholds
fdr <- 0.05
threshold <- quantile(con$V4, (1-fdr))
# filter STARR regulatory regions
filtered <- subset(starr, starr$V4 >= threshold)
# estimate fraction of retained regions
frac <- signif(nrow(filtered)/nrow(starr), digits=4)
# set up multipanel plot area
pdf("Density_eFDR_STARR_Peak_Filtering.pdf", width=5, height=5)
# plot control/observed enhancer activities for STARR peaks with duplicates
den.starr <- density(starr$V4)
den.con <- density(con$V4)
plot(NA,
xlab="Enhancer Activity",
ylab="Density",
xlim=c(range(range(den.starr$x), range(den.con $x))),
ylim=c(range(range(den.starr$y), range(den.con$y))))
polygon(x=c(min(den.starr$x), den.starr$x, max(den.starr$x)),
y=c(0, den.starr$y, 0), col=alpha("darkorchid4", 0.5), border=NA)
polygon(x=c(min(den.con$x), den.con$x, max(den.con$x)),
y=c(0, den.con$y, 0), col=alpha("grey80", 0.5), border=NA)
abline(v=threshold, col="red", lty=2)
mtext(paste0("STARR peaks pass = ",frac," (", nrow(filtered), "/", nrow(starr),")"))
legend("right", legend=c("STARR Peaks", "Control Peaks", paste0("eFDR = ", fdr)), col=c("darkorchid4", "grey75", "red"), border=c(NA, NA, "red"), pch=c(16, 16, NA), lty=c(NA, NA, 2))
# close device
dev.off()
# save filtered STARR regulatory regions
write.table(filtered, file="STARR_merged_peaks.enhancer_activity.eFDR05.bed", quote=F, row.names=F, col.names=F, sep="\t")
# exit R
q()
Figure 4. Analysis of self-transcribed active regulatory region (STARR) regulatory region enhancer activity. A. Distribution of enhancer activity for STARR peaks (purple) and random control regions (grey). Dashed red line indicates the 95% quantile of enhancer activity of random control regions. B. Average (top) and individual site heatmaps of reads per million (RPM) for ATAC-seq input (left), mRNA output (middle), and enhancer activity (right) for control regions, all STARR peaks, and the filtered STARR peak set.
We can now assess the relative enhancer activities across all regions for the filtered and unfiltered STARR peaks and controls using DeepTools (Figure 4B). A script to plot heatmaps via DeepTools can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step07_plot_enhancer_activity.sh
# load function
getmaps(){
# input
ina=$1
id=$2
dat=../BED_files/*.bw
# output
outa=$id.mat.gz
outm=$id.mat.txt
fig=$id.pdf
# parameters
threads=48
window=2000
bin=20
cols=YlGnBu
# create matrix
computeMatrix reference-point --referencePoint center \
-S $dat \
-b $window -a $window \
-R $ina \
--missingDataAsZero \
-o $outa \
--outFileNameMatrix $outm \
-p $threads --binSize $bin
# plot heatmap
plotHeatmap --matrixFile $outa \
--colorMap YlGnBu \
-out $id.heatmap.pdf
}
export -f getmaps
# run for each file
getmaps STARR_merged_peaks.enhancer_activity.bed STARR_peaks
getmaps STARR_merged_peaks.enhancer_activity.eFDR05.bed STARR_peaks_filtered
getmaps STARR_CONTROL.enhancer_activity.bed control_regions
Identification of large regulatory domains in the maize genome
One question these data allow us to ask is whether a relationship exists between the size of a regulatory region and its enhancer activity. The so called super enhancers in mammalian systems describe hyperactive transcription-activating regulatory domains associated with cell identity that exhibit increased density of TF binding sites compared to typical enhancers (Hnisz et al., 2013). Integration of the STARR peaks and enhancer activities with other datasets allows us to determine whether similar hyperactive regulatory domains exist in maize. To query TF binding site density, we first download the position weight matrices of known TFs from the MEME database and identify putative TF binding sites using fimo (also from the MEME suite) conditioning on a P-value threshold less than 1-5. A script to identify large regulatory domains can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/step08_identify_large_regulatory_domains.sh
# make a new directory for the TFBS analysis
cd ../
mkdir 02_Hyperactive_Regulatory_Region_Analysis
cd ./02_Hyperactive_Regulatory_Region_Analysis
# download and decompress motif databases
wget https://meme-suite.org/meme/meme-software/Databases/motifs/motif_databases.12.23.tgz
tar -xvzf motif_databases.12.23.tgz
rm motif_databases.12.23.tgz
# variables
threads=16
ref=../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0.fa
peaks=../01_Peak_Analysis/STARR_merged_peaks.enhancer_activity.eFDR05.bed
controls=../01_Peak_Analysis/STARR_CONTROL.enhancer_activity.bed
motifs=./motif_databases/ARABD/ArabidopsisDAPv1.meme
# extract fasta sequences
bedtools getfasta -bed $peaks -fi $ref -fo $peaks.fasta
bedtools getfasta -bed $controls -fi $ref -fo $controls.fasta
# identify putative TFBS
fimo --oc TFBS_peaks $motifs $peaks.fasta
fimo --oc TFBS_controls $motifs $controls.fasta
# reformat fimo output (filtering p-value > 1e-5) using the perl script provided in the github repository (https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/bin/convertMotifCoord.pl )
perl convertMotifCoord.pl TFBS_peaks/fimo.gff | sed -e 's/_tnt//g' - | sort -k1,1 -k2,2n - > TFBS_peaks.motifs.bed
perl convertMotifCoord.pl TFBS_controls/fimo.gff | sed -e 's/_tnt//g' - | sort -k1,1 -k2,2n - > TFBS_controls.motifs.bed
# annotate motif coverage/counts for STARR and control peaks
bedtools annotate -i ../01_Peak_Analysis/STARR_merged_peaks.enhancer_activity.eFDR05.bed -files TFBS_peaks.motifs.bed -both | sort -k1,1 -k2,2n - > STARR_merged_peaks.enhancer_activity.eFDR05.ann.bed
bedtools annotate -i ../01_Peak_Analysis/STARR_CONTROL.enhancer_activity.bed -files TFBS_controls.motifs.bed -both | sort -k1,1 -k2,2n - > STARR_CONTROL.enhancer_activity.ann.bed
# extract genes
perl -ne 'if($_ =~ /^#/){next;}chomp;my@col=split("\t",$_);if($col[2] eq 'gene'){print"$_\n";}' ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3 | sort -k1,1 -k4,4n - > ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.genes.gff3
# classify genomic context of STARR and control peaks (you can ignore the warnings from bedtools about inconsistent naming conventions, you can thank the genome assembly team for these annoying, but harmless warnings)
bedtools closest -a STARR_merged_peaks.enhancer_activity.eFDR05.ann.bed -b ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.genes.gff3 -D b > STARR_merged_peaks.enhancer_activity.eFDR05.ann2.bed
bedtools closest -a STARR_CONTROL.enhancer_activity.ann.bed -b ../Genome_Reference/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.genes.gff3 -D b > STARR_CONTROL.enhancer_activity.ann2.bed
# clean up
mv STARR_merged_peaks.enhancer_activity.eFDR05.ann2.bed STARR_merged_peaks.enhancer_activity.eFDR05.ann.bed
mv STARR_CONTROL.enhancer_activity.ann2.bed STARR_CONTROL.enhancer_activity.ann.bed
We can now investigate the relationship among regulatory region size, motif density, and enhancer activity to identify putative regulatory domains (Figure 5A–5F). To do so, we will start an interactive R session and load the annotated peak and control files from above. A script to automate the following code can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/bin/Characterize_Regulatory_Regions.R
# open R
> R
# Analyze regulatory regions
# load libraries
library(vioplot)
library(dplyr)
library(MASS)
library(RColorBrewer)
library(scales)
# load data
starr <- read.table("STARR_merged_peaks.enhancer_activity.eFDR05.ann.bed")
control <- read.table("STARR_CONTROL.enhancer_activity.ann.bed")
# select random control regions to match the filtered STARR peaks
control <- control[sample(nrow(starr)),]
# rename columns for clarity (frac_RR_motif = fraction of regulatory region covered by motifs)
starr[,7:15] <- NULL
control[,7:15] <- NULL
colnames(starr)[4:7] <- c("activity", "motif_counts", "frac_RR_motif", "gene_distance")
colnames(control)[4:7] <- c("activity", "motif_counts", "frac_RR_motif", "gene_distance")
# classify
starr$class <- ifelse((starr$gene_distance < 0 & starr$gene_distance > -200), "TSS", ifelse(starr$gene_distance < -200 & starr$gene_distance > -2000, "promoter", ifelse(starr$gene_distance > 0 & starr$gene_distance < 1000, "TTS", ifelse(starr$gene_distance == 0, "genic", "intergenic"))))
control$class <- ifelse((control$gene_distance < 0 & control$gene_distance > -200), "TSS", ifelse(control$gene_distance < -200 & control$gene_distance > -2000, "promoter", ifelse(control$gene_distance > 0 & control$gene_distance < 1000, "TTS", ifelse(control$gene_distance == 0, "genic", "intergenic"))))
# plot distribution
pdf("STARR_peak_control_genomic_distribution.pdf", width=10, height=5)
layout(matrix(c(1:2), nrow=1))
pie(table(starr$class))
pie(table(control$class))
dev.off()
# estimate regulatory region size (in log10 scale)
starr$size <- log10(starr$V3-starr$V2)
control$size <- log10(control$V3-control$V2)
# compare sizes between peaks and controls (sanity check)
pdf("STARR_peak_control_sizes.pdf", width=5, height=6)
vioplot(starr$size, control$size,
ylab="Interval size (log10)",
col=c("dodgerblue", "grey75"),
names=c(paste0("STARR peaks \n (n=",nrow(starr),")"),
paste0("Control regions \n (n=",nrow(control),")")))
dev.off()
# compare motif counts
pval <- wilcox.test(starr$motif_counts, control$motif_counts)$p.value
pval <- ifelse(pval==0, 2.2e-16, pval)
mean.peak <- mean(starr$motif_counts)
mean.cont <- mean(control$motif_counts)
# find 95% quantile for control motif count
upper.threshold <- quantile(control$motif_counts, 0.95)
# plot
pdf("STARR_peak_control_motif_counts.pdf", width=5, height=6)
vioplot(log1p(starr$motif_counts), log1p(control$motif_counts),
ylab="log2(Motif counts + 1)",
col=c("dodgerblue", "grey75"),
names=c(paste0("STARR peaks \n (n=",nrow(starr),")"),
paste0("Control regions \n (n=",nrow(control),")")),
ylim=c(0,8),
areaEqual=T,
h=0.25)
mtext(paste0("Wilcoxon Rank Sum P-value = ", signif(pval, digits=3)))
text(1, 7.5, labels=paste0("Mean = ", signif(mean.peak, digits=3)))
text(2, 7.5, labels=paste0("Mean = ", signif(mean.cont, digits=3)))
points(1, log1p(upper.threshold), col="red", pch="-")
points(2, log1p(upper.threshold), col="red", pch="-")
dev.off()
# split STARR regions by motif counts based on 95% quantile control dist
starr$group <- ifelse(starr$motif_counts >= upper.threshold, "high", "low")
pval <- kruskal.test(starr$activity, starr$group)$p.value
pdf("STARR_peak_activity_vs_group.pdf", width=5, height=6)
vioplot(starr$activity~starr$group,
ylab="Enhancer activity",
col=c("dodgerblue4", "dodgerblue"),
names=c(paste0("Motif-enriched \n STARR peaks \n (n=", nrow(starr[starr$group=="high",]),")"),
paste0("Motif-depleted \n STARR peaks \n (n=", nrow(starr[starr$group=="low",]),")")),
areaEqual=F,
xlab="",
h=0.25)
mtext(paste0("Kruskal-Wallis rank sum P-value = ", signif(pval, digits=3)))
dev.off()
# compare STARR region size
pval <- kruskal.test(starr$size, starr$group)$p.value
pval <- ifelse(pval==0, 2.2e-16, pval)
pdf("STARR_peak_size_vs_group.pdf", width=5, height=6)
vioplot(starr$size~starr$group,
ylab="Interval size (log10)",
col=c("dodgerblue4", "dodgerblue"),
names=c(paste0("Motif-enriched \n STARR peaks \n (n=", nrow(starr[starr$group=="high",]),")"),
paste0("Motif-depleted \n STARR peaks \n (n=", nrow(starr[starr$group=="low",]),")")),
areaEqual=F,
xlab="",
h=0.25)
mtext(paste0("Kruskal-Wallis rank sum P-value = ", signif(pval, digits=3)))
dev.off()
# compare motif coverage
pval <- kruskal.test(starr$frac_RR_motif, starr$group)$p.value
pval <- ifelse(pval==0, 2.2e-16, pval)
pdf("STARR_peak_motif_coverage_vs_group.pdf", width=5, height=6)
vioplot(starr$frac_RR_motif~starr$group,
ylab="Fraction motif coverage",
col=c("dodgerblue4", "dodgerblue"),
names=c(paste0("Motif-enriched \n STARR peaks \n (n=", nrow(starr[starr$group=="high",]),")"),
paste0("Motif-depleted \n STARR peaks \n (n=", nrow(starr[starr$group=="low",]),")")),
areaEqual=F,
xlab="")
mtext(paste0("Kruskal-Wallis rank sum P-value = ", signif(pval, digits=3)))
dev.off()
# split by group
starr.me <- subset(starr, starr$group=="high")
starr.md <- subset(starr, starr$group=="low")
write.table(starr.me, file="STARR_starrs_peaks.enhancer_activity.eFDR05.ann.high_motif.bed",
quote=F, row.names=F, col.names=F, sep="\t")
write.table(starr.md, file="STARR_starrs_peaks.enhancer_activity.eFDR05.ann.low_motif.bed",
quote=F, row.names=F, col.names=F, sep="\t")
Figure 5. Identification of motif-dense enhancer regulatory domains. A. Genomic distribution of self-transcribed active regulatory region (STARR) peaks (left) and control regions (right). B. Distribution of control region (grey) and STARR peak (blue) interval lengths. C. Distribution of motif counts in control regions (grey) and STARR peaks (blue). The dashed red line indicates the 95% quantile of motif counts from control regions used to classify STARR peaks into high- and low-motif count classes. D. Distribution of enhancer activity for STARR peaks with enriched (dark blue) and depleted (light blue) motif counts. E. Distribution of interval lengths for motif-enriched (dark blue) and motif-depleted (light blue) STARR peaks. F. Distribution of fraction of STARR peaks covered by motif for motif-enriched (dark blue) and motif-depleted (light blue) STARR peaks. G. Heatmap illustrating Z-score-transformed motif-enhancer activities across intergenic motif-enriched STARR peaks scaled by the relative chromatin accessibility in various maize cell types.
3. To determine if the large intergenic regulatory domain regions are associated with cell identity, we will compare enhancer activities vs. various cell type–specific ACRs leveraging a recent single-cell ATAC-seq (scATAC-seq) dataset from multiple maize organs (Marand et al., 2021). First, download the matrix containing normalized accessibility counts across accessible chromatin regions for each profiled cell type. We then extract ACR genomic coordinates (which are in version 4 of the B73 reference genome) and convert them to version 5 of the B73 reference genome using the CrossMap tool and chain file.
# download the counts matrix
wget -O maize_scATAC_atlas_ACR_celltype_CPM.txt.gz https://www.ncbi.nlm.nih.gov/geo/download/\?acc\=GSE155178\&format\=file\&file\=GSE155178%5Fmaize%5FscATAC%5Fatlas%5FACR%5Fcelltype%5FCPM%2Etxt%2Egz
# unzip
gunzip maize_scATAC_atlas_ACR_celltype_CPM.txt.gz
# download chain file
wget https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/chain_files/B73_RefGen_v4_to_Zm-B73-REFERENCE-NAM-5.0.chain
# extract coordinates and conform chromosome names to V4 reference
cut -f1 maize_scATAC_atlas_ACR_celltype_CPM.txt \
| grep '^chr' - \
| perl -ne 'chomp;my@col=split("_",$_);print"$col[0]\t$col[1]\t$col[2]\n";' - \
| sed -e 's/chrB73V4ctg/B73V4_ctg/g' - \
| sed -e 's/chr//g' - \
| sort -k1,1 -k2,2n - > maize_scATAC_atlas_ACRs.bed
# convert ACR coordinates from V4 to V5
CrossMap.py bed B73_RefGen_v4_to_Zm-B73-REFERENCE-NAM-5.0.chain maize_scATAC_atlas_ACRs.bed > maize_scATAC_atlas_ACRs.V4_to_V5.txt
# discard unmapped and split projections
grep -v 'Unmap\|split' maize_scATAC_atlas_ACRs.V4_to_V5.txt \
| perl -ne 'chomp; my@col=split("\t",$_); print"chr$col[0]_$col[1]_$col[2]\tchr$col[4]_$col[5]_$col[6]\n";' - \
| sort -k1,1 -k2,2n - \
| sed -e 's/chrscaf/scaf/g' - \
| sed -e 's/chrB73V4_ctg/chrB73V4ctg/g' - > maize_scATAC_atlas_ACRs.V4_to_V5.clean.txt
# update ACR coordinates in matrix file using R
> R
# read into data frames
conv <- read.table("maize_scATAC_atlas_ACRs.V4_to_V5.clean.txt")
mat <- read.table("maize_scATAC_atlas_ACR_celltype_CPM.txt")
# subset mat rows by retained ACRs after projection
shared <- intersect(rownames(mat), as.character(conv$V1))
mat <- mat[shared,]
rownames(conv) <- conv$V1
conv <- conv[shared,]
# update mat rowIDs
rownames(mat) <- conv$V2
# save output
write.table(mat, file="maize_scATAC_atlas_ACR_celltype_CPM.V5.txt", quote=F, row.names=T, col.names=T, sep="\t")
# exit R
q()
# remove temporary files
rm maize_scATAC_atlas_ACR_celltype_CPM.txt maize_scATAC_atlas_ACRs.bed maize_scATAC_atlas_ACRs.V4_to_V5.txt maize_scATAC_atlas_ACRs.V4_to_V5.clean.txt
4. Intersect the scATAC-seq ACRs with the STARR peaks with enriched motif counts. Load the scATAC-seq matrix and intersected ACRs/STARR peaks files into R to estimate enhancer activity enrichment scaled by relative accessibilities across cell types. As the STARR-seq data was derived from maize seedlings, we further restrict the analysis of scATAC-seq cell types to those derived primarily from maize seedlings. The following code written in R can be executed with the script named “motif_enhancer_activity_maize_celltypes.R” and provides estimates of enhancer activity over various motifs scaled by the relative cell type accessibility, allowing insights into cell type–specific transcription factor regulation of active enhancers (Figure 5G).
# extract ACR coordinates
cut -f1 maize_scATAC_atlas_ACR_celltype_CPM.V5.txt| grep -v 'unknown.5.50' | sed -e 's/scaf_/scaf/g' | perl -ne 'chomp;my@col=split("_",$_);print"$col[0]\t$col[1]\t$col[2]\n";' - | sed -e 's/scaf/scaf_/g' - | sort -k1,1 -k2,2n - > maize_scATAC_atlas_ACRs.V5.bed
# intersect scATAC ACRs with high motif counts STARR peaks
bedtools intersect -a STARR_starrs_peaks.enhancer_activity.eFDR05.ann.high_motif.bed -b maize_scATAC_atlas_ACRs.V5.bed -wa -wb > STARR_starrs_peaks.enhancer_activity.eFDR05.ann.high_motif.scATAC_ACRs.bed
# map enhancer activity over motifs
bedtools map -a TFBS_peaks.motifs.bed -b ../BED_files/B73_maize.enhancer_activity.bdg -c 4 -o max > TFBS_peaks.motifs.enhancer_activity.bed
# motifs to large regulatory regions
bedtools intersect -a TFBS_peaks.motifs.enhancer_activity.bed -b STARR_starrs_peaks.enhancer_activity.eFDR05.ann.high_motif.scATAC_ACRs.bed -wa -wb > TFBS_peaks.motifs.enhancer_activity.bed
# open R (alternatively, a script to automate the following code can be found here: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq/blob/master/workflow/bin/motif_enhancer_activity_maize_celltypes.R)
> R
# estimate enhancer activity cell type specificity
# load libraries
library(RColorBrewer)
library(gplots)
library(edgeR)
# load data
enh <- read.table("STARR_starrs_peaks.enhancer_activity.eFDR05.ann.high_motif.scATAC_ACRs.bed")
acrs <- read.table("maize_scATAC_atlas_ACR_celltype_CPM.V5.txt")
motifs <- read.table("TFBS_peaks.motifs.ENRICHED.enhancer_activity.bed")
# subset for representative leaf-derived clusters
keep <- c("bulliform.2.26",
"bundle_sheath.2.16",
"ground_meristem.7.69",
"guard_cell.7.74",
"guard_mother_cell.7.71",
"L1_SAM.4.46",
"leaf_provascular.7.67",
"mesophyll.2.14",
"parenchyma.10.90",
"protoderm.7.72",
"stomatal_precursor.7.75",
"subsidiary.7.68")
all.acrs <- acrs
# rescale acrs
acrs <- cpm(acrs, log=F)
acrs <- acrs[,keep]
# subset enhancers by genomic feature
enh <- subset(enh, enh$V8=="intergenic")
# get overlapping regions from the scATAC matrix
enh$ids <- paste(enh$V11,enh$V12,enh$V13,sep="_")
enh <- enh[order(enh$V11, decreasing=T),]
enh <- enh[!duplicated(enh$ids),]
shared <- intersect(enh$ids, rownames(acrs))
rownames(enh) <- enh$ids
enh <- enh[shared,]
enh$starrIDs <- paste(enh$V1, enh$V2, enh$V3,sep="_")
# filter motifs
motifs$starrIDs <- paste(motifs$V5, motifs$V6, motifs$V7, sep="_")
motifs <- motifs[motifs$starrIDs %in% unique(enh$starrIDs),]
# normalize acrs
acrs <- t(apply(acrs, 1, function(x){x/max(x)}))
# iterate over each cell type
cts <- colnames(acrs)
outs <- lapply(cts, function(x){
access <- acrs[rownames(enh),x]
names(access) <- enh$starrIDs
motif.scores <- access[motifs$starrIDs] * as.numeric(as.character(motifs$V15))
mtf <- data.frame(motif=motifs$V4, score=motif.scores)
aves <- aggregate(score~motif, data=mtf, FUN=mean)
score <- aves$score
names(score) <- aves$motif
return(score)
})
outs <- do.call(cbind, outs)
colnames(outs) <- cts
vars <- apply(outs, 1, var)
outs <- outs[vars > 0,]
z <- as.matrix(t(scale(t(outs))))
# # cluster columns
co <- hclust(dist(t(outs)))$order
# reorder rows
z <- z[,co]
row.o <- apply(z, 1, which.max)
z <- z[order(row.o, decreasing=F),]
# cap
z[z < -3] <- -3
z[z > 3] <- 3
# get family
tfs <- data.frame(do.call(rbind, strsplit(rownames(z), "\\.")))
cols2 <- colorRampPalette(brewer.pal(12, "Paired"))(length(unique(tfs$X1)))
tfs$cols2 <- cols2[factor(tfs$X1)]
# visualize
pdf("celltype_starr_motif_activity.pdf", width=10, height=10)
heatmap.2(z, scale="none", trace='none',
RowSideColors=tfs$cols,
col=colorRampPalette(rev(brewer.pal(9, "RdBu")))(100),
useRaster=T, Colv=F, Rowv=F, dendrogram="none", margins=c(9,9))
dev.off()
Acknowledgments
This study was funded by support from the National Science Foundation (DBI-1906869) and the National Institute of Health (1K99GM144742) to A.P.M. The ATAC-STARR-seq data analyzed in this study was originally generated by Ricci, Lu, Ji, and colleagues (Ricci et al., 2019).
Competing interests
A.P.M. declares no competing interests.
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Supplementary information
Data and code availability: All data and code have been deposited to GitHub: https://github.com/Bio-protocol/Maize_ATAC_STARR_seq
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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4,954 | https://bio-protocol.org/en/bpdetail?id=4954&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Identification of Accessible Chromatin Regions with MNase-seq
CK Chuizheng Kong *
HP Hongcui Pei *
ZL Zefu Lu
LG Lifeng Gao
GZ Guangyao Zhao
XL Xu Liu
JJ Jizeng Jia
(*contributed equally to this work)
Published: Mar 20, 2024
DOI: 10.21769/BioProtoc.4954 Views: 150
Reviewed by: Hassan Rasouli Anonymous reviewer(s)
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Abstract
The study of accessible chromatin, also known as open chromatin, is currently a hot spot in the research of chromatin non-coding cis-regulatory elements and cis-trans controls of gene expression. Compared to animals, the accessible chromatin is different and relatively conserved across plant species. The identification of accessible chromatin regions (ACRs) in plants promotes our understanding of gene regulation, plant development, and regulatory changes underlying phenotypic evolution. Here, we describe an approach to identify wheat ACRs using differential MNase-seq. Micrococcal nuclease (MNase) is highly sensitive to digestion degree; it tends to cut accessible regions in case of light digestion and more closed regions in case of heavy digestion. We set up gradients of high- and low-concentration MNase digestion and performed high-throughput sequencing of DNA fragments near the length of mononucleosomes in the fragments digested by the two gradients. By comparing the differences in read enrichment under the two concentrations, we defined wheat genome regions highly sensitive to the change of digestion degree as ACRs and regions highly insensitive to the change as closed chromatin regions and identified nucleosome occupancy profiles as well. In short, we modified and refined the method from Rodgers-Melnick et al. (2016) for identifying open chromatin in maize, optimizing the nuclei extraction and ACRs identification for polyploidy, making its application in plants more intuitive, fast, and easy to operate. This method allows us to use MNase-seq to more easily identify ACRs in polyploid plants or large-genome species and to make multiple comparisons with ACRs obtained by other methods, so as to better facilitate the study of plant ACRs.
Graphical overview
Keywords: MNase-seq Differential nuclease sensitivity Accessible chromatin Open chromatin Wheat
Background
Accessible chromatin regions (ACRs) of eukaryotes generally imply the functional genome of the species or the collection of cis-regulatory elements (CREs) (Lu et al., 2019), such as regulatory elements located in promoters and enhancers (Yan et al., 2019). By identifying ACRs, important non-coding CREs and their roles in the regulation of gene expression, species growth and development, environmental adaptation, and natural evolution can be explored. There are several methods to identify ACRs, the most common of which are DNase-seq, ATAC-seq, and MNase-seq (Tsompana and Buck, 2014; Klein and Hainer, 2020). Unlike the enzymes DNase I used in DNase-seq and Tn5 used in ATAC-seq, MNase has both endonuclease and exonuclease activities, which digest and degrade naked DNA, leaving only sequences bound by repressors such as nucleosomes or DNA-binding proteins. The process is highly sensitive to the degree of digestion (i.e., MNase dose or concentration) and can be used to study nucleosome occupancy and digestion sensitivity, indirectly obtaining ACRs. Compared with DNase-seq and ATAC-seq, MNase-seq can identify some ACRs that cannot be identified by the other methods (Zhao et al., 2020). For example, in Arabidopsis thaliana, 20% more ARCs were identified by MNase-sensitive sites, obtained by sequencing 20–100 bp fragments, than by DNase-seq or ATAC-seq reads coverage. Meanwhile, MNase-seq can be used to estimate and contrast histone and non-histone DNA-binding components (Chereji et al., 2017) and identify both open and closed chromatin regions (Vera et al., 2014). However, the identification of plant ACRs with this method has also the disadvantage of demanding more sequencing material and higher sequencing depth, which requires weighing the pros and cons.
There are multiple strategies for identifying ACRs by MNase-seq. One is to select and sequence small DNA fragments (generally significantly smaller than the length of a mononucleosome, such as <80 bp or <100 bp), which may be the binding sites of some non-histone DNA-binding proteins (e.g., TF), directly based on MNase light digestion and define enrichment location of these small fragments as ACRs (Zhao et al., 2020). An alternative strategy is to define ACRs by processing with MNase digestion concentration gradients and sequencing DNA fragments that show a length close to that of mononucleosomes, to detect loci with changes in susceptibility to digestion (Rodgers-Melnick et al., 2016). Another strategy is to use MNase-seq and then combine it with ChIP-seq of histone subunit H3 or H4 for capture, to indirectly calculate the ACRs (Cook et al., 2017). Using these strategies, ACRs have been identified, and cis-acting elements have been found in human and mammalian species, Drosophila, yeast, Arabidopsis, maize, rice, and wheat (Fang et al., 2016; Mieczkowski et al., 2016; Rodgers-Melnick et al., 2016; Brahma and Henikoff, 2019; Schwartz et al., 2019; Jordan et al., 2020; Zhao et al., 2020). There may be differences in the ACRs obtained by the above strategies or in different species. This is mainly due to different research objectives, with differences in the definition of accessible chromatin and the target ACRs (a typical example is the controversy about fragile nucleosomes). Here, we provide a detailed protocol for this MNase-seq method in the identification of ACRs using the strategy of differential nuclease sensitivity, illustrated by data from polyploid crop wheat, while optimized for plant cell nuclei extraction and polyploid genome-specific alignment (Figure 1).
Figure 1. Schematic overview of the protocol.The experimental flow is shown on the left and the data analysis flow isshown on the right. MSF: MNase-sensitive footprint; MRF: MNase-resistantfootprint; ACR: accessible chromatin region.
Materials and reagents
Materials
Wheat seedlings at the three-leaf stage (Aikang58, CAAS)
Miracloth (Calbiochem, catalog number: 475855-1R)
50 mL centrifuge tube (Corning, catalog number: 430290)
5 mL pipette (Corning, catalog number: 4487)
Reagents
PIPES (BBI, catalog number: A600719)
Sorbitol (BBI, catalog number: A610491)
EGTA (Millipore, catalog number: 324626)
DTT (Thermo Fisher, catalog number: R0861)
Spermine (Sigma-Aldrich, catalog number: S3256)
Spermidine (Sigma-Aldrich, catalog number: S2626)
37% Formaldehyde (Sigma-Aldrich, catalog number: 818708)
PMSF (Thermo Fisher, catalog number: 36978)
Glycine (Diamond, catalog number: A100167)
1 M Tris-HCl (pH 7.5) (Sangon, catalog number: B548124)
0.5 M EDTA (pH 8) (Sangon, catalog number: B540625)
Sucrose (Diamond, catalog number: A100335)
MgCl2 (Sigma-Aldrich, catalog number: M8266)
CaCl2 (Sigma-Aldrich, catalog number: C3306)
KCl (Sigma-Aldrich, catalog number: P9541)
NaCl (Sigma-Aldrich, catalog number: S3014)
SDS (Thermo Fisher, catalog number: 28364)
Phenol:Chloroform:Isoamyl alcohol 25:24:1 (Wako, catalog number: 311-90151)
RNase (Thermo Fisher, catalog number: EN0531)
Isopropanol (Sangon, catalog number: A507048)
1× TE buffer (Sangon, catalog number: B548106)
Glycerol (Diamond, catalog number: A100854)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
Percoll (GE, catalog number: 17-0891)
MNase (Thermo Fisher, catalog number: 88216)
Proteinase K (Solarbio, catalog number: 17-0891)
NEBNext UltraTM DNA Library Prep kit for Illumina (NEB, catalog number: E7645S)
Qiaex II gel extraction kit (Qiagen, catalog number: 20021)
Solutions
Nuclei isolation buffer (see Recipes)
Fixation buffer (see Recipes)
Percoll cushion solution (see Recipes)
MNase digestion buffer (see Recipes)
Recipes
Note: All concentrations listed are final concentrations.
Nuclei isolation buffer
15 mM PIPES (NaOH at pH 6.8)
0.32 M sorbitol
80 mM KCl
20 mM NaCl
0.5 mM EGTA
2 mM EDTA
1 mM DTT
0.15 mM spermine
0.5 mM spermidine
Fixation buffer
Nuclei isolation buffer, add 1% formaldehyde and 0.1 mM PMSF freshly
Percoll cushion solution
50% (vol/vol) Percoll in nuclei isolation buffer
MNase digestion buffer
50 mM Tris-HCl at pH 7.5
320 mM sucrose
4 mM MgCl2
1 mM CaCl2
Equipment
Mortar and pestle (Avantor, catalog number: HALDL55/1/G)
Beaker (30 mL) (PYREX, catalog number: 1000-30)
Magnetic stirrer (Thermo Fisher, catalog number: S194615)
Hybridization oven (Galanz, catalog number: P70J17L-V1)
4 °C centrifuge (Eppendorf, catalog number: 5804)
Nanodrop 2000 (Thermo Fisher, catalog number: 13-400-412)
Software
Deeptools v3.4.3 (Max Planck Institute for Immunobiology and Epigenetics; https://deeptools.readthedocs.io/en/latest/index.html) (February 2022)
Samtools v1.6 (Genome Research Limited; http://www.htslib.org/doc/samtools.html) (February 2022)
BEDTools v2.29.1 (University of Utah; https://bedtools.readthedocs.io/en/latest/content/bedtools-suite.html) (February 2022)
R v3.2.2 (Lucent Technologies; https://www.r-project.org/) (March 2022)
IGV v2.5.0 (University of California; http://software.broadinstitute.org/software/igv/) (March 2022)
Trimmomatic v0.36 (THE USADEL LAB; http://www.usadellab.org/cms/?page=trimmomatic) (February 2022)
Bowtie2 v2.3.4 (Johns Hopkins University; http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) (February 2022)
FastQC v0.11.8 (Babraham Bioinformatics; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (February 2022)
Procedure
Material fixation
Sampling: Harvest 10 g of leaves or roots (water rinsing) from the three-leaf-stage wheat seedlings after 21 days of seeds’ germination and immediately freeze the tissues in liquid nitrogen. Store at -80 °C.
Grinding: Ground the frozen materials under liquid nitrogen using a mortar and pestle (previously cooled with liquid nitrogen).
Fixation: Prepare 100 mL of pre-cooled fixation buffer in a beaker: add 2.78 mL of 37% formaldehyde to 1% and 102 μL of 100 mM PMSF to 0.1 mM. Pour the ground sample into the beaker and magnetically stir the sample for 10 min (100–200× g).
Termination: Stop the reaction by adding 6.87 mL of 2 M glycine stock to 125 mM and magnetically stir for another 5 min.
Nuclear extraction
Stirring: Slowly add 12 mL of 10% (vol/vol) Triton X-100 stock to 1%, gently agitate, and incubate for 10 min. Pre-cool the refrigerated centrifuge now.
Filter: Filter the solution into a beaker with two layers of Miracloth filter cloth and add 15 mL of Percoll cushion solution into a 50 mL centrifuge tube. Gently add 35 mL of the filtrate with a pipette on the top of the solution surface and ensure the liquid flows down along the tube wall without disturbing the boundary layer. Centrifuge at 3,000× g for 15 min at 4 °C (acceleration and deceleration should be slow) (see Note 1).
Transfer: The middle layer of the centrifuged Percoll filtrate contains the nuclei; transfer this layer to 50 mL centrifuge tubes (see Note 1) and add a 1-fold volume of MNase digestion buffer. Centrifuge at 2,000× g for 10 min at 4 °C.
Reconstitution: Re-dissolve the precipitate from each 35 mL in 2.5 mL of MNase digestion buffer and dispense the sample into five tubes with 500 μL each. Measure and record the DNA concentration by Nanodrop 2000 (approximately 50–150 ng/µL).
Microscopy: Observe the morphology of the nuclei with a fluorescence microscope. (The nuclei can be flash-frozen in liquid nitrogen and stored at -80 °C for up to one month.)
Digestion and de-crosslinking
Digestion: Thaw proteinase K, MNase, and five tubes of nuclei samples on ice (the nuclei can only be thawed once); then, treat two tube samples with 10 U/mL MNase (light), two tube samples with 100 U/mL MNase (heavy), and one tube sample as a control. Incubate all for 5 min at room temperature (see Note 2).
Termination: Add 10.2 μL of 0.5 M EGTA stock to 10 mM and vibrate the samples to stop the digestion reaction.
De-crosslinking: Add 57.5 μL of 10% SDS to 1% and 5.75 μL of 10 μg/μL proteinase K to 100 μg/mL, vibrate the samples, and incubate overnight in a 65 °C hybridization oven (>6.5 h).
Product extraction
DNA extraction: Add an equal volume of 600 μL of Phenol:Chloroform:Isoamyl alcohol 25:24:1 and mix thoroughly by inversion. Centrifuge at 12,000× g for 10 min at room temperature. Transfer the supernatant into new 1.5 tubes, add 5 μL of RNase to 40 μg/μL, mix by inverting, and incubate at 37 °C for 15 min. Add 400 μL of pre-cooled isopropanol and mix gently. Then, place tubes at -20 °C for 20 min to precipitate DNA. Pre-cool the centrifuge and centrifuge at 12,000× g for 10 min at 4 °C. Discard the supernatant, add 1 mL of 70% ethanol, mix by inverting, and wash twice. Dry the remaining ethanol for 20 min in a fume hood. Dissolve the precipitate in 30 μL of 1× TE buffer.
Fragment selection: Run 1% agarose gel (using single-color loading), select and extract the 100–200 bp DNA fragment (Figure 2, Note 2), and purify the DNA using the Qiaex II gel extraction kit.
Figure 2. Ladders of MNase-digested DNA bands. The nuclei are treated with different concentrations of MNase and the DNA fraction from 100 to 200 bp is purified from the gel and sequenced.
Library preparation and sequencing
Library construction: Construct the MNase-seq library using the NEBNext® UltraTM DNA Library Prep kit for Illumina® according to the instructions, where approximately 1 μg of total DNA is used.
High-throughput sequencing: Sequence the libraries on the Illumina platform HiSeq2000 system (or any Illumina sequencing instrument) to obtain 150 bp paired-end reads with 300 million reads (~90 G) for each sample of hexaploid wheat (genome size ~15 G) (see Note 3). Sequence the libraries for two biological replicates.
Data analysis
The analysis of arbitrary MNase-seq data can be achieved through the following instructions, with only minor modifications to the given code. Software used in this protocol can be easily installed via the conda command of anaconda ( https://www.anaconda.com/products/individual) or downloaded from the official website and installed by following the instructions. Make sure that the software is installed and the environment is set up normally in your Linux device before the program is executed.
Uploading data: Create a new working directory to store all the MNase-seq-related data and files and save all the raw data to a dedicated folder under that directory.
cd data1/user/path # enter your working directory
mkdir MNase_seq
cd MNase_seq
mkdir raw_data # move all your sequencing data to this directory
Quality control: Use FastQC to evaluate the quality of raw data obtained by sequencing, including basic statistics, base and sequence quality, GC content, sequence length distribution, adapter content, and other indicators of data.
mkdir ./fastqc_result
for i in ./raw_data/*.fq.gz
do
fastqc -o ./fastqc_result $i
done
Reads filtering: Use software such as Trimmomatic to filter the raw data to obtain clean data. The main goal of this step is to remove the adapter sequence, leading and trailing low-quality or N bases and sequences too short in length. When renaming the filtered data, tissue, digestion, and replicate should be considered. For example, “leaf_light_rep1” can be used to represent the first replicate with light digestion in leaves, and “root_heavy_rep2” can represent the second replicate with heavy digestion in roots.
mkdir clean_data
java -jar trimmomatic-0.36.jar PE -phred33 \
./raw_data/*.r1.fq.gz \
./raw_data/*.r2.fq.gz \
./clean_data/$name.1P.fq \
./clean_data/$name.1U.fq \
./clean_data/$name.2P.fq \
./clean_data/$name.2U.fq \
ILLUMINACLIP:data1/user/tools/Trimmomatic-0.36/adapters/\
TruSeq3-PE-2.fa:2:30:10:1:true \
LEADING:5 TRAILING:5 MINLEN:20 >trimmomatic.$name.log
Quality control: Use FastQC again to evaluate the quality of the clean data. If there is any undesirability, the parameters of the previous step can be adjusted and re-filtered until the reads can be used for the alignment in the next step.
for i in ./clean_data/*.fq.gz
do
fastqc -o ./fastqc_result $i
done
Sequence alignment: The filtered clean data is aligned to the reference genome using Bowtie2 software. Before that, library the genome with bowtie2-build and index the fasta file with Samtools faidx. Use the same parameters as suggested in maize for alignment.
bowtie2-build ref_genome.fa ref.genome
samtools faidx ref_genome.fa
bowtie2 --phred33 -p 10 --reorder -5 6 \
--no-mixed --no-discordant --no-unal --dovetail \
-x /index_path/ref.genome \
-1 ./$name.1P.fq \
-2 ./$name.2P.fq \
-S ./$name.sam >bowtie2.$name.log
Non-unique and duplicate alignments removal: Screen according to the MAPQ value in the alignment results. The larger the MAPQ value, the higher the confidence of the unique alignment. Here, we extract the part of MAPQ > 20. After that, remove the repeated reads due to PCR library amplification. Different software, like Picard, Sambamba, or Samtools, remove PCR duplicates. Here, we used Samtools markdup, before which the paired-end sequencing data were sorted by reads name and coordinates successively. A large index of the bam file should be established after that for the large genome.
function samtools_scripts(){
samtools view -q 20 -bhS $name.sam -o $name.Q20.bam
samtools sort -n $name.Q20.bam -o $name.Q20.nsort.bam
samtools fixmate -m $name.Q20.nsort.bam $name.Q20.nsort.ms.bam
samtools sort $name.Q20.nsort.ms.bam -o $name.Q20.ms.psort.bam
samtools markdup -r $name.Q20.ms.psort.bam $name.Q20.psort.markdup.bam
samtools index -c $name.Q20.psort.markdup.bam
}
samtools_scripts $name >samtools.$name.log
Differential nuclease sensitivity (DNS) identification: In the code here and later, we take “leaf_light_rep1” and “leaf_heavy_rep1” as examples for demonstration; you can replace the name when running other samples. The mapped reads of heavy digestion are subtracted from light digestion using bamCompare in the deepTools and are then normalized by the CPM method to output a DNS score file in bedgraph format. For this step, a 10-bp bin is used for division and statistics, and then a 50-bp window is used for smoothing.
bamCompare -b1 leaf_light_rep1.bam -b2 leaf_heavy_rep1.bam \
--scaleFactorsMethod None --normalizeUsing CPM --operation subtract \
--binSize 10 --smoothLength 50 --outFileFormat bedgraph \
-o leaf_diff_rep1.bdg
Bayes factor calculation: Here, reads coverage from both light-digested and heavy-digested samples is calculated, and the CPM method is used for standardizing; then, the Bayes factor is calculated for reads coverage in 10-bp intervals. You can speed up the program by running splitted files and calculating their Bayes values separately.
bamCoverage -b leaf_light_rep1.bam \
--binSize 10 --smoothLength 50 \
--normalizeUsing CPM --outFileFormat bedgraph \
-o leaf_light_rep1.bdg
bamCoverage -b leaf_heavy_rep1.bam \
--binSize 10 --smoothLength 50 \
--normalizeUsing CPM --outFileFormat bedgraph \
-o leaf_heavy_rep1.bdg
bedtools unionbedg \
-i leaf_light_rep1.bdg leaf_heavy_rep1.bdg \
-header -names light heavy \
>leaf_rep1.unionbdg
# run the R script to obtain a file named “leaf_rep1.unionbdg.bayes”
Rscript bayes_factor_caculator.R leaf_rep1.unionbdg
Accessible chromatin regions identification: Based on the Bayes values in the “leaf_rep1.unionbdg.bayes” file, identify MNase-sensitive footprints (MSFs) and MNase-resistant footprints (MRFs) in the “leaf_diff_rep1.bdg” file. Parts with DNS values greater than 0 and Bayes values greater than 0.5 are defined as significant MSFs; parts with DNS values lower than 0 and Bayes values greater than 0.5 are defined as significant MRFs (see Note 4). The significant MSF or MRF signals within 200 bp are merged into one MSF or MRF signal. The significant MSFs are also referred to as MNase-hypersensitive (MNase HS) regions or ACRs (see Note 5).
bedtools unionbedg \
-i leaf_diff_rep1.bdg leaf_rep1.unionbdg.bayes \
>leaf.diff_rep1_bayes
cat leaf.diff_rep1_bayes | awk '$4>0 && $5>0.5' | \
cut -f1-3 | bedtools merge -d 200 \
>leaf.MSF_rep1_bayes_0.5_merge_200.bed
cat leaf.diff_rep1_bayes | awk '$4<0 && $5>0.5' | \
cut -f1-3 | bedtools merge -d 200 \
>leaf.MRF_rep1_bayes_0.5_merge_200.bed
Signal visualization: The reads coverage depth of the samples obtained by different digestion treatments and the sensitivity imprint DNS values can be generated with bigwig files by deepTools and then loaded into the Integrative Genomics Viewer (IGV browser) for browsing. The MSF or MRF location files can also be loaded into the IGV browser.
bamCoverage -b leaf_light_rep1.bam \
--binSize 10 --smoothLength 50 \
--normalizeUsing CPM --outFileFormat bigwig \
-o leaf_light_rep1.bw
bamCoverage -b leaf_heavy_rep1.bam \
--binSize 10 --smoothLength 50 \
--normalizeUsing CPM --outFileFormat bigwig \
-o leaf_heavy_rep1.bw
bamCompare -b1 leaf_light_rep1.bam -b2 leaf_heavy_rep1.bam \
--scaleFactorsMethod None --normalizeUsing CPM --operation subtract \
--binSize 10 --smoothLength 50 --outFileFormat bigwig \
-o leaf_diff_rep1.bw
Attached R script for calculating Bayes factor: Upload the script to the same folder as “leaf_rep1.unionbdg” before use and then modify the working path, nh, nf, and other assignments according to your experiment.
### bayes factor caculator 1.0 ###
setwd("data1/user/path/MNase_seq")
M=100000
nh=2
nf=2
bayes_factor_caculator <- function(old, new) {
lkd.model1=function(y,n,lambda){ return(exp(-n*lambda+y*log(lambda))) }
lkd.model2=function(y1,n1,y2,n2,lambda1,lambda2){ return(exp(-n1*lambda1+y1*log(lambda1)-n2*lambda2+y2*log(lambda2))) }
BF_MC=function(a,b,y1,n1,y2,n2,M){
lambda1=rgamma(M,a,b)
m1=cumsum(lkd.model1(y1+y2,n1+n2,lambda1))/(1:M)
lambda2.1=rgamma(M,a,b)
lambda2.2=rgamma(M,a,b)
m2=cumsum(lkd.model2(y1,n1,y2,n2,lambda2.1,lambda2.2))/(1:M)
return(m2/m1)
}
con <- file(old,"rt")
line <- readLines(con,n=1)
while (length(line) > 0){
line <- unlist(strsplit(line,split = "\t"))
qh <- as.numeric(line[5])
qf <- as.numeric(line[4])
set.seed(1)
bayes <- BF_MC(10,1,qh,nh,qf,nf,M)[M]
newline=t(c(line[1],line[2],line[3],round(bayes,5)))
write.table(newline, new, col.names = F, row.names = F, sep = '\t', quote=F, append =T)
line <- readLines(con, n = 1)
}
close(con)
# BF_MC(10,1,qh,nh,qf,nf,M)[M]
# qh: total number of mapped reads for heavy-digestion
# nh: number of replicates for heavy-digestion
# qf: total number of mapped reads for light-digestion
# nf: number of replicates for light-digestion
}
args=commandArgs(T)
bayes_factor_caculator(args[1], paste0(args[1], '.bayes'))
Notes
Care should be taken when adding the filtrate to the Percoll cushion, slowly adding the filtrate along the top of the tube wall without causing violent fluctuations in the interface and mixing between liquids. When centrifuging, acceleration and deceleration must be slow. After centrifugation, a cloudy layer of liquid containing the nuclei at the interface between Percoll and the filtrate should be taken out, rather than the precipitation at the bottom of the centrifuge tube.
The timing of MNase and EGTA addition during the digestion reaction should be precisely controlled. Furthermore, the selection of the 100–200 bp fragment should be precise and consistent among different samples and replicates.
We recommend sequencing the library samples using PE150, in which case each fragment can be fully read. The sequencing depth can be determined according to the size of the genome, where the coverage of 6× is used for wheat, and the final actual performance is good. In a specific implementation, a portion of data can be preliminarily measured, and then the amount of deep sequencing data could be determined according to the effect.
When identifying significant MSF and MRF, if the background signal is too high, the threshold of the Bayes factor can be raised or the signals with too short length can be filtered directly to obtain more real and effective signals, which can be adjusted repeatedly in combination with the actual display of IGV browser.
While doing MNase-seq, we recommend performing ChIP-seq of histone modifications, such as activation modification H3K9ac, which are significant features of some ACRs and have good colocalization with ACR signals, which can be used to assist with the identification.
Acknowledgments
This protocol was applied in our open chromatin study in wheat (Kong et al., 2024), which was supported by the Central Public-interest Scientific Institution Basal Research Fund (Y2021YJ01), the National Natural Science Foundation of China (Major Program, 31991213), and the National Natural Science Foundation of China (31971882). We mainly referred to open chromatin research articles in maize (Vera et al., 2014; Rodgers-Melnick et al., 2016). In the development of this procedure, we received help from Eli Rodgers-Melnick and Daniel L. Vera. Professor Zou Cheng at Cornell University helped with our algorithms. Thanks a lot for their help!
Competing interests
There are no conflicts of interest or competing interests.
References
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Supplementary information
Data and code availability: All data and code have been deposited to GitHub: https://github.com/Bio-protocol/ACRs_by_MNase-seq.
Article Information
Copyright
© 2024 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 molecular biology > DNA
Molecular Biology > DNA > Chromatin accessibility
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4,955 | https://bio-protocol.org/en/bpdetail?id=4955&type=0 | # Bio-Protocol Content
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Classification of a Massive Number of Viral Genomes and Estimation of Time of Most Recent Common Ancestor (tMRCA) of SARS-CoV-2 Using Phylodynamic Analsysis
XH Xiaowen Hu *
SG Siqin Guan *
YH Yiliang He
GY Guohui Yi
LY Lei Yao
JZ Jiaming Zhang
(*contributed equally to this work)
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4955 Views: 1208
Reviewed by: Migla MiskinytePooja Verma Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS ONE Jun 2023
Abstract
Estimating the time of most recent common ancestor (tMRCA) is important to trace the origin of pathogenic viruses. This analysis is based on the genetic diversity accumulated in a certain time period. There have been thousands of mutant sites occurring in the genomes of SARS-CoV-2 since the COVID-19 pandemic started; six highly linked mutation sites occurred early before the start of the pandemic and can be used to classify the genomes into three main haplotypes. Tracing the origin of those three haplotypes may help to understand the origin of SARS-CoV-2. In this article, we present a complete protocol for the classification of SARS-CoV-2 genomes and calculating tMRCA using Bayesian phylodynamic method. This protocol may also be used in the analysis of other viral genomes.
Key features
• Filtering and alignment of a massive number of viral genomes using custom scripts and ViralMSA.
• Classification of genomes based on highly linked sites using custom scripts.
• Phylodynamic analysis of viral genomes using Bayesian evolutionary analysis sampling trees (BEAST).
• Visualization of posterior distribution of tMRCA using Tracer.v1.7.2.
• Optimized for the SARS-CoV-2.
Graphical overview
Graphical workflow of time of most recent common ancestor (tMRCA) estimation process
Keywords: Viral origin Genome classification Genetic linkage Bayesian phylodynamic analysis SARS-CoV-2 Redundant genome Diversity
Background
Revealing the origins of pathogenic viruses, crucial for cutting them off from the root and preventing future spillover, requires long-term hard work from scientists all around the world [1]. Although some infectious pathogens can be traced back decades, the debate on their origin continues. For example, AIDS was officially reported on June 5, 1981, by the Centers for Disease Control and Prevention of the USA. Five years later, HIV infection was detected in a human serum sample collected in Léopoldville in early 1959 [2]. Bayesian phylodynamic analyses using recovered viral gene sequences from decades-old paraffin-embedded tissues traced the most recent common ancestor (MRCA) of the M group of HIV back to approximately 1908 (CI 1884–1924), suggesting that HIV has been circulating in the human population for approximately 100 years [3]. MERS-CoV is another example, as it was first reported in a Saudi Arabian man in 2012 [4]. Bats are thought to be the reservoir hosts of MERS-CoV, and dromedary camels are considered to be the major intermediate host [5]; however, the transmission route from animals to humans is not well understood. Researchers tested 189 camel serum samples from 1983 to 1997 and found that 81% had neutralizing antibodies against MERS-CoV, suggesting long-term virus circulation in these animals [6]. Similarly, COVID-19 was first reported on December 27, 2019, in Wuhan, China [7,8], and the Huanan seafood market was suspected to be the place of origin [9]; however, disputes remain. Pekar and colleagues explored the evolutionary dynamics of the first wave of SARS-CoV-2 infections in China using a strict clock Bayesian phylodynamic analysis but failed to capture the index case [10], probably because the redundant sequences were not removed, which usually influences the accuracy of time of MRCA (tMRCA) estimation, as indicated in two recent tMRCA analysis [11,12]. Genome classification plays a critical role in tracing the origin of pathogenic viruses [3,12]. We have previously classified SARS-CoV-2 genomes based on two amino acids, Spike-614 and Orf8-84, and revealed 16 haplotypes. From those, three major haplotypes were found to separately drive the development of the pandemic in China and the world. However, genome classification based on amino acid mutations did not rule out recombination and reverse mutations. In this paper, we provide detailed protocols to filter and classify the massive number of viral genomes according to six highly linked mutations that happened in the early phase of the epidemic by custom scripts and common programs and estimate tMRCA of non-redundant genome subpools.
Materials and reagents
SARS-CoV-2 genome sequences were obtained from the GISAID database [13] on May 1, 2022. Genomes that were collected from hosts other than humans and/or had a length of less than 2,900 nucleotides and more than 0.05% of unknown nucleotides were filtered out. More than 5 million genomes were retained for further analysis.
Equipment
Bioinformatic platform (CentOS, 7.2.1511)
Windows desktop computers (v11, 6/6/2021)
Software and datasets
Perl (v5.34.0, 20/5/2021)
Python (v3.9.0, 5/10/2020)
Gcc (v8.4.1, 28/9/2020)
SeqKit (v2.3.0, 12/8/2022)
minimap2 (v2.24, 26/12/2021)
ViralMSA (v3, 29/6/2007)
cd-hit (v4.8.1, 1/9/2018)
ALTER (v1.3.4, 30/10/2016)
BEAST (v1.10.4, 9/11/2018)
Jdk (v17_windows-x64_bin, 16/6/2021)
Jre (8u341-windows-x64, 5/6/2021)
Tracer (v1.7.2, 5/1/2018)
Global Initiative of Sharing All Influenza Data (GISAID) (https://gisaid.org/) (Access date, 1/5/2022)
All personalized scripts have been deposited in GitHub: https://github.com/XiaowenH/SARS-CoV-2-Classification (Access date, 5/9/2023). Figure 1 is a screenshot of the files in GitHub.
Figure 1. Screenshot of the personalized scripts deposited in GitHub
Procedure
Set up the working environment
Create a Python environment of conda.
$ wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
$ bash Miniconda3-latest-Linux-x86_64.sh -b -p ~/miniconda
$ export PATH="/$HOME/miniconda/bin:$PATH"
$ conda create -n myenv python=3.9
$ conda activate myenv
#Please note that "$HOME" corresponds to the PATH you want to install in the conda environment (the same hereafter).
Install the following software:
Install Seqkit in a Linux platform.
$ conda install -c bioconda seqkit
Install minimap2 in a Linux platform.
$ git clone https://github.com/lh3/minimap2
$ cd minimap2 && make
Install ViralMSA [14] in a Linux platform.
$ wget "https://raw.githubusercontent.com/niemasd/ViralMSA/master/ViralMSA.py"
$ chmod a+x ViralMSA.py
$ sudo mv ViralMSA.py /usr/local/bin/ViralMSA.py
Install CD-HIT in a Linux platform.
$ git clone https://github.com/weizhongli/cdhit.git
$ cd cd-hit
$ make
$ cd cd-hit-auxtools
$ make
Install Alter in a Linux platform.
$ git clone https://github.com/sing-group/ALTER.git
$ cd ALTER
$ /$HOME/apache-maven-3.8.4/bin/mvn
Install BEAST [15] in a Windows platform.
# Download BEAST.v1.10.4 at
http://beast.community/programs
Install BEAGLE in a Windows platform.
# Download BEAGLE at
https://github.com/beagle-dev/beagle-lib
Install Tracer v1.7.2 in a Windows platform.
# Download BEAGLE at
http://beast.community/tracer
Download the viral genomes and metadata
Download SARS-CoV-2 genomes and meta.tsv files from the GISAID database [16] after login (https://gisaid.org/). Note that the content in each column in the meta.tsv may change (e.g., the accession numbers were put in column 3 in the metadata downloaded on May 1, 2022, but in column 5 in the metadata downloaded on May 1, 2023).
Unpack the files.
$ xz -d sequences_fasta_2022_04_29.tar.xz
$ tar -xvf sequences_fasta_2022_04_29.tar
$ xz -d metadata_tsv_2022_04_29.tar.xz
$ tar -xvf metadata_tsv_2022_04_29.tar
Rename the taxa in the Fasta file of the genomes by accession numbers (e.g., EPI_ISL_4405694) using a custom script
$ perl rename_fasta_taxa_to_tsv_acc_column3.pl -t meta.tsv -f sequence.fasta
Fetch GISAID reference sequence
Create a file named “accession.txt,” paste in EPI_ISL_402124, and save.
$ echo ‘EPI_ISL_402124’ >accession.txt
# EPI_ISL_402124 is the reference sequence used by the GISAID database and in many researches.
Fetch the reference sequence from total sequence file.
$ cat sequence.fasta | /$HOME/seqkit grep -f accession.txt -t dna -j 10 -o reference_wiv04.fasta
Retrieve genomes with complete, high coverage sequences and accurate dates and sampled from human hosts
The accession number, host, completeness, and coverage of the genomes are located in columns 3, 8, 18, and 19, respectively, in the metadata of April 29, 2022. The sample collection date is located in column 4. The column number may be different in the metadata downloaded at a different day.
Filter metadata.tsv for accessions with complete genomes, with high coverage, and from human hosts.
$ awk -F '\t' '{if($18 == "True" && $19 == "True" && $8 == "Human") print}' metadata.tsv >global_2022_04_29_human_complete_metadata.tsv
Filter metadata.tsv for accessions with accurate sample collection date using a custom script: dates_filter.pl.
$ perl dates_filter.pl -t global_2022_04_29_human_complete_metadata.tsv -o global_2022_04_29_human_complete_dates_metadata.tsv
Print selected accession numbers to a file from metadata.
$ awk -F '\t' '{print $3}' global_2022_04_29_human_complete_dates_metadata.tsv > global_2022_04_29_human_complete_dates_accessions.txt
Retrieve genome sequences with complete and high coverage and accurate dates.
$ cat sequence.fasta | /$HOME/seqkit grep -f global_2022_04_29_human_complete_dates_accessions.txt -t dna -j 10 -o global_2022_04_29_human_complete_dates_genome.fasta
Align the genome sequences
$ /$HOME/ViralMSA-master/ViralMSA.py -e [your email] -s global_2022_04_29_human_complete_dates_genome.fasta -o output -r reference_wiv04.fasta --omit_ref
All genome classification
Retrieve the six highly linked sites using the custom script fetch_nucleotides_from_alignments.pl (Figure 2).
$ perl fetch_nucleotides_from_alignments.pl -f global_2022_04_29_human_complete_dates_genome.fasta.aln
-r 241-241,3037-3037,8782-8782,14408-14408,23403-23403,28144-28144 -o global_2022_04_29_human_complete_dates_genome.fasta_six_sites.txt
Figure 2. Output format of the six linked sites in the genomes. The six nucleotides of sites 241, 3037, 8782, 14408, 23403, and 28144 were retrieved from SARS-CoV-2 genomes aligned to reference genome wiv04 EPI_ISL_402124.
Classify the genomes into haplotypes by the six linked mutation sites (Figure 3).
$ cat global_2022_04_29_human_complete_dates_genome.fasta_six_sites.txt | /$HOME/seqkit grep -s -i –p CCTCAC > global_2022_04_29_human_complete_dates_DS.txt
$ cat global_2022_04_29_human_complete_dates_genome.fasta_six_sites.txt | /$HOME/seqkit grep -s -i -p CCCCAT > global_2022_04_29_human_complete_dates_DL.txt
$ cat global_2022_04_29_human_complete_dates_genome.fasta_six_sites.txt | /$HOME/seqkit grep -s -i -p TTCTGT > global_2022_04_29_human_complete_dates_GL.txt
$ /$HOME/seqkit stats global_2022_04_29_human_complete_dates_DS.txt >haplotype.statistics.txt
$ /$HOME/seqkit stats global_2022_04_29_human_complete_dates_DL.txt >>haplotype.statistics.txt
$ /$HOME/seqkit stats global_2022_04_29_human_complete_dates_GL.txt >>haplotype.statistics.txt
Figure 3. Pie chart of haplotypes in global SARS-CoV-2 genomes. DS (CCTCAC), DL (CCCCAT), and GL (TTCTGT) are the three main haplotypes classified by the six linked sites.
Fetch accession numbers of each haplotype.
$ /$HOME/seqkit seq global_2022_04_29_human_complete_dates_DS.txt -n > global_DS.accessions.txt
$ /$HOME/seqkit seq global_2022_04_29_human_complete_dates_DL.txt -n > global_DL.accessions.txt
$ /$HOME/seqkit seq global_2022_04_29_human_complete_dates_GL.txt -n > global_GL.accessions.txt
Retrieve aligned genome sequences of each haplotype.
$ cat global_2022_04_29_human_complete_dates_genome.fasta.aln | /$HOME/seqkit grep -f global_DS.accessions.txt -t dna -j 10 -o global_DS.aln
$ cat global_2022_04_29_human_complete_dates_genome.fasta.aln | /$HOME/seqkit grep -f global_DL.accessions.txt -t dna -j 10 -o global_DL.aln
$ cat global_2022_04_29_human_complete_dates_genome.fasta.aln | /$HOME/seqkit grep -f global_GL.accessions.txt -t dna -j 10 -o global_GL.aln
Classification of early genomes collected in the early phase of the pandemic (from beginning to end of April 2020)
The sample collection date is located in column 4 in the metadata of April 29, 2022.
Retrieve accession numbers of early genomes.
$ awk -F '\t' '{if($4~/2019-12/) print}' global_2022_04_29_human_complete_dates_metadata.tsv > global_early_meta.tsv
$ awk -F '\t' '{if($4~/2020-01/) print}' global_2022_04_29_human_complete_dates_metadata.tsv >> global_early_meta.tsv
$ awk -F '\t' '{if($4~/2020-02/) print}' global_2022_04_29_human_complete_dates_metadata.tsv >> global_early_meta.tsv
$ awk -F '\t' '{if($4~/2020-03/) print}' global_2022_04_29_human_complete_dates_metadata.tsv >> global_early_meta.tsv
$ awk -F '\t' '{if($4~/2020-04/) print}' global_2022_04_29_human_complete_dates_metadata.tsv >> global_early_meta.tsv
$ awk -F '\t' '{print $3}' global_early_meta.tsv >global_early_accessions.txt
Retrieve the aligned sequences of the early genomes.
$ cat global_2022_04_29_human_complete_dates_genome.fasta.aln | /$HOME/seqkit grep -f global_early_accessions.txt -t dna -j 10 -o global_early_all_haplotype.fasta.aln
Retrieve the six highly linked sites using the custom script fetch_nucleotides_from_alignments.pl.
$ perl fetch_nucleotides_from_alignments.pl -f global_early_all_haplotype.fasta.aln
-r 241-241,3037-3037,14408-14408,23403-23403,28144-28144 -o global_early.aln_six_sites.txt
Classify the genomes.
$ cat global_early.aln_six_sites.txt | /$HOME/seqkit grep -s -i -p CCTCAC > global_early_DS.txt
$ cat global_early.aln_six_sites.txt | /$HOME/seqkit grep -s -i -p CCCCAT > global_early_DL.txt
$ cat global_early.aln_six_sites.txt | /$HOME/seqkit grep -s -i -p TTCTGT > global_early_GL.txt
Fetch accession numbers of each haplotype.
$ /$HOME/seqkit seq global_early_DS.txt -n > global_early_DS.accessions.txt
$ /$HOME/seqkit seq global_early_DL.txt -n > global_early_DL.accessions.txt
$ /$HOME/seqkit seq global_early_GL.txt -n > global_early_GL.accessions.txt
Retrieve aligned genome sequences of each haplotype.
$ cat global_early_all_haplotype.fasta.aln | /$HOME/seqkit grep -f global_ early_DS.accessions.txt -t dna -j 10 -o global_ early_DS.aln
$ cat global_early_all_haplotype.fasta.aln | /$HOME/seqkit grep -f global_ early_DL.accessions.txt -t dna -j 10 -o global_ early_DL.aln
$ cat global_early_all_haplotype.fasta.aln | /$HOME/seqkit grep -f global_ early_GL.accessions.txt -t dna -j 10 -o global_ early_GL.aln
Bayesian phylodynamic analysis using the early genomes of three haplotypes as examples
Filter out genomes with unknown higher than 0.05%.
Filter out genomes of DS (CCTCAC) haplotypes with unknown nucleotides higher than 0.05%.
# Calculate nucleotide composition of each sequence in each haplotype
$ perl base_stats.pl global_early_DS.aln
# Pick up accessions with unknowns < 0.05%
$ awk -F '\t' '{if($6 < 0.0005) print$1}' global_early_DS.aln.stats > global_early_DS.aln_0.0005N_ID.txt
# Fetch aligned genome sequences with unknowns <0.05%
$ cat global_early_DS.aln |/$HOME/seqkit grep -f global_early_DS.aln_0.0005N_ID.txt -t dna -j 10 -o global_early_DS_0.0005N.aln
Filter out genomes of DL (CCCCAT) haplotypes with unknown nucleotides higher than 0.05%.
# Calculate nucleotide composition of each sequence in each haplotype
$ perl base_stats.pl global_early_DL.aln
# Pick up accessions with unknowns < 0.05%
$ awk -F '\t' '{if($6 < 0.0005) print$1}' global_early_DL.aln.stats > global_early_DL.aln_0.0005N_ID.txt
# Fetch aligned genome sequences with unknowns <0.05%
$ cat global_early_DL.aln |/$HOME/seqkit grep -f global_early_DL.aln_0.0005N_ID.txt -t dna -j 10 -o global_early_DL_0.0005N.aln
Filter out genomes of GL (TTCTGT) haplotypes with unknown nucleotides higher than 0.05%.
# Calculate nucleotide composition of each sequence in each haplotype.
$ perl base_stats.pl global_early_GL.aln
# Pick up accessions with unknowns < 0.05%
$ awk -F '\t' '{if($6 < 0.0005) print$1}' global_early_GL.aln.stats > global_early_GL.aln_0.0005N_ID.txt
# Fetch aligned genome sequences with unknowns <0.05%
$ cat global_early_GL.aln | /$HOME/seqkit grep -f global_early_GL.aln_0.0005N_ID.txt -t dna -j 10 -o global_early_GL_0.0005N.aln
Remove redundant genomes with a threshold of 0.9997.
$ /$HOME/cd-hit-v4.8.1-2019-0228/cd-hit-est -i global_early_DS_0.0005N.aln -o global_early_DS_0.0005N_CDHit0.9997.fas -M 2500 -c 0.9997 -aL 1 -aS 1 -d 0
$ /$HOME/cd-hit-v4.8.1-2019-0228/cd-hit-est -i global_early_DL_0.0005N.aln -o global_early_DL_0.0005N_CDHit0.9997.fas -M 2500 -c 0.9997 -aL 1 -aS 1 -d 0
$ /$HOME/cd-hit-v4.8.1-2019-0228/cd-hit-est -i global_early_GL_0.0005N.aln -o global_early_GL_0.0005N_CDHit0.9997.fas -M 2500 -c 0.9997 -aL 1 -aS 1 -d 0
Change format to NEX.
$ java -jar /$HOME/ALTER/alter-lib/target/ALTER-1.3.4-jar-with-dependencies.jar -i global_early_DS_0.0005N_CDHit0.9997.fas -ia -o global_early_DS_0.0005N_CDHit0.9997.fas.nex -of NEXUS -oo Windows -op MrBayes
$ java -jar /$HOME/ALTER/alter-lib/target/ALTER-1.3.4-jar-with-dependencies.jar -i global_early_DL_0.0005N_CDHit0.9997.fas -ia -o global_early_DL_0.0005N_CDHit0.9997.fas.nex -of NEXUS -oo Windows -op MrBayes
$ java -jar /$HOME/ALTER/alter-lib/target/ALTER-1.3.4-jar-with-dependencies.jar -i global_early_GL_0.0005N_CDHit0.9997.fas -ia -o global_early_GL_0.0005N_CDHit0.9997.fas.nex -of NEXUS -oo Windows -op MrBayes
Fetch dates for accessions from metadata.
$ awk -F "\t" -v OFS="\t" '{print $3,$4}' global_early_meta.tsv > global_early_dates.txt
Convert dates to decimal dates using a custom script date_convertor.pl.
$ perl date_convertor.pl global_early_dates.txt > global_early_decimal_dates
Create BEAST XML file in Windows platform.
# Open BEAUti-v1.10.4 by double-click its the icon
# Load genome alignment file in NEX format
# Import dates (sample collection dates) in the file global_early_decimal_dates
# Set options for analysis, e.g. set ‘Clocks’ to either strict, or relaxed, set ‘Tree Prior’ to either ‘Bayesian Skyline’ or ‘Bayesian SkyGrid’, set MCMC ‘Length of Chain’ to 1,200 million generations. The run may be stopped when the Explained Sum of Squares (ESS) of all parameters are significant (>200). When the parameters are all set, click ‘Generate BEAST file’.
Run BEAST.
# Open BEAST v1.10.4 by double-click the icon
# Load BEAST XML file, click ‘Run’. The run may take a few days until all ESSs are significant.
Visualize tMRCA by Tracer (Figure 4).
# Open Tracer v1.7.2 by double click the icon
# Import the log file created by BEAST. Posterior distribution of tMRCA can be shown by clicking ‘age(root)’ and ‘Marginal Density’. The mean tMRCA and 95% HPD interval are provided in ‘Estimates’.
Figure 4. Screenshot of time of most recent common ancestor (tMRCA) estimation of three main haplotypes of the early SARS-CoV-2 genomes as summarized with Tracer. The dates are shown in decimal.
Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:
Hu et al. [17]. Genome characterization based on the Spike-614 and NS8-84 loci of SARS-CoV-2 reveals two major possible onsets of the COVID-19 pandemic. PLoS ONE 18(6): e0279221. https://doi.org/10.1371/journal.pone.0279221 (Figure 4, panel 1; Figure 6).
Guan et al. [12]. Genome analysis reveals much earlier separation and parallel evolution of major haplotypes of SARS-CoV-2 than its occurrence in China. Science in One Health 2(2023), https://doi.org/10.1016/j.soh.2023.100041 (Figure 1; Figure 3; Figure 4).
Acknowledgments
We gratefully acknowledge all data contributors, including the authors and their originating laboratories responsible for obtaining the specimens, and their submitting laboratories for generating the genetic sequence and metadata, and sharing via the GISAID Initiative, on which this research is based. This research was supported by grants from National Key R&D Program of China and the Central Public-interest Scientific Institution Basal Research Fund to J.Z. (1630052020022), and the Project of Science and Technology Department of Sichuan Provincial of China to L.Y. (2019JDJQ0035). This protocol was partially described in PLoS ONE 18(6): e0279221. Doi: 10.1371/journal.pone.0279221 (Hu et al. [17]) and in Science in One Health (2023), Doi: 10.1016/j.soh.2023.100041) (Guan et al. [12]).
Competing interests
The authors declare no competing interests.
Ethical considerations
This protocol is not involved in any experiments.
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Hu, X., Mu, Y., Deng, R., Yi, G., Yao, L. and Zhang, J. (2023). Genome characterization based on the Spike-614 and NS8-84 loci of SARS-CoV-2 reveals two major possible onsets of the COVID-19 pandemic. PLoS One 18(6): e0279221.
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4,956 | https://bio-protocol.org/en/bpdetail?id=4956&type=0 | # Bio-Protocol Content
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Efficient Genetic Transformation and Suicide Plasmid-mediated Genome Editing System for Non-model Microorganism Erwinia persicina
TC Tingfeng Cheng §
TG Tongling Ge
XZ Xinyue Zhao
ZL Zhu Liu
LZ Lei Zhao
(§ Technical contact)
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4956 Views: 584
Reviewed by: Samik Bhattacharya Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Applied Microbiology and Biotechnology Nov 2023
Abstract
Erwinia persicina is a gram-negative bacterium that causes diseases in plants. Recently, E. persicina BST187 was shown to exhibit broad-spectrum antibacterial activity due to its inhibitory effects on bacterial acetyl-CoA carboxylase, demonstrating promising potential as a biological control agent. However, the lack of suitable genetic manipulation techniques limits its exploitation and industrial application. Here, we developed an efficient transformation system for E. persicina. Using pET28a as the starting vector, the expression cassette of the red fluorescent protein–encoding gene with the strong promoter J23119 was constructed and transformed into BST187 competent cells to verify the overexpression system. Moreover, suicide plasmid–mediated genome editing systems was developed, and lacZ was knocked out of BST187 genome by parental conjugation transfer using the recombinant suicide vector pKNOCK-sacB-km-lacZ. Therefore, both the transformation and suicide plasmid–mediated genome editing system will greatly facilitate genetic manipulations in E. persicina and promote its development and application.
Key features
• Our studies establish a genetic manipulation system for Erwinia persicina, providing a versatile tool for studying the gene function of non-model microorganisms.
• Requires approximately 6–10 days to complete modification of a chromosome locus.
Graphical overview
Keywords: Erwinia persicina Genetic transformation Genome editing Non-model microorganism
Background
Erwinia persicina belongs to the phylum Proteobacteria, class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae, and genus Erwinia [1]. E. persicina has been identified as a soft rot pathogen that causes pink-pigmented soft rot on plant hosts including garlic, onions, barley, Cucurbita pepo, celery, and alfalfa [2–8]. However, E. persicina has exhibited great potential as a biological control agent to inhibit Salmonella enterica contamination in the sprout food industry [9], Fusarium head blight (FHB) severity in wheat [10], and a variety of soil-borne pathogens (Alternaria solani, Sclerotinia sclerotiorum, Rhizoctonia solani, and Fusarium proliferatum) in potato dextrose agar plates [11]. Recently, E. persicina BST187 was isolated from the rhizosphere of tomato and exhibited broad-spectrum antibacterial activity due to its inhibitory effects on bacterial acetyl-CoA carboxylase [12].
The construction and application of chassis modification of E. persicina has not yet been studied, except by Zhao et al. [13]. Efficient genetic transformation and genome editing systems will be beneficial for exploring the pathogenic mechanisms of E. persicina to provide a fundamental basis for control strategies. Moreover, tools developed for the genetic manipulation of E. persicina will provide excellent cell factories for diverse industrial applications such as andrimids.
In this study, the details of the BST187 chassis modification system are described. We initially identify the design flaws with the pET series of expression plasmids used for BST187, as it lacks T7 RNA polymerase-encoding genes and T7 promoter cannot be used for the overexpression. Then, we demonstrate that the constitutive promoter J23119 could be used for the overexpression of the red fluorescent protein (RFP)-encoding gene. Finally, the whole preparation method and transformation parameters of E. persicina competent cells are determined. Moreover, the knockout and integration system of target genes into the genome of E. persicina strains are conducted by double-crossover homologous recombination using the suicide vector. A schematic diagram of suicide plasmid–mediated genome editing is shown in Figure 1.
Figure 1. Schematic diagram of suicide plasmid–mediated genome editing. A. Construction of lacZ knockout plasmid. B. Parental conjugation transformation and recombination exchanges.
Materials and reagents
Biological materials
Erwinia persicina BST187 [13]
pGD plasmid [13]
pET28a-Cas9 plasmid [13]
Escherichia coli DH5α chemically competent cells (Biomed, catalog number: BC102-02)
pKNOCK-sacB-km [13]
Escherichia coli DH5α λpir chemically competent cells (ZOMANBIO, catalog number: ZK227)
Escherichia coli S17-1 λpir chemically competent cells (ANGYUBIO, catalog number: G6055-10)
Reagents
KOD OneTM PCR Master Mix (TOYOBO, catalog number: KMM-201)
Agarose (GenStar, catalog number: VA10252)
Nucleic acid dye (Biosharp, catalog number: BS354B)
StarMarker D5000 (GenStar, catalog number: M030-10)
StarMarker D8000 (GenStar, catalog number: M031-05)
New Gel Mini Purification kit (ZOMANBIO, catalog number: ZPN202-3)
EZ-HiFi Seamless Cloning kit (GenStar, catalog number: T196-20)
Super Taq DNA Polymerase (GenStar, catalog number: A012-100)
Fast Plasmid Miniprep kit (ZOMANBIO, catalog number: ZPK101-3)
Chemicals
Tryptone (OXOID, catalog number: LP0042B)
Yeast extract powder (OXOID, catalog number: LP0021B)
Agar powder (Solarbio, catalog number: A8190)
Kanamycin sulfate (Solarbio, catalog number: BS-K8020-5)
Ampicillin (Solarbio, catalog number: BS-A8180-5)
Sucrose (Sigma-Aldrich, catalog number: V900116)
NaCl (SCR, catalog number: 10019318)
KCl (SCR, catalog number: 10016318)
Na2HPO4·12H2O (Macklin, catalog number: S818120-500 g)
KH2PO4 (Macklin, catalog number: P815662-500 g)
Tris-base (Solarbio, catalog number: SJ025)
Glacial acetic acid (Macklin, catalog number: A801295-500 mL)
EDTA (Aladdin, catalog number: E116428-500 g)
CaCl2 (Macklin, catalog number: C804986-500 g)
Glycerol (DAMAO, catalog number: 2294)
Solutions
Kanamycin (50 mg/mL) (see Recipes)
Ampicillin (100 mg/mL) (see Recipes)
50% glycerol (see Recipes)
0.1 M CaCl2 (see Recipes)
0.85% NaCl (see Recipes)
50× TAE electrophoresis buffer (see Recipes)
LB medium (see Recipes)
10× Phosphate-buffered saline (PBS) (see Recipes)
Recipes
Kanamycin (50 mg/mL)
Dissolve 500 mg of kanamycin sulfate in 10 mL of ddH2O. Filter sterilize using a 0.22 µm syringe filter and store at -20 °C before use.
Ampicillin (100 mg/mL)
Dissolve 1,000 mg of ampicillin in 10 mL of ddH2O. Filter sterilize using a 0.22 µm syringe filter and store at -20 °C before use.
50% glycerol
Make the 50% glycerol solution by diluting 100% glycerol in ddH2O and autoclave.
0.1 M CaCl2
Dissolve 1.47 g of CaCl2·2H2O in ddH2O to a final volume of 100 mL and autoclave the solution.
0.85% NaCl
Dissolve 0.85 g of NaCl in ddH2O to a final volume of 100 mL and autoclave the solution.
50× TAE electrophoresis buffer (1,000 mL)
Note: Use 1 M HCl or 1 M NaOH to adjust the pH to 8.5 and dilute to 1× with ddH2O before use.
Reagent Final concentration Quantity
Tris-base 2 M 242 g
Glacial acetic acid 1 M 57.1 mL
EDTA 100 mM 37.2 g
H2O n/a To 1,000 mL
LB medium (1,000 mL)
Add 1 mL of kanamycin (50 mg/mL) or ampicillin (100 mg/mL) into 1 L of LB broth or agar after autoclaving when the media has cooled to 50–55 °C.
Note: Sucrose solution should be autoclaved at 115 °C for 30 min.
Reagent Final concentration Quantity
Tryptone 1% 10 g
Yeast extract powder 0.5% 5 g
NaCl 1% 10 g
Agar powder 1.5% 15 g (agar plate)
Sucrose 15% 150 g (sucrose plate)
H2O n/a To 1,000 mL
10× Phosphate-buffered saline (PBS) (1,000 mL)
Use 1 M HCl or 1 M NaOH to adjust the pH to ~6.8. The pH of solution should be adjusted to 7.4 when diluted to 1× PBS.
Dilute 10× PBS to 1× PBS and then sterilize the solution by autoclaving for 15 min at 121 °C. Store it at 4 . Dissolve 1 mL of 1× PBS in 99 mL of ddH2O (0.01 M) and sterilize by autoclaving for 15 min at 121 °C before use.
Reagent Final concentration Quantity
Na2HPO4·12H2O 100 mM 35.814 g
KH2PO4 18 mM 2.4496 g
NaCl 1.37 M 80.0669 g
KCl 27 mM 2.0129 g
H2O n/a To 1,000 mL
Laboratory supplies
Petri plates, 90 mm × 15 mm (Biosharp, catalog number: BS-90-D)
1.5 mL Eppendorf tubes (Biosharp, catalog number: BS-15-M)
0.2 mL PCR tubes (LABSELECT, catalog number: PST-0208-FT-C)
Pipette tips (Biosharp, catalog numbers: BS-10-T, BS-200-T, BS-1000-T)
Cell spreader (Biologix, catalog number: 65-1001)
Parafilm (Parafilm, catalog number: PM996)
12 mL Polypropylene test tube (Biosharp, catalog number: BS-PPT-12-S)
10 mL syringes (MOTUO, catalog number: MT-ZSQ-04)
Syringe filter (0.22 µm) (Merck Millipore, catalog number: C134954)
Sterile 50 mL conical tubes (LABSELECT, catalog numbers: CT-002-50A)
96-well cell culture plate (Corning, catalog numbers: CLS351172)
Blue cap reagent bottle (SHUNIU, catalog numbers: 250 mL, 500 mL)
Equipment
Pipettes (Eppendorf, model: 3121000023, 3121000090, 3120000267)
Ultrapure Water Systems (ZHIANG, model: Best-D)
PCR instruments (BIO-GENER, model: GET3XG)
Microwave oven (Midea, model: 2105210600)
DNA electrophoresis apparatus (Tanon, model: EPS-300)
Gel image system (Tanon, model: Tanon 1600R)
UV glue cutting instrument (Miulab, model: DUT-48)
Electrothermal constant temperature (MIULAB, model: DTC-100)
Water bath pot (Shanghai Yiheng, model: DK-8D)
Liquid nitrogen container (DONGYA, model: YZD-50)
Clean bench (AIRTECH, model: SW-CJ-1FD)
Ice machine (XUEKE, model: IMS-70)
Constant temperature oscillation incubator (Shanghai Zhichu, model: ZQZY-78AES)
Vortex (DLAB, model: VJ218AG0003297)
4 °C refrigerator (Zhongke Duling, model: MPC-5V656)
-20 °C ultra-low temperature freezer (Zhongke Duling, model: MDF-25V278W)
-80 °C ultra-low temperature freezer (Haier, model: DW86L626)
High-pressure sterilization pot (BioNation, model: CT-90A)
Centrifuge 5424R (Eppendorf, model: CENTRIFUGE 5424R)
High-speed freezing centrifuge (Eppendorf, model: CENTRIFUGE 5810R)
Electronic balance (RADWAG, model: WTC-2000)
Ultra-micro ultravioletvisible spectrophotometer (BIO-DL, model: Micro drop 5803032204)
Baking oven (CIMO, model: DHG-9143BS-II)
Oven incubator (CIMO, model: SPX-250BSH-II)
Microplate reader (Molecular Devices, model: SMP7)
pH meter (Sartorius, model: PB-10)
Software and datasets
SnapGene® (SnapGene, https://www.snapgene.com/)
Tanon 1600 Gel Image System (Tanon)
SoftMax Pro 7 (Molecular Devices, http://www.moleculardevices.com)
Procedure
Genetic transformation
Construct red fluorescent protein (RFP) overexpression cassette
Use Gibson assembly (a method for seamlessly joining multiple DNA fragments with overlapping ends, enabling the construction of larger and more complex DNA molecules [14]) to construct the pET28a-PJ23119::rfp plasmid, which contains an RFP reporter gene under the control of the constitutive promoter J23119. The vector map was generated by SnapGene, and the detailed procedure is as follows:
Amplify the target fragments by polymerase chain reaction (PCR) using KOD. Amplify the RFP gene (765 bp) from pGD plasmid with the primers F1/R1. Amplify the plasmid backbone (3,651 bp) from pET28a plasmid with the primers F2/R2. The components and procedures of PCR are described as follows (Table 1 and Table 2).
Note: Colony PCR.
Table 1. Components for PCR reaction of KOD
Components Volume Final concentration
KOD OneTM PCR Master Mix 10 µL 1×
Forward primer (10 µM) 1 µL 0.5 µM
Forward primer (10 µM) 1 µL 0.5 µM
Template 1 µL -
ddH2O to 20 µL -
Table 2. Thermocycling conditions for PCR reaction of KOD
Step Temperature (°C) Duration No. of cycles
Initial denaturation 98 3 min 1
Denaturation 98 10 s 35
Annealing Ta 5 s
Extension 68 10 s per kb
Final extension 68 2 min 1
Hold 4 ∞ -
Ta: The annealing temperature (Ta) chosen for PCR depends directly on the length and composition of the primers. The details of the primers are listed in Table S1.
Purify PCR products by 1% agarose gel electrophoresis in 1× TAE electrophoresis buffer followed by gel extraction using a gel purification kit.
Measure the absorbance at 260 nm and estimate DNA concentration.
Use EZ-HiFi Seamless Cloning kit to construct the plasmid. The recombination reaction mixture (10 µL) contains 5 µL of 2× HiFi Seamless cloning mix and 5 µL of target fragment mix (vector: insert molar ratio of 1:3; the details of the calculations are listed in Table S2). Incubate the combined reaction mixture at 50 °C for 30 min and immediately place on ice or hold at -20 °C until use.
Transform 10 µL of the ligation reaction into 100 µL of E. coli DH5α competent cells by the heat-shock method following the manufacturer’s instructions. This heat treatment (usually approximately 42 °C) increases the permeability of the bacterial cell membrane, allowing the foreign DNA to enter the cells. Plate the transformation mix on LB agar medium (K+) and incubate the plate at 37 °C overnight.
Pick colonies with a 2.5 µL pipette tip and transfer the picked colony into 500 µL of liquid LB (K+). Incubate the 1.5 mL tubes on a rotary shaker (200 rpm) at 37 °C for 12 h.
Confirm the transformants through PCR using Super Taq DNA Polymerase with primers F3/R3 and capture the gel image with Tanon 1600 Gel Image System (Tanon). The expected fragment size generated by PCR is 1,131 bp (Figure 2). The components and procedures of the PCR are described as follows (Table 3 and Table 4).
Note: A volume of 2–5 µL of PCR product should be loaded on the gel.
Figure 2. PCR amplification from the E. coli DH5α transformant carrying pET28a-PJ23119::rfp
Table 3. Components for PCR reaction of Super Taq DNA Polymerase
Components Volume Final concentration
2× SuperTaq PCR StarMix 5 µL 1×
Forward primer (10 µM) 0.5 µL 0.5 µM
Forward primer (10 µM) 0.5 µL 0.5 µM
Template 1 µL -
ddH2O to 10 µL -
Table 4. Thermocycling conditions for PCR reaction of Super Taq DNA Polymerase
Step Temperature (°C) Duration No. of cycles
Initial denaturation 95℃ 2 min 1
Denaturation 95℃ 15 s 35
Annealing 58℃ 15 s
Extension 72℃ 30 s per kb
Final extension 72℃ 5 min 1
Hold 4 ∞ -
Use sanger sequencing to evaluate the accuracy of the assembly (GENEWIZ Company). The sequencing data can be analyzed by SnapGene.
Cultivate the correct colonies and extract plasmids using the Fast Plasmid Miniprep kit. Store the pET28a-PJ23119::rfp plasmid at -20 °C until use.
E. persicina competent cells preparation
E. persicina BST187 was initially grown in 100 mL of LB broth and incubated at 28 °C for 12 h under shaking at 200 rpm to an optical density at 600 nm (OD600) of 0.4–0.8 (use SoftMax Pro 7).
Transfer the cultures into two precooled 50 mL conical sterile polypropylene centrifuge tubes and place them on ice for 20 min.
Centrifuge the cultures at 1,503× g for 5 min at 4 °C.
Resuspend the cell pellets in 5 mL of precooled 0.1 M CaCl2 solution and incubate on ice for 20 min.
Centrifuge the cells at 1,503× g for 5 min at 4 °C.
Resuspend the cell pellets in 2 mL of precooled 0.1 M CaCl2 and 1 mL of precooled 50% glycerol solution. Both components were added together in a single step.
Mix gently by pipetting up and down or flicking the tube 4–5 times. Store 100 µL of the yielding competent cells in 1.5 mL centrifuge tubes at -80 °C for a further transformation step.
Cell transformation
Take competent cells from -80 °C and thaw on ice (approximately 3–5 min). Add 10 µL of overexpression plasmids (approximately 50 ng/µL) into 100 µL of competent cells suspension and mix by tapping the tube gently.
Place competent cells on ice for 30 min.
Heat the mixture at 42 °C for 90 s; then, immediately transfer it to ice and incubate for 2 min.
Add 500 µL of LB broth into the mixture and incubate in a shaker at 200 rpm for 60 min at 28 °C.
Centrifuge the mixture at 1,878× g for 1 min.
Discard 500 µL of supernatant and spread 100 µL of the mixture on LB agar medium (K+). Incubate the plates for more than 24 h at 28 °C to obtain transformants.
Pick colonies into 1 mL of LB broth (A++K+) and incubate on a rotary shaker (200 rpm) at 28 °C for 12–24 h. Centrifuge the cells at 4,509× g for 5 min and examine them under a UV glue-cutting instrument for easy visibility with the naked eye (Figure 3).
Figure 3. BST187 transformant carrying pET28a-PJ23119::rfp
Suicide plasmid–mediated genome editing
Construction of the lacZ knockout plasmid
Gene knockout was conducted by replacing the target genes in the chromosome with NdeI digestion site fragment (CATATG CATATG). A schematic diagram of vector construction for gene knockout and chromosomal integration is shown in Figure 1. The vector map was generated by SnapGene and detailed procedure is as follows:
Amplify the three fragments individually (upstream of lacZ, downstream of lacZ, and pKNOCK-sacB-km) by PCR using KOD. The specific primer pairs used to amplify each of the fragments are listed in Table S1, and PCR is described above.
Gibson assembly method is described above; transform 10 µL of the ligation reaction into 100 µL of E. coli DH5α λpir competent cells by the heat-shock method. See General note 3.
Verify the transformants via PCR with primers F7/R7 (use Tanon 1600 Gel Image System). The expected fragment size generated by PCR is 2,081 bp (Figure 4). The components and procedures of PCR are described above.
Cultivate the correct colonies and extract plasmids using the Fast Plasmid Miniprep kit. Store the pKNOCK-sacB-km-lacZ plasmid at -20 °C until use.
Figure 4. PCR amplification from E. coli DH5α λpir transformants carrying pKNOCK-sacB-km-lacZ
Construction of the donor strains
Transform 10 µL of the lacZ knockout plasmid into 100 µL of E. coli donor strain S17-1 λpir competent cells by the heat-shock method following the manufacturer’s instructions. See General note 3.
Parental conjugation transformation
Preparation of donor strain: Inoculate E. coli S17-1 λpir in 5 mL of LB broth (K+) overnight at 37 °C with shaking at 200 rpm.
Preparation of recipient: Inoculate E. persicina BST187 in 5 mL of LB broth (A+) overnight at 28 °C with shaking at 200 rpm.
Centrifuge both cultures at 2,254× g for 5 min and remove the supernatant.
Resuspend the cell pellets with 3 mL of 0.01 M PBS buffer and centrifuge again to remove the supernatant. Repeat twice the steps of PBS resuspension and centrifugation to remove the supernatant.
Resuspend the cell pellets in 0.01 M PBS buffer solution to adjust the OD600 to 0.6–0.8 (use SoftMax Pro 7).
Combine donor strain S17-1 and recipient strain BST187 at a 1:1 volume ratio and incubate the mixture at 28 °C for approximately 10–12 h at 200 rpm.
Centrifuge 1 mL of conjugation suspension at 1,878× g for 2 min and reserve 100 µL of the supernatant to resuspend the pellets.
Plate the 100 µL resuspend pellets on LB agar medium (A++K+) and incubate the plate at 28 °C for more than 24 h.
Recombination exchanges
First recombination exchange: Pick single colonies (Figure 5A) inoculated in 500 µL of LB broth (A++K+) with shaking at 200 rpm for 10–12 h at 28 °C. See General note 4.
Figure 5. Colony of the first recombination exchange screening and resistance validation. A. First recombination exchange. B. Resistance validation.
Confirm the first crossover strains through PCR using primers F7/R8 (use Tanon 1600 Gel Image System). The expected fragment size generated by PCR is 1,794 bp or 4,866 bp (Figure 6A).
Figure 6. PCR amplification from BST187 that completed recombination exchange. A. First recombination exchange. B. Second recombination exchange.
Second recombination exchange: Centrifuge the single-crossover strains at 1,878× g for 2 min and remove the supernatant.
Resuspend the cell pellets with 0.85% NaCl solution and for gradient dilution (10,000-fold). Plate the 100 µL dilutions on LB agar medium (15% sucrose) and incubate the plate at 28 °C for more than 24 h.
Pick single colonies into 500 µL of liquid LB (15% sucrose) and incubate on a rotary shaker (200 rpm) at 28 °C for 12 h. See General note 5.
Confirm the second recombination exchange strains through PCR using primers F8/R8 (use Tanon 1600 Gel Image System). The expected fragment size generated by PCR is 1887 bp or 4,971 bp (Figure 6B).
Aspirate 2.5 µL of the culture with a band size of 1,887 bp and dispense it onto LB agar medium (K+) at 28 °C for 24 h. A knockout strain (ΔlacZ) is obtained that was not grown on K+ LB agar medium (Figure 5B). See General note 6.
Data analysis
To verify the genome editing events, amplify the target region via colony PCR.
Editing efficiency = No. of correct mutants/No. of colonies tested.
Validation of protocol
For complete details on the use and execution of this protocol, please refer to Zhao et al. [13]. Fine-tuning gene expression of regulator AdmX for improved biosynthesis of andrimid in Erwinia persicina BST187. Applied Microbiology and Biotechnology (Figure 3).
General notes and troubleshooting
General notes
The T7 promoter does not work when used for the construction of overexpression cassette in E. persicina BST187.
Genes that can affect bacterial growth are prone to mutations when overexpressed with the J23119 promoter. It is recommended to use inducible or moderately strong promoter J23116.
Recombinant suicide vector (Replicon: R6K) is dependent on the λpir gene in competent cells.
E. coli S17-1 could grow on A+ plates and pick abnormally large colonies (Figure 5A).
BST187 will grow rapidly on sucrose plates and, after picking colonies, the plates will be placed at 4 °C.
Successful knockout of strains requires resistance verification in LB agar medium (K+) to avoid residual resistance fragments (Figure 5B).
Knockout efficiency is related to the target gene and, according to our experience, the editing efficiency is approximately 10%–80%.
DpnI is not usually necessary when gel purifying fragments.
Troubleshooting
Problem 1: After the first exchange, there was no growth of the target strain on LB agar medium (A++K+).
Possible cause(s): Instability of ampicillin causing the S17-1 strain to cover BST187.
Solution(s): Ready-to-use ampicillin plates.
Problem 2: After the second exchange, knockout strains contain residual resistance.
Possible cause(s): Incomplete suicide and sucrose selection is not entirely effective.
Solution(s): Selection of positive strains and repeat screening in 15% sucrose LB.
Acknowledgments
This work was financially supported by the Hundred Talents Program of the Chinese Academy of Sciences to LZ (E3J56201), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-CXRC-027) and the international postdoctoral exchange fellowship program (Talent-Introduction Program) of the office of China postdoc council (YJ20210438).
We would like to acknowledge our original publication [13].
Competing interests
Authors have no competing interests.
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Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A. and Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6(5): 343–345.
Supplementary information
The following supporting information can be downloaded here:
Table S1. Information primers used in this study
Table S2. Ligation of homologous recombination
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Microbiology > Microbial genetics > Genome editing
Molecular Biology > DNA > Transformation
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4,957 | https://bio-protocol.org/en/bpdetail?id=4957&type=0 | # Bio-Protocol Content
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Mesenchymal Stromal Cell (MSC) Functional Analysis—Macrophage Activation and Polarization Assays
Hazel Y. Stevens
AJ Angela C. Jimenez
BW Bryan Wang
YL Ye Li
SS Shivaram Selvam
AB Annie C. Bowles-Welch
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4957 Views: 1708
Reviewed by: Vivien J. Coulson-Thomas Anonymous reviewer(s)
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Abstract
Stem cell–based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies.
Key features
• The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells–derived macrophages.
• The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores.
• The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages.
• This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.
Graphical overview
Keywords: Immunomodulation MSCs Macrophage activation Macrophage phenotype M1 M2
Background
Mesenchymal stromal cell (MSC) transplants, through their immunomodulatory and regenerative properties, have emerged as a viable therapeutic approach in a wide range of diseases. MSCs isolated from bone marrow, umbilical cord, and adipose tissue have been used in clinical trials to treat conditions as far-reaching as traumatic brain injury, autism spectrum disorder, osteoarthritis, immune system disorders, and cardiovascular diseases [1]. To standardize such treatments and attempt to ensure successful outcomes, various approaches have been suggested to characterize the cells in functional or potency assays in vitro, to better predict treatment responses, e.g., DOSES [donor; origin; separation method; exhibited characteristics (cell behavior); and site of delivery] [2]. A known property of MSCs is their ability to modulate the phenotype of macrophages.
Monocyte-derived macrophages are important cells in the inflammatory cascade, key to tissue healing and regeneration. In the early stages of tissue injury or throughout chronic inflammatory pathologies, macrophages are driven to polarize to an M1 state, referred to as classically activated, and will secrete a range of proinflammatory cytokines and chemokines such as tumor necrosis factor-alpha (TNFα), interleukin-12 (IL-12), and CC-chemokine receptor-7 (CCR7), and express major histocompatibility complex (MHC) II cell surface receptor (HLA-DR) [3]. As the inflammation subsides, the predominant macrophage phenotype shifts to M2 (alternative activation) and is associated with the release of anti-inflammatory cytokines such as interleukin-10 (IL-10), interleukin-13 (IL-13), and interleukin-1 receptor antagonist (IL-1RA) [4]. Polarization to M2 reparatory macrophages, effected by MSCs in situ, can result in the restoration of homeostasis and tissue repair as well as the reduction of inflammation [5].
MSCs in culture are responsive to culture conditions and secrete both free and exosome-encapsulated cytokines and microRNA. To establish the cell culture techniques that best prepare MSCs for in vivo delivery and subsequent immunomodulation of macrophage phenotype and function in the body, in vitro co-culture assays can be used to study the crosstalk between these cell types to recapitulate the complex interactions in vivo. However, studies with macrophage cultures can be challenging due to the difficulties in attaining a pure population of macrophages and in the harvesting of these cells, as they strongly adhere to tissue culture surfaces when differentiated/activated. The Tohoku Hospital Pediatrics-1 (THP-1) cell line has been widely used to study monocyte and macrophage function in vitro, in the presence of a wide range of other cell types such as intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells [6].
The proposed mechanisms of action of MSCs [7–13] center on the abatement of the inflammatory condition by the reduction in a number of proinflammatory cytokines such as TNFα, IL-6, IL-8, and IL1-β. In the first part of this assay, the ability of delivered MSCs to counter this proinflammatory condition is measured by the suppression of the release of TNFα and other cytokines from M1-like macrophages. In the clinic, the mobilization of MSCs to counter inflammation, as seen with oxygen therapies administered to patients with respiratory and cognitive disorders, may mimic a similar endogenous response by MSCs [14,15].
MSC administration generates concomitant decreases in proinflammatory cytokine release and increases in anti-inflammatory mediator production by macrophages [16,17]. These changes are accompanied by the polarization of M1 towards M2 macrophages [18]. This shift is examined in the second part of the assay.
In consideration of the role of cell therapy and, in particular, the delivery of allogeneic MSCs in ameliorating disease conditions, the study of the interactions of MSCs and macrophages is pertinent. Herein, we have established an in vitro assay system to study both macrophage activation and polarization in the presence of delivered MSCs. Freshly thawed and culture-rescued cells can differ in their ability to activate and polarize macrophages. Freshly thawed cells have disturbed membrane physiology and display a differential release of paracrine mediators and microparticles (exosomes, microvesicles, and apoptotic bodies) compared with cultured cells. Although freshly thawed cells are less likely to persist in the body due to innate immune cascades, coagulation response, etc., their passive release of microparticles is likely augmented compared with culture-rescued cells. However, stronger immunomodulatory capacity has been attributed to freshly cultured cells [19]. These assays establish key parameters for studying the interaction of MSCs and macrophages in vitro.
Materials and reagents
Biological materials
THP-1 cell vial, which has been stored in liquid nitrogen (LN2) at vapor phase (ATCC, catalog number: TIB-202)
Human MSCs derived from bone marrow or umbilical cord tissue, which have been stored frozen in a cryovial and maintained in LN2 at vapor phase (RoosterBio Inc., Frederick, MD, catalog number: RoosterVial-hBM or supplied by Duke University MC3)
Reagents
RPMI 1640 containing L-glutamine and 10 mM HEPES (ATCC, catalog number: 30-2001)
RoosterNourishTM -MSC (RoosterBio Inc, catalog number: KT-001)
Prime-XV MSC Expansion XSFM (Irvine Scientific, catalog number: 91149)
Penicillin/Streptomycin (P/S) (Millipore Sigma, catalog number: P4333)
Fetal bovine serum (FBS), heat inactivated (H.I.) and characterized (Cytiva, catalog number: SH30071.01)
β-mercaptoethanol (BME) (Thermo Fisher Scientific, catalog number: 21985023)
Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: C6164)
Phorbol 12-myristate 13 acetate (PMA) (Sigma-Aldrich, catalog number: P1585)
PLASMA-LYTE A (Baxter Intl Inc., catalog number: 2B2543Q)
Human serum albumin (HSA) (Albutein) 25% (Grifols, catalog number: 68516-5216-02)
Phosphate buffered saline (PBS) Ca2+/Mg2+ free, low endotoxin (Millipore Sigma, catalog number: TMS-012-A)
Interferon-gamma (IFN-γ) (Thermo Fisher Scientific, catalog number: 300-02)
Lipopolysaccharide (LPS) (Millipore Sigma, catalog number: L4391)
Sterile endotoxin-free water (VWR, catalog number: 75799-280)
TrypLE Express 1× (Millipore Sigma, catalog number: 12605028)
Zombie UV Fixable Viability kit (BioLegend, catalog number: 423107)
Human TruStain FcXTM (BioLegend, catalog number: 422302)
Antibodies (BioLegend, see Table 1)
Table 1. Antibody information for cell phenotype panel
Surface marker Final dilution Conjugate Vendor Catalog Clone
Human macrophage
/MSC panel antibodies
HLA-DR 1:20 (see General note 6) FITC BioLegend 307620 L243
CD163 1:20 PE BioLegend 333606 GHI/61
CD73 1:20 PerCP/Cy5.5 BioLegend 344014 AD2
CD206 1:20 APC BioLegend 321110 15-2
CD14 1:20 PE/Cy7 BioLegend 367112 63D3
Zombie UV 1:1,000 - BioLegend 423107 -
Compensation beads Anti-Mouse Ig +/- control beads One drop/500 µL FC buffer - BD 552843 -
ArC reactive beads for use with Zombie UV One drop/500 µL FC buffer - Thermo Fisher A10346 -
Anti-mouse negative and positive control beads (Thermo Fisher Scientific, catalog number: 552843)
ArC reactive beads (Thermo Fisher Scientific, catalog numbers: A10346 and A10628)
Cytofix fixation buffer (BD Biosciences; catalog number: 554655)
ELISA kit TNFα (e.g., Thermo Fisher Scientific, catalog number: PO1375)
Luminex Cytokine Kit Magnetic 30 Plex Panel (Thermo Scientific, catalog number: LHC6003M)
Solutions
THP-1 expansion medium (see Recipes)
THP-1 differentiation medium (see Recipes)
Diluting buffer (see Recipes)
PMA diluted (see Recipes)
LPS stock (see Recipes)
IFN-γ stock (see Recipes)
Co-culture medium (see Recipes)
Zombie-UV (see Recipes)
Blocking buffer (see Recipes)
FC buffer (see Recipes)
Antibody cocktail (see Recipes)
Recipes
THP-1 expansion medium
Reagent Final concentration Volume
RPMI 1640 79% 394.5 mL
FBS H.I. 20% 100 mL
P/S 1% 5 mL
BME 0.1% 0.5 mL
Total 100% 500 mL
THP-1 differentiation medium
Reagent Final concentration Volume
RPMI 1640 98% 48.5 mL
FBS H.I. 1% 0.5 mL
P/S 1% 0.5 mL
PMA diluted 100 ng/mL 0.5 mL
Total 100% 50 mL
Diluting buffer
Reagent Final concentration Volume
PLASMA-LYTE A 96% 48 mL
HSA (25%) 4% (1%) 2 mL
Total 100% 50 mL
PMA diluted
Make a stock solution of 5 mg of PMA in 1 mL of DMSO. Then, add 5 µL of stock to 2.5 mL of RPMI 1640 (no additives). Filter sterilize through a 0.2 µm filter and then add to 1% FBS media as above.
Reagent Final concentration Volume
PMA stock (5 mg/mL in DMSO) 10 µg/mL 5 µL
Medium RPMI 1640 2.5 mL
Total 100% 2.5 mL
LPS stock
Reagent Final concentration Quantity
LPS 1 mg/mL 1 mg
Sterile water 1 mL
Total 100% 1 mL
IFN-γ stock
Reagent Final concentration Quantity
IFN-γ 100 µg/mL 100 µg
Sterile water 1 mL
Total 100% 1 mL
Co-culture medium
Reagent Final concentration Volume
RPMI 1640 98% 49 mL
FBS H.I. 1% 0.5 mL
P/S 1% 0.5 mL
LPS 100 ng/mL 5 µL
IFN-γ 100 ng/mL 50 µL
Total 100% 50 mL
Zombie- UV
Reagent Final concentration Volume
Zombie-UV (reconstituted) 0.1% 5 µL
PBS Ca2+/Mg2+ free (1×) 99.9% 5 mL
Total 100% 5 mL
Blocking buffer
Reagent Final concentration Volume
TruStain FcX 10% 1 mL
FC buffer 90% 9 mL
Total 100% 10 mL
FC buffer
Reagent Final concentration Volume
FBS H.I. 2% 2 mL
PBS Ca2+/Mg2+ free (1×) 98% 98 mL
Total 100% 100 mL
Antibody cocktail (70 wells)
Reagent Final concentration Volume
Antibodies (350 µL each) 50% 1.75 mL
FC buffer 50% 1.75 mL
Total 100% 3.5 mL
Laboratory supplies
Non-treated T-25, T-75, and T-175 suspension flasks (e.g., Corning, ultralow attachment, catalog number: 4616, 3814)
Tissue culture treated T-225 flasks (e.g., Corning, CELLBIND, catalog number: 3293)
Cryogenic vials for vapor phase (e.g., Corning, Fisher Scientific, catalog number: 431386)
Freezing container (e.g., Corning, Millipore Sigma, model: CoolCell LX, catalog number: CLS432002)
Serological pipettes [e.g., VWR, catalog numbers: 414004-266 (5 mL); 414004-267 (10 mL); 414004-268 (25 mL)]
Micropipette tips, P10P1000 µL [e.g., Fisher Scientific, Biotix uTIP filter, catalog numbers: 12-111-000 (10 µL); 12-111-004 (300 µL); 12-111-132 (1,000 µL)]
Via-1-CassetteTM cartridges (Chemometec, catalog number: 941-0012)
CELLBIND 96-well cell culture plate (Corning, Millipore Sigma, catalog number: CLS3300)
96-well culture plate for media storage (e.g., Corning, tissue culture treated, catalog number: 3894)
V-bottom 96-well plate (Corning, Millipore Sigma, catalog number: CLS3894)
Parafilm (e.g., Sigma-Aldrich, catalog number: P7793)
Pre-saturated alcohol wipes (e.g., VWR, catalog number: 33503-136)
Equipment
Biosafety cabinet (BSC) level A2 (e.g., Thermo Fisher Scientific, catalog number: 1377)
Incubator set to 5% CO2 and 37 °C (e.g., Thermo Fisher Scientific, model: HeraCell VIOS)
Cell counter, (e.g., Chemometec, model: NucleoCounter NC-200TM)
Refrigerated centrifuge capable of reaching 500× g and using 50 mL conical tubes and microplates (e.g., Thermo Fisher Scientific, model: Megafuge 8R, catalog number: 75007214)
Micropipettes P10–1,000 µL (e.g., Eppendorf, Research Plus range, catalog number: 312300020)
Pipette controller (e.g., Eppendorf, model: Easypet, catalog number: 2231000955)
Inverted microscope (e.g., Thermo Fisher Scientific, model EVOS M5000, catalog number: AMF5000)
Microplate absorbance reader (e.g., Cytation 5)
Flow cytometer, e.g., CytoFlex (Beckman Coulter), LSRFortessa (BD)
Luminescence microplate reader (e.g., Bio-Rad, model: Bioplex 200)
Software and datasets
Flow cytometry software is needed for data analysis. This can be either FlowJoTM (BD Biosciences) or CytoBank (Beckman Coulter) software
Prism 9 (GraphPad, San Diego, CA)
JMP statistical software (Pro 17)
Procedure
Establishment of macrophage cultures
Thaw a vial of THP-1 cells (7 × 106 cells). Place the cell vial in a foam float in a water bath at 37 °C and time for 2 min. Check that 90% of the ice has disappeared before removing it from the water bath.
Wipe the vial with a pre-saturated alcohol wipe. Add the cells using a 1,000 µL micropipette and tip to 9 mL of THP-1 expansion medium that has been prewarmed to 37 °C in a 50 mL conical tube and resuspend the cells using a 10 mL serological pipette.
Count the cells by taking up 60 µL of cell suspension into a Via-1 Cassette and placing the cassette into the NucleoCounter. Record the cell count and cell viability.
Centrifuge the cells at 300× g for 5 min at 4 °C. Aspirate the supernatant and add 30 mL of expansion medium to the pellet. Resuspend the pellet using a 10 mL serological pipette.
Add 30 mL of fresh prewarmed expansion medium to a T-175 suspension culture flask and add the 30 mL of cell suspension to give 60 mL total. This will seed cells at a cell density of 1.2 × 105/mL.
Incubate the flasks at 37 °C and 5% CO2 in an incubator. Feed the cell cultures twice a week by removing all media from the flask into 2 × 50 mL conical tubes using a 25 mL serological pipette, centrifuging the tubes at 300× g for 5 min at 4 °C, and resuspending each cell pellet in 30 mL of fresh media. Use a total of 60 mL of media that has been prewarmed to 37 °C for each media change.
Check the cell density at each feeding by taking up 60 µL of cell suspension in a Via-1 cassette as above. Once the cultures have reached 1 × 106/mL, they can be further divided as below (see Figure 1 and Troubleshooting 1 and 2).
Figure 1. THP-1M loosely adherent cells after 48 h of phorbol 12-myristate 13 acetate (PMA) treatment (20×). Scale bar = 150 µm.
Split the THP-1 cell suspension 1:5 into T-175 suspension culture flasks (with 60 mL of media in each) and further culture the cells to 1 × 106/mL.
Harvest the THP-1 cells by removing all the media from the flasks using a 25 mL serological pipette, centrifuging to obtain pellets, and resuspending the pellets in 1 mL of cryopreservation medium comprising 10% DMSO: 90% FBS H.I. at 10 M cells/vial. The vials can be cryopreserved by placing them in a freezing container at -80 °C overnight before storing in vapor phase LN2.
Five days before the start of the co-culture (day -5), thaw THP-1 cells in the expansion medium as above.
Centrifuge at 300× g for 5 min at 4 °C.
Count cells. Resuspend THP-1 cells in 6 mL of expansion media at 8 × 105 /mL in T-25 suspension flasks, standing upright to restrict the surface area available. Incubate for 72 h at 37 °C and 5% CO2.
On day -2, harvest THP-1 cells from T-25 flasks. Count the cells to check viability (> 90%) and cell size (~13–14 µm).
Centrifuge at 300× g for 5 min at 4 °C and resuspend the THP-1 cells at 5 × 105/mL in differentiation media (PMA treatment).
Seed 200 µL of cell suspension in each well (105/well) of a CELLBIND 96-well flat-bottom plate.
Note: Set up extra wells for the THP-1 macrophage without MSCs (THP-1M) and the fluorescence minus one (FMO) controls.
Incubate at 37 °C and 5% CO2 for 48 h to generate naïve macrophages (now termed THP-1M for macrophages).
Preparation of MSC cultures
On the same day of PMA treatment (day -2), thaw the MSCs in diluting buffer or the media of choice (RoosterNourish/XSFM) for the culture-rescued (CR) group.
Thaw one vial of cells of each MSC donor in a water bath for 2 min. Then, add the cells from the vial to 9 mL of diluting buffer or media prewarmed to 37 °C and resuspend using a 10 mL serological pipette.
Count as before and seed MSC at 2,000 (XSFM) or 3,700 (RoosterNourish) cells/cm2 in T-225 flasks with MSC growth medium. The volume of media required is typically 40–42 mL.
Note: Required MSC seeding density may vary by manufacturer/supplier. Follow the supplier recommendations.
Incubate at 37 °C and 5% CO2 for 48 h.
On day 0, harvest the MSC by washing the flask with 10 mL of PBS Ca2+/Mg2+ free, aspirating the PBS, and adding 10 mL of TrypLE solution.
Incubate the flask for 5 min at 37 °C to allow for cell detachment. Check under a microscope to see that all the cells are detached from the surface of the flask.
Transfer the cell suspension to a tube and wash the flask with MSC growth medium. Count the MSC.
Centrifuge at 500× g for 10 min at 4 °C and resuspend CR MSC at 2.5 × 106/mL or 1.25 × 106/mL in diluting buffer/media of choice.
On the day of MSC harvest (day 0), thaw extra MSC in diluting buffer or media of choice for the out-of-thaw (OOT) group.
Centrifuge at 500× g for 10 min at 4 °C and resuspend OOT MSC at 2.5 × 106/mL or 1.25 × 106/mL in diluting buffer/media of choice (see General note 1).
Establishment of co-cultures
On day 0, centrifuge the naïve macrophages plate at 300× g for 5 min at 4 °C. Gently aspirate PMA media without disturbing the cells.
Add 40 µL of MSC cell suspension (CR or OOT) into each well, resulting in MSC:THP-1M of 1:1 or 1:2 (1 × 105 MSC:1 × 105 macrophages or 0.5 × 105 MSC: 1 × 105 macrophages). Set up extra wells containing both cell types for FMOs for flow staining (see General note 2).
Prepare co-culture medium by adding LPS and IFNγ to 1% FBS RPMI 1640, prewarmed to 37 °C.
Caution: Take care to avoid skin exposure to LPS.
Add 200 µL of co-culture medium to THP-1 M-only wells. Add 160 µL of co-culture medium to wells containing MSCs.
Critical: Mix by triturating the cells three times to ensure uniform distribution of MSCs and macrophages. The macrophage plate will have the loosely adherent cells distributed on one side of the well due to centrifugation.
Incubate the co-culture plate at 37 °C and 5% CO2 for 24 h.
Removal and storage of conditioned media
On day 1, bring the co-culture plate to the BSC but do not centrifuge.
Collect 60 µL of supernatant from each well (not FMO) and place into the corresponding wells of another 96-well plate so that the plate map is the same. Collect another 40 µL supernatant from each well and place into a second 96-well plate. Secure the plate with a plate sealer and parafilm. Store the plates at -20 °C for Luminex and ELISA assays.
Add 100 µL of fresh co-culture medium containing LPS and IFNγ (see General note 3).
Incubate co-culture plate at 37 °C and 5% CO2 for a further 48 h.
Harvest of cells after co-culture
On day 3, aspirate 100 µL of co-culture supernatant from each well and discard (see General note 4).
Triturate the remaining supernatant and move to a 96-well V-bottom plate.
Wash wells once with 100 µL of PBS and transfer PBS to the V-bottom plate following the plate map.
Centrifuge the V-bottom plate at 500× g for 5 min at 4 °C and decant the supernatant. Decanting is best performed by immediately removing the plate from the centrifuge after spinning and removing the plate lid with one hand while flicking the plate liquid into the sink/biohazard container with the other hand in a swift but firm movement. The plate is then righted, and the lid replaced. The pellets should remain intact at the bottom of the plate.
Add 50 µL of TrypLE to each well in the co-culture plate and incubate at 37 °C for 5 min to detach the cells (see General note 5).
Collect and transfer cell suspension to the V-bottom plate.
Use 50 µL of fresh co-culture medium (w/o LPS or IFNγ) to wash each well and transfer the medium to the V-bottom plate.
Repeat step E7 for an additional wash.
Centrifuge the V-bottom plate at 500× g for 5 min at 4 °C and decant the supernatant.
Add 200 µL of PBS to sample wells in the V-bottom plate. Add 50 µL of PBS to FMO wells, pool all samples together in a microcentrifuge tube, and then divide between eight wells for FMO and unstained samples.
Flow staining for macrophage phenotype
Centrifuge the V-bottom plate at 500× g for 5 min at 4 °C and decant the supernatant.
Prepare Zombie-UV live-dead dye in PBS (see Recipes 8).
Add 100 µL of Zombie UV to each of the cell pellets except unstained and Zombie FMO.
Add 100 µL of PBS to unstained and Zombie FMO.
Resuspend the cells in the staining solution in each well.
Incubate in the dark for 15 min at room temperature (RT).
Centrifuge the V-bottom plate at 500× g for 5 min at 4 °C and decant the supernatant.
Wash each well with 200 µL of FC buffer.
Centrifuge the V-bottom plate at 500× g for 5 min at 4 °C and decant the supernatant.
Prepare Fc block staining solution. Add 50 µL of Fc block staining solution to all wells and resuspend three times (see Recipes 9).
Incubate in the dark for 5 min at RT.
Prepare the antibody cocktail using FC buffer (see Recipes 10) plus 10% v/v of HLA-DR, CD163, CD73, CD206, and CD14 (see Table 1 for dilutions, conjugates, and catalog numbers).
Without removing Fc block, add 50 µL of antibody cocktail to each of the sample wells.
Treat the FMOs by adding 5 µL of each antibody except the one listed (FITC FMO has no FITC antibody).
Add 50 µL of FC buffer to the unstained well. Resuspend all wells three times.
Incubate in the dark for 20 min at 4 °C.
Add 100 µL of FC buffer for washing, centrifuge the V-bottom plate at 500× g for 5 min at 4 °C, and decant the supernatant.
Repeat step F17 for an additional wash.
Add 100 µL of CytoFix fixation buffer to the pellets and resuspend three times. Incubate in the dark for 15 min at 4 °C.
Add 100 µL of FC buffer to each well to wash, centrifuge the V-bottom plate at 500× g for 5 min at 4 °C, and decant the supernatant.
Resuspend the cell pellet in 100 µL of FC buffer. Keep the plate at 4 °C and analyze using a flow cytometer within three days. If the flow cytometer does not have a high throughput system for plates, transfer the samples to the appropriate tubes for your system prior to flow.
Prepare compensation beads for the antibody panel by adding one drop of positive and one drop of negative anti-mouse beads (see Table 1) to each of the six tubes and adding 1–5 µL of single antibody into each single stain tube, leaving one as unstained. Vortex for 5 s and then incubate in the dark at RT for 15–30 min. Add 2 mL of FC buffer to wash. Centrifuge at 500× g for 5 min. Carefully remove the supernatant and resuspend in 400 µL of FC buffer.
Prepare compensation beads for Zombie staining by using the ArC reactive beads in place of the anti-mouse beads and only incubate Zombie dye with the positive beads (component A). Once washed as above, add the negative beads into the tube. Use all beads for flow compensation.
Analysis of growth factors, cytokines, and chemokines in conditioned media
Thaw conditioned media plates and use aliquots for the TNFα ELISA and the Luminex assay (see manufacturers’ instructions).
Data analysis
The results for the TNFα ELISA comprise absorbance values, which are converted to concentrations of TNFα via the standard curve generated in the assay. It is recommended to set up six replicate wells for THP-1M only and each sample group of MSCs and THP-1M (biological replicates). The results are normalized to the TNFα release from macrophages in the absence of MSCs and are calculated as % inhibition of the THP-1M response. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using Prism 9 (GraphPad, San Diego, CA) using one-way analysis of variance (ANOVA) with post-hoc Tukey’s tests on paired comparisons.
The results for the Luminex 30 Plex Panel comprise fluorescence intensity measurements, which are converted to concentrations based on the standard curves generated for the 30 analytes. It is recommended to set up four replicate wells for THP-1M only and each sample group of MSCs and THP-1M (biological replicates). Readings are considered outliers if they fall outside of the linear portion of the standard curves. The concentrations can be standardized using JMP statistical software (Pro 17) to give a Z-score heatmap.
Since the polarization assay is downstream of the activation assay, the same number of biological replicates (n = 6) is used for each MSC donor. The polarization assay generates flow data, which can be compensated on the instrument (using single stains) and gated using FMOs and unstained samples using FlowJo or CytoBank software. Once populations have been established, the data are presented as HLA-DR+ cells as a percentage of CD14+ live cells (M1) or CD206+/CD14+ (single positive M2), CD163+/CD14+ (single positive M2), or CD206+/CD163+/CD14+ (double positive M2) cells.
Validation of protocol
Macrophage activation assay
Previous work in which the activation assay was described and validated is found in Medrano-Trochez et al. ([20], Figures 6 and 7), and Pradhan et al. ([21], Figures 2 and 6).
Evidence that inhibition of TNFα has occurred can be sought by a relatively straightforward ELISA. Once that has been determined, the conditioned media can then be subjected to a Luminex for comparison of multiple analytes. Two THP-1 cell vials from different culture batches (B1 = batch 1 and B2 = batch 2) were used in this assay. The profiles of the THP-1 B1 and B2 are similar. The protocol is robust and reproducible since each co-culture is compared to its THP-1M-only control. The activation assay was validated using bone marrow–derived and umbilical cord tissue–derived MSCs [21,20] (Pradhan et al., 2020; Medrano-Trochez et al., 2021). Separate data on three MSC donors are shown in Figure 2. Overall, the culture-rescued cells showed less inhibition of the TNFα release from macrophages than freshly thawed MSCs.
Figure 2. Percent inhibition of tumor necrosis factor-alpha (TNFα) by mesenchymal stromal cells (MSC) donor and Z-score of analytes in conditioned media. A. Values of TNFα concentration in the media are measured using ELISA and used as a baseline to calculate the % inhibition of the response when MSCs are added. THP-1M is macrophage only; OOT = out-of-thaw/freshly thawed; CR = culture rescued. All donor samples are significantly different from THP-1M. B. For six donors OOT and CR, a panel of analytes present in the co-culture media is presented as Z scores, where orange is high, and blue is low concentration.
Macrophage polarization assay
Phenotypic analysis of cells was performed at harvest by flow cytometry. The percentage of cells expressing surface markers outlined in the methods section was analyzed following an initial gating strategy of forward and side scatter > singlets > live cells (Zombie UV live/dead discrimination) on > 30K acquired events. In wells containing MSCs, there was a high surface marker expression of CD73, and this was used to gate out MSCs. The cell population that expressed CD14 was then identified and used to measure levels of HLA-DR for THP-1M and THP-1M and MSC donor. The gating strategy is shown in detail in Figure 3 as cell plots and in Figure 4 as gating hierarchy. This protocol was validated using bone marrow–derived and umbilical cord tissue–derived MSCs for both out-of-thaw and culture-rescued cells. Data for five donors, added at a ratio of 1:2 (MSCs: THP-1M), are shown in Figure 5. Overall, MSCs showing a greater loss of HLADR positivity showed a larger extent of polarization to M2, at least for CD163 positivity.
Figure 3. Illustration of gating steps needed to establish macrophage phenotype (HLA-DR+, CD163+/CD14+, CD206+/CD14+, or CD163+/CD206+/CD14+ cells) and presence of mesenchymal stromal cells (MSCs) (CD73+). Gating strategy shown in the sequence of panels from CytoBank.
Figure 4. Illustration of gating hierarchy for separation of cell populations. The final gate is a quadrant for CD163 and CD206 marker analysis.
Figure 5. Macrophage phenotype determined at harvest by flow cytometry. Macrophage phenotype determined for THP-1M and with mesenchymal stromal cells (MSC) in co-culture at harvest. A. HLA-DR positive (M1-like) macrophages as a percentage of CD14+ live cells, measured with and without MSCs, with all groups showing significant M1 marker reduction apart from donor 2. B. CD206 positive (M2-like) macrophages as a percentage of CD14+ live cells. C. CD163 positive (M2-like) macrophages as a percentage of CD14+ live cells. Donors 4 and 5 showed lower HLA-DR positivity. D. Double positive CD206/CD163 (M2-like) macrophages as a percentage of CD14+ live cells. The statistical test applied was a one-way ANOVA with Tukey’s post-hoc test; *(p < 0.05), **(p < 0.001), ***(p < 0.001), ****(p < 0.0001); * denotes significance compared to THP-1M group.
General notes and troubleshooting
General notes
MSCs can be delivered in diluting buffer or media. Both have been used successfully for this protocol. Importantly, the same delivery vehicle should be in all MSC samples, with the same cell concentration and the same volume for comparisons to be made.
This protocol demonstrates the assay for bone marrow–derived MSCs and umbilical cord tissue–derived MSCs. Additional optimization may be required for thaw, culture, and harvest if using MSCs derived from another tissue source as well as ratios of MSCs to THP-1M.
In our hands, the maintenance of the THP-1M M1 state required the sustained presence of LPS and IFNγ in the culture media. The release of TNF-α, coupled with the establishment of M1 phenotype, can be used to optimize the concentration of LPS needed in the assay.
A successful harvest from the co-culture plate requires a two-stage process. The loosely adherent macrophages will be in the media at the bottom of the well, so removing media without disturbing the lowest layer is important. After collecting the macrophages in a wash step, enzyme digestion is needed to detach the MSCs from the plate.
This step can be avoided if the phenotype of the THP-1Ms is the only cell type under investigation. The TrypLE can be used if MSCs are required in the flow analysis.
As 50 µL of Fc block is left in each well before the antibody cocktail is added, the final dilution is 1/20 for 5 µL of antibody. It is recommended to first titrate antibody concentrations to test detection limits for particular cytometers. A concentration of as little as 1 µL of antibody per well (1:100), for each of the five-panel antibodies, has been demonstrated to work well in this assay.
Troubleshooting
The method to culture THP-1 monocytes has been described before [22] and has been modified to deal with the many issues encountered with expanding and maintaining these cells [23]. The cells are freezer sensitive and can become exhausted if expanded too much or less proliferative if not dense enough. A rule of thumb is to observe the diameter of the cells. Healthy viable cells are 13–14 µm and dead/dying cells are 6–7 µm, determined using a cell counter. Once cell size decreased, they became irretrievable in our hands. Once expanded, however, they can provide a very consistent cell line for functional assays.
If THP-1 monocytes are slowing down in proliferation during the expansion phase, add 1:1 conditioned media to fresh media at each feeding.
Acknowledgments
This work was supported by the Billi and Bernie Marcus Foundation and the National Science Foundation Engineering Research Center for Cell Manufacturing Technologies (NSF EEC 1648035). Previous work in which the activation assay was described and validated is found in Medrano-Trochez et al. [20] and Pradhan et al. [21]. Graphical overview created with BioRender.com.
Competing interests
The authors have no competing interests that may influence the material presented in this protocol.
Ethical considerations
No human subjects were used for any experiments included in this study. All procedures using MSCs derived from umbilical cord tissue were supplied by collaborators at Duke University in accordance with the ethical standards and approved by the ethics committee of the institutional review boards at Georgia Institute of Technology and Duke University (IRB Protocol No. H17348).
References
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Preparation and Purification of β-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay
PP Pirjo Pärnänen *
TS Timo Sorsa *
TT Taina Tervahartiala *
PN Pirjo Nikula-Ijäs *
(*contributed equally to this work)
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4958 Views: 359
Reviewed by: Alba BlesaSuresh PantheeSascha Brunke
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Original Research Article:
The authors used this protocol in Microbial Pathogenesis Dec 2020
Abstract
Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify β-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes β-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration(charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC–MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species.
Key features
• Uses basic materials and laboratory equipment, enabling low-cost studies.
• Facilitates the selection and identification of proteases with certain molecular weights.
• Enables further functional studies with host proteins, such as structural or immune response–related, or enzymes and candidate protease inhibitors(e.g., from natural substances).
• This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.
Graphical overview
Keywords: Candida glabrata Cell wall proteases Ion-exchange chromatography Gel filtration Gelatin-zymography assay
Background
Candida glabrata is an opportunistic pathogen causing infections in vulnerable hosts. As C. glabrata is often resistant to the most used azole antimycotics, the search for novel and safe antimycotics is ongoing. Among the most difficult challenges in understanding yeast pathogenesis is identifying the yeast proteome and virulence factors that affect the course of infection. While the cell wall composition of Candida albicans, the most common opportunistic yeast found in candidiasis, has been quite extensively studied, less is known of C. glabrata’s cell wall proteins, whose key elements and interactions remain undiscovered. The cell wall proteome is the front line when contacting host cells. C. glabrata cell wall structure resembles that of C. albicans and, even more closely, of S. cerevisiae [1–5].
Glycosylphosphatidylinositol (GPI) proteins, covalently bound to the cell wall β-1,6-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to β-1,3-glucan are the two main classes of C. glabrata cell wall proteins, which participate in the interaction with the host [2]. To the last group belong Candida proteins that are delivered to the cell wall from their original intracellular location, named as “moonlighting” proteins. They lack an N-terminal signal sequence guiding formed proteins into the cell wall and are transported there in extracellular vesicles; these have extracellular enzymatic activities in addition to their intracellular housekeeping roles and play multiple functions in host cell interactions. The role of these moonlighting proteins as virulence factors is complex and partially unknown.
The presented protocol describes the enzymatic separation and purification of proteases from C. glabrata cell wall attached to the glucan network (see workflow in Graphical overview). The main objective is to identify novel proteases from the C. glabrata cell wall and enable further analysis of their potential enzymatic activities in host protein degradation. The protocol retains the isolated proteases in their active forms, which is a crucial advantage compared to previous studies in proteomics, allowing the understanding of the functions and interactions with host proteins. The glycosylation of cell wall proteins poses challenges in their identification process: carbohydrates attached to proteins mask them, hampering enzymes used in assays from degrading the protein into a form that may be identified by mass analysis. By detaching the proteases from the cell wall glucan network, it is possible to identify isoenzyme forms by MS/MS. Although dithiothreitol or β-mercaptoethanol have been traditionally used in extracting yeast cell wall proteins, they may cause cell rupture or denaturation of proteins [4]. The use of the β-1,3-glucan specific enzyme lyticase (from Arthrobacter luteus) in cleaving is essential to maintain the proteases in their active form, contrary to conventional non-functional studies. Lyticase has traditionally been used in the degradation of the yeast cell wall, formation of spheroplasts, and transformation procedures, and Candida enzymatic activity may be determined by 7-amino-4-methylcoumarin (AMC)-labeled L-arginine fluorogenic substrate degradation [6].
2-Methoxy-2,4-diphenyl-3(2H)-furanone (MDPF)-gelatin zymography is a convenient method for monitoring the protein purification process. In MDPF-gelatin zymography, the C. glabrata proteases retain their enzymatic activity, because non-denaturing conditions are maintained through the process. The strength of gelatinolysis caused by C. glabrata proteases may be seen as the disappearance of the MDPF-labeled gelatin fluorescence in the SDS-PAGE gel, which may be monitored under UV light. MDPF is a more stable fluorophore compared with FITC or fluorescein [7,8]. MDPF-gelatin zymography is suitable for host protein degradation assays [9]. The lyticase extract is initially separated into coarse fractions with centrifugal devices having cut-off values of 10 and 50 kDa. Further protein charge–based ion-exchange chromatographic separation is optimized regarding pH, and a weak anion-exchange column is used to extract > 50 kDa proteins; further 10–50 kDa and > 50 kDa fractions are obtained by size-exclusion gel filtration. The refining of these fractions enables proper separation, extraction, and identification of the protein bands from silver-stained gels for further mass analyses. The methods used here are easily applied with modifications to study the cell wall proteome in other pathogenic species when searching proteins with specific linkage to the cell wall. In studying the entire proteome of the cell wall, a shotgun approach should be preferred.
Materials and reagents
Biological materials
C. glabrata T-1639, blood isolate (Helsinki University Central Hospital, Helsinki, Finland). Other Candida species may be studied by these methods if there are structural analogies with C. glabrata cell wall protein attachment molecules.
Reagents
Sabouraud dextrose agar (Lab M, catalog number: LAB009)
Lyticase, 10 kU (Sigma-Aldrich, catalog number: 37340-57-1)
2-Methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) (Sigma-Aldrich, catalog number: 50632-57-0)
Precision Plus ProteinTM standards (Bio-Rad, catalog number: 161-0373)
Tris base (Sigma, catalog number: T-1378)
ZnCl2 (Sigma, catalog number: 208086)
CaCl2 (Sigma, catalog number: 10035-04-8)
Tween 80, BioXtra viscous liquid (Sigma, catalog number: P8074)
BactoTM yeast extract (BD, catalog number: 212750)
Peptone, BactoTM peptone (BD, catalog number: 211677)
Glucose (Merck, catalog number: 108337)
Sodium dodecyl sulphate (SDS) (Fisher Scientific, catalog number: BP166-500)
Sodium azide (NaN3) (Merck, catalog number: 6688), for long-term preservation of ion-exchange columns
Coomassie Brilliant Blue, Serva Blue R (Serva Electrophoresis GmbH, catalog number: 35051)
Sodium tetraborate (STB) (Merck, catalog number: 6308)
Gelatin (Merck, catalog number: 1.04070)
Acetone (Merck, catalog number: 1.00658)
Na2HPO4 (Sigma, catalog number: 137036)
KCl (Sigma, catalog number: PHR1329)
NaCl (Fisher Scientific, catalog number: FLS271500)
KH2PO4 (Merck, catalog number: 104871)
β-mercaptoethanol (Merck, catalog number: M6250)
TEMED (Merck, catalog number: 411019)
Acrylamide 30% (Merck, catalog number: A3574)
EtOH, Ethanolum anhydricum, Aa (Berner, catalog number: 13110124)
HCl 1 M (Riedel-de-Haen, catalog number: 30721)
HCl 37% (Sigma, catalog number: 258148)
NaOH 1 M (Acros Organics, catalog number: 15634890)
Methanol (Fisher, catalog number: M/4057/PB17)
Acetic acid (Fisher, catalog number: A/0400/PB17)
Ammonium persulfate (APS) 10% (Sigma, catalog number: A3678), store in 100 µL aliquots at -20 °C
4x Laemmli Sample Buffer (Bio-Rad, catalog number 1610747)
Glycine (Merck, catalog number: G8898)
Solutions
Yeast extract peptone glucose (YPG) (see Recipes)
MDPF-gelatin (see Recipes)
Separating gel (lower) buffer for zymo gels (see Recipes)
Stacking gel (upper) buffer for zymo gels (see Recipes)
8% MDPF-gelatin SDS-PAGE (see Recipes)
SDS-PAGE running buffer 5× (see Recipes)
Zymo buffer 10× (see Recipes)
Zymo gel incubation buffers (see Recipes)
Coomassie Brilliant Blue (CBB) 0.1% (see Recipes)
IEC start buffer (see Recipes)
IEC elution buffer I (see Recipes)
IEC elution buffer II (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
10% SDS (see Recipes)
Lyticase stock (see Recipes)
Recipes
Perform pH adjustments with 1 M NaOH or 1 M HCl. Prepare zymogels as needed and solutions according to Recipes beforehand. Preparing double amounts of Recipes 10–13 is advised.
Yeast extract peptone glucose (YPG)
0.5% yeast extract
1% peptone
1% glucose
Add Milli-Q water (MQ) up to 4 L
Sterilize by autoclave at 121 °C for 15 min
MDPF-gelatin
20 mM STB solution: 0.3814 g of STB per 50 mL of MQ, pH 9.2.
Add 500 mg of gelatin into STB in warm water bath (approximately 37 °C) with magnetic stirring.
Dissolve 25 mg of MDPF into -20 °C acetone on ice and add MDPF into gelatin-STB at room temperature (RT) with magnetic stirring for 1 h protected from light. Divide into 500 µL aliquots and store at -20 °C protected from light.
Separating gel (lower) buffer for zymo gels
800 mL of MQ
187 g of Tris base
Adjust pH to 8.8
4 g of SDS
Add MQ up to 1 L
Stacking gel (upper) buffer for zymo gels
800 mL of MQ
60.5 g of Tris base
Adjust pH to 6.8
4 g of SDS
Add MQ up to 1 L
8% MDPF (1 mg/mL)-gelatin SDS-PAGE (4 gels)
Prepare gels in a fume hood:
Bottom: separating gel
Separating buffer 5 mL (Recipe 3)
30% Acrylamide 5.3 mL
MQ 7.3 mL
MDPF-gelatin (1 mg/mL) 2 mL (Recipe 2)
10% SDS 200 µL
TEMED 20 µL
10% APS 200 µL
Top: stacking gel
Stacking gel buffer 2.5 mL (Recipe 4)
30% Acrylamide 1.35 mL
MQ 5.95 mL
10% SDS 100 µL
TEMED 10 µL
10% APS 100 µL
SDS-PAGE running buffer 5×
Add 4 L of MQ into a Duran bottle
723 g of glycine
150 g of Tris base
20 g of SDS
Adjust pH to 8.3
Add MQ up to 5 L
Zymo buffer 10×
Add 800 mL of MQ into a Duran bottle
60.55 g of Tris base
2 g of NaN3
33 mL of 37% HCl
Adjust pH to 7.5
Add MQ up to 1 L
Zymo gel incubation buffers for 4 gels
Zymo buffer I
360 mL of 1× zymo buffer (dilute from 10× zymo buffer with MQ)
40 mL of 25% Tween 80
Zymo buffer II
270 mL of 1× zymo buffer
30 mL of 25% Tween 80
30 µL of 10 mM ZnCl2
1530 µL of 100 mM CaCl2
Zymo buffer III
300 mL of 1× zymo buffer
30 µL of 10 mM ZnCl2
1530 µL of 100 mM CaCl2
Coomassie Brilliant Blue (CBB), 0.1% (staining and destaining in fume hood)
1 g of Coomassie Brilliant Blue, Serva Blue R
600 mL of MQ
300 mL of methanol
100 mL of acetic acid
Destaining: 40% MeOH + 10% acetic acid in MQ
IEC start buffer (20 mM Tris HCl, pH 8.2)
2.4 g of Tris base
Add 800 mL of MQ
Adjust pH to 8.2
Add MQ up to 1 L
Sterile filter and degas
IEC elution buffer I (20 mM Tris HCl-0.5 M NaCl, pH 8.2)
29.22 g of NaCl
2.4 g of Tris base
Add 800 mL of MQ
Adjust pH to 8.2
Add MQ up to 1 L
Sterile filter and degas
IEC elution buffer II (20 mM Tris HCl- 1 M NaCl, pH 8.2)
14.61 g of NaCl
0.6 g of Tris base
Add 200 mL MQ
Adjust pH to 8.2
Add MQ up to 250 mL
Sterile filter and degas
Phosphate-buffered saline (PBS), pH 7.4
800 mL of MQ
8.0 g of NaCl
1.44 g of Na2HPO4
0.2 g of KCl
0.24 g of KH2PO4
Adjust pH to 7.4 using HCl
Add MQ up to 1 L
Filter and sterilize by autoclavation
10% SDS
80 mL of MQ
10 g of SDS
Add MQ up to 100 mL
Lyticase stock
1.6 mL of MQ
Add 10 kU lyticase
Store at -20 °C in 400 µL aliquots
Laboratory supplies
Sterile plastic inoculation loops (Greiner Bio-One, catalog number: 731171)
Millipore ExpressTM Plus or Filtration unit Stericup® with MILLIPORE Express® PLUS (PES), 0.22 µm (Merck Millipore, catalog number: X342.1, optional method: centrifugation)
Millipore Millex, 0.22 µm (Merck, catalog number: 051581)
Agar plates, plastic Petri dishes, PS (Sigma, catalog number: P5731)
Glass chromatography column (9.5 mL) with stopcock and lower silicon tubing (or Econo Alpha column, Bio-Rad, catalog number: 12009430) and column stand
Glass Erlenmeyers, 2× 3 L, 2× 200 mL
Amicon® Ultra-15 Centrifugal Filter Devices, 10 K and 50 K (Merck Millipore, catalog numbers: UFC901024, UFC905024)
Glass decanters 500 mL
EppendorfTM Snap-Cap Microcentrifuge Safe-LockTM Tubes [Fisher, catalog number: 05-402-18 (1.5 mL) and 05-402-18 (0.5 mL)]
FalconTM tubes, 15 mL, 50 mL (Fisher, catalog numbers: 14-959-53A, 14-432-22)
Micropipettes and sterile pipette tips, 10 µL–5 mL
Aluminum foil
Tube racks for FalconTM and EppendorfTM tubes
HiTrap® IEX Selection Kit (Merck, GE17-6002-33)
Sephadex G-100 and G-200 (Pharmacia Fine Chemicals, catalog numbers: 01-900-1-1861-03, 01-900-1-1863-03) 1 g/40 mL of buffer
10 mL sterile plastic syringes (Fisher, catalog number: 14955459)
Ice, 2 × 5 L plastic dish
Duran bottles, 1 L, 5 L
Funnel that fits gel filtration column
Equipment
Heating cabinet, 31 °C, 37 °C, optional: Melag Incubat® 85 (or Merck My Temp Mini Digital Incubator, catalog number: Z763357) or SureTemp® Dual Convection Incubator (Merck, catalog number: Z742696)
Nanodrop 1000 (or NanoDrop Lite Plus Spectrophotometer, catalog number: NDLPLUSGL)
pH meter (VWR® phenomenal® MU 6100 H, catalog number: 665-0311)
PAGE-running device, 20 µL well volume, 4–6 combs of 10 wells (Bio-Rad, model: Mini-Protean II, catalog number: 165-2940)
Plastic containers (500 mL) for zymo buffer incubation, disposable PP, flat bottom (Marjukka, catalog number: 6410416249018)
Light box for gel visualization, LX104, Lammex (or X-ray viewer; Quirumed, catalog number: 482-6001.S)
Gel scanner, GS-700 densitometer (Bio-Rad, catalog number: 170-7601 with QUANTITY ONE PROGRAM)
Light microscope, 10–40× magnification
Vacuum suction system for degassing buffers and Sephadex gel
UV light for gel visualization and photography (Uvidoc, model: M03 0103 or GelDoc Go Gel Imaging System with Image Lab Touch Software, catalog number: 12009077)
Level mixer (Heidolph Vibramax 100, catalog number: 544-21200-00)
Centrifuge SL16R and adapters for 15 mL tubes (Thermo Scientific, catalog number: 75004030)
Autoclave (Systec, model: D45)
Fume hood (Visukaluste Ltd, model: VISU ProFocus EN 14175-2)
Procedure
Preparation of yeast cells
Transfer C. glabrata cells from -80 °C onto a Sabouraud dextrose agar (SDA) plate with a sterile inoculation loop. Incubate the plate at 37 °C for 24 h. This incubation time is optimal to obtain enough stationary-phase yeast cells (expected OD > 2), as C. glabrata does not form true hyphae. However, the 24 h incubation time might be a problem in hyphae-forming species, such as C. albicans [try overnight (o/n) culturing].
Check purity of growth visually from the culture plate and with light microscopy (10–40× magnification) regarding colony and cell morphology. Prepare a pure culture from this by picking one colony and incubate as previously described.
Transfer a loopful of cells with a sterile inoculation loop from the pure culture SDA plate (check purity as above) into 2 × 2 L of YPG (see Recipe 1) divided into two Erlenmeyers. Cap the Erlenmeyers loosely with aluminum foil, place onto a level mixer, and incubate at low speed (65 rpm) for 24 h at 37 °C.
Filter the cell suspension into 2 L aliquots with a Stericup with MILLIPORE Express PLUS 0.22 µm filter with vacuum suction. Use a second filter unit for the rest of the cell suspension, as the filter may clog. Recover the cells from the filter membranes with 150 mL of PBS/filter into eight 50 mL Falcon tubes in equal volumes.
Centrifuge at 1,753× g for 10 min at RT. Discard supernatant.
Add 40 mL of PBS onto the cells in each tube and suspend to even composition. Centrifuge at 1,753× g for 10 min at RT. Discard supernatants, add 40 mL of PBS to each tube, centrifuge at 1,753× g, and discard supernatants.
Recover all cells from the bottom of each Falcon tube with 2.5 mL of PBS by suspending cells with a pipette and transfer them into a 100 mL Erlenmeyer. Add 40 mL of PBS (total suspension volume = 80 mL).
Lyticase treatments
Add 400 µL of prepared lyticase solution onto the cell suspension (final concentration approximately 30 U/mL), cap loosely with aluminum foil, and incubate on a level shaker (at 65 rpm) for 22 h at 31 °C. (Here, the heating cabinet was placed and strapped onto a level mixer; optionally, place level mixer into a larger incubator cabinet.)
Divide cell suspension equally into two 50 mL FalconTM tubes and centrifuge at 1,753× g for 10 min at RT. Recover supernatants (total volume = 35 mL) and store at -20 °C.
Recover cells from Falcon tubes with a total of 40 mL of PBS into a 200 mL Erlenmeyer. Add 400 µL of lyticase solution, cap loosely with aluminum foil, and incubate on a level shaker (65 rpm) for 22 h at 31 °C.
Repeat step B3. Freeze supernatants or continue pooling with supernatants from the freezer. Take a 500 µL sample for zymography and protein concentration measurement and store the rest at -20 °C.
Resuspend cells in residual volume in the Falcons and verify that cells are intact with light microscopy (see Figure 1 ).
Figure 1. Intact C. glabrata spheroplasts after lyticase treatments. Light microscopy, 40× magnification.
Separation of 10–50 kDa and > 50 kDa fractions
Place PBS on ice. Thaw and filter pooled supernatants (V = 70 mL) with a Filtration unit Stericup with MILLIPORE Express PLUS 0.22 µm filter with vacuum suction or, optionally, by centrifugation. Take a 500 µL sample for protein concentration analysis and zymography. Divide the supernatant into eight Amicon® Ultra-15 Centrifugal Filter Devices 10 K. Centrifuge at 1,753× g for 20 min at 4 °C. Remove and pool flowthroughs.
Add 10 mL of cold PBS to each tube and centrifuge at 1,753× g for 20 min at 4 °C.
Pool flowthroughs with earlier flowthroughs. Take a 500 µL sample for zymography of the pooled total flowthrough and store it and the rest in 50 mL Falcon tubes at -20 °C (< 10 kDa fraction). Transfer upper supernatants equally into two Amicon® Ultra-15 Centrifugal Filter Devices 50 K. Centrifuge at 1,753× g for 20 min at 4 °C.
Recover flowthrough (10–50 kDa fraction).
Add 12 mL of PBS/filter and centrifuge at 1,753× g for 20 min at 4 °C. Pool flowthrough with the 10–50 kDa fraction (approximately 40 mL). Take a 500 µL sample and store it and the rest in 10 mL aliquots in Falcon tubes at -20 °C.
Recover > 50 kDa fraction by suspending 3 × 5 mL of PBS to obtain approximately 30 mL of > 50 kDa fraction. Take a 500 µL sample and store it and the rest in 8 mL aliquots in Falcon tubes at -20 °C.
Measure protein concentrations (A280 nm) with Nanodrop 1000 and run 8% MDPF-gelatin zymography to evaluate protein recovery. See Figure 2.
Figure 2. UV photograph of 8% MDPF-gelatin zymography after separation of 10–50 kDa and > 50 kDa proteases with centrifugation devices. Lanes: 1. Molecular weight standard in kDa. 2. Lyticase-treated fraction. 3. Lyticase-treated fraction with 1 µL of β-mercaptoethanol. 4. Lyticase-treated 10–50 kDa fraction. 5. Lyticase-treated > 50 kDa fraction. 6. Lyticase-treated > 50 kDa fraction with 1 µL of β-mercaptoethanol. Fraction in lane 5 was chosen for ion-exchange chromatography because of desired strong gelatinolytic activity of > 50 kDa sized proteases. The activating effect of β-mercaptoethanol as a reducing agent breaking disulfide bonds in the > 50 kDa fraction in lane 6 may be seen.
Preparation for gel filtration
Measure 1 g of Sephadex G-200 and swell in 40 mL of 20 mM Tris HCl, 0.5 M NaCl, pH 8.2 at RT for 3 days in a sterile decanter (covered with aluminum foil).
Degas gel and buffers with vacuum suction until no bubbling of gases. Store these at 4 °C until used.
MDPF-gelatin zymography
Prepare 8% MDPF-gelatin-labeled zymogels. Prepare bottom separating gel. Add 4.45 mL of this solution between glasses in the casting device and add MQ on top of the gel to prevent drying and to obtain a linear top level of the gel. Let it settle for 30 min. Remove MQ by pouring and gently absorbing with cellulose blotting paper before casting the stacking gel. Prepare top stacking gel. Add stacking gel and comb and cover from light and drying while setting of gels (30 min). Store at 4 °C in MQ-moistened paper wrap and plastic bag covered from light until used. Shield from light during and after setting. Gels may be stored at 4 °C wrapped in MQ-moistened paper in a plastic bag shielded from the light up to one week.
Incubate 15 µL of samples with 5 µL of 4× Laemmli sample buffer in 0.5 mL Eppendorf tubes at RT for 2 h in non-shaking conditions.
Fill the running chamber using 1× SDS-PAGE running buffer (diluted from 5× running buffer with MQ). Use pre-stained SDS–polyacrylamide gel electrophoresis standards (10 µL, Precision Plus ProteinTM standards) for evaluation of protein size, pipette the standard, and incubated samples into gel wells.
Perform electrophoresis at 110 V with the device shielded from light and on ice water.
Incubate gels in zymo buffer I for 30 min at RT and then in zymo buffer II for 30 min at RT on a platform swing. Then, incubate in zymo buffer III o/n or for 1–7 days at 37 °C in a heat cabinet, all protected from light with aluminum foil.
Follow the gelatinolytic reaction for 1–2 days with UV light (photograph), stop at the desired gelatin degradation stage, and stain the gel with 0.1 CBB, destain, scan image, and analyze. Gels may be stored in MQ until scanned.
Choose fractions with the highest gelatinolytic activity and protein concentration. Pool and/or concentrate fractions if needed for the next step (see Figure 2).
Ion-exchange chromatography (IEC)
Evaluate cation and anion exchangers with pH optimization (tested pH for the 10–50 kDa fraction was 6.4; for the > 50 kDa fraction, pH was 7.5 and 8.2) with both weak and strong anion and cation exchangers to find the best combination to capture desired proteins (HiTrap® IEX Selection Kit). This process depends on the isoelectric point of the proteins and affects the bonding to and elution from the column depending on the net charge of the proteins. Aggregation of proteins is maximal at the isoelectric point (pI). We had prior knowledge of the estimated pIs from earlier studies using isoelectric focusing and 2D-MDPF-gelatin zymography. DEAE FF (weak anion exchanger) with buffer pH 8.2 is described in the rest of the protocol as an example for obtaining best recovery of the > 50 kDa fraction.
Prepare IEC start buffer, IEC elution buffer I, and IEC elution buffer II (see Recipes). Filter buffers (0.22 µm), degas, and place on ice.
Centrifuge 8 mL of > 50 kDa fraction at 1753× g for 10 min at 4 °C (fraction C6). Discard supernatant and recover volume to 8 mL with IEC start buffer. Concentrate sample as needed by centrifugation at 1753× g for 20 min at 4 °C. Keep sample on ice.
Wash the DEAE FF column (HiTrap® IEX Selection Kit) with 5 mL of IEC start buffer at 1 mL/min, then with 5 mL of IEC elution buffer II, and then with 5 mL of IEC start buffer again. Avoid air bubbles.
Apply sample with syringe at 1 mL/min. Start gathering 0.5 mL fractions into 1.5 mL Eppendorf tubes. Wash the column with 5 mL of IEC start buffer and then add 5 mL of IEC elution buffer I.
Store the column according to manufacturer’s instructions.
Measure protein concentration with Nanodrop 1000 and run zymography gels to locate fractions with gelatinolytic activity and highest protein concentration. See Figure 3.
Figure 3. UV photograph of 8% MDPF-gelatin zymography (A) and after Coomassie brilliant blue stain (B) for evaluation of ion-exchange chromatography. Lanes: 1. Molecular weight standard in kDa. 2. Protease sample before ion-exchange chromatography. 3 and 5. Flowthroughs. 4 and 6. Elution fractions. Elution fraction in lane 6 was chosen for gel filtration because of strongest gelatinolytic activity and highest A 280 in the desired protein sizes.
Gel filtration
The setup of the equipment for gel filtration is shown in Figure 4.
Figure 4. Example of setting up the equipment for gel filtration. Keep samples on ice. Remember the safety loop in silicon tubing.
Fill a 500 mL decanter with IEC elution buffer I. Enable buffer flow by placing the decanter so high that its silicon tubing end is at a higher level than the silicon tubing end attached to the lower part of the glass column. Ensure there is a safety loop in the tubing to avoid gel dehydration if buffer runs out (see Figure 4).
Take a 9.5 mL column (15 cm length, radius = 4.5 cm) with lower silicon tubing. Open the lower valve of the column and, with a funnel, pour gel until the column is 4/5 full in one continuous motion to avoid air bubbles. Let it settle so that the upper level of the gel does not drop. Fill the column halfway from its lower end with 10% EtOH with a syringe to exclude air bubbles.
Adjust the flow rate of the buffer with 2 × 10 mL of IEC elution buffer I to one drop per 30 s by adjusting decanter position or lower tube length. Drain the buffer to just above the top level of the gel. Shut the lower valve. The column may be stored at 4 °C for approximately one week if not immediately used. For longer storage, add 0.02% sodium azide and wash with approximately 2× column volumes before use.
Recommended sample volume is 1%–5% of bed volume. Choose best elution fraction from ion exchange chromatography (see Figure 3) and concentrate 5 mL to 500 µL with Amicon® Ultra-15 Centrifugal Filter Device 50 K.
Apply 500 µL of the > 50 kDa fraction gently along the edge, avoiding splashing onto the top of the gel bed. Open the lower valve and let the sample sink in. Fill the column all the way to the top with IEC elution buffer I, close upper cap of the column, and attach the silicon tubing from the decanter to the top of the column. Open the lower valve and observe if buffer flow is continuous.
Collect approximately 24 × 0.5 mL fractions. Number the tubes. Measure protein concentration and select the highest concentration (pool fractions if necessary). Run zymography gels to detect highest enzymatic activity for further analyses. See Figure 5. Chosen fraction may be further purified with Sephadex G-100 (as described above) and run on traditional 8% SDS-PAGE and parallel silver-stained gel for LC−MS/MS analyses (Figure 6).
Figure 5. UV photograph of 8% MDPF-gelatin zymography after gel filtration. Lanes: 1. Molecular weight standard in kDa. 2. > 50 kDa fraction after ion-exchange chromatography. 3–8. Elution fractions 1–6 after G-200 gel filtration. Gel filtration of > 50 kDa fraction with Sephadex G-100 did not show any activity with zymography. Fraction 3 in lane 5 was further used in LC−MS/MS analyses.
Figure 6. Overview of the purification results, 8% SDS-PAGE gel, silver stained. Lanes: 1. Molecular weight standard in kDa. 2. > 50 kDa fraction before IEC. 3. > 50 kDa fraction before gel filtration. 4−10. > 50 kDa gel filtration fractions.
Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:
Pärnänen, P., Sorsa, T., Tervahartiala, T., and Nikula-Ijäs, P. (2020). Isolation, characterization, and regulation of moonlighting proteases from Candida glabrata cell wall. Microb. Pathogen. 149: 104547. https://doi.org/10.1016/j.micpath.2020.104547 (Figures 1 and 2).
Pärnänen, P., Meurman, J.H., and Nikula-Ijäs, P. (2015). A novel Candida glabrata cell wall associated serine protease. Biochem. Biophys. Res. Commun. 457: 676–680. https://doi.org/10.1016/j.bbrc.2015.01.047 (Figures 1 and 2).
Pärnänen, P., Kari, K., Virtanen, I., Sorsa, T., and Meurman, J. (2008). Human laminin-332 degradation by Candida proteinases. J. Oral Pathol. Med.37: 329-35. https://doi.org/10.1111/j.1600-0714.2008.00638.x (Figure 1).
General notes and troubleshooting
General notes
Keep fractions and samples on ice when appropriate to not lose activity.
Sterile filter or autoclave and degas buffers and store at 4 °C.
Ion exchange chromatography and gel filtration may be conducted also for the 10–50 kDa proteins in the sample with the same principles as for the > 50 kDa fraction.
Use sterile laboratory equipment to avoid contaminations.
Getting to know the principles of the methods in advance is advised.
Troubleshooting
Preparation of yeast cells
Twenty-four hours has proven to be the best incubation time to ensure enough cells for the study. C. glabrata does not form true hyphae, but in other Candida species the incubation time should be shorter to keep cells in non-hyphal form; anti-hyphae substances may have to be used in addition. The OD 600 should be well over two. Too long incubation periods often cause cells to start breaking or transforming.
Lyticase treatment
If yeast cells are not intact when visualized by light microscopy, the lyticase concentration may be too high or treatment time too long. In this case, the intracellular proteins have leaked out and new cells must be cultured.
If there is no or low gelatinolytic activity (zymography) left after lyticase treatment, check if incubation temperature is too low /too high. Other Candida species than C. glabrata may have a different bonding of the cell wall gelatinolytic proteases, which are not hydrolyzed by lyticase.
Separation of 10–50 kDa and > 50 kDa fractions
If fractions show no or low gelatinolytic activity (zymography) left after centrifugal filtering devices, the proteins might not have been recovered from the filter. Try to gently suspend the buffer with a pipette tip (avoiding foaming) on the filter to detach stuck proteins.
Too high salt concentration may precipitate proteins.
Keep fractions on ice to avoid gradual loss of enzymatic activity.
MDPF-gelatin zymography
If CBB-stained gels are not blue, check MDPF-gelatin concentration or expiration date. Keep MDPF-gelatin in the dark; otherwise, fluorescence is lost.
Do not boil zymography samples prior to electrophoresis or enzymatic activity will be lost.
Proteins < 50 kDa may be seen in Figures 3, 4, 6, and 7. This is assumed to be caused by autodegradation or dissociation of smaller subunits of proteins.
IEC
Filter and degas buffers to avoid clogging of column by impurities or air bubbles.
No or low activity in zymography: protein concentration too low (concentrate sample but remember that too much concentration aggregates proteins); wrong pH in buffer or unsuitable ion-exchange column matrix.
Gel filtration
If proper size-dependent separation does not occur, the filtration column height or diameter may be wrong. You may also have to pack the column with matrix carefully and wait until the matrix settles to a horizontal position on the top part of the column. Gentle application of the sample into the column (not disturbing the settled matrix) ensures sharper fraction separation.
If there is no buffer flowing through the column: impurities or air bubbles in gel matrix (caused by non-continuous packing of the column) or buffer vial located too low relative to the column (no hydrostatic pressure).
Collect enough fractions and run zymograms of all fractions to recover/detect desired proteins.
Competing interests
The authors declare no competing interests.
References
Gow, N. A. R., Latge, J. P. and Munro, C. A. (2017). The Fungal Cell Wall: Structure, Biosynthesis, and Function. Microbiol. Spectr. 5. https://doi.org/10.1128/microbiolspec.FUNK-0035-2016
de Groot, P. W. J., Kraneveld, E. A., Yin, Q. Y., Dekker, H. L., Gross, U., Crielaard, W., de Koster, C. G., Bader, O., Klis, F. M., and Weig, M. (2008). The Cell Wall of the Human Pathogen Candida glabrata: Differential Incorporation of Novel Adhesin-Like Wall Proteins. Eukaryotic Cell 7(11): 1951–1964. https://doi.org/10.1128/ec.00284-08
Pardini, G., De Groot, P. W., Coste, A. T., Karababa, M., Klis, F. M., de Koster, C. G. and Sanglard, D. (2006). The CRH Family Coding for Cell Wall Glycosylphosphatidylinositol Proteins with a Predicted Transglycosidase Domain Affects Cell Wall Organization and Virulence of Candida albicans. J. Biol. Chem. 281(52): 40399–40411. https://doi.org/10.1074/jbc.m606361200
Satala, D., Bras, G., Kozik, A., Rapala-Kozik, M. and Karkowska-Kuleta, J. (2023). More than Just Protein Degradation: The Regulatory Roles and Moonlighting Functions of Extracellular Proteases Produced by Fungi Pathogenic for Humans. J. Fungi 9(1): 121. https://doi.org/10.3390/jof9010121
Klis, F. M., de Jong, M., Brul, S. and de Groot, P. W. J. (2007). Extraction of cell surface‐associated proteins from living yeast cells. Yeast 24(4): 253–258. https://doi.org/10.1002/yea.1476
Klinke, T., Rump, A., Pönisch, R., Schellenberger, W., Müller, E. C., Otto, A., Klimm, W. and Kriegel, T. M. (2008). Identification and characterization of CaApe2-- a neutral arginine/alanine/leucine-specific metallo-aminopeptidase from Candida albicans. FEMS Yeast Res. 8(6): 858–869. https://doi.org/10.1111/j.1567-1364.2008.00411.x
Snoek-van Beurden, P. A. and Von den Hoff, J. W. (2005). Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. BioTechniques 38(1): 73–83. https://doi.org/10.2144/05381rv01
O'Grady, R. L., Nethery, A. and Hunter, N. (1984). A fluorescent screening assay for collagenase using collagen labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone. Anal. Biochem. 140(2): 490–494. https://doi.org/10.1016/0003-2697(84)90199-4
Sorsa, T., Salo, T., Koivunen, E., Tyynelä, J., Konttinen, Y. T., Bergmann, U., Tuuttila, A., Niemi, E., Teronen, O., Heikkilä, P., et al. (1997). Activation of Type IV Procollagenases by Human Tumor-associated Trypsin-2. J. Biol. Chem. 272(34): 21067–21074. https://doi.org/10.1074/jbc.272.34.21067
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup
MD Margarida Dantas
DV Débora Vareiro
JF Jorge G. Ferreira
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4959 Views: 1493
Reviewed by: Rajesh RanjanSevgi OnalAnkur Garg
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Cited by
Original Research Article:
The authors used this protocol in The Journal of Cell Biology Oct 2022
Abstract
Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells.
Key features
• Protocol for microfabrication of confinement devices.
• Single-cell dynamic confinement coupled with high-resolution microscopy.
• Static cell confinement protocol that can be combined with super-resolution STED microscopy.
• Analysis of the mechanical control of mitotic entry in a time-resolved manner.
Graphical overview
Keywords: Cell confinement Cyclin B1 G2-M transition Hydrogels Live-cell microscopy Mechanical forces Mitotic entry Nucleus
Background
As proliferating cells prepare to enter mitosis, they undergo a series of biochemical and morphological changes that contribute to the assembly of the mitotic machinery and the efficiency of chromosome segregation [1]. Many of these changes are regulated by the activity and subcellular localization of the cyclin B1-CDK1 complex [2,3]. Notably, nuclear translocation of cyclin B1 seems to be essential to ensure full activity of the complex, as it stimulates its own nuclear import [3] through the nuclear pore complexes, triggering a positive feedback mechanism [4]. While the biochemical pathways that regulate the activity and localization of the cyclin B1-CDK1 complex have been well established [5], the contribution of mechanical forces to these processes remained unknown. We have recently identified a tension-dependent signal on the prophase nucleus that fine-tunes cyclin B1 translocation across the nuclear pores, setting the time for mitotic entry [6]. We further show that this signal relies on actomyosin contractility that leads to an unfolding of the nucleus and increased tension on the nuclear envelope (NE). Overall, we propose that mechanical forces are an important contributor for mitotic entry.
Here, we describe a protocol that enables the mechanical manipulation of cells during mitotic entry with high temporal and spatial resolution in single cells undergoing mitosis. This method allows the implementation of cell confinement techniques [7] in combination with high- (spinning-disk) and super-resolution (STED) microscopy to probe how mitotic entry is mechanically regulated. We provide step-by-step instructions on cell seeding, preparation of the confinement slides, assembly of the confinement devices, live-cell imaging, and data analysis. Moreover, we explain how to generate coverslips coated with polyacrylamide (PAA) hydrogels of calibrated stiffness that can be used in combination with high-resolution spinning-disk live-cell imaging. Finally, we describe an experimental setup that allows the long-term confinement of cell populations for fixed-cell immunofluorescence analysis. While these are used in the context of cell division, these methods could be easily adapted to other experimental settings.
Materials and reagents
Biological materials
RPE-1 parental cell line, already available in the lab
RPE-1 expressing endogenously tagged with H2B-GFP and tubulin mRFP, already available in the lab
RPE-1 cell line expressing exogenous cyclinB1-Venus (gift from Jonathon Pines)
RPE-1 cell line expressing cyclinB1-Venus/tubulin-mRFP, generated in our lab by transduction with lentiviral vectors containing pRRL-mRFP-α-tubulin
shRNAi-ROCK1-LV to deplete ROCK1 (gift from João Relvas)
RPE-1 cell line expressing exogenous cGAS-GFP (gift from Matthieu Piel)
RPE-1 cell line expressing GFP-NLS/tubulin-mRFP, created by lentiviral transduction using a pCDJ-NLS-copGFP-EF1-BlastiS plasmid (Addgene, catalog number: 132772)
RPE-1 cell lines expressing exogenous LBR-mCherry and endogenous cyclinB1-Venus/LBR-mCherry, generated by lentiviral transfection using a plasmid pWPT LBR-mCherry (gift from Stephen Royle)
DN-KASH plasmid (gift from Edgar Gomes)
Rap1Q63E plasmid (gift from Jean de Gunzburg)
Reagents
RO-3306 (Santa Cruz Biotechnology, catalog number: sc-358700A), used at 9 mM for 16 h
Importazole (gift from Helder Maiato), used at 40 mM for 2 h
Y-37632 (Sigma-Aldrich, catalog number: Y0503), used at 5 mM for 30 min
AACOCF3 (TOCRIS, catalog number: 1462), used at 20 μM for 30 min
BAPTA-AM (Abcam, catalog number: ab120503), used at 10 μM for 15 min
2APB (Abcam, catalog number: ab120124), used at 10 μM for 30 min
Para-nitro-blebbistatin (MotorPharma, catalog number: 1621326-32-6), used at 50 μM for 30 min
ML-7 (Merck, catalog number: I2764), used at 50 μM for 30 min
Cytochalasin D (Biogen Cientifica, catalog number: TO-1233), used at 0.5 μM for 30 min
Nocodazole (Merck, catalog number: M1404), used at 3.3 μM for 30 min
BI2536 (Axon Med Chem, catalog number: AXON 1129), used at 200 mM for 2 h
Etoposide (Selleck Chemicals CO., catalog number: S1225), used at 1 μM for 2 h
Thymidine (Sigma-Aldrich, catalog number: T1895), used at 2 mM for 16 h followed by a 10 h release
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540)
Lipofectamine 2000 (Invitrogen, catalog number: 11668019)
Lipofectamine RNAiMAX (Invitrogen, catalog number: 13778150)
Dulbecco’s Modified Eagle Medium (DMEM) (Corning, catalog number: 10-013-CV)
Fetal bovine serum (FBS) (Gibco, catalog number: 11550356)
Fibronectin (FBN) (Sigma, catalog number: F1141)
Leibovitz’s-L15 medium (Life Technologies, catalog number: 11415064)
Antibiotic-Antimycotic 100× (Life Technologies, catalog number: 15240096)
Bind-Silane (Sigma, catalog number: M6514)
Acetic acid (glacial) 100% (Merck, catalog number: 1.00063)
40% (w/v) acrylamide stock solution (Bio-Rad, catalog number: 1610140)
2% (w/v) bis-acrylamide stock solution (Bio-Rad, catalog number: 1610142)
PDMS crosslinker A + B (Momentive, catalog number: RTV615)
Isopropanol (Merck, catalog number: 1.09634)
dH2O
Trypsin (TrypLE Express Enzyme 1×) (Thermo Fisher Scientific, catalog number: 12605010)
Trypan Blue solution 0.4% (Gibco, catalog number: 15250061)
Sodium hydrogen carbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434)
Potassium chloride (KCl) (Merck, catalog number: 1.04936)
Dissodium hydrogen phosphate (Na2HPO4) (Merck, catalog number: 1.06586)
Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 1.04873)
Absolute ethanol (Sigma-Aldrich, catalog number: E7148)
Hydrochloric acid (HCl) 37% (Merck, catalog number: 1.00317)
Sodium hydroxide (NaOH) (Merck, catalog number: 1.06498)
Solutions (see note 1)
10% acetic acid (see Recipes)
Bind-silane solution (see Recipes)
Phosphate buffered saline (PBS) 10× (see note 2) (see Recipes)
Fibronectin (FBN) solution (see note 3) (see Recipes)
70% ethanol (see Recipes)
Recipes
10% acetic acid
Reagent Final concentration Quantity or Volume
Acetic acid (glacial) 100% 10% (v/v) 10 mL
Absolute ethanol n/a 90 mL
Total n/a 100 mL
Bind-silane solution
Reagent Final concentration Quantity or Volume
Acetic acid 10% 5% 2.5 mL
Bind silane 0.3% 15 μL
Absolute ethanol n/a 2.485 mL
Total n/a 5 mL
Phosphate buffered saline (PBS) 10×
Reagent Final concentration Quantity or Volume
NaCl 1.37 M 80 g
KCl 0.027 M 2 g
Na2HPO4 0.1 M 14.4 g
KH2PO4 0.018 M 2.4 g
H2O n/a Top up volume for 1 L
Total n/a 1 L
Fibronectin (FBN) solution
Reagent Final concentration Quantity or Volume
FBN 25 mg/mL 25 mg
NaHCO3 pH 8.6a 100 mM Top up volume for 1 mL
Total n/a 1 mL
a Dilute 84 mg of NaHCO3 (MW = 84.01 g/mol) in 10 mL of ddH2O and adjust the pH to 8.6. Store at room temperature.
Ethanol 70% solution
Reagent Final concentration Quantity or Volume
Absolute ethanol 70% 700 mL
dH2O n/a Top up volume for 1 L
Total n/a 1 L
Laboratory supplies
FluoroDishes (WPI, catalog number: FD-35-100)
Round glass coverslips 10 mm thickness #1.5 (Fisher Scientific, catalog number: NC1272767)
Square glass coverslips 22 mm × 22 mm thickness #1.5 (Corning, catalog number: 2850-22)
Microfabricated coverslips with confining pillars
6-well cell culture plate (Corning, catalog number: CLS3516)
6-well static confiner (4DCell, catalog number: CSOW 620)
T25 cell culture flasks (Sarstedt, catalog number: 83.3910)
Scotch Magic tape (3M, catalog number: 8-1933)
Parafilm M (Fisher Scientific, catalog number: 10018130)
Scalpel blade or razor blade
Fine forceps (Fine Science Tools, catalog number: 11254-20)
SU-8 mold with micropillar design (either custom-designed or ordered from MesoBioTech, France)
Harris Uni-Core puncher (0.75 mm) (WPI, catalog number: 504529)
Disposable 5 mL sterile pipettes (nerbe plus, catalog number: 12-441-9105)
Disposable 10 mL sterile pipettes (nerbe plus, catalog number: 12-461-9108)
Disposable 50 mL centrifuge tubes (Corning, catalog number: 734-1868)
Disposable 15 mL centrifuge tubes (Corning, catalog number: 734-1812)
Cell counting slides (Bio-Rad, catalog number: 1450011)
Equipment
Temperature-controlled TE2000 microscope equipped with a modified Yokogawa CSU-X1 spinning-disk head (Yokogawa Electric), an electron multiplying iXon+ DU-897 EM-CCD camera (Andor), and a filter wheel (Nikon)
Immersion oil 60× 1.4NA Plan-Apo DIC objective (Nikon)
Dynamic cell confiner (adapted from Le Berre et al. [7])
AF-1 dual vacuum/pressure controller (Elveflow)
6-well confinement device (4DCell, France)
Low-pressure plasma cleaner (Zepto, Diener Electronics, Germany)
Centrifuge
Hot plate
pH meter
Electronic TC10TM/TC20TM cell counter device
Vacuum degasser (optional)
37 °C humidified CO2 (5%) incubator
Laminar flow hood
Software and datasets
NIS Elements AR software (Nikon)
ImageJ (version 1.54)
Custom-designed MATLAB algorithm for nuclear pore analysis (The MathWorks Inc, USA; v2018b)
Procedure
Preparation of cell confinement setup (see note 4)
Preparing the PDMS suction cup (Figure 1A)
Mix the PDMS crosslinker A with the PDMS crosslinker B in a 10/1 mixture (w/w).
Mix until it is homogenous and centrifuge (150× g) for 5 min to remove all air bubbles. Alternatively, degas the PDMS mixture in a vacuum degasser for 5 min.
Pour the mixture on top of the custom-made mold (design instructions obtained from Le Berre et al. [7]) and let it bake for 1 h on a hot plate at 80 °C.
Create a hole on the side of the PDMS suction cup using a Harris Uni-Core puncher (0.75 mm).
Figure 1. Overall workflow for preparation of a dynamic cell confinement setup. A. Prepare a PDMS suction cup using a mold (design instructions available from Le Berre et al. [7]). B. Prepare either a PDMS confinement slide for rigid confinement with the desired micropillar height or a polyacrylamide (PAA) confinement slide for elastic confinement. C. Bind confinement slide to the PDMS suction cup. D. Seed cells on a 35 mm dish, previously coated with fibronectin (FBN). E. Remove the lid of the 35 mm dish and assemble the confinement device on top of the dish. Turn on the vacuum pump to secure the confinement device in place and confine cells. F. Representative time-lapse images of a cell undergoing multiple rounds of dynamic compression during mitosis. Timelapse is 2 min and scale bar is 10 mm.
Preparing the PDMS confinement slide for rigid confinement (Figure 1B)
Prepare a mix of PDMS with a crosslinker A/B mixture with a ratio of 10/1 (w/w).
Mix until it is homogenous and centrifuge (150× g) for 5 min to remove all air bubbles. Alternatively, degas the PDMS mixture in a vacuum degasser for 5 min.
Plasma-treat the 10 mm glass coverslips for 2 min. This step will activate one side of the coverslips and promote the binding of the glass with the PDMS layer in the next step.
Distribute drops of the PDMS mix on the SU-8 wafer mold with the micropillars of desired height (in this case, 8 μm) and place a coverslip on top of each drop, making sure the plasma-treated side is facing the PDMS. It is important to ensure a very thin layer of PDMS under the coverslips.
Bake the wafer with the coverslips on a hot plate at 95 °C for 15 min. Remove excess PDMS by washing with isopropanol and using a scalpel and forceps, being careful not to scratch the SU-8 mold.
Place a drop of isopropanol on the wafer next to the slides to peel off the confinement slides. Using a razor blade or scalpel, carefully lift the glass coverslips with the PDMS pillars (8 μm height) from the wafer.
Preparing the confinement slide with PAA hydrogels for elastic confinement (Figure 1B)
The confinement slides are prepared on 10 mm coverslips. The coverslips are initially treated with air plasma for 2 min using a plasma cleaner.
After cleaning, incubate the coverslips with a solution of 0.3% Bind-silane + 5% acetic acid in ethanol. Spread the mixture on top of the coverslips and make sure the entire surface of coverslip is covered in solution. Let the solution dry by evaporation.
Rinse the coverslips once with 70% ethanol and leave to dry again.
Prepare a gel mixture corresponding to approximately 15 kPa of stiffness. Full details are available in Le Berre et al. [7]. Place a drop of the mixture (25 μL) on a parafilm strip and place a silanized coverslip on top (silanized side facing the mixture). Allow the gel to polymerize.
Carefully lift the coverslip with the gel from the parafilm using a razor blade or scalpel, making sure the gel remains attached to the coverslip.
Hydrate gels with at least 1 mL of PBS for 5 min and then incubate with cell culture medium for at least 30 min (see notes 5 and 6).
Dynamic cell confinement
Seeding RPE-1 cells for confinement
Plasma-treat a fluorodish using the plasma cleaner (2 min air exposure).
Coat the clean fluorodish with 50–100 mL of FBN for at least 30 min. Gently rinse with 2 mL of dH2O.
Wash cells growing on a T25 flask with 5 mL of room-temperature PBS. Aspirate the PBS, add 2 mL of trypsin, and incubate the cells at 37 °C for 5 min (see notes 7, 8, and 9).
Resuspend cells by gently pipetting up and down, collect the cell suspension, and add 5 mL of fresh medium.
Determine the number of cells in your sample using an electronic cell counter. Calculate the volume of cell suspension needed for a recommended seeding density of 1.5 × 105 cells per fluorodish.
Place the corresponding number of cells in the previously coated fluorodish (see note 10) and add 2 mL of cell culture medium supplemented with Antibiotic-Antimicotic (Figure 1D, E).
Let the cells grow overnight at 37 °C in a humidified incubator with 5% CO2.
Once RPE-1 cells are adherent and whenever necessary, transfections with the DN-KASH, Rap1Q63E, or shRNAi-ROCK1-LV plasmids are done using Lipofectamine2000 or Lipofectamine RNAiMAX, according to the manufacturer's instructions.
Using the dynamic cell confiner (Figure 1E, F)
Before imaging, change the medium of the RPE-1 cells to Leibovitz’s-L15 medium (500 μL, see note 11) supplemented with antibiotics. If using any of the drugs described in the Reagents section (Reagents 1–13) or their corresponding DMSO control, they should be added at this point and cells should be incubated for the corresponding time as described in the reagents list.
Carefully dry the back of the confinement slide with absorbing paper and attach it to the piston of the suction cup device. If the glass side of the confinement slide is not completely dry, it will not stick to the PDMS piston of the suction cup, causing it to detach during imaging.
Attach the PDMS suction cup to the vacuum generator apparatus and set the pressure to zero (see notes 12 and 13).
Place the 35 mm dish with the RPE-1 cells on the microscope. To start the experiment, carefully remove the lid of the fluorodish, place the suction cup on top of the fluorodish, and lower the pressure of the vacuum generator to -30 mbar until the suction cup is firmly attached to the bottom of the fluorodish, without compressing the cells.
Start imaging.
Lower the pressure on the vacuum generator until it reaches -100 mbar.
Confirm on the microscope if the cells remain in focus and compression is visible.
When adding drugs to the cells, slowly increase the pressure on the vacuum generator to allow the PDMS suction cup to detach.
Without removing the PDMS suction cup, add the drug to the imaging medium and mix carefully by gently pipetting up and down.
To compress cells again, decrease the pressure on the vacuum generator to -100 mbar.
Confinement can be modulated at any time by either decreasing or increasing the pressure on the vacuum line (see notes 12 and 13).
Static cell confiner
For static confinement experiments, we used a commercially available 6-well confinement device with custom-designed confinement slides (see note 4) that were prepared using 10 mm standard round microscope coverslips and designed with regular hole arrays (diameter 449 μm, 1 mm spacing), similar to what was published previously in Le Berre et al. [7].
Preparing the confinement slides
To prepare the confinement slides, use the same method outlined above in step A2 or A3, depending on whether you require rigid or elastic confinement, respectively.
Seeding cells for confinement
Plasma-treat a 6-well plate or 22 mm × 22 mm square coverslips with air plasma for 2 min, depending on your need.
Coat the 6-well plate or the coverslips with FBN (see note 10) and incubate at room temperature for at least 30 min. Rinse with dH2O.
If your experiment requires the use of 22 mm × 22 mm square coverslips, place one coverslip in each well of the 6-well plate using a fine forceps.
Wash cells with 5 mL of room-temperature PBS. Discard the PBS, add 2 mL of trypsin, and incubate the cells at 37 °C for 3–5 min (see notes 7, 9, and 14).
Add fresh DMEM medium supplemented with FBS, resuspend, and collect the cellular suspension.
Determine the number of cells in your sample using an electronic cell counter (see note 15). Calculate the volume of cell suspension needed for a recommended seeding density of 1.5 × 105 cells per well.
Place the corresponding number of cells in the wells and add 2 mL of cell culture medium supplemented with antibiotics (see note 8).
Allow cells to adhere to the substrate at 37 °C in a humidified incubator with 5% CO2.
Assembly of the 6-well confinement device
Rinse the microfabricated confinement slide with 70% ethanol and air-dry with compressed air.
Clean the PDMS pistons supplied with the static confiner (4DCell, CSOW 620) with 70% ethanol. If the PDMS pistons have dust particles, remove them with Scotch Magic tape and rinse once again with 70% ethanol (see note 16).
Place the PDMS pistons on the lid of the 6-well confiner device. Ensure that the PDMS spacers are aligned with the holes in the lid (see note 17).
Attach each confinement slide to the PDMS piston by carefully pressing the glass side of the confinement slide against the PDMS piston. Ensure that the slides are well centered on the pistons and avoid creating air bubbles between the two surfaces.
To equilibrate the PDMS, place the lid of the confinement device containing the PDMS pistons and the confinement slides on a regular 6-well plate. Ensure the confinement device is set to “resting position.” Add sufficient cell culture medium to submerge the slides and incubate for at least 1 h at 37 °C in a humidified incubator with 5% CO2 (see note 18).
Imaging with the static confiner
Approximately 30 min before using the cells, change the medium of the RPE-1 cells to Leibovitz’s-L15 medium supplemented with antibiotics.
After equilibrating the PDMS confinement slide (see step C3e), transfer the lid of the confinement device to the 6-well plate where your cells are seeded. Gently pull down the metal springs on the confinement lid, one by one, to attach the lid to the bottom of the 6-well plate.
Place the cells on the microscope and start imaging.
To confine the cells, gently and simultaneously press the locks on both sides of the confinement lid down. At this point, the pressure imposed by the lid on the PDMS pistons will induce confinement of the cells.
If the experiments require addition of drugs (Reagents 1–13) or the corresponding DMSO control, they can be added through the holes present on the lid of the confinement device (see notes 17 and 18). After addition of the drugs, mix by carefully shaking the 6-well plate.
To release the confinement, gently pull out the locks on the lid of the 6-well confinement device. This will release the pressure on the PDMS pistons.
Using the static confiner for fixed cell analysis
For experiments involving fixed-cell imaging, follow the instructions provided in the previous sections (steps C1–C4), until the moment of adding fixative to the cells.
Fixative is added through the holes on the lid of the confinement device. Alternatively, the lid can be removed and cells fixed immediately after release of confinement.
Incubate cells with fixative for 10 min at room temperature.
After fixation, use your standard immunofluorescence protocol to label cells.
Data analysis
Quantification of cellular parameters after dynamic, single-cell confinement was done using ImageJ. Initially, the efficiency of the confinement was determined by measuring cell height.
Open the images using ImageJ.
Adjust brightness and contrast by clicking Image > Adjust > Brightness and Contrast > Auto.
Prepare the lateral projections. Click Image > Stacks > 3D project. Do the lateral projections for both the xz and yz axes.
Combine the lateral projections in the SUM projection of the movie.
Measure cell height by determining the size occupied by the cells in the lateral projections. Select the cells that were confined to a maximum of 8 mm of height (see note 19).
In our specific case, we wanted to analyze the effect of mechanical confinement on the nuclear translocation of cyclin B1. To quantify the levels of cyclin B1 inside the nucleus, ImageJ was used:
Draw a small region of interest (ROI) and add it to the manager (Edit > Selection > Add to manager).
Measure cyclin B1 intensity by selecting Analyze > Measure. Copy the values to an Excel sheet.
Using the same ROI from the Manager, select a region outside of the cell and measure the background fluorescence intensity. Copy the values to an Excel sheet.
Background-correct the fluorescence intensity values by subtracting the background fluorescence intensity from the cyclin B1 fluorescence intensity measured in 2b.
Normalize nuclear cyclin B1 fluorescence intensities by dividing the fluorescence intensity of each time point with the lowest intensity level of cyclin B1.
To obtain a measure of cyclin B1 nuclear translocation rate, align fluorescence values for all cells relative to the lowest nuclear intensity. The time point corresponding to the lowest nuclear fluorescence intensity was defined as zero.
To correlate cyclin B1 translocation with the time of nuclear envelope permeabilization (NEP), the fluorescence intensity values were normalized and aligned relative to the accumulation of tubulin in the nucleus, which acts as a proxy for the loss of the nuclear barrier function.
A minimum of three independent experiments is recommended, with at least 30 cells analyzed for each condition. Normality of the samples was assessed using the Kolmogorov-Smirnov test. In comparing multiple groups with a normal distribution, a parametric one-way analysis of variance (ANOVA) was done. In other cases, a nonparametric ANOVA (Kruskal-Wallis) was performed. No power calculations were used. All specific details regarding data analysis can be found in the original manuscript [6].
Validation of protocol
This protocol was used and validated in Dantas et al. [6]. doi:10.1083/jcb.202205051. The dynamic cell confiner was used in Figures 1, 2, 3, 5, S1, S2, and S3. The static cell confiner was used in Figure 4.
In addition, this protocol or parts of it have been used and validated in the following research articles:
Le Berre et al. [7] Methods for two-dimensional cell confinement. Methods Cell Biol.
Lancaster et al. [8] Mitotic rounding alters cell geometry to ensure efficient bipolar spindle formation. Dev Cell (Figures 1, 4, and 6).
Liu et al. [9] Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell.
Maiuri et al. [10] Actin flows mediate a universal coupling between cell speed and cell persistence. Cell.
Le Berre et al. [11] Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes. Integr Biol (Camb).
General notes and troubleshooting
General notes
pH of solutions should be carefully adjusted using either HCl or NaOH, whenever necessary. For the NaHCO3 solution, this is extremely relevant as the basic pH facilitates the adhesion of FBN to the coverslip.
The pH of the PBS 10× solution should be adjusted to 7.4 and the solution should be autoclaved afterwards.
Once reconstituted, the FBN solution should be stored at 4 °C for a maximum of two weeks.
The cell confiner and the cell confinement slides used for the static confiner can be acquired commercially (4DCell). The dynamic confiner was microfabricated in the lab using an adaptation of the protocol described previously [7] to adjust the diameter of the confiner slide to a 10 mm diameter coverslip. Adjust the equipment and laboratory supplies according to the purpose of your experiments.
The PAA hydrogels used to create the confining slides can be stored at 4 °C for a week, covered in PBS and sealed with parafilm.
When using drugs, prepare a new suction cup for each experimental condition, as the PDMS can uptake hydrophobic molecules from the cell medium. Washing is not sufficient to remove them efficiently from the PDMS.
The continuous use of antibiotic in the cell culture medium should be avoided. Antibiotics are advised only when preparing cells for imaging or fixed-cells analysis.
Use a laminar flow hood to perform all cell manipulation protocols. Prepare, use, store, and dispose of all the reagents according to the Material Safety Data Sheet (MSDS) guidelines. All the mediums and buffers used for cell culture should be prewarmed unless otherwise stated.
Cells should be used at a low passage number and frequently tested for possible mycoplasma infections.
For 22 mm × 22 mm coverslips and fluorodishes, use 100 μL of FBN 25 mg/mL; for 6-well plates, use 500 μL of FBN 25 mg/mL per well. For other sizes, adjust the volume accordingly.
Add a small volume of cell culture medium (usually less than 500 mL) when using the dynamic cell confiner to avoid suction of culture medium into the vacuum line attached to the pressure/vacuum generator.
Frequently check the tubing of the vacuum generator to avoid the trapping of air bubbles. This will influence the generation of vacuum and therefore affect compression efficiency.
Frequently calibrate the vacuum/pressure controller.
Different volumes can be used according to the dimension of your cell culture dish. For example, for a T75 flask, use approximately 10 mL of PBS to wash, 3 mL of trypsin to detach the cells, and at least 6 mL of fresh medium supplemented with 10% FBS to resuspend the cells.
Dust particles interfere with the attachment of the glass to PDMS. Ensure that the glass is cleaned thoroughly. The use of Scotch Magic tape is highly recommended, as it removes dust efficiently without leaving residues.
To count cells using the automated cell counter, prepare a mixture of 10 μL of the cellular suspension and 10 μL of the Trypan Blue solution. Mix gently and apply 10 μL on the cell counting slide. Then, place the counting slide onto the electronic counter device.
The holes that are present in the lid of the cell confinement device allow the access for pipettes through the glass plate and can be used to add or aspirate medium and/or drugs. If necessary, these holes can be closed with the caps provided in the kit.
Whenever drugs are used in the experiment, they should be added during this incubation step, as PDMS can absorb small molecules from the medium.
Confinement experiments were performed in cells confined to a minimum of 8 μm height. This is the minimum height that RPE-1 cells can cope without inducing cell death. Other cell types need to be tested.
Troubleshooting
Problem 1: Suction cup not attaching to the 35 mm dish.
Possible cause: Vacuum is not being generated.
Solution: Check the tubing for air bubbles. Calibrate the vacuum/pressure generator. Check integrity of the suction cup as it needs a flat surface to work.
Problem 2: Cell height varies and is not accurately controlled.
Possible cause: Micropillar height is not correct or PDMS layer is not flat.
Solution: Check the integrity of the SU-8 mold as it can deteriorate with use. Make sure that you use a very thin layer of PDMS when preparing the confinement slide and press homogeneously over the entire coverslip to ensure it is lying flat.
Problem 3: Confinement slide detaching from suction cup.
Possible cause: Slide is not dry.
Solution: Make sure the glass side of the confinement slide is thoroughly dried before attaching it to the PDMS piston. Any moisture will prevent binding of glass to PDMS.
Acknowledgments
This protocol is related to the paper: Dantas et al. [6] “Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation,” originally published in the Journal of Cell Biology (DOI: 10.1083/jcb.202205051).
This work was funded by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BIA-CEL/6740/2020.
Competing interests
The authors have no competing interests.
Ethical considerations
There are no ethical considerations associated with this protocol.
References
Champion, L., Linder, M. I. and Kutay, U. (2017). Cellular Reorganization during Mitotic Entry. Trends Cell Biol. 27(1): 26–4.
Gavet, O. and Pines, J. (2010). Activation of cyclin B1–Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J. Cell Biol. 189(2): 247–259.
Gavet, O. and Pines, J. (2010). Progressive Activation of CyclinB1-Cdk1 Coordinates Entry to Mitosis. Dev. Cell 18(4): 533–543.
Santos, S. D., Wollman, R., Meyer, T. and Ferrell, J. E. (2012). Spatial Positive Feedback at the Onset of Mitosis. Cell 149(7): 1500–1513.
Lindqvist, A., Rodríguez-Bravo, V. and Medema, R. H. (2009). The decision to enter mitosis: feedback and redundancy in the mitotic entry network. J. Cell Biol. 185(2): 193–202.
Dantas, M., Oliveira, A., Aguiar, P., Maiato, H. and Ferreira, J. G. (2022). Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation. J. Cell Biol. 221(12): e202205051.
Le Berre, M., Zlotek-Zlotkiewicz, E., Bonazzi, D., Lautenschlaeger, F. and Piel, M. (2014). Methods for Two-Dimensional Cell Confinement. Methods Cell Biol.: 213–229.
Lancaster, O. M., Le Berre, M., Dimitracopoulos, A., Bonazzi, D., Zlotek-Zlotkiewicz, E., Picone, R., Duke, T., Piel, M. and Baum, B. (2013). Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation. Dev. Cell 25(3): 270–283.
Liu, Y. J., Le Berre, M., Lautenschlaeger, F., Maiuri, P., Callan-Jones, A., Heuzé, M., Takaki, T., Voituriez, R. and Piel, M. (2015). Confinement and Low Adhesion Induce Fast Amoeboid Migration of Slow Mesenchymal Cells. Cell 160(4): 659–672.
Maiuri, P., Rupprecht, J. F., Wieser, S., Ruprecht, V., Bénichou, O., Carpi, N., Coppey, M., De Beco, S., Gov, N., Heisenberg, C. P., et al. (2015). Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence. Cell 161(2): 374–386.
Le Berre, M., Aubertin, J. and Piel, M. (2012). Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes. Integr. Biol. 4(11): 1406.
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496 | https://bio-protocol.org/en/bpdetail?id=496&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Primary Culture of Cortical Neurons
Rieko Muramatsu
Toshihide Yamashita
Published: Vol 3, Iss 8, Apr 20, 2013
DOI: 10.21769/BioProtoc.496 Views: 25005
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Original Research Article:
The authors used this protocol in Nature Medicine Nov 2012
Abstract
Primary culture of neurons from cerebral cortex is a popular model to study neuronal function in vitro and to explore the molecular mechanism of neurite outgrowth in the developing and adult central nervous system. This protocol is for preparing a culture of cerebral cortical neurons from postnatal rodent brain (Muramatsu et al., 2012). One day after cell plating, we can observe neurite outgrowth by microscope.
Keywords: Brain Postnatal Mouse Vitro Dissociate
Materials and Reagents
Poly-L-lysine (Sigma-Aldrich, catalog number: P2636 )
Dulbecco's Modified Eagle Medium, DMEM (Life Technologies, InvitrogenTM, catalog number: 12800-017 )
Fetal bovine serum (Life Technologies, Gibco®, catalog number: 10437 )
2.5% Trypsin solution (Life Technologies, Gibco®, catalog number: 15090-046 )
DNase (Sigma-Aldrich, catalog number: DN25 )
Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
NaH2PO4.2H2O
Na2HPO4.12H2O
NaCl
DMEM
FBS
Serum-containg culture medium (see Recipes)
PBS (see Recipes)
Equipment
Cell culture incubator
Centrifuge
Stereoscopic microscope
Scissors, forceps, and knives
Sterile filter (0.45 μm)
Cell strainer 70 μm nylon (BD Biosciences, catalog number: 352350 )
24 well culture plate
Procedure
Coating and washing culture plates.
Dilute poly-L-lysine stock solution with sterile PBS to 100 μg/ml. Add optimal volume of solution to each well of culture plates. In case of 24 well culture plate, 0.5 ml solution is enough. Coating allows neurons to adhere to the culture plates.
Leave the plates in incubator for at least 1 h. After that, wash the wells twice with 1 ml/well of sterile PBS. Completely remove the PBS before use. It is not necessary to dry the plate after PBS washing.
Choose appropriate anesthesia according to the recommendations of your Institutional Animal Care and Use Committee. After anesthetization, cut the scalp and remove the skull. We use postnatal day 1 mice to measure the length of corticospinal neuron. Pick up whole brain from pups and place it in cold PBS on ice (Chen et al., 2007).
Under a microscope, dissect out the cerebral cortex of brains. Remove meninges and blood vessels. The tissues are cut into as small piece (1 mm cubes) as can with sterile knives.
Transfer tissues to a 50 ml tube. The tissues are trypsinized in 10 ml of the trypsin solution (0.25% trypsin, 100 μg/ml DNase in PBS), and are incubated at 37 °C for 15 min.
Add an equal amount of 10% FBS-DMEM, and pipettes up and down a few times to help with tissues dissociation.
Centrifuge at 300 x g for 3 min at room temperature, discard the supernatant, and resuspend the pellet in fresh DMEM (not 10% FBS-DMEM).
The cells are filtered through a 70 μm nylon cell strainer to obtain single cell suspension. Centrifuge the resulting cell suspension at 300 x g for 3 min at room temperature. Resuspend the cell pellet in fresh DMEM (not 10% FBS-DMEM).
Count the number of cells and adjust the concentration using 10% FBS-DMEM. To measure the neurite length, plate the cells on a 4-well chamber slide at a density of 5 x 104 cells/well. Transfer the cell suspension to culture plates and incubate for 24 h at 37 °C with 5% CO2. To investigate the effect of pharmacological agents, change to the medium containing each agent 2 h after cell plating. Representative image shows the neuronal class III β-Tubulin (Tuj-1) labeled cortical neuron obtained 1 day culture.
Figure 1. Image of cultured cortical neuron. Representative image shows the neuronal class III β-Tubulin (Tuj-1) labeled cortical neuron obtained 1 day culture.
Recipes
Poly-L-lysine: Dissolve of sterile distilled water to make stock solution (10 mg/ml). Keep aliquots at -20 °C. Dilute with sterile PBS right before use.
Serum-containg culture medium
Reagents
Volume
DMEM
445 ml
FBS
50 ml
Penicillin/streptomycin
5 ml
PBS
Reagents
Volume
NaH2PO4·2H2O
0.312 g
Na2HPO4·12H2O
2.8652 g
NaCl
8.5 g
DW
1 L
Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientists (A) (25710006) from the Japan Society for the Promotion of Sciences to RM, the Osaka University Program for the Support of Networking among Present and Future Researchers to RM, and a Grant-in-Aid for Scientific Research (S) from JSPS (25221309) to TY.
References
Chen, Y., Balasubramaniyan, V., Peng, J., Hurlock, E. C., Tallquist, M., Li, J. and Lu, Q. R. (2007). Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protocols 2(5): 1044-1051.
Muramatsu, R., Takahashi, C., Miyake, S., Fujimura, H., Mochizuki, H. and Yamashita, T. (2012). Angiogenesis induced by CNS inflammation promotes neuronal remodeling through vessel-derived prostacyclin. Nat Med 18(11): 1658-1664.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Muramatsu, R. and Yamashita, T. (2013). Primary Culture of Cortical Neurons. Bio-protocol 3(8): e496. DOI: 10.21769/BioProtoc.496.
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Category
Neuroscience > Development > Neuron
Cell Biology > Cell isolation and culture > Cell differentiation
Neuroscience > Neuroanatomy and circuitry > Animal model
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4,960 | https://bio-protocol.org/en/bpdetail?id=4960&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing
BN Beverly Naigles §
JS Jan Soroczynski
NH Nan Hao
(§Technical contact: [email protected])
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4960 Views: 2173
Reviewed by: David PaulManoj B. Menon Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Biological Chemistry Sep 2023
Abstract
CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time.
Key features
• CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci.
• This protocol is optimized for the use of the Neon transfection system.
• Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting.
• Designed for knocking in fluorescent proteins and screening transfected cells byFACS, but modification for non-fluorescent knock-ins may be possible.
Graphical overview
Keywords: RAW 264.7 Macrophage CRISPR knock-in Endogenous tagging Genome editing
Background
The use of CRISPR/Cas9 genome editing to insert DNA into the genome at a specific locus using the cell’s endogenous homology-directed repair (HDR) pathway is a valuable approach for modifying the genome of a cell [1,2]. One specific use of this technology is to endogenously tag genes with fluorescent proteins, enabling the study of gene expression and protein localization in live single cells. In contrast to the use of viral vectors to randomly insert transgenes into the genome, endogenous tagging ensures that the tagged protein is expressed using all endogenous regulation, including any trans-acting regulatory factors, which is useful for studying the mechanisms of gene expression. Endogenous tagging also ensures that the copy number of the protein remains the same in tagged and untagged cells, in contrast to the overexpression that occurs when using transgenes. There are existing knock-in protocols for many common easy-to-transfect cells lines, such as human RPE1, HCT116 [3], HeLa, and U2-OS cells [4], human iPSCs [5], and mouse embryonic stem cells [6]. In macrophages, there are existing protocols for knock-out CRISPR screens in mouse bone marrow–derived macrophages (BMDMs) [7] and in mouse RAW 264.7 cells [8], as well as for targeted knock-outs using Cas9-ribonucleoprotein complex nucleofection in mouse BMDMs [9]. A recent study reported a protocol for inserting transgenes into the Rosa26 locus of RAW 264.7 cells using CRISPR/Cas9 technology coupled to electroporation of plasmid DNA and growing up a bulk population of edited cells [10]. We developed the protocol that we report here to use CRISPR/Cas9 genome editing to create clonal populations of macrophages with knock-in fluorescent tags at diverse genomic loci in order to study stimulus-responsive gene expression at a single-cell level by imaging the fluorescent protein over time in single cells [11]. We base our CRISPR design on the strategy first developed by Ran and colleagues and use their pSpCas9 plasmid and one-step restriction-ligation cloning strategy for the guide RNA (gRNA) [12]. In this protocol, we use the Neon transfection system to deliver the gRNA, Cas9, and HDR donor sequences as circular plasmid DNA, coupled to a simple strategy for growing up clonal populations following a single single-cell sorting step. In this paper, we use a knock-in of YFP at the C-terminus of the IRF1 protein as our example, but we have used this approach for at least six different loci. This protocol works best for fluorescent proteins because FACS is used to sort positive cells, and positive cells are quite rare. However, it may be possible to adapt this protocol to non-fluorescent knock-ins in the future. At the end of this protocol, we also describe how we prepare an imaging experiment with the edited cells.
Materials and reagents
Biological materials
RAW 264.7 mouse macrophage-like cells (ATCC, catalog number: TIB-71)
NIH3T3 mouse embryonic fibroblasts (ATCC, catalog number: CRL-1658)
pSp-Cas9(BB)-2A-Puro (PX459) plasmid (Addgene, catalog number: 62988)
pUC19 plasmid (Addgene, catalog number: 50005)
Reagents
Neon Transfection System 100 μL kit (Thermo Fisher, catalog number: MPK10025)
HyCloneTM classical liquid media, Dulbecco’s modified Eagles medium, high glucose, GE healthcare cell culture (DMEM)/high: with 4500 mg/L glucose and 4.0 mM L-Glutamine, without sodium pyruvate (VWR, catalog number: 16750-072)
Fetal bovine serum (FBS) (Fisher, catalog number: MT35010CV)
DPBS, no calcium, no magnesium (Thermo Fisher, catalog number: 14190250)
Penicillin-Streptomycin solution, 100×, 10,000 IU penicillin, 10,000 μg/mL streptomycin (Fisher, catalog number: MT30002CI)
Gibco DMEM, high glucose, no glutamine, no phenol red (Thermo Fisher, catalog number: 31053036)
Gibco L-Glutamine (200 mM) (Thermo Fisher, catalog number: 25030081)
T4 DNA ligase reaction buffer (NEB, catalog number: B0202S)
T4 polynucleotide kinase (PNK) (NEB, catalog number: M0201S)
10× Tango buffer (Thermo Fisher, catalog number: BY5)
Dithiothreitol (DTT) (Millipore Sigma, catalog number: D9799-5g)
10 mM ATP (NEB, catalog number: P0756S)
Bbs1-HF (NEB, catalog number: R3539S)
T7 DNA ligase (enzymatics, catalog number: L6020L)
Autoclaved MilliQ water
NEB 5-alpha competent E. coli (high efficiency) (NEB, catalog number: C2987I)
Opti-MEM reduced serum medium (Thermo Fisher, catalog number: 31985070)
Lipofectamine 2000 transfection reagent (Thermo Fisher, catalog number: 11668027)
Puromycin dihydrochloride (Millipore Sigma, catalog number: P8833-25MG)
Macherey-Nagel Nucleobond Xtra Midi Plus EF kit (Macherey Nagel, catalog number: 740422.5)
QIAprep Spin Miniprep kit (Qiagen, catalog number: 27104)
Zymo Quick-gDNA MiniPrep kit capped columns (Zymo Research, catalog number: D3024)
QIAquick Gel Extraction kit (Qiagen, catalog number: 28704)
Zymo DNA Clean & Concentrator-5 kit capped columns (Zymo Research, catalog number: D4013)
Phusion High-Fidelity DNA Polymerase-100u (NEB, catalog number: M0530S)
NEBuilder HiFi DNA Assembly Master Mix (NEB, catalog number: E2621S)
EDTA powder (Sigma, catalog number: E9884)
HEPES 1 M (Gibco, catalog number: 15630080)
Luria Broth (LB)-carbenicillin agar plates, with carbenicillin at 100 μg/mL
Luria Broth (LB)
Carbenicillin (Fisher BioReagents, catalog number: BP26485), stock at 100 mg/mL
Corning 0.25% Trypsin, 0.1% EDTA in HBSS w/o calcium, magnesium, and sodium bicarbonate; 6/PK 25-053-CI (Fisher, catalog number: MT25053CI)
Solutions
Complete DMEM (See Recipes)
Conditioned DMEM (See Recipes)
Antibiotic-free DMEM (See Recipes)
0.1 M EDTA solution (See Recipes)
FACS sorting buffer (See Recipes)
Phenol Red–free DMEM (See Recipes)
1,000× Puromycin solution (See Recipes)
Recipes
Complete DMEM
Reagent Final concentration Quantity or Volume
HyCloneTM DMEM N/A 445 mL
FBS 10% 50 mL
Penicillin-Streptomycin solution, 100× 1% 5 mL
Total n/a 500 mL
Combine all ingredients and pass through a 0.2 μm filter bottle (Corning).
Conditioned DMEM
Reagent Final concentration Quantity or Volume
Supernatant from RAW 264.7 culture (RAW 264.7 cells split from a confluent plate at 1:8 density in complete DMEM with 10% FBS, 1% pen/strep, supernatant is collected after 24 h) 50% 100 mL
HyCloneTM DMEM n/a 69 mL
FBS 20% 30 mL
Penicillin-Streptomycin solution, 100x 1% 1 mL
Total n/a 200 mL
Combine all ingredients and pass through a 0.2 μm filter bottle (Corning).
Antibiotic-free DMEM
Reagent Final concentration Quantity or Volume
HyCloneTM DMEM n/a 450 mL
FBS 10% 50 mL
Total n/a 500 mL
Combine all ingredients and pass through a 0.2 μm filter bottle (Corning).
0.1 M EDTA
Reagent Final concentration Quantity or Volume
Autoclaved MilliQ water n/a Make up to 50 mL
EDTA powder 0.1 M 1.46 g
Total n/a 50 mL
To make the 0.1 M EDTA, dissolve the EDTA powder in 45 mL of autoclaved MilliQ water, then top off to 50 mL once totally dissolved for a total volume of 50 mL. Filter this solution through a 0.2 μm syringe filter (Acrodisc) before use.
FACS sorting buffer
Reagent Final concentration Quantity or Volume
DPBS n/a 47.75 mL
EDTA 0.1 M (from Recipe 4) 1 mM 500 µL
HEPES 1 M 25 mM 1.25 mL
FBS 1 % 500 µL
Total n/a 50 mL
To make the FACS sorting buffer, combine all reagents and then pass through a 0.2 μm syringe filter (Acrodisc).
Note: Other compositions of FACS sorting buffer will likely also work.
Phenol Red–free DMEM
Reagent Final concentration Quantity or Volume
Gibco DMEM, no phenol red N/A 440 mL
FBS 10% 50 mL
Penicillin-Streptomycin solution, 100× 1% 5 mL
L-Glutamine (200 mM) 2 mM 5 mL
Total n/a 500 mL
Combine all ingredients and pass through a 0.2 μm filter bottle (Corning).
1,000× Puromycin solution (1 mg/mL)
Reagent Final concentration Quantity or Volume
Autoclaved MilliQ water n/a Make up to 10 mL
Puromycin dihydrochloride 1 mg/mL 10 mg
Total n/a 10 mL
To make the 1,000× puromycin, dissolve the puromycin dihydrochloride in 9 mL of autoclaved MilliQ water, then top off to 10 mL once totally dissolved for a total volume of 10 mL. Filter this solution through a 0.2 μm syringe filter (Acrodisc) before use and store at -20 °C in 1 mL aliquots.
Laboratory supplies
Falcon 5 mL round bottom polystyrene test tube, with cell strainer snap cap (Falcon, catalog number: 352235)
GenClone 96-well cell culture plates flat bottom wells, TC treated (Genesee, catalog number: 25-109)
Falcon tissue culture plates 24 well (Fisher, catalog number: 087721H)
GenClone 6-well TC treated plates (Genesee, catalog number: 25-105MP)
GenClone TC treated dishes, 100 × 20 mm vented (Genesee, catalog number: 25-202)
Acrodisc syringe filter 0.2 µm Supor membrane, low protein binding, non-pyrogenic, PN 4612 (VWR, catalog number: 28143-310)
Pipette tips, serological pipettes, Eppendorf tubes, PCR tubes, 50 mL conical tubes, 15 mL conical tubes, 50 mL syringes
Cell scraper (e.g., VWR, catalog number: 75799-934)
Corning® 500 mL vacuum filter/storage bottle system, 0.22 µm pore 33.2 cm PES membrane, sterile (Corning, catalog number: 431097)
Equipment
Neon transfection system (Thermo Fisher, model: MPK5000)
PCR thermocycler (e.g., Thermo Fisher, model: ProFlex PCR System Thermocycler)
Hemacytometer (e.g., Hausser Scientific Hemacytometer Thermo Fisher, model: S17040)
Agarose gel electrophoresis rigs
Fluorescence microscope (e.g., Nikon, model: Eclipse Ti)
FACS machine (e.g., BD, model: FACSAria Fusion)
Benchtop mini centrifuge (e.g., MyFuge, model: Southern Labware C1012)
Benchtop microcentrifuge (e.g., Eppendorf, model: 5420)
Software and datasets
ApE (plasmid editor) v3.1.3 November 11, 2022. Free, but you can equally well use a paid cloning software such as SnapGene if you prefer
Procedure
Guide RNA (gRNA) design and testing (following the procedure from Ran et al. [12]) (Figure 1)
Figure 1. Diagram summarizing what will be accomplished in step A of this procedure. We will design multiple gRNAs, in this case targeting the C-terminal end of mouse IRF1, will clone each of them into a gRNA-Cas9 expression plasmid, and will test them to determine the optimal guide RNA (in this example, gRNA 1). The light pink or blue underlines in the sequence indicate the gRNA sequences, and the dark pink underlines indicate the PAM sequences for each possible gRNA. The desired insertion site is marked with yellow arrows, and the cleavage site for gRNA 1 is marked with purple arrows.
Design guide RNAs
Identify where in the mouse genome you want to insert your knock-in sequence. For endogenous gene tagging, this should either be immediately after the endogenous ATG or immediately before the endogenous stop codon.
Select the genomic DNA sequence from 50 base pairs before to 50 base pairs after your desired insertion site.
Copy this sequence into crispor.tefor.net (http://crispor.tefor.net/) to design guides. For the genome, choose Mus musculus mm10 or mm39. For selecting a PAM, select “20bp-NGG – Sp Cas9, SpCas9-HF1, eSpCas9 1.1.”
Select guides with high specificity scores and as high as possible cutting efficiency scores. You should additionally optimize for being as close to your desired insertion site as possible. You should select the three most promising guides (Figure 2).
Note: Rarely do you have guides that look perfect here. This is part of why we screen three guides, so that you can choose several that look promising and then test them. The cutting efficiency prediction algorithms are not accurate for all cell types or loci, and this is also why we test the guides.
Figure 2. Example output from crispor.tefor.net (http://crispor.tefor.net/). Select the 100 bp region from 50 bp upstream to 50 bp downstream of your desired knock-in insertion position, enter it into cirspor.tefor.net, and then analyze the possible gRNA results to choose the three most promising guides based on having a high predicted efficiency, high specificity score, and being as close to your desired insertion site as possible. In this example, gRNAs 1, 2, and 3 best meet these criteria.
Order two oligos (one forward and one reverse) per guide (from Eurofins, IDT, etc.). For the forward oligo, append CAACG to the 5’ end of the guide sequence. For the reverse oligo, take the reverse-complement of the forward guide sequence (not including the added CAACG) and add AAAC to the 5’ end and C to the 3’ end. For example, if your guide sequence was ACCCTT…GGGCT, then you would order forward oligo caacgACCCTT….GGGCT and reverse oligo aaacAGCCC…AAGGGTc.
Clone the gRNA oligos into the pSpCas9-puro plasmid
Anneal oligos.
i. Dilute oligos to 100 µM each in autoclaved MilliQ water based on instructions from the manufacturer.
ii. In a PCR tube, combine 1 µL of each gRNA oligo (forward and reverse), 1 µL of T4 DNA ligase reaction buffer, 1 µL of T4 PNK, and 6 µL of autoclaved MilliQ water. Flick the tube and spin it down in a benchtop mini centrifuge.
iii. Put this tube in the thermocycler with a cycle of 37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 0.1 °C/s, hold 1 min at 25 °C, hold at 4 °C. This takes approximately 1 h to run.
iv. Pause point: Annealed oligos can be frozen at -20 °C for at least a year before use.
Clone the annealed gRNA oligos into the pSpCas9(BB)-2A-puro plasmid using the standard one-step restriction-ligation protocol described in Ran et al. [12].
i. Dilute the annealed oligo 1:200 by adding 1 µL of annealed oligo (from above) to 199 µL of autoclaved MilliQ water.
ii. Prepare the gRNA-Cas9 cloning reaction in a PCR tube on ice. Once the reagents are combined, flick the tube with your fingernail and spin it down in a benchtop mini centrifuge.
Component Volume (µL)
pSpCas9(BB)-2A-puro plasmid (100 ng/µL) 1
1:200 diluted oligo 2
10× Tango buffer 2
DTT (10 mM) 1
ATP (10 mM) 1
Bbs1-HF 0.5
T7 DNA ligase 0.5
Autoclaved MilliQ water 12
Total 20
iii. Incubate this tube in a thermocycler for six cycles of (37 °C for 5 min followed by 21 °C for 5 min) and then hold at 4 °C. This cycle takes approximately 1 h.
iv. Pause point: After removal from the thermocycler, this can be stored at -20 °C for at least a year.
Transform 5 µL of this gRNA-Cas9 reaction into 50 μL of NEB 5-alpha competent E. coli (high efficiency).
i. Place competent cells on ice for 10 min.
ii. Add 5 µL of gRNA-Cas9 reaction to the cells and mix gently by tapping the tube.
iii. Incubate cells + DNA on ice for 15–30 min.
iv. Heat shock the cells + DNA tube for 30 s at 42 °C.
v. Incubate the tube for 2 min on ice.
vi. Add 200 μL of LB to the tube.
vii. Shake at 300 rpm for 30–60 min at 37 °C.
viii. Plate on LB-carbenicillin agar plate.
ix. Place LB-carbenicillin plate in a 37 °C incubator overnight.
Note: Other types of competent cells will likely also work.
Pick three colonies from the plate that has grown up overnight and grow up each picked colony in a 5 mL culture of LB + carbenicillin. Perform minipreps using the QIAprep Spin Miniprep kit to extract the plasmids from the bacteria and sequence the plasmids using a universal U6 primer to confirm correct gRNA insertion. Re-streak the minipreps with the correct plasmid sequence onto new LB-carbenicillin plates and grow up overnight.
Pick a colony from the re-streak plate that had the correct sequencing and grow up this colony in a midiprep culture of 100 mL of LB + carbenicillin (so adding 100 μL of 1,000× carbenicillin). The culture should be grown overnight at 37 °C with shaking at 300 rpm.
Use the Macherey Nagel NucleoBond Midi Plus EF kit to perform a midiprep to extract the plasmid from the midiprep culture. Follow the kit instructions for midipreps of low-copy plasmids. For clarification and loading, we load the entire mix onto the filter (rather than spinning it out first); for DNA concentration at the end, we use the finalizers. We elute in 600 µL of provided water. This resulting plasmid is the pSp-Cas9-puro gRNA plasmid that you will use in subsequent steps.
Note: Other midiprep kits could likely be used, but it is important for the final DNA concentration to be high, ideally over 1 μg/μL. For transfection later into macrophages, it is also important for the DNA to be endotoxin-free, which this kit accomplishes.
Test gRNA cutting efficiency
On day 1, use your hemocytometer to count the cells and seed 2 × 105 NIH3T3 cells per well in a 6-well plate in complete DMEM. Seed the same number of wells as gRNAs that you are testing (usually three) plus one well as a control for uncut genomic DNA and one well as a control for puromycin selection.
Note: We use NIH3T3 cells to test the gRNA cutting efficiency because they can be transiently transfected with a high efficiency and therefore selected for transfected cells while the cells are still expressing the transient selectable marker. RAW 264.7 cells transfected using the Neon system are too sick immediately after transfection to be selected using puromycin. We find that gRNAs selected for high efficiency in NIH3T3 cells are very effective for creating knock-ins in RAW 264.7 cells.
The next day (day 2), for each gRNA to test, transfect one well.
i. Add 2.5 μg of pSpCas9-puro gRNA plasmid DNA to 125 μL of Opti-MEM in an Eppendorf tube.
ii. Add 7.5 μL (for a 1:3 ratio with the DNA) of Lipofectamine 2000 transfection reagent to 125 μL of Opti-MEM in a second Eppendorf tube.
iii. Add the Lipofectamine + Opti-MEM solution to the DNA + Opti-MEM solution, pipette up and down gently, and incubate for 5 min at room temperature.
Note: Be gentle with this solution, as the DNA–lipofectamine complex can be fragile.
iv. Add the transfection solution dropwise onto the well, gently tilt the plate to mix, and return the plate to the incubator.
On day 3, trypsinize the wells and transfer the entire contents of each well into its own 10 cm plate in complete DMEM. For the gRNA test plates and the puromycin control plate, add 10 μL of 1 mg/mL puromycin in 10 mL of total media.
On day 5, inspect the puromycin control plate and confirm that all cells are dead. Wash the gRNA test plates with DPBS and add fresh complete DMEM (without puromycin) onto the plate. Split the uncut genomic DNA control plate to be confluent the next day (usually 1:3).
On day 6, collect the cells using trypsin (each condition of gRNA test cells and the uncut control), spin down into cell pellets, and store at -80 °C.
Extract genomic DNA from cell pellets
We use the Zymo Quick-gDNA Miniprep kit to extract gDNA; whichever gDNA extraction kit you prefer should work.
Amplify the region where the cut should occur
Design PCR primers to amplify from ~300–600 bp upstream of the desired cut to ~300–600 bp downstream of the desired cut.
Note: We use PrimerBlast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) from NCBI to identify primer pairs, aiming for 20 bp primers and an annealing temperature of 60 °C.
Use these primers to amplify this region using PCR from the DNA you extracted from the gRNA test cells as well as the control uncut cells.
Note: We use the NEB Phusion DNA polymerase and associated reagents, but alternative polymerase systems will likely also work. As the Phusion polymerase requires a different Tm for primers than is generally calculated, we use the NEB Tm calculator (https://tmcalculator.neb.com/#!/main) to calculate the Tm we will use in the PCR program. We use a thermocycler program of 98 °C for 30 s, then 30 cycles of (98 °C for 10 s, annealing temp for 30 s, 72 °C for 1 min per kilobase of PCR product), then 72 °C for 10 min, hold at 4 °C.
Run the PCR product on a 1% agarose gel to confirm band size.
Gel extract the PCR product from the gel using the QIAquick Gel Extraction kit.
Note: Any gel extraction kit should work.
Send the uncut control and gRNA test amplicons that you gel extracted for Sanger sequencing with the primers you used for the PCR.
Note: Occasionally, these primers will be bad sequencing primers; in this case, you can design and order an additional internal primer to sequence across the cut site.
Use the online Synthego ICE tool (https://ice.synthego.com/#/) to assess gRNA cutting efficiency.
Note: This tool compares the .ab1 Sanger sequencing files in uncut (control) cells and cells transfected with a gRNA to determine the fraction of cells with an indel at the CRISPR site. The software also requires you to input the guide sequence used for that sample. The fraction of cells with an indel correlates with the gRNA cutting efficiency, which is also calculated by the software. The software also shows exactly which indels were formed (Figure 3).
Figure 3. Sample output from the online Synthego ICE tool (https://ice.synthego.com/#/) showing the indel percentage and knock-out score
Choose the gRNA with the best cutting efficiency that is as close to your desired insertion site as possible.
Note: We select the gRNA with the highest cutting efficiency (indel percentage) that is as close to our desired insertion site as possible. In our experience, any indel percentage above 50% is generally sufficient, though we have not specifically tested moving forward with a gRNA with a score of 40% vs. 60%, for example. We have seen gRNAs with an indel score below 30% fail, though we cannot necessarily attribute their failure specifically to their low cutting efficiency. In the IRF1 example, we chose gRNA 1.
Design and build HDR donor plasmid
Use NEB Phusion polymerase to amplify ~1 kb homology arms on each side of the desired insertion site from RAW 264.7 genomic DNA. We use the Zymo Quick-gDNA Miniprep kit to extract RAW 264.7 genomic DNA from wild-type RAW 264.7 cells. For N-terminal tagging, the insertion site should be immediately after the endogenous ATG start codon and, for C-terminal tagging, it should be immediately before the endogenous stop codon. Once the best gRNA has been identified, its PAM sequence needs to be mutated in the donor plasmid to avoid re-cutting. We do this using primers with overhangs to add new sequence. If the PAM cannot be mutated synonymously, then at least three synonymous mutations should be made in the seed sequence of the gRNA so that Cas9 re-binding is prevented in that way. If the PAM is in a UTR, we do a literature search to identify if it is in a region of the UTR known to be important for regulation; if not, we simply mutate the PAM. If there is known regulation in the UTR, we try to avoid mutating that region. The desired fluorescent protein insertion sequence also needs to be amplified via PCR from a plasmid containing that fluorescent protein. We assemble the plasmids using Gibson assembly in a pUC19 backbone and so add the appropriate Gibson overhangs on our primers as well when needed. This generally results in a four-part Gibson assembly reaction with the plasmid backbone, left homology arm, right homology arm, and fluorescent protein (Figure 4).
Figure 4. Diagram of construction of homology-directed repair (HDR) donor plasmid. The plasmid consists of the left homology arm, linker sequence, fluorescent protein tag, and right homology arm. Each of these sequences must be PCR-amplified out of either the genomic DNA (for the homology arms) or a fluorescent protein plasmid (for the fluorescent protein). As this is a C-terminal tag, the fluorescent protein is inserted directly before the endogenous stop codon. The grey shading indicates the process of mutating the gRNA recognition sequence synonymously to prevent further gRNA binding without changing the protein sequence. The homology arm incorporated in the donor plasmid contains this mutated sequence, and the mutated bases are marked with asterisks in this diagram. Each homology arm should be ~1 kb long.
Notes:
You can use a different PCR chemistry, but we recommend a high-fidelity PCR enzyme because it is important for the entire plasmid to have the correct sequence.
For N-terminal tagging, be sure to not include a stop codon on the fluorescent protein tag.
We use PrimerBlast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design our cloning primers, aiming for 20 bp primers and an annealing temperature of 60 °C.
We use restriction enzymes to digest 2 μg of the pUC19 backbone. We then run this digested backbone as well as our three PCR fragments (left homology arm, right homology arm, and fluorescent protein tag) on an agarose gel, gel extract the bands using the QIAquick gel extraction kit where we elute in 30 µL, and then run that product through a Zymo DNA Clean & Concentrator-5 kit and elute in 12 μL of the Zymo elution buffer. This two-step purification results in cleaner, higher-concentration DNA that leads to better efficiency with Gibson assembly in our hands. We do our Gibson assembly reactions using 100 ng of each DNA piece and use the NEBuilder HiFi DNA Assembly Master Mix, incubating for 1 h at 50 °C. We design our Gibson cloning to have ~23 bp overlaps between each two DNA pieces.
If you are adding linker or T2A/P2A sequences into the plasmids between the endogenous protein and the fluorescent tag, these can also be introduced on primer tails when designing your PCR. We use the T2A sequence from Nora et al. [6] and a GDGAGLIN linker with DNA sequence GGCGACGGCGCCGGCCTGATCAAC.
Transform the cloned plasmid (after Gibson assembly) into NEB 5-alpha competent E. coli (high efficiency) as above.
Grow up four minipreps, sequence the minipreps, and identify a miniprep with the correct sequencing. You want to be sure the entire homology arm and insertion sequences are correct.
Grow up a midiprep culture of the plasmid with the correct sequencing and perform the midiprep using the Macherey Nagel NucleoBond Midi Plus EF kit in the same way as described for the gRNA-Cas9 plasmid above.
Note: Plasmid concentration is best if above 1 μg/μL. If it is below 500 ng/μL, we suggest repeating the midiprep.
Transfect gRNA-Cas9 plasmid and donor plasmid into RAW 264.7 cells (Figure 5)
Note: We use the Neon transfection system rather than any form of lipofection because, in our hands, the Neon transfection system gives much higher transfection efficiency than any lipofection method in RAW 264.7 cells. See General Note 6.
Figure 5. Graphical outline of steps C and D. We transfect the gRNA-Cas9 and homology-directed repair (HDR) donor plasmids into the cells, grow up transfected cells, sort single positive transfected cells into individual wells using FACS, and grow up clones for further screening.
Have a confluent 10 cm plate of RAW 264.7 cells ready on the day of transfection.
Fill three wells of a 6-well plate with 2 mL each of antibiotic-free DMEM.
Note: It is important to use the antibiotic-free DMEM here, as having antibiotics in the media decreases cell viability after transfection.
In an Eppendorf tube, combine 45 μg of gRNA-Cas9 plasmid and 45 μg of donor plasmid. Ideally, both the donor and gRNA-Cas9 plasmid concentrations are over 1 μg/μL.
In this protocol, we transfect in triplicate, so each single transfection is 15 μg of each plasmid.
Note: We attempted various ratios of gRNA-Cas9 DNA to donor DNA, different volumes of DNA, pretreating the cells with DMSO, pretreating with interferon-gamma (as our genes were interferon-gamma inducible and the idea was to open up the chromatin at their loci), and also plating post-transfection into media containing 2 μM M3814 (M3814 is a DNA-PK inhibitor that can increase the ratio of HDR edits to NHEJ edits [13]). Adding more DNA decreased the cell viability after transfection, and we saw little effect of either DMSO pretreatment or M3814 post-treatment. Therefore, we chose not to either pretreat or post-treat our cells for our experiments.
Note: We do not linearize our plasmids before transfection, but we did not test how linearization might affect transfection or knock-in efficiency. One reason we did not attempt plasmid linearization was concern over the possible introduction of endotoxins in this process. Therefore, we are transfecting circular plasmid DNA.
Set up the Neon transfection system machine, program it for 1,680 V, 20 ms, 1 pulse, and set up the Neon cuvette with 3 mL of buffer E2 from the Neon electroporation reagent kit.
Prepare an Eppendorf tube with 1 mL of DPBS for washing the Neon tip.
Spin down 7.5 × 106 RAW 264.7 cells at 200× g for 3 min and aspirate the supernatant.
In this protocol, we transfect in triplicate, so each single transfection is 2.5 × 106 cells.
Resuspend cells in 1 mL of DPBS, spin down again, and aspirate the supernatant.
Resuspend the cells in 300 μL of R buffer from the Neon electroporation reagent kit.
Note: The kit says that extended R buffer exposure can be harmful for cells, so move quickly after this step.
Add the cells in R buffer to the tube with your plasmid DNA and pipette up and down gently 3–5 times.
Use a 100 μL tip on the Neon pipette to pick up 100 μL of the cells–buffer–DNA solution. Make sure there are no bubbles in the tip.
Put the tip in the Neon cuvette and push down until it clicks.
Press Start on the program (1,680 V, 20 ms, 1 pulse).
Once the program has completed, gently eject the contents of the tip into one well of a 6-well plate with antibiotic-free DMEM.
Insert the pipette tip into the tube of DPBS and pipette the DPBS up and down five times to wash.
Repeat steps C10–14 twice more, for a total of three transfections, plating into a new well for each transfection.
Once all transfections are complete, rock the cell plate back and forth to evenly distribute cells.
Place the cell plate in your tissue culture incubator.
Put away the Neon machine. According to Thermo Fisher, the tips are not reusable, and the cuvettes should only be reused a few times. In our experience, tips can be reused at least five times, but we use a new tip for each new cell line that we construct to avoid contamination. To store tips, we place them in a 15 mL conical tube. At least 24 h before reuse, we use forceps to separate the internal gold plunger from the external plastic, put both the plunger and plastic back in the 15 mL conical tube, and add filtered 100% ethanol for at least 2 h. We then dry them by placing them on a Kimwipe in a tissue culture hood with the UV light on for 1 h. We also reuse cuvettes at least five times, though we do sometimes see a decrease in cell viability after transfection using older cuvettes. We store cuvettes in a 50 mL conical tube with filtered 100% ethanol, and before reuse we remove them from the conical tube and dry them by placing them on a Kimwipe in a tissue culture hood with the UV light on for 1 h.
Grow up cells, sort single positive cells, and grow up clonal colonies from single cells
Two days after electroporation, inspect cell viability. There may be many floating cells, many of which are dead but some of which may be alive.
Note: Cell viability after transfection can vary greatly based on a number of factors. It can depend on cell health before electroporation, age of reagents (buffers, cuvettes, etc.), as well as the nature and purity of the DNA. We have done electroporations of the same DNA into two different RAW 264.7 lines in parallel on the same day and had very different post-transfection viability between them. We have also electroporated different DNAs into the same cell line on the same day and had different viability. At two days post-transfection, an electroporation that had good viability will look like a ~50% confluent well of cells, but the cells are more elongated than they normally are, so 50% confluence here is fewer cells than 50% confluence of normally growing RAW 264.7 cells. An electroporation that had poor viability will look like sparse single cells on the bottom. However, we have still seen successful genome editing in cases of poor viability after transfection.
Use a cell scraper followed by strongly pipetting media against the bottom of the well to collect all cells in all three wells. Combine these into one 15 mL conical tube, spin down the conical tube at 200× g for 3 min, and aspirate the supernatant.
Resuspend the cells in 2 mL of complete DMEM and place this suspension in a new well of a 6-well plate (so you have now combined three wells into one well).
Monitor the well until it reaches confluence; this generally takes 2–5 days depending on cell viability.
Expand these cells into a confluent 10 cm plate.
We like to first inspect the cells under a microscope to see if they express the knocked-in fluorescent protein before sorting them. To do this, we plate two wells of a 6-well plate with these bulk transfected cells and image 60 positions per well every hour for 24 h, adding our stimulus after 2 h for stimulus-inducible genes. If you see the fluorescence expression, that is great! If not, sometimes the actual knock-ins are so rare that you can only pick up on them via FACS; we have had cases where we do not see fluorescence via microscopy but do pick up correctly edited cells via FACS. Be sure to only image a fraction of your bulk transfected population and keep most of it in the incubator to prepare for sorting (Figure 6).
Figure 6. Sample field of view from imaging bulk transfected cells after they have grown to confluency where a gene has been tagged with YFP. In this field of view, there is a group of cells that express YFP after stimulus, which are the edited cells we are looking for. There are also many cells that do not express YFP.
Sort single positive cells into a 96-well plate.
Note: We generally sort 7–9 days after transfection. Occasionally, it will take longer for the cells to grow up sufficiently; that is ok too.
Induce a 10 cm plate of transfected cells with whatever stimulus should express your fluorescent knock-in. If it is constitutively expressed, then skip this step.
Prepare two 96-well plates with 100 μL of conditioned DMEM per well.
Note: Using conditioned rather than regular DMEM results in the single cells growing into colonies faster after sorting as well as a higher fraction of sorted single cells surviving and growing into colonies. This is a key step in this protocol.
Collect your induced transfected cells, as well as the cells you made them from as a control for FACS gating.
Spin cells down at 200× g for 3 min, resuspend in ~3 mL of FACS sorting buffer, and pass through a filter into a FACS tube (Falcon 5 mL round bottom polystyrene test tube, with cell strainer snap cap).
Bring the FACS tubes with the cells on ice to a FACS machine. Gate for positive cells compared with the pre-transfection control. It is ok to get some false positives here because you will screen the clones further in subsequent steps. Sort cells that express your fluorescent protein higher than the control into the 96-well plates with one cell per well (Figure 7 ).
Note: The fraction of positive cells is often very small, generally between 2% and 0.01% of the sample (see below for more discussion of this). However, this fraction is often enough to recover the knock-in cells.
Figure 7. Sample FACS plots when sorting for positive cells. In this example, iRFP+ parent cells were transfected to knock in a YFP tag. These plots show YFP vs. iRFP for each cell being sorted. On the left are the parent cells, and on the right are the bulk transfected cells. P5 is the gate used to collect positive cells that have higher YFP than any cells in the parent population. In this example, in the bulk transfected cells, P5 is 0.3% of the alive, single-cell population and 0.2% of the total population. The true YFP-positive population likely extends to the left of the P5 gate shown here, but we drew the gate here to try to minimize false positives. In other situations, it may be worth it to take some false positives to avoid throwing out true positives and to screen out the false positives later when the clones are screened by microscopy.
Grow up single colonies into clonal populations.
Seven to nine days after sorting, look at each plate under a regular tissue culture inverted microscope and identify wells with colonies growing in them.
Note: We usually have between 30% and 60% of wells with growing colonies.
Using a fluorescence microscope, image each growing colony in the channel where you expect to see your knock-in fluorescence.
If you are looking for stimulus-responsive expression, add stimulus, wait the appropriate time for your gene to turn on, and then image on the fluorescence microscope again.
Based on the fluorescence images, identify clones that are positive for fluorescence. We generally also screen by colony size and take the colonies on the bigger side. Choose 12 colonies to grow up (Figure 8).
Note: Generally, if the editing worked, most (~70%–100%) clones that you screen will be positive for the fluorescence. If the editing did not work and you ended up sorting false positives, then no clones will be positive, or those that are will be positive even without induction for inducible genes.
Figure 8. Sample images from screening clones in a 96-well plate eight days after sorting, showing both a positive clone and a negative clone for the knocked-in YFP tag in a nuclear marker (iRFP+) background
When the colony gets large in the 96-well plate (generally 10–12 days after sorting; it is ok to wait until the media turns yellow, but if you choose to wait that long, you should move the colony promptly after that), use forceful pipetting of media using a P200 pipette to dislodge the colony and move the cell suspension to a 24-well plate.
Continue to grow up and transfer the cells when confluent from a 24-well plate to a 6-well plate to a 10 cm plate.
Screen clones
For the 12 colonies that you grew up, spin down two cell pellets from a confluent 10 cm plate and store at -80 °C. Additionally, freeze cryovials of cells so that you can recover ones you want to use later.
Extract the genomic DNA from the cell pellet for each clone; we use the Zymo Quick-gDNA Miniprep kit.
Use PCR to amplify the region across the insertion site from the left into the right homology arm. We use the primers that we designed for determining gRNA cutting efficiency above. Use a long enough elongation time to elongate across the inserted DNA as well. We also run a control PCR on unedited wild-type DNA.
Run this PCR product on a gel. Note the bands: if there are two bands, then the clone is likely heterozygous for the knock-in; if there is only one large band, it is likely homozygous for the knock-in; if there is only one small band, it likely does not have the knock-in (Figure 9).
Figure 9. Cartoon example of a screening gel. Wild-type unedited cells have one small band, clones that do not have the knock-in inserted have one small band, homozygous knock-ins have one large band, and heterozygous knock-ins have one large band and one small band.
Gel extract each band of DNA using the QIAquick gel extraction kit and send each band for sequencing to sequence the entire amplicon. We sequence with the same primers we used for the PCR.
Analyze this sequencing data. For small bands, the goal is that the DNA sequence is identical to the uncut genomic DNA, rather than having been cut and repaired using non-homologous end joining to result in an indel. For large bands, the goal is to see precisely the endogenous sequence with your fluorescent protein inserted as you designed. In homozygous knock-ins, if the two alleles are slightly different, this will sometimes result in an .ab1 sequencing file with overlapping peaks as there are two sequences there. In this case, sequencing from the other side or an internal primer can help. It is also the case that you want any homozygous knock-ins to be identical, and so clones with overlapping-peak sequencing may not be the best choice and you may be able to discard them (Figure 10 ).
Figure 10. Example of a Sanger sequencing chromatogram with these overlapping peaks. This example is from tagging CXCL10 (instead of the IRF1 example used for the rest of this protocol), as we had no homozygous knock-ins in IRF1 and never saw this overlapping peak phenomenon there. Based on this chromatogram, we chose to not move forward with this particular clone. Arrows point to specific regions of overlapping peaks.
Select the clones with the correct insertion sequences to move forward with further screening.
For the rest of the screening, how you screen depends on what your tagged gene function is and what you are looking for. For inducible genes, we screen for fluorescence induction and choose a clone with representative fluorescence across all clones. We also do qPCR and Western blots where relevant to confirm near-endogenous RNA and protein expression level and timing for the tagged allele. If the tagged gene has a function (for example, as a transcription factor), we screen for gene function; for transcription factors, that means confirming the gene expression downstream of the transcription factor is at near-endogenous levels.
Based on all these data, choose your preferred clone and work with that clone.
Set up an imaging experiment
The day before (ideally, ~20 h before) you wish to image, seed 4 × 104 of your edited RAW 264.7 cells into each well of a 24-well plate in complete DMEM. We do this by preparing a tube with 104 × 104 cells in 13 mL of media, mixing that tube well, and then adding 500 μL of that mix to each well. After adding the cells to all wells, shake the plate back and forth on the floor of the TC hood enough to really agitate the liquid but not enough for it to spill over between wells. This is important to ensure an even distribution of cells. Incubate the plate in a tissue culture incubator for ~20 h for the cells to settle on the plate.
Note: This seeding density is dense enough for the cells to be happy, but relatively sparse so that the cells can sit for 20 h in the incubator and then be imaged for 48 h without starting to grow on top of each other. You may want to modify this density based on your imaging time.
Right before imaging, aspirate the media in each well and replace it with 500 μL of Phenol Red–free DMEM for imaging.
Bring the plate to the microscope and set up on the microscope, including the temperature and CO2 incubation.
Select exposure time and frequency; we take phase and iRFP images every 10 min for tracking and fluorescence in other channels every sixth cycle (every 60 min) to minimize phototoxicity.
Select positions, ensuring that you are in the middle of each well (which should look like the part with the darkest background). We take two images per well for a total of 48 images; our microscope cannot take more images per well within a 10 min image frequency. We also move through the plate in an “S” pattern to avoid the stage moving the plate back and forth more than necessary.
Run your experiment!
Data analysis
As this protocol describes creation of cell lines, there is no data analysis.
Validation of protocol
We have used this protocol to insert several different constructs into both the Tigre and Rosa26 safe harbor loci in RAW 264.7 cells. Additionally, we have used it to tag the genes IRF1, CXCL10, CXCL9, IRF8, and GBP1, all on the first try. We have tried and been unsuccessful in using this protocol (and its associated troubleshooting steps) to tag NOS2 and FCGR1, and when we tagged STAT1, its function was perturbed. A paper describing a cell line with EF1alpha-NLS-iRFP knocked into the Tigre locus and tags on IRF1, CXCL10, and CXCL9 is described in Fig 1A of Naigles 2023 and used extensively there [11].
General notes and troubleshooting
General notes
There is a pSpCas9(BB)-2A-GFP plasmid that can be used in place of the pSpCas9(BB)-2A-puro plasmid. This would allow for visual assessment of cell transfection efficiency by the fraction of GFP-expressing cells two days after Neon transfection. This can be useful for troubleshooting if there is concern about low transfection efficiency. Depending on your experimental design, this may also be useful if you want to select for transfected cells at that time without using puromycin. Using the pSpCas9-GFP plasmid will not interfere with downstream screening (even of GFP insertions) because the transient expression of GFP will have ended by the 7–9 day timepoint when cells are screened by FACS in this protocol. However, a cell line that constitutively expresses GFP would not be a suitable background to use if you are trying to use the pSpCas9-GFP plasmid to assess transfection efficiency, as the constitutive GFP will mask the GFP transiently expressed by the Cas9-GFP construct. If you are knocking in a GFP construct without a functional promoter, you should have no issues with expression from your donor plasmid interfering with assessing transfection efficiency via screening for the GFP expression from the Cas9-GFP plasmid. However, if your donor plasmid expresses GFP and contains a promoter for the GFP, then at the 2–3 day post-transfection timepoint you will see expression from both the Cas9-GFP and donor GFP construct.
In this protocol, cells are not selected for successful transfection before being selected for successful knock-in of the fluorescent protein. This means that the fraction of the cells that are positive for successful knock-in is very low, as only a fraction of cells is successfully transfected and then only a fraction of those has successful CRISPR cutting and HDR repair. Our preliminary experiments showed that transfection efficiency using the Neon system as described here is approximately 40%–60%, but that the fraction of cells with the fluorescent protein knocked into the locus ranges from 2% to 0.01% and is most commonly around 0.4%–0.1%. This 2% to 0.01% range is high enough to obtain positive cells from FACS. It may be possible to use puromycin to select for transfected RAW 264.7 cells, as they will transiently express puromycin resistance. This approach would need to be optimized to not further decrease cell viability after transfection but could be worth trying if attempting to knock-in a non-fluorescent construct.
We did not try linearizing the donor DNA, so cannot comment on how that would affect HDR efficiency. We did not do this due to concerns over introduction of endotoxins and because the protocol worked sufficiently for our purposes without it.
In our experience, there are two genes that we have been unable to tag despite extensive efforts. While one of these genes is in a closed chromatin environment, we have successfully tagged other closed-chromatin genes. Pretreating the cells with DMSO or interferon-gamma to attempt to open the chromatin at this locus prior to transfection did not lead to successful tagging. One gene had the correct genomic sequence that would indicate successful tagging, but the fluorescence was too weak to image. This leads us to conclude that a certain minimal level of protein expression is needed for fluorescent tagging to be a useful strategy.
We attempted many variations on the Neon transfection described here, including varying the electroporation settings, quantity of cells, and quantity of DNA. The protocol described here works best for maximizing knock-in efficiency and minimizing cell death after electroporation. After we had settled on the electroporation setting and cell number per electroporation (2.5 × 106), we tried pretreating the cells with DMSO for 24 h, pretreating with IFNγ for 2 h, increasing the donor DNA to be 30 μg rather than 15 μg, and plating the cells post-transfection into media containing 2 μM A3814, which inhibits DNA-dependent protein kinase (DNA-PK). DNA-PK plays a role in non-homologous end joining (NHEJ), and some studies have shown that its inhibition decreases NHEJ and so promotes HDR as the resolution pathway after double-strand breaks [13–15]. Overall, we found that pretreatment with IFNγ or using 30 µg of donor DNA decreased cell viability after transfection, while DMSO pretreatment did not. The DMSO pretreatment + 30 μg donor and the no pretreatment + 15 μg donor + plate into media with M3814 both had slightly higher knock-in rates than the no pretreatment + 15 μg donor case we present here. We chose to not use the higher quantity of donor DNA due to the cell toxicity, and later experiments showed less difference between plating into media with or without M3814, which also contributed to our choice to stop using the M3814. However, if there is a need to increase HDR efficiency specifically, then plating into media with M3814 is worth trying.
We tried a variety of other transfection methods for the RAW 264.7 cells, but in all cases had worse transfection efficiency than the Neon system. When using a constitutive fluorescent protein expression plasmid to test transfection efficiency, we got 5%–10% transfection efficiency with FuGene, up to 30% but more commonly 10% with TransIT-X2, 10% with polyethylenimine (PEI), 20% with Lipofectamine 2000, and <10% with GeneJet. For all these approaches, we tried a variety of conditions and DNA:reagent ratios, including those suggested by the supplier for RAW 264.7 cells. We used the Neon due to its much higher transfection efficiency (40%–60%); however, if another transient transfection approach works for RAW 264.7 cells in your hands, then it may also be usable for delivering plasmids for this endogenous tagging approach.
Troubleshooting
The main issue that occurs with this protocol is that you do the whole procedure and have no positive clones. In this situation, it is best to go back and make sure each previous step is working. Below are some of the intermediate steps that need to work to obtain the final knock-in. However, as discussed above, there are a couple of loci that we have been unable to tag using this protocol even after troubleshooting.
Step How to test Possible ways to fix
gRNA-Cas9 cutting of DNA Go back to the data where you test cutting efficiency in NIH3T3 cells; you can also do the same protocol to test in RAW 264.7 cells in case there are single nucleotide polymorphisms (SNPs) that lead to different efficiencies in each cell type. Choose a different guide sequence.
Neon transfection Confirm transfection efficiency using a plasmid that constitutively expresses a fluorescent protein. Adjust quantity of cells or DNA, ensure cells are happy before transfection, minimize time in R buffer, minimize time cells are in a tube rather than a plate, try a new Neon tip or cuvette, replace Neon buffers.
HDR at the locus There is no direct way to test for poor HDR because we only screen after both transfection and HDR, but if you have good cutting and transfection efficiency and still no knock-in cells, this may be due to a lack of HDR at the locus. It is worth trying to pretreat with DMSO or a stimulus that opens the chromatin at your locus or to plate into media with M3814. It may also be worth trying to move to tag the other terminus if that is an option for your gene, as some termini just seem hard to tag for reasons we do not understand. You could also grow up some of the relatively brighter single-cell clones, amplify the locus using PCR, and analyze that sequencing, as it is possible that HDR was successful, but fluorescence intensity is just low.
Some additional issues that may occur are described below.
Issue Suggested solution
You only have heterozygous clones and want homozygous clones Try to screen more clones. In our experience, most knock-ins are either all homozygous or all heterozygous, but we have had one case (in a safe harbor locus) where there has been a mix that can be seen by screening more clones.
All the intermediate steps work and you still have no clones There are a number of reasons this might be. While there are some genes that we have never been able to tag, here we include some options to try. 1) The chromatin at this locus may be very closed, and so pretreatment with something that would open the chromatin at that locus (DMSO or a specific inducer if the gene is inducible) may help. 2) Consider trying to tag the other terminus. 3) The gene may be expressed too weakly to see fluorescence or the protein may be so dispersed in the cell that the fluorescence is not visible. In this case, one option is to make a transcriptional reporter by adding a T2A sequence between the endogenous protein and the fluorescent protein and adding an NLS to the fluorescent protein to concentrate the fluorescent protein signal in the nucleus. Another option is to use a brighter fluorescent protein or multiple copies of the fluorescent protein. You can generally assess if there is correct tagging but weak fluorescence by sorting the relatively brighter cells and screening those colonies by PCR to see if the DNA sequence is or is not inserted at the locus—if the correct DNA is there but you do not see fluorescence, it is likely too dim.
Your tagged gene seems to have perturbed function Consider tagging the other terminus, adding a longer/different linker, or adding a T2A so it becomes a transcriptional reporter. You could also investigate smaller tags.
Acknowledgments
We thank the Murre lab for use of their Neon transfection system and many friends for suggesting and letting us borrow different transfection reagents while developing this protocol. We thank the staff at the Sanford Consortium Human Embryonic Stem Cell Core for help with cell sorting. This work was supported by the NIH-sponsored Quantitative Integrative Biology Training Grant (T32GM127235) on which BN was a trainee, and NIH F31AI161903 (to BN), NIH R01GM111458 (to NH) and NIH R01 GM144595 (to NH). JS is supported by a PhD Fellowship from Boehringer Ingelheim Fonds. We developed this protocol for Naigles et al [11] (Quantifying dynamic pro-inflammatory gene expression and heterogeneity in single macrophage cells) and use a cell line created with this protocol extensively in that paper [11].
Competing interests
The authors declare no competing interests.
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Proximity Labelling to Quantify Kv7.4 and Dynein Protein Interaction in Freshly Isolated Rat Vascular Smooth Muscle Cells
JH Jennifer van der Horst
TJ Thomas A. Jepps
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4961 Views: 507
Reviewed by: Pilar Villacampa AlcubierreWilliam C. W. Chen Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of General Physiology Feb 2021
Abstract
Understanding protein–protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.
Keywords: Proximity ligation assay Vascular smooth muscle cells Spatial co-localisation Subcellular localisation Fluorescence microscopy Protein interactions
Background
Studying protein–protein interactions is crucial for unravelling the subcellular distribution of proteins, contributing to our understanding of cellular organisation. Besides revealing the spatial arrangement of proteins, interaction or co-localisation studies provide insights into the functional relationships between proteins and cellular processes. These studies play a role in clarifying signalling pathways, as the interaction partners of certain proteins often indicate their involvement in specific signalling cascades. Additionally, co-localisation studies can contribute to the elucidation of mechanisms governing the trafficking of proteins within cells. For example, identifying whether a certain protein interacts with the microtubule network or specific motor proteins, such as kinesin or dynein that are responsible for cargo movement along the microtubule network in opposite directions, provides insights into trafficking dynamics, including the direction of trafficking.
Various techniques exist for detecting protein–protein interactions in different cellular contexts, including live and fixed cells, tissues, and protein lysates. These techniques typically involve the use of antibodies or direct fluorescent protein fusions. Förster resonance energy transfer (FRET) is a well-established technique for measuring co-localisation in live cells by expressing two proteins of interest, each fused to different fluorophores (donor and acceptor) [1,2]. Proximity of the two proteins induces energy transfer between the fluorophores, which can be measured. However, FRET has limitations, requiring cultured cells and molecular cloning for the fluorophore fusion to proteins. Co-immunoprecipitation, another method for studying protein–protein interactions, involves using antibodies to pull out targeted proteins from cell or tissue lysates, along with some of the direct interacting partners [3]. Analysis using western blotting or mass spectrometry can identify the binding partners of the targeted protein. However, the success of co-immunoprecipitation can be affected by the choice of lysis buffer since this can easily disrupt several protein–protein interactions. Thus, this technique is not optimal for low-affinity interactions. Additionally, co-immunoprecipitation lacks detailed information about the spatial co-localisation of proteins within a cell. Fluorescence microscopy is a widely used technique, which involves labelling proteins with specific antibodies and fluorescent labels, with co-localisation assessed by evaluating the spatial overlap of two fluorescent signals. Although this technique does not detect direct physical protein–protein interactions, advanced light microscopy techniques, such as stimulated emission depletion or structured illumination microscopy, enhance the resolution, providing a more precise determination of protein co-distribution.
The in situ proximity ligation assay (PLA) enables accurate, spatial detection of protein–protein interactions in fixed cells and tissues [4]. This technique involves using two primary antibodies for the targeted proteins, raised in different species. The species-specific secondary PLA probes, each linked to a unique DNA oligonucleotide, bind to their respective primary antibodies. When the targeted proteins are within 40 nm of each other, the DNA oligonucleotides on the PLA probes interact and serve as a template for the hybridising connector oligonucleotide, resulting in the formation of circular DNA through enzymatic ligation. This circular DNA template undergoes rolling circle amplification, producing DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides are added, which hybridise to the complementary circle DNA. The resulting fluorescent spots can be visualised using fluorescent microscopy. These detected signals can be quantified and attributed to specific subcellular locations, to determine the co-localisation pattern within cells. Overall, PLA offers a precise way to study the spatial relationships between proteins in cells. Moreover, PLA enables the application of pharmacological treatments to isolated vascular smooth muscle cells prior to fixation, allowing exploration of treatment impacts on the co-localisation of specific proteins. In our study, we employed PLA to visualise the interaction of the motor protein dynein with voltage-gated potassium Kv7.4 channels in freshly isolated vascular smooth muscle cells from rat mesenteric arteries [5]. Our goal was to explore dynein's impact on the microtubule-dependent trafficking of Kv7.4 channels. The Kv7.4 and Kv7.5 channels are important regulators of the resting membrane potential in smooth muscle cells, particularly from arteries [6]. Additionally, these channels contribute to the β-adrenoceptor-mediated vasodilatation in vascular smooth muscle cells [7] and other cAMP and cGMP-derived relaxations [8,9]. In arteries from hypertensive rodents, the Kv7.4 channel is downregulated and function is attenuated [10], which contributes to the reduced β-adrenoceptor-mediated vasodilatation observed in these arteries [11]. Our previous studies indicate that dynein motor proteins facilitate the trafficking of Kv7.4 channels along the microtubule network, away from the plasma membrane, in rat vascular smooth muscle cells [5,12]. Furthermore, manipulating this trafficking system by inhibiting the dynein-mediated trafficking of Kv7.4 channels enhances Kv7.4 membrane abundance, restoring the β-adrenoceptor-mediated vasodilatation in arteries from hypertensive rats [13].
Materials and reagents
Biological materials
Rat (Janvier Labs, France)
This protocol has been employed on male and female rats, including strains of Wistar Kyoto, Wistar Hannover, and Spontaneously Hypertensive rats, aged between 10 and 18 weeks. It is anticipated that this protocol is applicable to rats of other strains and ages.
Reagents
Milli-Q water
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888), stored at room temperature
Sodium gluconate (C6H11NaO7) (Sigma-Aldrich, catalog number: S2054), stored at room temperature
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 12636), stored at room temperature
Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670), stored at room temperature
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016), stored at room temperature
Glucose (C6H12O6) (Sigma-Aldrich, catalog number: G7021), stored at room temperature
HEPES (Sigma-Aldrich, catalog number: H3375), stored at room temperature
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647), stored at 4 °C
1,4-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: DTT-RO), stored at 4 °C
Papain from papaya latex (Sigma-Aldrich, catalog number: P4762), stored at -20 °C
Collagenase from Clostridium histolyticum Type F (Collagenase F) (Sigma-Aldrich, catalog number: C7926), stored at -20 °C
Collagenase from Clostridium histolyticum Type H (Collagenase H) (Sigma-Aldrich, catalog number: C8051), stored at -20 °C
Triton-X (Sigma-Aldrich, catalog number: T8787), stored at room temperature
4% paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: HT501128), stored at 4 °C
Phosphate buffered saline (PBS) (Gibco, catalog number: 10010023, or any other PBS without magnesium and calcium), stored at room temperature
Rabbit anti-Kv7.4 antibody (Abcam, catalog number: ab65797), stored at -20 °C
Mouse anti-Dynein antibody (Abcam, catalog number: ab23905), stored at -20 °C
Duolink in situ PLA probe anti-mouse PLUS (Sigma-Aldrich, catalog number: DUO92001-100RXN), stored at 4 °C
Duolink in situ PLA probe anti-rabbit MINUS (Sigma-Aldrich, catalog number: DUO92005-100RXN), stored at 4 °C
Duolink in situ detection reagents red, with 5× ligation buffer and5× amplification buffer (Sigma-Aldrich, catalog number: DUO92008-100RXN), stored at -20 °C
Duolink in situ wash buffer A and wash buffer B (Sigma-Aldrich, catalog number: DUO82049-4L), stored at room temperature or at 4 °C once dissolved (see Recipes)
Anti-dynein (Mouse; 1:500: ab23905; Abcam) and anti-Kv7.4 (Rabbit; 1:200; ab65797; Abcam)
Solutions
Calcium chloride (1 M) (see Recipes)
Calcium chloride (100 mM) (see Recipes)
Magnesium chloride (1 M) (see Recipes)
HEPES-Krebs solution (see Recipes)
Smooth muscle cell dissection solution (SMDS) (see Recipes)
Stock solutions (see Recipes)
Tube 1 (see Recipes)
Tube 2 (see Recipes)
Recipes
Prepare all solutions in volumetric flasks up to the specified volume. Using volumetric flasks will contribute to precise measurements and reliable results in your experiments.
Calcium chloride (1 M)
Reagent Final concentration Quantity or Volume
Calcium chloride 1 M 11.098 g
Dissolve the calcium chloride in Milli-Q water, add the volume up to exactly 100 mL, and store at 4 °C.
Calcium chloride (100 mM)
Dilute 100 μL of calcium chloride 1 M in 900 μL of Milli-Q water and store in a 1.5 mL tube at room temperature.
Magnesium chloride (1 M)
Reagent Final concentration Quantity or Volume
Magnesium chloride hexahydrate 1 M 20.33 g
Dissolve the magnesium chloride hexahydrate in Milli-Q water, add the volume up to exactly 100 mL, and store at 4 °C.
HEPES Krebs solution
Reagent Final concentration Quantity or Volume
Sodium chloride 134 mM 1.593 g
Potassium chloride 6 mM 89.46 mg
Glucose 7 mM 252.21 mg
HEPES 10 mM 476.6 mg
Calcium chloride (1 M) 2 mM 400 μL
Magnesium chloride (1 M) 1 mM 200 μL
Dissolve the reagents in Milli-Q water, adjust the pH to 7.4, and add the volume up to exactly 200 mL. The solution should be freshly prepared on the day of experiment.
SMDS solution
Reagent Final concentration Quantity or Volume
Sodium chloride 60 mM 1.75 g
Sodium gluconate 80 mM 8.73 g
Potassium chloride 5 mM 186.38 mg
Magnesium chloride hexahydrate 2 mM 203.3 mg
Glucose 10 mM 900.75 mg
HEPES 10 mM 1.19 g
Dissolve the reagents in Milli-Q water, adjust the pH to 7.4, and add up to exactly 500 mL. After preparation, distribute the solution into 50 mL tubes and store them at -20 °C. Thaw one 50 mL SMDS solution on the day of the experiment before use.
Wash buffer A and wash buffer B
Prepare PLA wash buffer A and B ahead by dissolving the PLA buffer sachets each in 1 L of Milli-Q. Filter sterilise both buffers using a syringe and a 0.2 μm pore filter and store at 4 °C.
Stock solutions
Reagent Concentration Minimum volume needed
BSA 10 mg/mL 200 μL
DTT 10 mg/mL 150 μL
Papain 5 mg/mL 100 μL
Collagenase F 10 mg/mL 70 μL
Collagenase H 5 mg/mL 40 μL
Tube 1
Stock solution Final concentration Quantity or Volume
BSA (10 mg/mL) 1 mg/mL 100 μL
DTT (10 mg/mL) 1.5 mg/mL 150 μL
Papain (5 mg/mL) 0.5 mg/mL 100 μL
SMDS n/a 650 μL
Total n/a 1,000 μL
Tube 2
Stock solution Final concentration Quantity or Volume
BSA (10 mg/mL) 1 mg/mL 100 μL
Collagenase F (10 mg/mL) 0.7 mg/mL 70 μL
Collagenase H (5 mg/mL) 0.2 mg/mL 40 μL
*Calcium Chloride (100 mM) 100 µM 1 μL
SMDS n/a 790 μL
Total n/a 1000 μL
*A stock solution of calcium chloride can be pre-made and stored at room temperature for repeated use.
Laboratory supplies
Surgical scissors (Fine Science Tools, Mayo-Stille Scissors, catalog number: 14101-14)
Dissection forceps (Fine Science Tools, Fine Forceps, catalog number: 11252-00)
Dissection scissors (Fine Science Tools, Spring scissors, catalog number: 15020-15)
Dissection dish (Silicone coated)
Sewing pins
1.5 mL microcentrifuge tube (Eppendorf, catalog number: 0030120086)
50 mL Falcon tube (Falcon)
100/200 mL glass beaker
50 mL syringe (BD Plastipak)
0.2 μm pore syringe filter (Sigma-Aldrich, catalog number: WHA9914)
Disposable Pasteur pipette (Thermo Fisher, catalog number: 202-1SPK)
24-well cell culture plate (Thermo Fisher, catalog number: 144530)
Styrofoam ice box
Fire-polished glass pipette (custom-made, see Figure 1)
Figure 1. Fire-polishing of the glass Pasteur pipette. A. Remove and discard the long end of the pipette. B. Rotate the end of the pipette in a Bunsen flame until its edges are polished and the opening is smoothed and narrowed. Comparison of the opening before (left) and after (right) fire polishing.
Humidified chamber (custom-made)
Cover glass 12-mm circles (VWR, catalog number: 76355-906)
Microscope slides (VWR, catalog number: 16004-422)
Clear nail polish (any)
Equipment
Pipettes (ranging from 1 to 1,000 μL)
Heat block (37 °C)
Bunsen burner
Plate rocker at room temperature
37 °C, 5% CO2 cell culture incubator
Dissecting microscope (Olympus SZX7 Stereomicroscope)
Cell microscope (Leitz Labovert inverted light microscope, 10× objective)
ZEIS LSM780/LSM900 microscope or any other laser scanning confocal microscope
Software
ImageJ (version 1.53k, July 2021)
GraphPad PRISM (version 9, October 2020)
Procedure
Part I: Isolation of rat vascular smooth muscle cells
Preparation
Take a 50 mL Falcon tube with SMDS solution out of the -20 °C freezer and thaw on ice.
Prepare the HEPES-Krebs solution (see Recipes)
Take the enzymes out of the -20 °C freezer and thaw at room temperature.
Isolation of the rat mesenteric arteries
Sacrifice the rat (in accordance with annex IV of the EU Directive 2010/63EU on the protection of animals used for scientific purposes).
Make the rat unconscious by a single percussive blow to the head. Immediately after the onset of unconsciousness, perform cervical dislocation to complete the killing.
Perform a laparotomy using surgical scissors and elevate the intestines out of the abdominal cavity (Figure 2A–2D).
Figure 2. Dissection process of rat mesenteric arteries. A. The male Wistar rat is euthanised via cervical dislocation. B–D. A laparotomy is performed, and the rat intestines are isolated. E. The intestines are pinned onto a dissection dish using sewing pins to expose the mesenteric vessels. F–H. The main mesenteric artery and vein, surrounded by adipose tissue, are visualised. I–K. The surrounding tissue is removed, and 3–4 branches of the mesenteric arteries are isolated for the extraction of arterial smooth muscle cells.
Cut the intestines just above the rectum and below the stomach and place the intestines in a beaker filled with HEPES-Krebs placed on ice (Figure 2A –2D).
Place the intestines into a dissection dish filled with cold HEPES-Krebs buffer and pin the jejunum and ileum along the side of the dissection dish forming a circle, allowing visualisation of the mesenteric vessels, with the main arterial branch in the centre (Figure 2E).
Place the dish under a 10× dissecting microscope for visualisation of the arteries and veins (Figure 2F–2G).
Arteries can be differentiated from veins by their thicker smooth muscle layer, which makes them more rigid compared to veins. Furthermore, the thicker wall creates the appearance of a reduced blood volume compared to veins.
Remove all tissue surrounding the arteries (Figure 2G–2H).
Begin by locating the larger veins and carefully tear them using two forceps; then, remove them all the way down to the lower-order branches.
Cut through the mesentery connecting the arterial branches and remove all adipose tissue surrounding the arteries.
Remove the mesenteric arteries by first cutting at the lower-order branches and then free the mesenteric arteries by cutting the central branch (Figure 2I –2K).
Take a small section of the main artery, including 3–4 branches of lower-order mesenteric arteries and place it in a 1.5 mL tube filled with SMDS solution on ice.
Remove the remaining intestines from the dissection dish for disposal.
Isolation of the mesenteric arterial smooth muscle cells
Have a fire-polished glass Pasteur pipette ready. To make a fire-polished pipette, break off the long end of the pipette and throw it out. Expose the pipette with its broken end to a Bunsen flame, constantly rotating it. Continue with this process until the edges are polished and the opening is smoothed and narrowed to an inner diameter of roughly 0.7–1 mm (Figure 1). Wash the pipette with ethanol after each experiment so it can be used for subsequent future experiments.
The following solutions used for smooth muscle cell isolation have been adapted from Zhong et al. [14] and Chadha et al. [11]. Prepare the following stock solutions (Recipes 7–9) by dissolving them in SMDS solution (Recipe 5) in 1.5 mL tubes and keep them at room temperature.
These stock solutions have to be prepared fresh on the day of the experiment and cannot be stored for future experiments.
Place the tube with the mesenteric artery branch in SMDS solution in a 37 °C heat block for 10 min to equilibrate the arteries at 37 °C.
In the meantime, prepare Tube 1 (Recipe 8) by combining the stock solutions (Recipe 7) and SMDS solution in a 1.5 mL tube.
Transfer the mesenteric arteries to Tube 1. Gently invert the tube 4–5 times to ensure proper mixing of the arteries with the solution and incubate at 37 °C for 10 min.
In the meantime, prepare Tube 2 (Recipe 9) by combining the stock solutions (Recipe 7) and SMDS solution in a 1.5 mL tube.
Take the arteries from the heat block and wash five times in ice-cold SMDS solution using a glass Pasteur pipette with a rubber bulb.
Transfer the mesenteric arteries to Tube 2. Gently invert the tube 4–5 times to mix the arteries with the solution, and incubate at 37 °C for 10 min.
The arterial branch may appear slightly blurry or fluffy during this step; this means that the enzymatic digestion is effectively isolating the cells.
Gently wash five times in ice-cold SMDS solution using a glass Pasteur pipette with a rubber cap. During the last wash, replace the solution with 500 μL of SMDS.
Liberate the myocytes by gently triturating the arterial branch using the custom-made fire-polished glass pipette.
Perform approximately 30 up-and-down triturations.
Store the supernatant containing liberated myocytes in a separate tube on ice and add 500 μL of fresh SMDS to the arterial branch in the tube. Place a droplet of the supernatant on a glass slide and assess the cell density using a 10× microscope.
Repeat the trituration process and save the supernatant in separate tubes on ice, until no more cells are released within the supernatant.
This process can take approximately 1–5 rounds of trituration.
Note: Consider shortening the artery incubation time (1–3 min shorter) with enzymes in tube 1 and tube 2, if the enzymes are newly opened, to prevent over-digestion of the arteries and enhance myocyte liberation.
Combine the supernatants that have liberated myocytes and adjust the concentration of cells by adding more SMDS to achieve the desired cell density. By placing 50 µL of the cells on a glass coverslip, it is possible to check the cell density under a light microscope. There is no defined cell density necessary for undertaking this protocol, but smooth muscle cells should be visible under a light microscope’s 20× objective, and the cells should not be crowded and touching one another.
Add 1 μL of 100 mM calcium chloride for every 1 mL of cell suspension (resulting in a final concentration of 100 μM calcium chloride). Calcium is required for the cells to adhere to the coverslips.
Note: Consider increasing the calcium chloride concentration by 10%–20% if there is significant loss of cells from the coverslip after fixing the cells.
Add 100 μL of cell suspension on a 12 mm coverslip in a 24-well plate and keep at room temperature for 30 min to let the cells adhere to the coverslips (Figure 3A ).
After 30 min, the cells can be incubated with a pharmacological agent. To do this, dissolve the compound at the desired concentration in SMDS solution supplemented with 100 μM calcium chloride, replace the solution on the coverslip with 100 μL of compound solution, and incubate at 37 °C.
Figure 3. Adhesion and primary antibody incubation for isolated vascular smooth muscle cells. A. Place 100 µL of cell suspension onto each coverslip in the well. B. Create a humidified chamber using a Styrofoam icebox and parafilm. C. Incubate the cells with the primary antibody by applying a drop of the diluted antibody on the parafilm and placing the coverslip with adhered cells on top. D. Seal the humidified chamber with cling film for incubation at 4 °C.
Fix the cells by gently aspirating the SMDS solution from the coverslip and apply 400 μL of 4% PFA to each well. Place the 24-wells on the rocker for 15 min at room temperature.
Wash three times with PBS, 400 μL per well.
Use the coverslips immediately for PLA experiment or store coverslips in the 24-wells plate (with each well containing 1 mL of PBS and sealed with parafilm) at 4 °C for up to four weeks until used for the PLA experiment. The duration of storage depends on the specific cell type.
Part II: PLA
Preparation
Allow the PLA wash buffers A and B to reach room temperature prior to use, as cold wash buffers can lead to nonspecific background signal.
Permeabilization, blocking, and primary antibody incubation
Prepare a sufficient volume of 0.1% Triton-X in PBS to add 400 μL of the solution to each coverslip within the well.
Add 400 μL of 0.1% Triton-X to each well, place on the rocker, and incubate for 5 min to permeabilise the cells.
Wash two times for 5 min each in PBS.
Remove the PBS and apply three drops of the blocking buffer (provided in the PLA probe kit) onto the cells on the coverslip in each well. Incubate at 37 °C for 30 min.
Prepare the primary antibody mix. It is very important that the two primary antibodies targeting the protein of interest are raised in different species.
In our experiment, to look at Kv7.4 and dynein co-localisation, we prepared a primary antibody mixture consisting of anti-dynein and anti-Kv7.4 antibodies.
Dilute the primary antibodies in the antibody diluent provided in the PLA probe kit, following the specific requirements of each antibody.
Prepare enough to use 40 μL of the primary antibody mix for each coverslip.
Create a humidified chamber (Figure 3B).
Use the lid of a Styrofoam ice box; the lid must have a recessed area in the middle.
Place a piece of parafilm in the centre of the lid, where the coverslips will be placed for incubation.
Create four wells using parafilm, each well containing a wet tissue inside (see Figure 3B). Place the four wet tissues in parafilm wells around the flat piece of parafilm in the middle of the Styrofoam lid. This will help maintain moisture and prevent the coverslips from drying out during the overnight incubation with the primary antibodies.
Add a 40 μL drop of primary antibody mix to the parafilm.
Using forceps, lift the coverslip from the blocking solution, removing it from the well. Gently tap the side of the coverslip on a paper tissue to remove excess blocking buffer and place it over the antibody drop with the side containing cells making contact with the drop and facing downward (Figure 3C).
Cover the Styrofoam lid with cling film and leave it overnight at 4 °C (Figure 3D).
Note: This primary antibody concentration and incubation time may require optimisation.
Probe incubation
Note: Each coverslip represents one reaction, and 40 µL of solution is used per reaction. The calculations provided in the following steps are for a single reaction.
The following day, take the PLA PLUS and MINUS probe out of the fridge and vortex.
Prepare the PLA probe mix by diluting the PLA PLUS and MINUS probes 1:5 in the antibody diluent provided in the kit. Make sufficient PLA probe solution to use 40 μL per coverslip.
For one coverslip (40 μL): dilute 8 μL of PLUS and 8 μL of MINUS in 24 μL of antibody diluent.
Place the coverslips incubated with the primary antibodies back in the 24-well plate and wash two times for 5 min each in PLA wash buffer A at room temperature on a rocker.
Place a new piece of parafilm in the handcrafted humidified chamber and add 40 μL drops of PLA probe mix for every coverslip.
Add the coverslips on top of the droplets with the side containing cells facing downward.
Place the humidified chamber in a 37 °C incubator for 1 h.
Ligation
Place the coverslips back in the 24-well plate and wash two times for 5 min each in PLA wash buffer A at room temperature on a rocker.
Prepare the PLA ligation mix by diluting the 5× ligation buffer with Milli-Q water (1:5 dilution) and add the ligase (1:40 dilution) to the mix.
For one coverslip (40 μL): dilute 7.8 μL of 5× ligation buffer in 31.2 μL of Milli-Q water and 1 μL of ligase.
Wait to add the ligase until immediately prior to incubation.
Place a new piece of parafilm in the handcrafted humidified chamber and add 40 μL droplets of PLA ligation mix for every coverslip.
Add the coverslips on top of the droplets with the side containing cells facing downward.
Place the humidified chamber in a 37 °C incubator for 30 min.
Amplification
Note: The fluorophore in the Amplification Red detection kit has an excitation/emission wavelength of 594 nm/624 nm, respectively, and can be detected using the Texas Red filter (see Part III.). Alternative detection fluorophores are also available, such as Amplification Green, Orange, and Far Red.
Place the coverslips back in the 24-well plate and wash twice for 2 min each time in PLA wash buffer A at room temperature on a rocker.
Prepare the PLA amplification mix by diluting the 5× amplification buffer with Milli-Q water (1:5 dilution) and add the polymerase (1:80 dilution) to the mix.
For one coverslip (40 μL): dilute 7.9 μL of 5× amplification buffer in 31.6 μL of Milli-Q water and 0.5 μL of polymerase.
Wait to add the polymerase until immediately prior to incubation.
Place a new piece of parafilm in the handcrafted humidified chamber and add 40 μL droplets of PLA amplification mix for every coverslip.
Add the coverslips on top of the droplets with the side containing cells facing downward.
Place the humidified chamber in a 37 °C incubator for 100 min, protected from light.
Mounting
Place the coverslips back in the 24-well plate and wash twice for 10 min each time in PLA wash buffer B at room temperature on a rocker.
Protect the samples from light by placing aluminium foil over the 24-well plate.
Wash for 1 min in 0.01× PLA wash buffer B.
Mount coverslips on microscope slides using 3 μL of mounting media containing DAPI for each coverslip.
Let the coverslips dry at room temperature protected from light for 10 min.
Use clear nail polish to seal the edges of the coverslip to the slide. Avoid getting air bubbles caught under the coverslip.
Store the slides in the dark at 4 °C for up to four days or at -20 °C for six months.
Part III: Imaging and analysis
Imaging
Visualise fluorescent signal by a confocal microscopy (Figure 4).
Figure 4. Representative images of a single vascular smooth muscle cell displaying a number of proximity ligation assay (PLA) dots. Each red dot represents a site where the two primary antibodies have bound to their respective protein within 40 nm of each other. A. Texas Red channel (excitation wavelength 592), in which the PLA dots are visible. B. Brightfield image of the vascular smooth muscle cell. C. DAPI staining of the nucleus (excitation wavelength 353). D. Merged image of A, B, and C. Scale = 5 µm
Use appropriate microscope settings.
In our specific setup, we capture images using a 63× oil immersion objective on either a ZEIS LSM780 or LSM900 laser scanning confocal microscope.
Choose a DAPI filter for nucleus detection and a Texas red filter for PLA dot detection. To visualise cell contours and contrast, include a brightfield imaging channel in your microscopy setup (Figure 4).
Ensure consistent laser settings are applied for each experiment.
Capture full z-stack images of individual cells. Multiple cells can be captured per coverslip.
For quantitative comparisons, PLA signals of at least 30 cells are captured from at least three biological replicates.
Include a negative control sample as part of the experiment. A suitable negative control may involve the use of two protein-targeting antibodies that are known to have no co-localisation and, therefore, should not produce a signal. Alternatively, consider using protein knockdown cells for additional control measures. Another appropriate control, as implemented in our study, involves the use of only one of the primary antibodies.
Data analysis
Note: Analyse the number of fluorescent PLA dots within each cell. Depending on the desired approach, PLA dots can be quantified on a single mid-cell z-plane or conducted on a maximum intensity projection encompassing the entire cell.
Open the image file in ImageJ. Make sure to open the images for PLA dots, DAPI, and brightfield in separate windows instead of using the merged channels file. You can achieve this by selecting the Split channel option (Figure 5A ).
Figure 5. Data analysis in ImageJ. A. Specific settings for opening a microscopy image in ImageJ. B. Example of threshold parameters for visualizing clear dots while minimizing background. C. Example of the data output, showing the number of dots per z-stack, with each row corresponding to a different stack.
Three individual windows, representing the PLA dots, DAPI, and brightfield will now open. Close the DAPI and brightfield windows so that only the PLA dots window remains visible.
In the ImageJ browser, select Image > Adjust > Threshold.
Adjust the threshold parameters by clicking and dragging the upper bar from the left to the right until the dots become clearly visible and the background is removed. Ensure that the lower bar is set all the way to the right (Figure 5B ).
To count the number of dots, select in the ImageJ browser Analyze > Analyze particles.
Adjust the size parameters for particle analysis, adjusting both the minimum and maximum size.
For our experiments, we typically select a particle size range from 0.3 to 3 micron^2. Particles outside of this range will be excluded from the count. Adjust these parameters as per specific requirements.
Select the Summarize option and click OK.
To mark the counted particles in your PLA image window, select the Add to manager option before clicking OK.
ImageJ will now give an option to process all images. By selecting Yes, ImageJ will count the PLA dots for each stack and generate an output data file for the number of dots per stack (Figure 5C). In case the number of PLA dots for one specific stack should be counted, select No, and ImageJ will count the dots for the stack you have currently selected in your PLA image window.
To compare the average number of PLA dots for each cell under various conditions (such as control or pharmacologically treated cells), add the dot counts for each biological replicate into GraphPad PRISM. Analyse the data set using a nested t-test to test whether pharmacological treatment has an impact on the number of interactions between two proteins.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
• van der Horst et al. (2021). Dynein regulates Kv7.4 channel trafficking from the cell membrane. J. Gen. Physiol. (Figures 3F, 6C, 7B&C and 8A).
Acknowledgments
This work was supported by funding from the Lundbeck Foundation (grant R323-2018-3674 to T.A. Jepps). We acknowledge the Core Facility of Integrated Microscopy at the University of Copenhagen for their technical assistance with fluorescent microscopes. Additionally, this protocol is adapted from the prior work of van der Horst et al. [5].
Competing interests
No competing interests to declare.
References
Day, R. N. and Davidson, M. W. (2012). Fluorescent proteins for FRET microscopy: Monitoring protein interactions in living cells. BioEssays 34(5): 341–350. https://doi.org/10.1002/bies.201100098
Bajar, B., Wang, E., Zhang, S., Lin, M. and Chu, J. (2016). A Guide to Fluorescent Protein FRET Pairs. Sensors 16(9): 1488. https://doi.org/10.3390/s16091488
Kaboord, B. and Perr, M. (2008). Isolation of Proteins and Protein Complexes by Immunoprecipitation. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. In Methods in Molecular Biology™, Humana Press. vol 424. https://doi.org/10.1007/978-1-60327-064-9_27
Söderberg, O., Leuchowius, K. J., Gullberg, M., Jarvius, M., Weibrecht, I., Larsson, L. G. and Landegren, U. (2008). Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45(3): 227–232. https://doi.org/10.1016/j.ymeth.2008.06.014
van der Horst, J., Rognant, S., Abbott, G. W., Ozhathil, L. C., Hägglund, P., Barrese, V., Chuang, C. Y., Jespersen, T., Davies, M. J., Greenwood, I. A., et al. (2021). Dynein regulates Kv7.4 channel trafficking from the cell membrane. J. Gen. Physiol. 153(3): e202012760. https://doi.org/10.1085/jgp.202012760
Stott, J. B., Jepps, T. A. and Greenwood, I. A. (2014). KV7 potassium channels: a new therapeutic target in smooth muscle disorders. Drug Discov. 19(4): 413–424. https://doi.org/10.1016/j.drudis.2013.12.003
van der Horst, J., Greenwood, I. A. and Jepps, T. A. (2020). Cyclic AMP-Dependent Regulation of Kv7 Voltage-Gated Potassium Channels. Front. Physiol. 11: e00727. https://doi.org/10.3389/fphys.2020.00727
Stott, J. and Greenwood, I. (2015). Complex role of Kv7 channels in cGMP and cAMP-mediated relaxations. Channels 9(3): 117–118. https://doi.org/10.1080/19336950.2015.1046732
van der Horst, J., Manville, R. W., Hayes, K., Thomsen, M. B., Abbott, G. W. and Jepps, T. A. (2020). Acetaminophen (Paracetamol) Metabolites Induce Vasodilation and Hypotension by Activating Kv7 Potassium Channels Directly and Indirectly. Arterioscler., Thromb., Vasc. Biol. 40(5): 1207–1219. https://doi.org/10.1161/atvbaha.120.313997
Jepps, T. A., Chadha, P. S., Davis, A. J., Harhun, M. I., Cockerill, G. W., Olesen, S. P., Hansen, R. S. and Greenwood, I. A. (2011). Downregulation of Kv7.4 Channel Activity in Primary and Secondary Hypertension. Circulation 124(5): 602–611. https://doi.org/10.1161/circulationaha.111.032136
Chadha, P. S., Zunke, F., Zhu, H. L., Davis, A. J., Jepps, T. A., Olesen, S. P., Cole, W. C., Moffatt, J. D. and Greenwood, I. A. (2012). Reduced KCNQ4-Encoded Voltage-Dependent Potassium Channel Activity Underlies Impaired β-Adrenoceptor–Mediated Relaxation of Renal Arteries in Hypertension. Hypertension 59(4): 877–884. https://doi.org/10.1161/hypertensionaha.111.187427
Lindman, J., Khammy, M. M., Lundegaard, P. R., Aalkjær, C. and Jepps, T. A. (2018). Microtubule Regulation of Kv7 Channels Orchestrates cAMP-Mediated Vasorelaxations in Rat Arterial Smooth Muscle. Hypertension 71(2): 336–345. https://doi.org/10.1161/hypertensionaha.117.10152
van der Horst, J., Rognant, S., Hellsten, Y., Aalkjær, C. and Jepps, T. A. (2022). Dynein Coordinates β2-Adrenoceptor-Mediated Relaxation in Normotensive and Hypertensive Rat Mesenteric Arteries. Hypertension 79(10): 2214–2227. https://doi.org/10.1161/hypertensionaha.122.19351
Zhong, X. Z., Harhun, M. I., Olesen, S. P., Ohya, S., Moffatt, J. D., Cole, W. C. and Greenwood, I. A. (2010). Participation of KCNQ(Kv7) potassium channels in myogenic control of cerebral arterial diameter. J. Physiol. 588(17): 3277–3293. https://doi.org/10.1113/jphysiol.2010.192823
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4,962 | https://bio-protocol.org/en/bpdetail?id=4962&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs
LE Lauren E.-A. Eyssen
SR Siva Ramadurai
SA Sahar Abdelkarim
IB Imogen Buckle
KC Katy Cornish
HL Hong Lin
AJ A.K. Jones
GS Gary J. Stephens
RO Raymond J. Owens
Published: Vol 14, Iss 6, Mar 20, 2024
DOI: 10.21769/BioProtoc.4962 Views: 3195
Reviewed by: Luis Alberto Sánchez VargasJaveena HussainDogan Can KirmanThirupugal Govindarajan
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Abstract
Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain–only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification.
Key features
• Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment.
• Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.
Graphical overview
Keywords: Camelid heavy chain–only antibody Nanobody Phage display Nanobody labelling
Background
The fact that camelids produce a unique heavy-chain antibody was discovered serendipitously some thirty years ago by researchers at the Vrije Universiteit Brussel [1]. Pioneering work from the Belgian group showed that the variable heavy chain domain of these antibodies, termed VHH, could be produced as a single domain binding protein, referred to as a nanobody. Subsequently, nanobodies have been generated to a wide variety of antigens for applications in cell and structural biology, including as crystallization chaperones for high-value membrane and unstructured proteins and as probes for super-resolution microscopy [2]. Typically, nanobodies are generated by screening phage display libraries of VHH domains cloned from the peripheral blood cells of immunised llamas and alpacas with the target immunogen [3]. Since 2019, our group have generated nanobodies to 75 different antigens. The antigens varied from complexes, membranes, and soluble proteins. Some of the nanobodies that we have identified have been applied as structural chaperones [4], diagnostics [5], and anti-viral therapeutics for SARS-CoV-2 [6] and for in vitro and in vivo cell biology [7]. Although several protocols for producing nanobodies have been published previously [8], we have identified areas for streamlining the process, e.g., incorporating ligation-independent cloning to facilitate the construction of VHH domain libraries and small-scale expression screening to identify high-producing clones.
Furthermore, by using generic VHH cloning primers, we have designed a suite of expression vectors that enable the functionalisation of any cloned VHH with a variety of carboxy terminal tags, e.g., Flag, Avi-tag®, or SNAP-tag® for subsequent site-specific labelling. Overall, our aim is to make this technology readily accessible to any research group with an appropriately equipped laboratory.
Materials and reagents
Biological materials
Post-immune heparinised llama blood
Pre- and post-immune llama sera
Electrocompetent TG1 (Agilent, catalog number: 200123)
CM13K trypsin-sensitive helper phages (Antibody Design Laboratories, catalog number: PH050L)
StellarTM competent cells (Takara Bio, catalog number: 636766)
Escherichia coli (Migula) Castellani and Chalmers, strain WK6 (ATCC, catalog number: 47078)
BL21(DE3)-R3-pRARE2-BirA E. coli cells for in vivo biotinylation, SGC (Structural Genomics Consortium)
HeLa cells (ATCC, catalog number: CCL-2)
Reagents
Gerbu adjuvant F (Biotechnik GmbH, catalog number: 3030). Store at 4 °C
NeutrAvidinTM biotin-binding protein (Invitrogen, catalog number: A2666). Store at -20 °C
10× PBS (Fisher BioReagents, catalog number: BP399-20), for general use. Store at room temperature (RT)
Skim milk powder (Oxoid, catalog number: LP0031). Store at RT
Tween-20 (Sigma-Aldrich, catalog number: P1379). Store at RT
BSA (Sigma-Aldrich, catalog number: A2153). Store at 4 °C
Goat anti-llama IgG (H+L) HRP (Invitrogen, catalog number: A16060). Store at -20 °C in 5 µL aliquots
KPL ABTS peroxidase solution A (SeraCare, catalog number: 5120-0034). Store at 4 °C
KPL peroxidase substrate solution B (SeraCare, catalog number: 5120-0037). Store at 4 °C
ChemgeneTM (Chemgene&Trade, catalog number: XTM308). Store at RT
Caution: This chemical is corrosive.
Ethanol (EtOH) (Fisher BioReagents, catalog number: E/0650DF/17). Store at RT in a flammable liquid storage cupboard
Caution: This chemical is flammable.
Virkon® (Rely+onTM, catalog number: 12358667). Store at RT
Caution: This chemical is corrosive.
PBS, pH 7.4 for polymorphonuclear cell (PMNC) isolation (Gibco, catalog number: 10010015). Store at 4 °C
Histopaque®-1077 (Sigma-Aldrich, catalog number: 10771). Store at 4 °C
RNaseZapTM RNase decontamination solution (Invitrogen, catalog number: AM9782). Store at RT
Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154-20ML). Store at RT
Caution: This chemical is potentially carcinogenic.
TRIzolTM reagent (Invitrogen, catalog number:15596026). Store at 4 °C
Caution: This chemical is corrosive and toxic.
Chloroform (Fluorchem, catalog number: D007F). Store at RT in a flammable liquid storage cupboard
Caution: This chemical is flammable.
2-propanol (Sigma-Aldrich, catalog number: I9516). Store at RT in a flammable liquid storage cupboard
Caution: This chemical is flammable.
Nuclease-free water (not DEPC treated) (Invitrogen, catalog number: AM9932). Store at RT
SuperScriptTM IV one-step RT-PCR system (Invitrogen, catalog number: 12594025). Store at -20 °C
CALL_001 primer 5′ GTCCTGGCTGCTCTTCTACAAGG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
CALL_002 primer 5′ GGTACGTGCTGTTGAACTGTTCC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
6× DNA gel loading buffer (New England Biolabs, catalog number: B7024S). Store at 4 °C
10× TBE buffer (Thermo Scientific, catalog number: B52). Store at RT
Agarose (Fisher BioReagents, catalog number: BP1356-500). Store at RT
1,000× SYBRTM safe DNA gel stain (Invitrogen, catalog number: S33102). Store at RT
GeneRuler 1 kb DNA ladder (Thermo Scientific, catalog number: SM0311). Store at 4 °C
Nucleospin Gel and PCR clean-up kit (Macherey-Nagel, catalog number: 740609.50). Store at RT
Purelink PCR purification kit (Invitrogen, catalog number: K310002). Store at RT
HyperLadderTM 1 kb DNA ladder (Meridian Bioscience®, Bioline, catalog number: BIO-33053). Store at 4 °C
Phusion flash high fidelity PCR master mix (Invitrogen, catalog number: F548L). Store at -20 °C
VHHFor2 primer 5′ TACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCT GGRGGA 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
VHHRev primer 5′ GTGATGGTGTTGGCCTCCTGAGGAGACGGTGACCTGG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
pADL23c vector (Antibody Design Laboratories, catalog number: PD0111). Store at -20 °C
SfiI (New England Biolabs, catalog number: R0123S). Store at -20 °C
10× rCutSmartTM buffer (New England Biolabs, catalog number: B6004S). Store at 4 °C
ClonExpress II one step cloning kit, which contains 5× CEII buffer and Exnase II (Vazymbiotech, catalog number: C112-02). Store at -20 °C
Recovery medium (Sigma-Aldrich, catalog number: S1797). Store at 4 °C
LB medium mix (Formedium, catalog number: LBL0103). Store at RT
Bacto tryptone (Melford, catalog number: T60065-2000.0). Store at RT
Yeast extract (Melford, catalog number: Y20020-1000.0). Store at RT
Bacto agar (Formedium, catalog number: AGR10). Store at RT
Ampicillin (Formedium, catalog number: AMP100). Store at 4 °C
Caution: Ampicillin is a sensitiser.
25 mM dNTP (Thermo Scientific, catalog number: R1122). Store at -20 °C in 30 µL aliquots
Taq polymerase with standard Taq reaction buffer (New England Biolabs, catalog number: M0273X). Store at -20 °C
PhD_seq_Fwd primer 5′ GCTTCCGGCTCGTATGTTG 3′. Store at -20 °C
PhD_seq_Rev primer 5′ GTCGTCTTTCCAGACGTTAG 3′. Store at -20 °C
Glycerol (Sigma-Aldrich, catalog number: G5516-1L). Store at RT
PEG 6000 (Sigma-Aldrich, catalog number: 81260). Store at RT
NaCl (Sigma-Aldrich, catalog number: S9888). Store at RT
Kanamycin (Sigma-Aldrich, catalog number: K1377). Store at 4 °C
Caution: Kanamycin is a sensitiser and may damage fertility.
StartingBlockTM (PBS) blocking buffer (Invitrogen, catalog number: 37538). Store at 4 °C
DynabeadsTM M-280 streptavidin (Invitrogen, catalog number: 11205D). Store at 4 °C
Tris base (Melford, catalog number: T60040-100.0). Store at RT
CaCl2·2H2O (Sigma-Aldrich, catalog number: C3306). Store at RT
Trypsin (Sigma-Aldrich, catalog number: T1426). Store at -20 °C
Anti-M13 HRP (Sino Biological, catalog number: 11973-MM05T-H). Store at -20 °C in 5 µL aliquots
Highprep PCR magnetic beads (Magbio, catalog number: AC60050). Store at 4 °C
Glucose (Sigma-Aldrich, catalog number: G8270). Store at RT
MgCl2·6H2O (Sigma-Aldrich, catalog number: M2670). Store at RT
IPTG (NeoBiotech, catalog number: NB-45-00030). Store at -20 °C
Polymyxin B sulfate (Gibco, catalog number: 21850029). Store at RT
Caution: Polymyxin B sulfate is toxic.
2× Laemmli buffer (Sigma-Aldrich, catalog number: S3401). Store at 4 °C
20× NuPAGETM MES SDS running buffer (Invitrogen, catalog number: NP000202). Store at RT
Mark12TM unstained standard (Invitrogen, catalog number: LC5677). Store at 4 °C
InstantBlue® Coomassie protein stain (Abcam, catalog number: ab119211). Store at 4 °C
Ni-NTA spin columns (QIAGEN, catalog number: 31014). Store at 4 °C
Imidazole (Sigma-Aldrich, catalog number: I202-500g). Store at RT
Caution: Imidazole is toxic, corrosive, and an irritant and may damage fertility.
ZebaTM spin desalting columns, 7K MWCO, 0.5 mL (Thermo Scientific, catalog number: 89882). Store at 4 °C
Rabbit anti-camelid VHH HRP (GenScript, catalog number: A01861-200). Store at -20 °C in 5 µL aliquots
pOPINVHH_his vector (Addgene, catalog number: 210405). Store at -20 °C
pOPINVHH_cys_his vector (Addgene, catalog number: 210403). Store at -20 °C
pOPINVHH_BAP_his vector (Addgene, catalog number: 210402). Store at -20 °C
pOPINVHH_sort_his vector (Addgene, catalog number: 210406). Store at -20 °C
pOPINVHH_flag_his vector (Addgene, catalog number: 210404). Store at -20 °C
pOPINVHH_myc_his vector (Addgene, catalog number: 210526). Store at -20 °C
pOPINVHH_snap_his vector (Addgene, catalog number: 210407). Store at -20 °C
pOPINE-3C-eGFP vector (Addgene, catalog number: 41125). Store at -20 °C
pOPINE-3C-eYFP vector (Addgene, catalog number: 214028). Store at -20 °C
pOPINE-3C-mCherry vector (Addgene, catalog number: 214060). Store at -20 °C
Common Fwd primer 5′ GCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
His FLAG Rev primer 5′ GTGATGGTGGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
Cys Rev primer 5′ ATGGTGACAGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
BAP Rev primer 5′ ATCATTCAAGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C.
Sort Rev primer 5′ CGGCAGGCCGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
Myc Rev primer 5′ GTGATGGTGGTGGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
SNAP Rev primer 5′ GTCCTTGTCGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
pOPINE common Fwd primer 5′ AGGAGATATACCATGCAGGTGCAGCTGGTGGAG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
pOPINE common Rev primer 5′ CAGAACTTCCAGTTTAGGGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
Bsu36I (New England Biolabs, catalog number: R0524S). Store at -20 °C
NcoI (New England Biolabs, catalog number: R01093S). Store at -20 °C
X-gal ready to use (Thermo Scientific, catalog number: R0941). Store at 4 °C
QIAprep spin miniprep kit (QIAGEN, catalog number: 27104). Store at RT
DNA sequence of anti-GFP nanobody [9] after codon optimisation for expression in E. coli. CAG GTC CAA TTA GTG GAG TCC GGT GGG GCA CTT GTC CAG CCT GGA GGT TCA CTT CGC TTG TCT TGC GCA GCG TCT GGA TTC CCG GTG AAC CGC TAT AGT ATG CGT TGG TAC CGT CAA GCT CCG GGG AAA GAA CGT GAA TGG GTA GCA GGG ATG TCT TCC GCC GGT GAC CGC TCT TCA TAC GAG GAC TCG GTC AAG GGG CGC TTC ACA ATC TCT CGT GAT GAT GCC CGT AAC ACC GTT TAC TTG CAA ATG AAC AGC CTG AAA CCG GAA GAC ACT GCG GTG TAT TAC TGC AAT GTT AAT GTA GGG TTT GAA TAC TGG GGT CAA GGT ACA CAA GTT ACA GTT TCG TCA. Ordered gBlock from IDT. Store at RT prior to reconstitution in TE buffer, incubate at 50 °C for 20 min, then store at -20 °C
Chloramphenicol (Sigma-Aldrich, catalog number: C1919). Store at 4 °C
Caution: Chloramphenicol is toxic, can cause eye damage, and is a carcinogen.
Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014). Store at 4 °C
Caution: Spectinomycin is an irritant.
Carbenicillin disodium salt (Sigma-Aldrich, catalog number: C3416). Store at 4 °C
Caution: Carbenicillin is a sensitiser.
Biotin (Sigma-Aldrich, catalog number: B4639). Store at 4 °C
Streptavidin, Alexa FluorTM 488 conjugate (Invitrogen, catalog number: S11223). Store at -20 °C
BenchMarkTM fluorescent protein standard (Invitrogen, catalog number: LC5928). Store at -20 °C
DNA sequence of anti-vimentin (VB3) nanobody [10] after codon optimisation for expression in E. coli: CAG GTC CAA CTT GTA GAG TCA GGA GGT GGA AGC GTG CAA GCT GGG GAC TCT CTG CGC CTG TCT TGT GCT TCG AGC GGA AAT ACC TTC TCG ATC AAA GTC ATG GGA TGG TAC CGC CAG GCA CCT GGA AAG CAA CGT GAA TTA GTC GCG GTT TCA ACC AAT AGC GGG GCC TCT GTT AAT TAT GCC AAC TCT GTG AAG GGA CGC TTT ACC ATT TCT ATT GAT TCA GTA AAA AAA ACA ACC TAC TTA CAG ATG AAT TCC TTG AAG CCA GAA GAT ACA GCC GTC TAC TTT TGC AAT GCA TAT GAT GGG CGT TAT GAG GAC TAT TAC GGT CAG GGG ACC CAA GTG ACA GTA TCA TCA. Ordered gBlock from IDT. Store at RT prior to reconstitution in TE buffer, incubate at 50 °C for 20 min, then store at -20 °C
TCEP, hydrochloride (Merck, Millipore®, catalog number: 580567-5GM). Store at RT
Alexa FluorTM 647 C2-maleimide (Invitrogen, catalog number: A20347). Store at -20 °C
ZebaTM dye and biotin removal columns (Thermo Scientific, catalog number: A44296). Store at 4 °C
PageRulerTM prestained protein ladder (Thermo Scientific, catalog number: 26616). Store at 4 °C
DMEM, high glucose, HEPES, no phenol red (Gibco, catalog number: 21063-029). Store at 4 °C
Fetal bovine serum (FBS) (Biowest, catalog number: S00NB1001Y). Store at -20 °C
100× GlutaMAXTM (Gibco, catalog number: 35050-038). Store at -20 °C
100× penicillin-streptomycin (Gibco, catalog number: 15140-122). Store at -20 °C
4% paraformaldehyde in PBS (Thermo Scientific chemicals, catalog number: J61899.AK). Store at 4 °C
Caution: Paraformaldehyde is an eye irritant, skin sensitiser, and carcinogen.
TritonTM X-100 (Sigma-Aldrich, catalog number: T8787-250mL). Store at RT
Fluoroshield with DAPI (Abcam, catalog number: ab104139). Store at RT
Nail varnish. Store at RT
Type F immersion liquid (Leica, catalog number: 11513859). Store at RT
Fugene transfection reagent (Promega, catalog number: E2311). Store at 4 °C
Solutions
0.5 mg/mL neutrAvidin biotin-binding protein (see Recipes)
10 µg/mL neutrAvidin for coating ELISA plate (see Recipes)
1× PBS (see Recipes)
Blocking solution (see Recipes)
Washing buffer (PBST) (see Recipes)
0.1% (w/v) BSA-PBS (see Recipes)
5% (v/v) Chemgene (see Recipes)
70% (v/v) ethanol (EtOH) (see Recipes)
2% (w/v) Virkon (see Recipes)
75% (v/v) EtOH (see Recipes)
1× TBE buffer (see Recipes)
0.7% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain (see Recipes)
1% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain (see Recipes)
2× YT medium (see Recipes)
LB medium (see Recipes)
100 mg/mL ampicillin (see Recipes)
1% (w/v) agar LB plates containing 100 µg/mL ampicillin (see Recipes)
2% (w/v) agar LB plates containing 100 µg/mL ampicillin (see Recipes)
50% glycerol (see Recipes)
2× YT medium containing 25% glycerol (see Recipes)
1% (w/v) agar LB plates without antibiotic (see Recipes)
2× YT containing 100 µg/mL ampicillin (see Recipes)
50 mg/mL kanamycin (see Recipes)
2× YT containing 25 µg/mL kanamycin (see Recipes)
PEG/NaCl precipitation solution (see Recipes)
2× YT containing 100 µg/mL ampicillin and 25 µg/mL kanamycin (see Recipes)
TBSC (see Recipes)
1 mg/mL trypsin (see Recipes)
250 µg/mL trypsin (see Recipes)
Terrific broth (see Recipes)
20% (w/v) glucose (see Recipes)
1 M MgCl2·6H2O (see Recipes)
Terrific broth containing 100 µg/mL ampicillin, 0.1% glucose, 2 mM MgCl2·6H2O (see Recipes)
1 M IPTG (see Recipes)
1 mg/mL polymyxin B sulfate in PBS (see Recipes)
1× NuPAGETM MES SDS running buffer (see Recipes)
Equilibration buffer (see Recipes)
Elution buffer (see Recipes)
Octet dilution buffer (see Recipes)
1% (w/v) agar LB plate containing 100 µg/mL ampicillin, 2 mM IPTG, 40 µg/mL X-gal (see Recipes)
34 µg mL chloramphenicol (see Recipes)
50 µg/mL spectinomycin (see Recipes)
50 µg/mL carbenicillin (see Recipes)
1% (w/v) agar LB plate containing 34 µg/mL chloramphenicol, 50 µg/mL spectinomycin, 50 µg/mL carbenicillin (see Recipes)
0.2 M Biotin (see Recipes)
Terrific broth containing 50 µg/mL spectinomycin and 50 µg/mL carbenicillin (see Recipes)
1 mg/mL Streptavidin Alexa FluorTM 488 conjugate (see Recipes)
50 µg/mL Streptavidin Alexa FluorTM 488 conjugate (see Recipes)
500 mM TCEP, pH 8.0 (see Recipes)
1 mM TCEP, pH 8.0 (see Recipes)
10 mg/mL Alexa FluorTM 647 C2-maleimide (see Recipes)
DMEM medium without phenol red, 10% (v/v) FBS, 1× GlutaMAXTM, 1× Penicillin-streptomycin (see Recipes)
Confocal blocking buffer (see Recipes)
Confocal dilution buffer (see Recipes)
Recipes
0.5 mg/mL neutrAvidin biotin-binding protein
Prepare 200 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
neutrAvidinTM 0.5 mg 5 mg
ddH2O n/a 10 mL
Total n/a 10 mL
10 µg/mL neutrAvidin for coating ELISA plate
Prepare just before use.
Reagent Final concentration Quantity or Volume
neutrAvidinTM (Recipe 1) 10 µg/mL 200 µL
PBS (1×) 1× 9.8 mL
Total n/a 10 mL
1× PBS
Prepare both sterile (by autoclaving) and non-sterile PBS. Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
PBS (10×) 1× 100 mL
ddH2O n/a 900 mL
Total n/a 1,000 mL
Blocking solution
Prepare on day of use.
Reagent Final concentration Quantity or Volume
Milk 2% (w/v) 2 g
PBS (Recipe 3) (or buffer that is compatible with the protein) n/a 100 mL
Total n/a 100 mL
Washing buffer (PBST)
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
PBS (Recipe 3) 1× 1 000 mL
Tween-20 0.05% (v/v) 500 µL
Total n/a 1,000 mL
0.1% (w/v) BSA-PBS
Prepare on day of use.
Reagent Final concentration Quantity or Volume
BSA 0.1 % (w/v) 50 mg
PBS (Recipe 3) 1× 50 mL
Total n/a 50 mL
5% (v/v) Chemgene
Store at RT.
Reagent Final concentration Quantity or Volume
Chemgene (100%) 5% (v/v) 50 mL
H2O n/a 950 mL
Total n/a 1,000 mL
70% (v/v) EtOH
Store at RT.
Reagent Final concentration Quantity or Volume
Ethanol (absolute) 70% (v/v) 70 mL
H2O n/a 30 mL
Total n/a 100 mL
2% (w/v) Virkon
Prepare weekly and store at RT.
Reagent Final concentration Quantity or Volume
Virkon powder 2% (w/v) 100 g
H2O n/a 5,000 mL
Total n/a 5,000 mL
75% (v/v) EtOH
Store at RT.
Reagent Final concentration Quantity or Volume
Ethanol (absolute) 75% (v/v) 75 mL
H2O n/a 25 mL
Total n/a 100 mL
1× TBE buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
TBE (10×) 1× 100 mL
H2O n/a 900 mL
Total n/a 1,000 mL
0.7% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain
Heat in a microwave until all the agarose has dissolved and allow to cool to 55 °C before the addition of the gel stain. Pour into the DNA casting apparatus and allow to solidify. Prepare on day of use.
Reagent Final concentration Quantity or Volume
Agarose 0.7 % (w/v) 0.7 g
TBE (Recipe 11) 1× 100 mL
Total n/a 100 mL
SYBRTM Safe DNA gel stain (1,000×) 1× 10 µL
1% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain
Heat in a microwave until all the agarose has dissolved and allow to cool to 55 °C before the addition of the gel stain. Pour into the DNA casting apparatus and allow to solidify. Prepare on day of use.
Reagent Final concentration Quantity or Volume
Agarose 1% (w/v) 1 g
TBE (Recipe 11) 1× 100 mL
Total n/a 100 mL
SYBRTM Safe DNA gel stain (1,000×) 1× 10 µL
2× YT medium
Autoclave and store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
Bacto tryptone 16 g/L 4 g
NaCl 5 g/L 1.25 g
Yeast extract 10 g/L 2.5 g
ddH2O n/a 250 mL
Total n/a 250 mL
LB medium
Autoclave and store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
LB medium mix 25 g/L 6.25 g
ddH2O n/a 250 mL
Total n/a 250 mL
100 mg/mL ampicillin
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Caution: Ampicillin is a sensitiser.
Reagent Final concentration Quantity or Volume
Ampicillin 100 mg/mL 2 g
ddH2O n/a 20 mL
Total n/a 20 mL
1% (w/v) agar LB plates containing 100 µg/mL ampicillin
Autoclave LB medium and bacto agar mix and allow to cool to 55 °C before the addition of antibiotic. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool at RT. Prepare on day of use.
Reagent Final concentration Quantity or Volume
LB medium mix 25 g/L 6.25 g
Bacto agar 1% (w/v) 2.5 g
ddH2O n/a 250 mL
Total n/a 250 mL
Ampicillin (Recipe 16) 100 µg/mL 250 µL
2% (w/v) agar LB plates with 100 µg/mL ampicillin
Autoclave LB medium and bacto agar mix and allow to cool to 55 °C before the addition of antibiotic. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool at RT. Prepare on day of use.
Reagent Final concentration Quantity or Volume
LB medium mix 25 g/L 6.25 g
Bacto agar 2% (w/v) 5 g
ddH2O n/a 250 mL
Total n/a 250 mL
Ampicillin (Recipe 16) 100 µg/mL 250 µL
50% (v/v) glycerol
Autoclave and store at RT. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Glycerol 50% (v/v) 100 mL
ddH2O n/a 100 mL
Total n/a 200 mL
2× YT medium containing 25% (v/v) glycerol
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume
2× YT medium n/a 20 mL
Glycerol (Recipe 19) 25% (w/v) 20 mL
Total n/a 40 mL
1% (w/v) agar LB plates without antibiotic
Autoclave and allow to cool to 55 °C. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool and set at RT. Store at 4 °C for two weeks.
Reagent Final concentration Quantity or Volume
LB medium mix 25 g/L 6.25 g
Bacto agar 1% (w/v) 2.5 g
ddH2O n/a 250 mL
Total n/a 250 mL
2× YT containing 100 µg/mL ampicillin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume
2× YT n/a 100 mL
Ampicillin (Recipe 16) 100 µg/mL 100 µL
Total n/a 100 mL
50 mg/mL kanamycin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Kanamycin 50 mg/mL 0.5 g
ddH2O n/a 10 mL
Total n/a 10 mL
2× YT containing 25 µg/mL kanamycin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume
2× YT n/a 100 mL
Kanamycin (Recipe 23) 25 µg/mL 50 µL
Total n/a 100 mL
PEG/NaCl precipitation solution
Autoclave and store at RT. Stable for at least one year.
Reagent Final concentration Quantity or Volume
PEG 6000 20% (w/v) 200 g
NaCl 2.5 M 146.1 g
ddH2O n/a Make up to 1,000 mL
Total n/a 1,000 mL
2× YT containing 100 µg/mL ampicillin and 25 µg/mL kanamycin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume
2× YT n/a 100 mL
Ampicillin (Recipe 16) 100 µg/mL 100 µL
Kanamycin (Recipe 23) 25 µg/mL 50 µL
Total n/a 100 mL
TBSC
Autoclave and store at RT. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Tris base 10 mM 0.788 g
NaCl 137 mM 4 g
CaCl2·2H2O 1 mM 73.5 mg
ddH2O n/a 500 mL
Total n/a 500 mL
1 mg/mL trypsin
Prepare 130 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Trypsin 1 mg/mL 50 mg
ddH2O n/a 50 mL
Total n/a 50 mL
250 µg/mL trypsin
Prepare just before use.
Reagent Final concentration Quantity or Volume
Trypsin (Recipe 28) 250 µg/mL 125 µL
TBSC (Recipe 27) n/a 375 µL
Total n/a 500 µL
Terrific broth
Autoclave and store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
Bacto tryptone 12 g/L 3 g
Yeast extract 24 g/L 6 g
Glycerol 0.4% (v/v) 1 mL
KH2PO4 0.17 M 0.5775 g
K2HPO4 0.72 M 3.135 g
ddH2O n/a 250 mL
Total n/a 250 mL
20% (w/v) glucose
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Glucose 20% (w/v) 20 g
ddH2O n/a Make up to 100 mL
Total n/a 100 mL
1 M MgCl2·6H2O
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
MgCl2·6H2O 1 M 4.066 g
ddH2O n/a 20 mL
Total n/a 20 mL
Terrific broth containing 100 µg/mL ampicillin, 0.1% glucose, 2 mM MgCl2·6H2O
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume
Terrific broth (Recipe 30) n/a 99.2 mL
Ampicillin (Recipe 16) 100 µg/mL 100 µL
Glucose (Recipe 31) 0.1% (v/v) 500 µL
MgCl2·6H2O (Recipe 32) 2 mM 200 µL
Total n/a 100 mL
1 M IPTG
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
IPTG 1 M 4.766 g
ddH2O n/a 20 mL
Total n/a 20 mL
1 mg/mL polymyxin-B sulfate in PBS
Prepare 1 mL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Polymyxin-B sulfate 1 mg/mL 2 mg
PBS (Recipe 3) 1× 2 mL
Total n/a 2 mL
1× NuPAGETM MES SDS running buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
MES (20×) 1× 100 mL
H2O n/a 1,900 mL
Total n/a 2,000 mL
Equilibration buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
Imidazole 30 mM 0.51 g
PBS (Recipe 3) 1× 250 mL
Adjust pH to 7.4
Total n/a 250 mL
Elution buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
Imidazole 300 mM 2.04 g
PBS (Recipe 3) 1× 100 mL
Adjust pH to 7.4
Total n/a 100 mL
Octet dilution buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume
BSA 0.1% (w/v) 50 mg
PBS (Recipe 3) (or buffer that is compatible with the protein) 1× 50 mL
Total n/a 50 mL
1% (w/v) agar LB plate containing 100 µg/mL ampicillin, 2 mM IPTG, 40 µg/mL X-gal
Autoclave and allow to cool to 55 °C before the addition of additives. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool and set at RT. Store at 4 °C for two weeks.
Reagent Final concentration Quantity or Volume
LB medium mix 25 g/L 6.25 g
Bacto agar 1% (w/v) 2.5 g
ddH2O n/a Make up to 250 mL
Total n/a 250 mL
Ampicillin (Recipe 16) 100 µg/mL 250 µL
IPTG (Recipe 34) 2 mM 500 µL
X-gal (20 mg/mL) 40 µg/mL 500 µL
34 mg/mL chloramphenicol
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Chloramphenicol 34 mg/mL 34 mg
EtOH 100% 1 mL
Total n/a 1 mL
50 mg/mL Spectinomycin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Spectinomycin dihydrochloride pentahydrate 50 mg/mL 50 mg
ddH2O n/a 1 mL
Total n/a 1 mL
50 mg/mL Carbenicillin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Carbenicillin disodium salt 50 mg/mL 50 mg
ddH2O n/a 1 mL
Total n/a 1 mL
1% (w/v) agar LB plate containing 34 µg/mL chloramphenicol, 50 µg/mL spectinomycin, 50 µg/mL carbenicillin
Autoclave LB medium and bacto agar mix and allow to cool to 55 °C before the addition of additives. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool and set at RT. Store at 4 °C for two weeks.
Reagent Final concentration Quantity or Volume
LB medium mix 25 g/L 2.5 g
Bacto agar 1% (w/v) 1 g
ddH2O n/a Make up to 100 mL
Total n/a 100 mL
Chloramphenicol (Recipe 41) 34 µg/mL 100 µL
Spectinomycin (Recipe 42) 50 µg/mL 100 µL
Carbenicillin (Recipe 43) 50 µg/mL 100 µL
0.2 M biotin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Biotin 0.2 M 0.488 g
NaOH (1 M) n/a Add to dissolve biotin
ddH2O n/a Make up to 10 mL
Total n/a 10 mL
Terrific broth containing 50 µg/mL spectinomycin and 50 µg/mL carbenicillin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume
Terrific broth (Recipe 30) n/a 998 mL
Spectinomycin (Recipe 42) 50 µg/mL 1 mL
Carbenicillin (Recipe 43) 50 µg/mL 1 mL
Total n/a 1 000 mL
1 mg/mL streptavidin Alexa FluorTM 488 conjugate
Protect from light, prepare 10 µL aliquots, and store at -20 °C. Stable for six months.
Reagent Final concentration Quantity or Volume
Streptavidin Alexa FluorTM 488 conjugate 1 mg/mL 1 mg
PBS (Recipe 3) 1× 1 mL
Total n/a 1 mL
50 µg/mL streptavidin Alexa FluorTM 488 conjugate
Protect from light and prepare just before use.
Reagent Final concentration Quantity or Volume
Streptavidin Alexa FluorTM 488 conjugate (1 mg/mL) 50 µg/mL 1 µL
PBS (Recipe 3) 1× 19 µL
Total n/a 20 µL
500 mM TCEP, pH 8.0
Store at 4 °C. Stable for at least one month.
Reagent Final concentration Quantity or Volume
TCEP 500 mM 1.433 g
ddH2O n/a 10 mL
Adjust to pH 8.0
Total n/a 10 mL
1 mM TCEP, pH 8.0
Prepare just before use.
Reagent Final concentration Quantity or Volume
TCEP (Recipe 49) 1 mM 2 µL
ddH2O n/a 998 µL
Total n/a 1 mL
10 mg/mL Alexa FluorTM 647 C2-Maleimide
Protect from light and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume
Alexa FluorTM 647 C2-Maleimide 10 mg/mL 1 mg
DMSO 1× 500 µL
Total n/a 500 µL
DMEM medium without phenol red, 10% (v/v) FBS, 1× GlutaMAXTM, 1× penicillin-streptomycin
Store at 4 °C. Stable for at least one month.
Reagent Final concentration Quantity or Volume
DMEM medium n/a 88 mL
FBS 10% (v/v) 10 mL
GlutaMAXTM (100×) 1× 1 mL
Penicillin-streptomycin (100×) 1× 1 mL
Total n/a 100 mL
Confocal blocking buffer
Store at 4 °C. Use within one month.
Reagent Final concentration Quantity or Volume
PBS (Recipe 3) 1× 10 mL
BSA 5% (w/v) 0.5 g
Triton X-100 0.25% (v/v) 25 µL
Total n/a 10 mL
Confocal dilution buffer
Store at 4 °C. Use within one month.
Reagent Final concentration Quantity or Volume
PBS (Recipe 3) 1× 10 mL
BSA 1% (w/v) 0.1 g
Triton X-100 0.25% (v/v) 25 µL
Total n/a 10 mL
Laboratory supplies
Vacuette® tube 4 mL CAT serum clot activator blood collection tubes (Greiner, Bio-one, catalog number: 454204)
Vacuette® tube 9 mL NH sodium heparin blood collection tubes (Greiner, Bio-one, catalog number: 455051)
Microcentrifuge tubes, 0.5 mL, 1.5 mL, 2 mL (Greiner, Bio-one, catalog numbers: 667201, 616201, 623201)
1.5 mL microcentrifuge tubes, RNase free (SARSTEDT, catalog number: 72.706.700)
PCR tubes and caps (VWR, catalog number: 20170-010)
50 mL conical centrifuge tubes (Greiner Bio-One, catalog number: 227261)
Serological pipettes, 2 mL, 5 mL, 10 mL, 50 mL (Greiner Bio-One, catalog numbers: 710180, 606180, 607180, 760180, 768160)
Screw cap microtubes (SARSTEDT, catalog number: 72.694.305)
MicrolanceTM 3 needles, 21G (BD, catalog number: 304432)
2.5 mL syringe (Greiner Bio-One, catalog number: SYR2)
8.5 cm Petri dishes (Greiner, Bio-one, catalog number: 633181)
Pipette tips, filtered, 20 µL, 200 µL, 1,000 µL (Mettler Toledo, Ranin, catalog numbers: 17005861, 17005863, 30389211)
Gene Pulser electroporation cuvettes, 0.1 cm gap (Bio-Rad, catalog number: 1652083)
Polystyrene semi micro cuvettes (Fisherbrand, catalog number: FB55147)
125 mL baffled flasks (VWR, catalog number: 214-0458)
250 mL baffled flasks (VWR, catalog number: 214-0460)
1 L baffled flasks (VWR, catalog number: 214-0464)
Micro quartz absorption cuvettes (Merck, Hellma®, catalog number: Z600210)
Square bioassay dish (Nunc, catalog number: 240845)
Reagent reservoirs, non-sterile (VWR, catalog number: 613-1176)
Reagent reservoirs, sterile (Axygen, catalog number: RES-V-25-S)
Deep-well 96-well 2 mL plate (Fisherbrand, catalog number: 11391555)
Deep-well 96-well 1 mL plate (Greiner, Bio-one, catalog number: 780215)
Deep-well 96-well 0.5 mL plate (Greiner, Bio-one, catalog number: 786261)
Cell culture adhesive seal (Azenta Life Sciences, catalog number: 4ti-0517)
Adhesive film for ELISA and general incubation (VWR, catalog number: 60941-062)
PCR foil seal (Azenta Life Sciences, catalog number: 4ti-0550)
Rigid semi-skirted 96-well PCR plate (Thermo Scientific, catalog number: AB-0990)
Framestar® 96-well skirted PCR plate (Azenta Life Sciences, catalog number: 4ti-0960)
96-well ELISA microplate (Greiner Bio-One, catalog number: 655061)
Octet SA biosensors (Sartorius, catalog number: 18-5019)
96-well black plate, polypropylene (Greiner Bio-One, catalog number: 655209)
96-well microtitre plate (Greiner Bio-One, catalog number: 65101)
250 mL PPCO centrifuge tube (Nalgene, catalog number: 3141-0250)
25 mL high speed PPCO centrifuge tube (Nalgene, catalog number: 3119-0010)
NuPage 4%–12% bis tris precast gel (Invitrogen, catalog number: NP0323BOX). A suitable alternative is homemade 12% running and 4% stacking SDS-PAGE gel
0.22 μm syringe filters (Agilent, catalog number: 5190-5116)
µ-Slide 8-well high polymer bottom chambered coverslip (ibidi, catalog number: 80807)
Microscope slides with ground edges (Fisherbrand, catalog number: 11572203)
Microscope coverslip, 12 mm diameter, no. 1.5 (Scientific Laboratory supplies, catalog number: MIC3334)
6-well cell culture dish (Greiner Bio-One, catalog number: 657160)
Dumont tweezer style 5 (Electron Microscopy Sciences, catalog number: 0203-5-PS)
Equipment
Pipetboy acu 2 pipette controller (Ingetra, catalog number: I155017)
Single-channel pipettes starter kit (Mettler Toledo, Ranin, catalog number: 30386738)
Multichannel pipettes 1–10 µL, 20–200 µL, 100–1,200 µL (Mettler Toledo, Ranin, catalog numbers: 17013802, 17013805, 17014496)
Microplate washer (Thermo Fisher, WellwashTM, catalog number: 5165000). If there is no well washer available, the wells can be washed manually using a multichannel pipette or using a wash bottle filled with PBST
CLARIOstar plus plate reader (BMG Labtech, catalog number: 430-501S-FL)
Class 2 microbiological safety cabinet (referred to as a safety cabinet and known as a laminar flow) (Contained Air solutions, catalog number: BioMAT 2). If no safety cabinet is available, working by a Bunsen burner is a suitable alternative. Working by an open flame with flammable EtOH should be done with caution
PCR workstation cabinet (Bigneat, catalog number: MW520-20)
NanoDropTM One/OneC Microvolume UV-Vis spectrophotometer (Thermo Scientific, catalog number: ND-ONE-W)
DynaMagTM 96-side magnet (Invitrogen, catalog number: 12331D)
PCR thermal cycler (Applied Biosystems, VeritiTM, catalog number: 4375305)
Eporator (Eppendorf, catalog number: 4309000027)
ChemiDocTM imaging system (Bio-Rad, catalog number: 12003154)
Cell density meter (Fisherbrand, catalog number: A0)
Multi Sub Electrophoresis System (Flowgen, catalog number: FMMS10)
SDS-PAGE equipment, mini gel tank (Invitogen, catalog number: A25977)
PowerPac basic power supply (Bio-Rad, catalog number: 1645050EDU)
JB Nova water bath (Grant, catalog number: JBN5)
Dry heating block (Grant, catalog number: QBD2)
Dual LED Blue/White Light Transilluminator (Thermo Fisher, catalog number: LB0100)
Safe ImagerTM viewing glasses (Thermo Fisher, catalog number: S37103)
HulaMixerTM sample mixer (Invitrogen, catalog number: 15920D). Agitation using this sample mixer is performed using the following settings: orbital = 5, rpm = 1
DynaMagTM-2 magnet (Invitrogen, catalog number: 12321D)
LSETM digital microplate shaker (Corning, catalog number: 4782-4). Agitation using the microplate shaker is performed at 500 rpm
Multifuge X4 Pro-MD (Thermo Scientific, catalog number: 75009500). In all centrifugation steps, maximum acceleration and deceleration rates are used unless otherwise specified
FrescoTM 21 microcentrifuge (Thermo Scientific, catalog number: 75002555)
Orbital shaking incubator (Shel Lab, catalog number: SI6/SI6R) (for 600 rpm agitation steps)
44R incubator shaker (New Brunswick, Innova®, catalog number: M1282-0006) (for 200 and 250 rpm agitation steps)
Octet® R8 (Sartorius, catalog number: 30-0518)
SP8 Lightning confocal microscope (Leica Microsystems Ltd.)
HeracellTM VIOS 160i CO2 Incubator (Thermo Scientific, catalog number: 51033559)
StuartTM SSM3 Gyratory Rocker (Cole-Parmer, catalog number: 51900-26)
Software and datasets
SnapGene (version 7.1.0, 2023, released 28 November 2023); requires a license
Octet® Analysis Studio Software (version 12.2.2.26); requires a license
LasX software for confocal microscope (version 5.2.1); requires a license
Procedure
Llama immunisation
Antibodies are raised in a llama by intramuscular immunisation with up to eight different proteins in parallel. The identity of the proteins depends on the intended application. We have raised nanobodies to integral membrane proteins (e.g., PEPT2 [4]), cell surface glycoproteins (e.g., GPC3 [7]) and viral antigens (e.g., SARS-CoV-2 spike protein [6]).
Store protein antigens at -80 °C in 3 × 200 µg aliquots, preferably 200 µL of 1 mg/mL per aliquot, in PBS or a buffer that is optimal for the protein (e.g., if in a detergent-solubilised membrane protein, then the required amount of appropriate detergent would be included in the buffer).
Before immunisation, collect 5 mL of blood in blood tubes without anticoagulant to prepare a sample of pre-immune serum. Preparation of the pre-immune serum sample is detailed in step C2.
Thaw an aliquot of each of the proteins that are to be included in the immunisation. Gently mix each protein with an equal volume of Gerbu adjuvant and inject subcutaneously (maximum 2 mL per site) in the neck base/shoulder of the llama.
The llama is immunised on day 0 followed by two boosts of antigen on days 28 and 56. Ten days following the final boost, 170 mL of blood from the jugular vein is collected into heparinised tubes (to prevent coagulation) for isolation of polymorphonuclear cells (PMNCs). In addition, collect 5 mL of blood in blood tubes without anticoagulant to prepare a sample of post-immune serum. Details for how to prepare the post-immune serum sample are described in step C2.
Seroconversion ELISA
A seroconversion ELISA is carried out to confirm if the immunisation of the llama was successful in generating a response to the injected antigen(s). A strong ELISA signal is strongly suggestive that binding nanobodies can be isolated from the created library by phage display.
Coat a 96-well ELISA microplate with 100 µL/well of 10 µg/mL neutrAvidinTM diluted in PBS and incubate overnight at 4 °C.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of 50 nM biotinylated target protein diluted in PBS and incubate for 1 h at RT on a microplate shaker. See General note 1.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 250 µL/well of blocking solution and incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 110 µL of either pre- or post-immunisation serum diluted 1:10 in blocking solution to the first well of the 96-well plate. Prepare a serial dilution of the sera in the plate by adding 100 µL of blocking solution to the second well and add 10 µL of the solution from the first well. Repeat for a third well so that the sera are diluted 1 in 10, 100, and 1,000; include a no-serum control. Incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of anti-llama-HRP diluted 1:2,500 in 0.1% BSA-PBS and incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of ABTS substrate, prepared by mixing solution A and solution B in a 1:1 ratio. Protect from light and measure the absorbance at 405 nm after 15 min. See General note 2.
VHH library preparation
Below (Table 1) is a suggested timetable of experiments required for the preparation of a VHH library. Instances where the protocol can be interrupted/held are indicated by “pause point.”
Table 1. Experimental timetable for the preparation of a VHH library
Task 1 Task 2 Task 3
Day 1
Isolation of PMNCs
(step C1)
Isolation of immune serum
(step C2)
RNA extraction (steps C3a–g) Pause point
Day 2
RNA extraction
(steps C3h–i)
Reverse transcription and PCR amplification of VHH1 (step C4) Production of VHH2 (step C5) Pause point
Day 3
Digestion of pADL23c (step C6)
Pause point
Day 4
Test of library size
(steps C7a–g)
Day 5
Test of library size
(steps C7h–n)
Pause point
Day 6 Scaled library preparation (steps C8a–i)
Day 7
Scaled library preparation (steps C8j)
Pause point
Isolation of PMNCs
The below instructions are based on receiving 170 mL of llama blood in 17 × 10 mL blood tubes without anticoagulant. Isolation of the PMNCs should be done on the day of the blood draw. All work is performed in a safety cabinet after spraying with 5% Chemgene followed by 70% EtOH.
Caution: Decontaminate all blood-contaminated consumables and liquid with 2% Virkon, followed by autoclaving.
Transfer blood from 2 × 10 mL blood tubes into 1 × 50 mL conical centrifuge tube using a 10 mL serological pipette.
Add 10 mL of PBS to each of the empty blood vials and transfer into the conical centrifuge tube with the 20 mL of blood (step C1a) to give a total volume of 40 mL of diluted blood per 50 mL conical centrifuge tube, or a total of 340 mL of diluted blood. Invert tubes gently to ensure a homogenous mixture.
In fresh 23 × 50 mL conical centrifuge tubes, add 15 mL of Histopaque®-1077 and then gently layer 15 mL of the diluted blood on top. See General note 3. Hold the tube at a 45° angle from the horizontal while adding the blood. See General note 4.
Critical: Avoid agitating the tubes at this point to maintain the density gradient that is forming.
Centrifuge the conical centrifuge tubes at 800× g for 20 min at 18 °C using an acceleration = 1 and deceleration = 0.
Critical: The acceleration and deceleration rates are very important, as they will create and maintain the desired gradient. The results of the density gradient and localisation of the PMNC layer is shown in Figure 1.
Figure 1. Isolation of polymorphonuclear cells (PMNCs) from llama blood using a density gradient. Diluted llama blood overlaid on Histopaque®-1007 and the resulting layer of PMNCs and red blood cells (RBCs) after centrifugation.
From the 23 prepared blood–Histopaque®-1077 tubes, collect the PMNC layer at the plasma–Histopaque®-1077 interface (red square in Figure 1). This is achieved by using a 25 mL serological pipette and placing it just above the interface, drawing the liquid up whilst moving across the surface interface. Any Histopaque®-1077 or serum that is drawn up with the PMNCs will be washed away in the next steps.
Transfer 2× conical centrifuge tubes’ worth of isolated PMNCs into a single 50 mL conical centrifuge tube. Typically, 15–20 mL of PMNCs is retrieved from each conical centrifuge tube. If more is collected, ensure that 20 mL of isolated PMNCs is added to fresh 50 mL conical centrifuge tubes. There should be between 23 and 25 × 50 mL conical centrifuge tubes at this point.
To each conical centrifuge tube containing isolated PMNCs, add PBS to a total volume of 40 mL. One can just pour from the bottle of PBS, as long as it does not touch the conical centrifuge tube. Gently invert the conical centrifuge tubes several times and centrifuge at 250× g for 10 min at 18 °C.
Pour off the supernatant and gently resuspend the pellet from each conical centrifuge tube in 5 mL PBS using a new 5 mL serological pipette per pellet. Centrifuge the cells at 100× g for 5 min at 18 °C.
Note: At this point, start using RNaseZapTM to wipe the work surface, gloves, and any items that are brought into the safety cabinet.
Use a 5 mL serological pipette to remove the supernatant from each 50 mL conical centrifuge tube. Gently resuspend the pellets from three conical centrifuge tubes in a total of 1 mL of PBS into a single 50 mL conical centrifuge tube using a 2 mL serological pipette. Use the final amount of liquid in this serological pipette and dispense it into a 1.5 mL microcentrifuge tube. There should be six 50 mL conical centrifuge tubes and six 1.5 mL microcentrifuge tubes at this point.
Remove the six 1.5 mL microcentrifuge tubes from the safety cabinet. In the lid of each of the 1.5 mL microcentrifuge tubes, add 20 µL of cells and 20 µL of trypan blue and mix gently. Determine cell number and viability using an automated cell counter. Expect approximately 1×107 cells at 98%–100% viability per sample taken from each conical centrifuge tube. Thus, an overall yield of 6×107 cells should be expected. See General note 5.
Note: The following steps can be performed at a lab bench or in a PCR cabinet after being sprayed with RNaseZapTM and using RNase-free tips and 1.5 mL microcentrifuge tubes.
Transfer the cell suspension from each 50 mL conical centrifuge tube (step C1i) into their own 1.5 mL microcentrifuge tube. There should be six 1.5 mL microcentrifuge tubes in total. Pellet the cells at 100× g for 5 min at 4 °C. Discard the supernatant. The RNA can now be isolated from the pellets (step C3a).
Critical: Perform RNA extraction (step C3) immediately.
Isolation of pre- and post-immune serum
Isolation of serum must be done on the day of the blood draw. These steps can be done during the isolation of PMNCs at a lab bench.
Incubate the coagulated blood at RT for 30 min.
Transfer the serum and the blood clot into a 50 mL conical centrifuge tube. This is achieved by using a 2 mL serological pipette to stab holes in the blood clot and using the stripette to guide the clot and serum into a 50 mL conical centrifuge tube. Centrifuge the conical centrifuge tube at 1,000× g for 10 min at 4 °C.
Transfer the serum into a 15 mL conical centrifuge tube. A second centrifugation at 1,000× g for 10 min at 4 °C may be required to obtain clear serum. Prepare 500 µL aliquots of serum in screw cap microtubes, which are then stored at -20 °C.
RNA extraction
Note: All work should be done at a lab bench or in a PCR cabinet after spraying with RNaseZapTM. Use RNase-free consumables and spray gloves with RNaseZapTM. See General note 6.
To each cell pellet in each of the six 1.5 mL microcentrifuge tubes (step C1k), add 1 mL of cold TRIzolTM reagent. Using a 21 G needle and 2.5 mL syringe, homogenise the contents of each 1.5 mL microcentrifuge tube by aspirating and dispensing ten times through the needle.
Caution: Use caution when working with needles.
Incubate the resulting lysate on ice for 5 min to permit complete dissociation of the nucleoproteins complex and then add 0.2 mL of chloroform to each 1.5 mL microcentrifuge tube.
Vortex each microcentrifuge tube vigorously for approximately 10 s. A homogenous pink milky solution should occur. Incubate on ice for 3 min and then centrifuge at 12,000× g for 15 min at 4 °C. The mixture separates into a lower red phenol-chloroform layer, an interphase layer, and a colourless upper aqueous phase. If the upper layer is still cloudy or has not formed properly, vortex and centrifuge again.
Transfer approximately 500 µL of the upper aqueous phase containing the RNA to a new 1.5 mL microcentrifuge tube using a 200 µL pipette. Some of the upper aqueous layer can be left behind to ensure that none of the precipitate from the interphase is included. Add 0.5 mL of isopropanol, gently invert each tube several times, and incubate on ice for 10 min.
Centrifuge at 12,000× g for 10 min at 4 °C to pellet the RNA. Keep the lid of the 1.5 mL microcentrifuge tube facing outwards in the rotor to aid in the visualisation of the white gel-like pellet after centrifugation. Remove the approximately 900 µL of the isopropanol containing supernatant using a 200 µL pipette to ensure that the pellet is not disturbed or accidently aspirated with the waste.
Add 1 mL of 75% ethanol to the 1.5 mL microcentrifuge tube, vortex briefly, and centrifuge at 7,500× g for 5 min at 4 °C. The RNA pellet will look a little whiter and slightly bigger than what it did at the end of the previous step. Remove the approximately 900 µL of the supernatant using a 200 µL pipette to ensure that the pellet is not disturbed or accidently aspirated.
Add 1 mL of 75% EtOH and store the purified RNA at -20 °C. See General note 7.
Pause point.
Remove one microcentrifuge tube of RNA from the -20 °C and centrifuge at 7,500× g for 5 min at 4 °C. Discard the EtOH using a 200 µL and then a 10 µL pipette to remove all EtOH without accidently aspirating the pellet. Allow to dry at RT for 10 min.
Dissolve the RNA pellet in 30 µL of nuclease-free water and measure the A260 using the Nanodrop. Expect a concentration between 500 and 800 ng/µL per 1.5 mL tube with values of approximately 2.0 for both A260/280 and A260/230.
Critical: Use RNase-free tips and pipette from the PCR cabinet to measure the RNA concentration to avoid the introduction of RNases from the communal tips and pipette used at the Nanodrop. Once the RNA has been isolated, proceed to step C4 as soon as possible to produce cDNA, which is more stable than RNA.
Reverse transcription and PCR amplification of VHH1
Note: Work at a lab bench or in a PCR cabinet after spraying with RNaseZapTM. Use RNase-free consumables and spray gloves with RNaseZapTM. See General note 6.
Prepare the following reaction in a PCR tube (1 × 50 µL) (Table 2).
Table 2. PCR reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 9.5 µL
10 µM CALL 001 primer 0.5 µM 2.5 µL
10 µM CALL 002 primer 0.5 µM 2.5 µL
2× PlatinumTM SuperFiTM RT-PCR Master Mix 1× 25 µL
1 µg Template RNA (step C3i) 200 ng 10 µL
SuperScriptTM IV RT Mix 0.5 µL
Total n/a 50 µL
Briefly vortex and centrifuge the reaction tube and place on ice.
Set up the thermal cycler as below and perform the PCR (Table 3).
Table 3. PCR cycling conditions
Step Temperature (°C) Duration Number of cycles
Reverse transcription 55 10 min 1
Reverse transcription inactivation/initial denaturation 98 2 min
Denaturation 98 10 s 30
Annealing 66 10 s
Extension 72 30 s
Final extension 72 5 min 1
Hold 12 Infinite hold -
Note: There is no need to maintain RNase-free conditions from this point onwards. Work at a lab bench.
Add 10 µL of 6× DNA gel loading buffer to the reaction tube and run alongside a lane of 1× GeneRuler 1 kb DNA ladder on a 0.7% agarose gel containing 1× SYBRTM safe DNA gel stain. Electrophorese in 1× TBE buffer at 80 V for 40 min.
Visualise the amplified 700 bp fragment (VHH-CH2) using a LED Blue transilluminator, excise from the gel using a scalpel knife, and gel extract using the Nucleospin Gel and PCR clean-up kit according to the manufacturer’s protocol. Elute in a final volume of 50 µL of NE buffer.
Caution: Use Safe ImagerTM viewing glasses when visualising the DNA during excision of the band from the agarose gel and use caution when using scalpel knives.
Using the Purelink PCR purification kit, add 4× volume of B2 buffer (200 µL) to the gel-extracted product and purify using one Purelink column from the Purelink PCR purification kit eluting in 25 µL E1 buffer. Measure the A280 and expect a concentration of approximately 40–50 ng/µL.
Mix 6× DNA gel loading buffer with 120 ng of VHH1 (step C4f) to a final concentration of 1× and run alongside a well of 1× HyperLadderTM 1 kb DNA ladder on a 1% agarose gel containing 1× SYBRTM safe DNA gel stain to determine the purity. Electrophorese in 1× TBE buffer at 80 V for 40 min. Expect a result as shown in Figure 2.
Figure 2. Sample of amplified VHH1 of approximately 700 bp visualised on a 1% agarose gel containing 1 × SYBRTM safe DNA gel stain. The sizes in base pairs of a DNA ladder run in parallel are shown.
Production of VHH2
Prepare the following PCR reaction in PCR tubes (6 × 50 µL reactions) (Table 4).
Table 4. PCR reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 19 µL
10 µM VHH For2 primer 0.5 µM 2.5 µL
10 µM VHH Rev primer 0.5 µM 2.5 µL
2× Phusion flash PCR master mix 1× 25 µL
5 ng/µL VHH1 (step C4f) 1 ng 10 µL
Total n/a 50 µL
Briefly vortex and centrifuge the reaction tubes.
Set up the thermal cycler as below and perform the PCR (Table 5).
Table 5. PCR cycling conditions
Step Temperature (°C) Duration Number of cycles
Denaturation 98 30 s 1
Annealing 98 10 s 30
Extension 55 30 s
Final extension 72 20 s
Hold 72 5 min 1
Denaturation 12 Infinite hold -
Combine all six reactions into a single 2 mL microcentrifuge tube. Using the Purelink PCR purification kit, add 4× volume of B2 buffer (1.2 mL) to the amplified PCR product and purify between two Purelink columns, eluting each with 25 µL of E1 buffer. Combine both eluates into one 1.5 mL microcentrifuge tube, measure the A280, and expect a concentration between 200 and 300 ng/µL.
Mix 6× DNA gel loading buffer with 120 ng of VHH2 (step C5d) to a final concentration of 1× and run alongside a well of 1× HyperladderTM 1 kb DNA ladder on a 1% agarose gel containing 1× SYBRTM safe DNA gel stain to determine the purity. Electrophorese in 1× TBE buffer at 80 V for 40 min. Expect a result as shown in Figure 3.
Pause point.
Figure 3. Sample of amplified VHH2 of approximately 400 bp visualised on a 1% agarose gel containing 1× SYBRTM safe DNA gel stain. The sizes in base pairs of a DNA ladder run in parallel are shown.
Digestion of pADL23c
Prepare the following restriction digestion reaction in PCR tubes (10 × 100 µL reactions) (Table 6).
Table 6. Restriction digestion reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 83 µL
rCutSmartTM buffer 1× 10 µL
SfiI 5 units 2.5 µL
400 ng/µL vector DNA 1.8 µg 4.5 µL
Total n/a 100 µL
Briefly vortex and centrifuge the reaction tubes.
Incubate at 50 °C for 2 h.
Add 20 µL of 6× DNA gel loading buffer to each reaction tube and run alongside a well of 1× GeneRuler 1 kb DNA ladder on a 0.7% agarose gel containing 1× SYBRTM Safe DNA gel stain. Electrophorese in 1× TBE buffer at 80 V for 40 min. Expect a result as shown in Figure 4.
Figure 4. Preparative 0.7% agarose gel containing 1× SYBRTM safe DNA gel stain of the vector backbone (upper band) and stuffer fragment (lower band) after SfiI digestion of pADL23c phagemid vector prior to gel extraction. The sizes in base pairs of a DNA ladder run in parallel are shown.
Visualise the digested, higher molecular weight, approximately 3,000 bp vector backbone using a LED Blue transilluminator, excise from the gel using a scalpel knife, and gel extract using 10 spin columns from the Nucleospin Gel and PCR clean-up kit according to the manufacturer’s protocol. Elute each column using 50 µL of NE buffer and pool all the eluates into a single 1.5 mL microcentrifuge tube.
Caution: Use Safe ImagerTM viewing glasses when visualising the DNA during excision of the band from the agarose gel and use caution when using a scalpel knife.
Using the Purelink PCR purification kit, add 4× volume of B3 buffer (2 mL) to the gel extracted product and purify using one Purelink column from the Purelink PCR purification kit eluting in 50 µL E1 buffer. Measure the A280 and expect a concentration between 50 and 80 ng/µL.
Mix 6× DNA gel loading buffer with 120 ng of VHH2 (step C5d) and digest pADL23c backbone (step C6f) to a final concentration of 1× and run alongside a well of 1× HyperLadderTM 1 kb DNA ladder on a 1% agarose gel containing 1× SYBRTM Safe DNA gel stain to determine the purity. Electrophorese in 1× TBE buffer at 80 V for 40 min. Expect a result as shown in Figure 5.
Pause point.
Figure 5. Samples of linearised and purified pADL23c (lanes 1 and 2) and purified VHH2 (lane 3) visualised on a 1% agarose gel containing 1× SYBRTM safe DNA gel stain. The sizes in base pairs of a DNA ladder run in parallel are shown.
Test of library size
Prepare the following in-fusion reaction in a PCR tube (1 × 10 µL) (Table 7).
Table 7. PCR reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 5 µL
5× CEII buffer 1× 2 µL
100 ng/µL VHH2 insert (step C5d) 100 ng 1 µL
20 ng/µL vector DNA (step C6f) 20 ng 1 µL
Exnase II 1 µL
Total n/a 10 µL
Briefly vortex and centrifuge the reaction tube.
Incubate at 42 °C for 30 min using a thermal cycler. Add 40 µL of TE buffer to terminate the reaction.
Using the Purelink PCR purification kit, add 4× volume of B3 buffer (200 µL) to the in-fusion product and purify using one Purelink column eluting in 20 µL nuclease-free water.
Note: Work in a safety cabinet.
Gently mix 5 µL of purified in-fusion reaction with 30 µL of competent TG1 in a 1.5 mL microcentrifuge tube and transfer to a chilled 1 mL electroporation cuvette.
Electroporate at 1.7 kV and immediately add 400 µL of recovery medium to the cuvette. Transfer contents to a 2 mL microcentrifuge tube and incubate for 1 h at 37 °C agitating at 600 rpm. See General note 8.
Using 50 µL of culture, prepare a 1:10, 1:100, and 1:1,000 dilution in 2× YT. Spread 100 µL on 1% agar LB plates containing 100 µg/mL ampicillin until dry. Incubate overnight at 37 °C.
Estimate the library size in terms of colony forming units per millilitre (CFU/mL) using the following formula: number colonies × 10 (to get to mL) × 0.4 (volume) × dilution. See General note 9.
Based on the estimated small-scale library size, set up the required number of reactions considering that 5 µL of in-fusion reaction yielded × size library. Calculate how many microlitres of in-fusion reactions would be needed to make a library with a size of 1 × 106. See General note 10.
To validate the number of full-length VHH clones in the small-scale library, a colony PCR to amplify the ~500 bp VHH gene in 48 randomly selected clones from a plate in step C7g is performed. Prepare the following PCR reaction master mixture in a 2 mL microcentrifuge tube (Table 8).
Table 8. PCR reaction composition
Reagent Final concentration Volume for single reaction Volume for master mixture
Nuclease-free water n/a 20.75 µL 1037.5 µL
10 µM PhD seq Fwd primer 0.2 µM 0.5 µL 25 µL
10 µM PhD seq Rev primer 0.2 µM 0.5 µL 25 µL
10× Taq buffer 1× 2.5 µL 125 µL
25 mM dNTP 0.25 mM 0.25 µL 12.5 µL
Taq polymerase 1 unit 0.5 µL 25 µL
Total n/a 25 µL 1250 µL
Briefly vortex and centrifuge the reaction tube.
Pipette 25 µL of the master mixture into 48 wells of a 96-well rigid semi skirted PCR plate. Pick 48 colonies with 48 × 10 µL tips and place into each filled well. Remove the tips using a multichannel pipette. Cover the PCR plate with PCR foil seal.
Set up the thermal cycler as below and perform the PCR (Table 9).
Table 9. PCR cycling conditions
Step Temperature (°C) Duration Number of cycles
Denaturation 95 7 min s 1
Annealing 95 15 s 35
Extension 55 30 s
Final extension 68 1 min 40 s
Hold 68 5 min 1
Denaturation 12 Infinite hold -
Add 20 µL of 2× DNA gel loading buffer to each well of the PCR plate and load 10 µL to each well alongside a well of 1× GeneRuler 1 kb DNA ladder on a 1% agarose gel containing 1× SYBRTM safe DNA gel stain. Electrophorese in 1× TBE buffer at 80 V for 40 min. In Figure 6, 46 out of the 48 clones that were subjected to colony PCR possessed the ~500 bp band corresponding to the amplified VHH gene. The two clones that were not positive are indicated by an asterisk.
Pause point.
Figure 6. Colony PCR of 48 randomly selected clones from a small-scale library preparation
Scaled library preparation
Prepare the following reaction in PCR tubes (20 × 20 µL) (Table 10).
Table 10. In-Fusion reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 9 µL
5× CEII buffer 1× 4 µL
50 ng/µL VHH2 insert (step C5c) 100 ng 2 µL
5 ng/µL vector DNA (step C6f) 20 ng 4 µL
Exnase II 1 µL
Total n/a 20 µL
Briefly vortex and centrifuge the reaction tube.
Incubate at 42 °C for 30 min in a thermal cycler.
Combine reactions into a single 2 mL microcentrifuge tube. Using the Purelink PCR purification kit, add 4× volume of B3 buffer (1.6 mL) to the in-fusion product and purify using one Purelink column eluting in 50 µL of nuclease-free water. Measure the A280 and expect a concentration of between 35 and 50 ng/µL.
Note: Work in a safety cabinet.
TG1 cells are transformed by electroporation in separate reactions to create the scaled library. Gently mix 6 µL of purified in-fusion reaction with 30 µL of competent TG1 in a 1.5 mL microcentrifuge tube and transfer to a chilled 1 mL electroporation cuvette. Electroporate at 1.7 kV, immediately add 960 µL of recovery medium to the cuvette, and transfer to a 50 mL conical centrifuge tube. See General note 8.
Repeat this a total of eight times with eight separate in-fusion-TG1 mixtures.
Combine two cuvettes worth of electroporated cells into a single 50 mL conical centrifuge tube and incubate at 37 °C for 1 h shaking at 200 rpm. You should have four conical centrifuge tubes in total.
Use 50 µL from one of the conical centrifuge tubes and prepare a 1:10, 1:100, and 1:1,000 dilution in 2× YT. Spread 100 µL of each dilution on 1% agar LB plates containing 100 µg/mL ampicillin until dry. Incubate overnight at 37 °C. Use these plates to estimate library size (CFU/mL) = number colonies × 10 (to get to mL) × 1.92 (volume) × dilution. See General note 9.
Plate the entire culture of each of the four conical centrifuge tubes onto separate bioassay dishes filled with 2% agar LB plates containing 100 µg/mL ampicillin. There should be four plates in total. Spread the entire volume until dry and incubate overnight at 37 °C. Expect a density similar to that shown in Figure 7, which is equivalent to a 1 × 106 CFU/mL sized library. See General note 11.
Figure 7. Expected density on a bioassay plate for a 1 × 106 CFU/mL library
Resuspend the lawn of colonies of each bioassay plate in 6 mL of 2× YT (containing a final concentration of 25% glycerol) and transfer to a single 50 mL conical centrifuge tube. Mix well by serological pipette and prepare 1 mL aliquots in screw cap microtubes, which are stored at -80 °C. This is the VHH library stock, which is used for the first round of panning.
Pause point.
Panning
Below is a suggested timetable for panning, hit identification by ELISA, and small-scale expression to maximise time in the lab and to allow space for overlap if multiple panning campaigns are being performed for multiple proteins by multiple people using shared resources (Table 11).
Table 11. Experimental timetable for the identification of nanobody binders through panning, ELISA, and small-scale expression
Monday Tuesday Wednesday Thursday Friday
Week 1 Amplify VHH library (step D3) Recover library phage (step D3)
First panning (step D4)
Recover lawn of colonies (step D4)
Amplify sub-library (step D5)
Recovery of sub-library phage (step D5)
Second panning (step D6)
Take plates out of the incubator and place in fridge.
Week 2 Pick 93 colonies and grow overnight (step D7)
Prepare master plates (step D7)
Start culture for anti-M13 ELISA (step D8)
Anti-M13 ELISA (step D8) PCR, clean up, sequencing (step D9)
Week 3 Overnight culture for small scale (step E1) Small-scale culture to OD and induce with IPTG (step E1)
Periplasm isolation (step E1)
Ni-NTA purification (step E2)
Desalting (step E3)
Titration ELISA (step E4)
Off-rate determination (step E5)
Prepare log-phase TG1
Note: Work in a safety cabinet.
Streak TG1 on a 1% agar LB plate without antibiotics and incubate overnight at 37 °C.
Inoculate 20 mL of 2× YT with a single TG1 colony in a 100 mL disposable baffled Erlenmeyer flask and incubate overnight at 37 °C shaking at 200 rpm.
Mix 20 mL of 50% glycerol with the overnight culture and store in 1 mL aliquots at -80 °C. This is the TG1 stock, which we use to prepare log-phase TG1 culture when needed.
To 25 mL of 2× YT in a 125 mL disposable baffled Erlenmeyer flask, add enough TG1 stock (step D1c) so that so that the starting OD600 is approximately 0.05. See General note 12.
Incubate the TG1 culture at 37 °C shaking at 200 rpm until the OD600 reaches 0.4–0.6. This takes approximately 1.5–2 h. This is the log-phase TG1, which should be kept on ice and used within 2 h.
Prepare the CM13K helper phage
Caution: Phage can infect all bacteria. If working in a protein expression lab, care must be taken not to release phage into communal areas. Wipe down surfaces with 5% Chemgene followed by 70% EtOH if the surface is susceptible to corrosion. The use of filtered tips is encouraged to reduce aerosols. All contaminated consumables must be decontaminated with 2% Virkon and autoclaved. If possible, use disposable Erlenmeyer flasks for all cultures outlined in steps D1–D5.
Note: Work in a safety cabinet.
Inoculate 20 mL of 2× YT with 200 µL of log-phase TG1 (step D1e) in a 125 mL disposable baffled Erlenmeyer flask. Add 1 µL of previously prepared 1 × 1013 PFU/mL CM13K helper phage glycerol stock (step D2h) and incubate at 37 °C for 4 h shaking at 250 rpm.
In two 1 L disposable baffled Erlenmeyer flasks, add 250 mL of 2× YT containing 25 µg/mL kanamycin and 10 mL of the prepared culture. Incubate overnight at 25 °C shaking at 250 rpm.
Pellet the cells at 4,500× g for 10 min at 4 °C and transfer approximately 170 mL of the phage containing supernatant into three 250 mL PPCO centrifuge tubes. Add approximately 35 mL of ice-cold PEG/NaCl to each tube, invert, and swirl gently to mix the PEG/NaCl solution with the phage supernatant. Incubate on ice for 1 h.
Centrifuge the tubes at 12,000× g for 15 min at 4 °C and resuspend each of the three pellets in 4 mL of ice-cold sterile PBS. Combine the resuspended pellets into a single 25 mL high-speed PPCO centrifuge tube and add 2.5 mL of ice-cold PEG/NaCl. Swirl and invert gently to mix the PEG/NaCl solution with the phage supernatant. Incubate on ice for 30 min.
Centrifuge at 12,000× g for 10 min at 4 °C and resuspend the pellet in 12 mL of ice-cold sterile PBS. Keep at 4 °C until the phage concentration, expressed as plaque forming units per millilitre (PFU/mL), has been determined either spectrophotometrically or by plaque assay, both of which should yield similar values.
For the spectrophotometric determination of phage concentration, in a 1.5 mL microcentrifuge tube, add 998 µL of PBS and 2 µL of phage (step D2e), transfer to a quartz cuvette, and measure the absorbance at 268 nm. Calculate the PFU/mL considering that an A268 of 1.0 is equivalent to 5 × 1012 PFU/mL [11].
For the determination of phage concentration using the plaque assay, prepare tenfold serial dilutions (10-1 to 10-25) of CM13K helper phage (step D2e) in log-phase TG1 (step D1e) and incubate at RT for 5 min. Plate 100 µL of 10-15 to 10-25 dilutions onto 1% agar LB plates without antibiotics. Add 3 mL of 0.7% agar LB (at 55 °C) per plate and swirl over the entire plate surface. Allow the top agar to solidify and incubate overnight at 37 °C. Calculate the PFU/mL using the following formula: number of plaques (clear areas) × 10 (to get to mL) × dilution.
Dilute the CM13K helper phage (step D2e) to 2 × 1013 PFU/mL using ice-cold sterile PBS, add an equal volume of ice-cold 50% glycerol, and mix gently to achieve a final concentration of 1 × 1013 PFU/mL. Store in 100 µL aliquots in 500 µL microcentrifuge tubes. This is the CM13K helper phage stock. Each time a new 100 µL aliquot is thawed, sub-aliquot 12 µL into 9 × PCR tubes to avoid freeze thawing of the original 100 µL aliquot.
Amplify and recover library
Note: Work in a safety cabinet.
To 50 mL of 2× YT containing 100 µg/mL ampicillin in a 250 mL disposable baffled Erlenmeyer flask, add enough VHH library stock (step C8j) so that the starting OD600 is approximately 0.05. See General note 13.
Incubate the culture at 37 °C shaking at 200 rpm until the OD600 reaches 0.4–0.6. This takes approximately 2 h. See General note 14.
Transfer 10 mL of this culture into a 50 mL conical centrifuge tube, add 10 µL of the CM13K helper phage stock (step D2h), and mix by gentle swirling. Incubate at 37 °C for 1 h without agitation.
Pellet the cells at 2,800× g for 10 min at 18 °C. Resuspend the pellet in 50 mL of 2× YT containing 100 µg/mL ampicillin and 25 µg/mL kanamycin and transfer to a 250 mL disposable baffled Erlenmeyer flask. Incubate overnight at 25 °C shaking at 250 rpm.
Transfer the overnight culture into a 50 mL conical centrifuge tube and pellet the cells at 3,200× g for 10 min at 4 °C.
Pour 40 mL of the phage containing supernatant into a new 50 mL conical centrifuge tube and add 10 mL of ice-cold PEG/NaCl. Invert several times and incubate on ice for 1 h.
Pellet the precipitated phage by centrifugation at 3,200× g for 10 min at 4 °C. Resuspend the phage in 1 mL of ice-cold sterile PBS and transfer to a 2 mL microcentrifuge tube.
After centrifugation at 20,000× g for 1 min at 4 °C, transfer the supernatant containing phage into a new 2 mL microcentrifuge tube. Add 250 µL of ice-cold PEG/NaCl and invert the tube until a homogeneous white suspension appears. Incubate on ice for 30 min.
Pellet the precipitated phage by centrifugation at 20,000× g for 15 min at 4 °C. Resuspend the pelleted phage in 1 mL of ice-cold sterile PBS and transfer to a 2 mL microcentrifuge tube.
After a final centrifugation at 20,000× g for 1 min at 4 °C, transfer the supernatant containing phage into a new 2 mL microcentrifuge tube. This is the isolated library phage; it should be kept either on ice or at 4 °C and is stable for a month.
Titrate 15 µL of the library phage using tenfold serial dilutions from 10-1 to 10-12 with log-phase TG1 cells (step D1e). After incubation at 37 °C for 15 min, spread 50 µL of the 10-4 to 10-12 dilutions on 1% agar LB plates containing 100 µg/mL ampicillin until dry and incubate overnight at 37 °C. Include a TG1-only control. See General note 15.
Use these plates to estimate how many phage can be produced from the library. This is calculated as PFU/mL = number colonies × 10 (to get to mL) × 0.05 (volume) × dilution. Expect 1 × 1010–1 × 1013 PFU/mL. See General note 16.
Panning first round
Note: Work at a lab bench.
In a 2 mL microcentrifuge tube, add 500 µL of StartingBlock buffer and 500 µL of isolated library phage (step D3j). Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 30 min at RT using a sample mixer.
Add an appropriate volume of biotinylated antigen to achieve 50 nM final concentration in 1.5 mL (which is achieved by step D4d). See General note 1. Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 1 h at RT using a sample mixer.
At this point, one can start growing the log-phase TG1 cells (steps D1d–e), which would ensure that the cells are at the required OD600 once the phage has been eluted (step D4h).
Thirty minutes into the incubation of phage with antigen (step D4b), in a new 2 mL microcentrifuge tube, add 100 µL of streptavidin DynabeadsTM M-280. Wash the beads twice with 500 µL of PBS and then add 500 µL of StartingBlock buffer. Incubate for 30 min at RT using a sample mixer.
Transfer the 1 mL of blocked phage with antigen (step D4b) to the blocked beads (step D4d). The total volume is 1.5 mL, and the final antigen concentration is now 50 nM. Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 15 min at RT using a sample mixer.
Place the microcentrifuge tube on a DynaMagTM-2 magnet. Once the beads have been pulled to the side, discard the unbound phage in the supernatant. Wash away loosely bound phage by resuspending the beads in 500 µL of PBST and then using the magnet to pull the beads to the side. Repeat this washing process a total of six times with PBST and then once with PBS.
Add 500 µL of 250 µg/mL trypsin to the beads. Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 30 min at RT using a sample mixer.
Collect the eluted phage from the first pan in the supernatant after using the DynaMagTM-2 magnet to pellet the beads.
Note: Work in a safety cabinet.
In a 50 mL conical centrifuge tube, add 500 µL of eluted phage from the first pan (step D4h) to 10 mL of log-phase TG1 cells (step D4c) and incubate at 37 °C for 30 min without agitation.
Pellet the cells at 2,800× g for 10 min at 18 °C and resuspend in 1 mL of 2× YT. Spread the entire volume until dry on one bioassay dish containing 1% agar LB with 100 µg/mL ampicillin and incubate overnight at 37 °C.
Resuspend the lawn of colonies in 6 mL of 2× YT (containing a final concentration of 25% glycerol) and store in 1 mL aliquots in cryotubes at -80 °C. This is the pan 1 sub-library stock.
Amplify and recover sub-library
Note: Work in a safety cabinet.
To 50 mL of 2× YT containing 100 µg/mL ampicillin in a 250 mL disposable baffled Erlenmeyer flask, add enough pan 1 sub-library stock (step D4k) so that the starting OD600 is approximately 0.05. See General note 13.
Incubate the culture at 37 °C shaking at 200 rpm until the OD600 reaches 0.4–0.6. This takes approximately 2 h. See General note 14.
Transfer 10 mL of this culture into a 50 mL conical centrifuge tube, add 10 µL of the CM13K helper phage stock (step D2h), and mix by gentle swirling. Incubate at 37 °C for 1 h without agitation.
Pellet the cells at 2,800× g for 10 min at 18 °C. Resuspend the pellet in 50 mL of 2× YT containing 100 µg/mL ampicillin and 25 µg/mL kanamycin and transfer to a 250 mL disposable baffled Erlenmeyer flask. Incubate overnight at 25 °C shaking at 250 rpm.
Transfer the overnight culture into a 50 mL conical centrifuge tube and pellet the cells at 3,200× g for 10 min at 4 °C.
Pour 40 mL of the phage containing supernatant into a new 50 mL conical centrifuge tube and add 10 mL of ice-cold PEG/NaCl. Invert several times and incubate on ice for 1 h.
Pellet the precipitated phage by centrifugation at 3,200× g for 10 min at 4 °C. Resuspend the phage in 1 mL of ice-cold sterile PBS and transfer to a 2 mL microcentrifuge tube.
After centrifugation at 20,000× g for 1 min at 4 °C, transfer the supernatant containing phage into a new 2 mL microcentrifuge tube. Add 250 µL of ice-cold PEG/NaCl and invert the microcentrifuge tube until a homogeneous white suspension appears. Incubate on ice for 30 min.
Pellet the precipitated phage by centrifugation at 20,000× g for 15 min at 4 °C. Resuspend the pelleted phage in 1 mL of ice-cold sterile PBS and transfer to a 2 mL microcentrifuge tube.
After a final centrifugation at 20,000× g for 1 min at 4 °C, transfer the supernatant containing phage into a new 2 mL microcentrifuge tube. This is the isolated pan 1 phage; it should be kept either on ice or at 4 °C and is stable for a month.
Panning second round
Note: Work in a lab bench.
In a 2 mL microcentrifuge tube, add 500 µL of StartingBlock buffer and 500 µL of isolated pan 1 phage (step D5j). Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 30 min at RT using a sample mixer.
Add an appropriate volume of biotinylated antigen to achieve 5 nM final concentration in 1.5 mL (which is achieved by step D6d). See General note 1. Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 1 h at RT using a sample mixer.
At this point, one can start growing the log-phase TG1 cells (steps D1d–e), which would ensure that the cells are at the required OD600 once the phage has been eluted (step D6h).
Thirty minutes into the incubation of phage with antigen (step D6b), in a new 2 mL microcentrifuge tube, add 10 µL of streptavidin DynabeadsTM M-280. Wash the beads twice with 500 µL of PBS and then add 500 µL of StartingBlock buffer. Incubate for 30 min at RT using a sample mixer.
Transfer the 1 mL of blocked phage with antigen (step D6b) to the blocked beads (step D6d). The total volume is now 1.5 mL, and the final antigen concentration is now 5 nM. Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 15 min at RT using a sample mixer.
Place the microcentrifuge tube on a DynaMagTM-2 magnet. Once the beads have been pulled to the side, discard the unbound phage in the supernatant. Wash away loosely bound phage by resuspending the beads in 500 µL of PBST and then using the magnet to pull the beads to the side. Repeat this washing process a total of six times with PBST and then once with PBS.
Add 500 µL of 250 µg/mL trypsin to the beads. Wrap parafilm around the microcentrifuge tube lid to avoid any accidental spillage and incubate for 30 min at RT using a sample mixer.
Collect the eluted phage from the second pan in the supernatant after using the DynaMagTM-2 magnet to pellet the beads.
Note: Work in a safety cabinet.
To obtain individual colonies for the master plate (step D7), titrate the eluted second pan phage (step D6h) using tenfold serial dilutions from 10-1 to 10-12 with log-phase TG1 cells (step D6c). After incubation at 37 °C for 15 min, spread 50 µL of the 10-4 to 10-12 dilutions on 1% agar LB plates containing 100 µg/mL ampicillin until dry and incubate overnight at 37 °C. Include a TG1 only control. See General note 15 and Troubleshooting 1.
Select the dilution that has approximately 100–300 individual colonies.
Preparation of master plates
Note: Work in a safety cabinet.
In a 1 mL deep-well block, add 180 µL/well of 2× YT containing 100 µg/mL ampicillin. Pick 93 individual clones from selected plate of titrated pan 2 phage in TG1 (step D6j) with 93 × 10 µL tips and place into each filled well. Remove the tips using a multichannel pipette. Leave wells F12, G12, and H12 without a colony, which serve as our controls.
Cover the plate with cell culture adhesive seal and incubate overnight at 37 °C shaking at 600 rpm. This overnight culture will be used to prepare two stock plates, the PCR template (step D7c) and the master plate (step D7d). Prepare the PCR plate first followed by the master plate.
Transfer 20 µL/well of overnight culture to a skirted PCR plate. Cover the plate with PCR foil seal and store at -80 °C. This is the PCR template plate.
To the remaining overnight culture, add 150 µL/well of 50% glycerol and mix by pipetting. Cover the plate with PCR foil seal and store at -80 °C. This is the master plate.
Anti-M13 phage ELISA
Note: Work in a safety cabinet.
In a 1 mL deep-well plate already filled with 150 µL/well of 2× YT, add 10 µL/well of the master plate (step D7d). Cover with cell culture adhesive seal and incubate for 3 h at 37 °C shaking at 600 rpm.
Prepare the CM13K helper phage mixture by adding 2 µL of CM13K helper phage stock (step D2h) to 10 mL of 2× YT. Add 30 µL/well of the prepared CM13K helper phage mixture and incubate for 1 h at 37 °C shaking at 600 rpm.
Pellet the cells at 2,800× g for 10 min at 18 °C and resuspend each well in 400 µL of 2× YT containing 100 μg/mL ampicillin and 25 μg/mL kanamycin.
Cover the plate with cell culture adhesive seal and incubate overnight at 25 °C shaking at 600 rpm.
After centrifugation at 4,000× g for 15 min at 4 °C, transfer the phage containing supernatant to a new 0.5 mL deep-well plate and cover with adhesive film. Store at 4 °C; this is stable for a month. This is the culture that is used for the anti-M13 ELISA (step D8l).
Note: Work in a lab bench.
Coat a 96-well ELISA microplate with 100 µL/well of 10 µg/mL neutrAvidinTM diluted in PBS and incubate overnight at 4 °C.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of 50 nM biotinylated target protein diluted in PBS and incubate for 1 h at RT on a microplate shaker. See General note 1.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 250 µL/well of blocking solution and incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 50 µL/well of blocking buffer followed by 50 µL/well of the anti-M13 ELISA culture (step D8e). In the control wells F12, G12, and H12, add 50 µL/well of blocking buffer and 50 µL/well of PBS. Incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of anti-M13-HRP diluted in 0.1% BSA-PBS (at a final concentration of 0.25 µg/mL) and incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of ABTS substrate, which is prepared by mixing solution A and solution B in a 1:1 ratio. Protect from light and measure the absorbance at 405nm. See General note 2.
Wells with absorbances above 1 are considered hits. See Troubleshooting 2 and 3.
PCR and sequencing of ELISA hits
Prepare the following PCR reaction master mixture in a 5 mL tube and set up 96 PCR reactions in a 96-well rigid PCR plate (Table 12). See General note 17. Cover the plate with PCR foil seal.
Table 12. PCR reaction composition
Reagent Final concentration Volume for single reaction Volume for master mixture
Nuclease-free water n/a 18.25 µL 1,825 µL
10 µM PhD seq Fwd primer 0.2 µM 0.5 µL 50 µL
10 µM PhD seq Rev primer 0.2 µM 0.5 µL 50 µL
10× Taq buffer 1× 2.5 µL 250 µL
25 mM dNTP 0.25 mM 0.25 µL 50 µL
Taq polymerase 1 unit 0.5 µL 50 µL
PCR template (step D7c) 2.5 µL -
Total n/a 25 µL 2275 µL
Set up the thermal cycler as below and perform the PCR (Table 13).
Table 13. PCR cycling conditions
Step Temperature (°C) Duration Number of cycles
Denaturation 95 7 min s 1
Annealing 95 15 s 35
Extension 55 30 s
Final extension 68 1 min 40 s
Hold 68 5 min 1
Denaturation 12 Infinite hold -
Add 45 µL of Highprep PCR magnetic beads/well and mix by pipette. Place on DynaMagTM 96-side magnet for 1 min or until the solution is clear and the beads have been pulled against the wall of the PCR tube and pipette off the supernatant.
Add 100 µL of 70% EtOH and mix by pipette. Place on DynaMagTM 96-side magnet for 1 min or until the solution is clear and the beads have been pulled against the wall of the PCR tube and pipette off the supernatant. Repeat this washing step a total of three times.
Leave beads to dry at RT for 10 min and then add 25 µL of EB buffer (from the QIAprep Spin Miniprep Kit) to the beads and mix by pipette. Place on DynaMagTM 96-side magnet for 1 min or until the solution is clear and the beads have been pulled against the wall of the PCR tube and transfer 20 µL of the purified PCR product containing supernatant to a new 96-well skirted PCR plate.
If a nanophotometer that can measure multiple samples at a time (such as the ImplenTM NanoPhotometer® N120) is available, measure the A280 of each well. If a single sample nanophotometer is available, measure the A280 of a random selection of clones. Expect around 15–25 ng/µL. Alternatively, 5 µL of purified PCR product of a random selection of clones can be mixed with 5 µL of 2× DNA gel loading buffer and run alongside a lane of 1× GeneRuler 1 kb DNA ladder on a 1% agarose gel containing 1× SYBRTM Safe DNA gel stain. Electrophorese in 1× TBE buffer at 80 V for 40 min. Image the gel and confirm the presence of amplified ~500 bp VHH genes.
Cover the plate with PCR foil seal and send for sequencing along with the PhD seq Fwd primer. See General note 18.
Small-scale expression and purification
The identification of nanobody clones that are expressed and purified well is achieved by performing a small-scale expression. The addition of a desalting step after purification allows for the confirmation of nanobody binding to the antigen using a titration ELISA or biolayer interferometry (BLI). The off-rate (kdis) of the nanobodies can be determined as well as preliminary epitope binning using BLI. These factors aid in the selection of which nanobodies will be taken forward for large scale expression.
Small-scale expression
Note: Work in a safety cabinet.
In a 1 mL deep-well plate already filled with 180 µL of 2× YT containing 100 µg/mL ampicillin per well, add 10 µL of selected clones based on sequencing results from the master plate (step D7d). Cover with cell culture adhesive seal and incubate overnight at 37 °C shaking at 600 rpm.
Add 10 µL of the overnight culture of each clone to a 24-well plate already filled with 4 mL/well of terrific broth containing 100 µg/mL ampicillin, 0.1% glucose, and 2 mM MgCl2. Cover with cell culture adhesive seal and incubate for 4 h at 37 °C, shaking at 600 rpm.
To each well, add 4 µL of 1 M IPTG (1 mM final concentration), cover with cell culture adhesive seal, and incubate overnight at 25 °C shaking at 600 rpm.
Note: Work in a lab bench.
Pellet the cells at 4,000× g for 15 min at 4 °C and gently resuspend the pellet in 300 µL of 1 mg/mL polymyxin B sulfate. Cover plate with adhesive film and agitate for 1 h at RT using a microplate shaker.
Pellet the cells at 4,000× g for 10 min at 4 °C and transfer the periplasm containing supernatant to a new 1.5 mL microcentrifuge tube.
Mix 10 µL of the isolated periplasm with 10 µL of 2× Laemmli sample buffer, incubate at 95 °C for 5 min, and run on a NuPage 4%–12% bis tris precast gel in 1× MES buffer alongside Mark12TM unstained standard at 200 V for 40 min.
Stain with InstantBlue® Coomassie protein stain. Expect prominent bands at approximately 14 kDa. Refer to the results from Expasy Protparam (Data analysis, step 1d) for the exact size of each clone.
Affinity purification
Note: Work in a lab bench.
Purification of the his-tagged nanobodies using Ni-NTA spin columns is performed according to the manufacturer’s instructions. Briefly, add 600 µL of equilibration buffer to the Ni-NTA spin column and centrifuge at 2,900 rpm (800× g) for 2 min at 4 °C.
Add the periplasm sample to the Ni-NTA spin column and centrifuge at 1,600 rpm (200× g) for 5 min at 4 °C.
Wash the Ni-NTA spin column three times by the addition of 600 µL of equilibration buffer and centrifuging at 2,900 rpm (800× g) for 2 min at 4 °C.
Transfer the Ni-NTA spin column into a new 2 mL microcentrifuge tube and add 100 µL of elution buffer. Incubate at RT for 2 min before centrifugation at 2,900 rpm (800× g) for 2 min at 4 °C. The flowthrough contains the purified nanobody.
Mix 5 µL of the purified nanobody with 5 µL of 2× Laemmli sample buffer, incubate at 95 °C for 5 min, and run on a NuPage 4%–12% bis tris precast gel in 1× MES buffer alongside Mark12TM unstained standard at 200 V for 40 min.
Stain with InstantBlue® Coomassie protein stain. Expect bands at approximately 14 kDa. Refer to the results from Expasy Protparam (Data analysis, step 1d) for the exact size of each clone.
Desalting
Note: Work in a lab bench.
Desalting of the Ni-NTA purified nanobodies using the ZebaTM Spin Desalting Columns is performed according to the manufacturer’s instructions. Briefly, break off the bottom cap of the desalting columns, loosen top cap, and place spin column in a new 2 mL microcentrifuge tube. Centrifuge at 1,500× g for 1 min at 4 °C.
Make a mark on the side of the desalting column where the resin is slanted upwards. Ensure that this mark is facing outwards in all centrifugation steps.
Wash the resin three times by gently adding 300 µL of PBS (or buffer of choice) to the resin bed and centrifuging at 1,500× g for 1 min at 4 °C.
Transfer the desalting column to a new 1.5 mL microcentrifuge tube and gently add 100 µL of purified nanobody (step E2d) to the centre of the resin bed. After the sample has been fully absorbed, gently add 15 µL of PBS (or buffer of choice) to the centre of the resin bed. Centrifuge at 1,500× g for 2 min at 4 °C and collect the desalted nanobody in the flowthrough.
Measure the A280 of the desalted nanobody and determine the molar concentration (µM = A280/ ϵ × 106) as well as the concentration (µg/mL = µM × kDa) using the kDa and extinction coefficient (ϵ) obtained from Expasy Protparam for the individual nanobodies (Data analysis, step 1d).
Titration ELISA
If an Octet R8 (or similar instrument) is not available, a titration ELISA will suffice to confirm binding of the purified nanobodies to the target protein.
Note: Work in a lab bench.
Coat a suitable number of wells of a 96-well ELISA microplate with 100 µL/well of 10 µg/mL neutrAvidinTM diluted in PBS and incubate overnight at 4 °C.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of 50 nM biotinylated target protein diluted in PBS and incubate for 1 h at RT on a microplate shaker. See General note 1.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 250 µL/well of blocking solution and incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 200 µL of 25 µg/mL desalted nanobody (step E3d) diluted in blocking solution to the first well. Prepare a serial dilution in the plate, i.e., add 100 µL of blocking solution to the second well and add 100 µL of the solution from the first well. Prepare a total of seven dilutions (25–0.39 µg/mL), leaving the last well with blocking solution only. Incubate for 1 h at RT on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of anti-camelid VHH-HRP diluted 1:5,000 in 0.1% BSA-PBS and incubate for 1 h at RT on a on a microplate shaker.
Using a plate washer, wash the plate five times with 300 µL/well of PBST.
Add 100 µL/well of ABTS substrate, which is prepared by mixing solution A and solution B in a 1:1 ratio. Protect from light and measure the absorbance at 405 nm. See General note 2.
Off-rate (kdis) determination using Biolayer interferometry (BLI)
Note: Work in a lab bench.
Preheat the Octet R8 at 25 °C overnight or at least 3 h before starting the experiment.
Typical starting concentrations of antigen and nanobody are 50 nM and 100 nM, respectively, prepared in octet dilution buffer. See General note 19.
Prepare all the dilutions in a 96-well black polypropylene plate. See General note 20.
A single replicate per nanobody is sufficient at this stage. Include a no nanobody control to rule out any non-specific interactions with the loaded antigen and to serve as the background.
The experimental run conditions using SA biosensors are as follows:
1,000 rpm shaking for all incubation steps.
Plate definition: Octet dilution buffer to be used in all baseline steps/wells.
Assay definition: Baseline 600 s into octet dilution buffer.
: Loading 300 s into antigen diluted in octet dilution buffer.
: Baseline 30 s in octet dilution buffer.
: Baseline 60 s in octet dilution buffer.
: Association 300 s in desalted nanobody diluted in octet dilution buffer (watch this step, time might need to be extended as we want the curves to reach a plateau).
: Dissociation 300 s into column where baseline 60 s was measured (this step should be the same duration as the association step).
Subcloning of the nanobody into the bacterial and mammalian vectors
We have developed two vector suites for the functionalisation of nanobodies in both bacterial and mammalian expression systems. They have been designed in such a way that any nanobody that has been identified from a library that has been constructed as detailed in section C can be cloned into them using a common forward primer and tag-specific reverse primers as well as using the same restriction sites (Figure 8). The characteristics, tags for functionalisation, and primers for each vector in the bacterial and mammalian toolboxes are detailed in Table 14.
Figure 8. Details of the primers, restriction enzymes, and tags present in each of the vectors in the bacterial and mammalian toolboxes. Abbreviations used: VHH, nanobody; BAP, biotin acceptor peptide/Avi-TagTM; wt, wild-type; huIg1Fc, human Ig1 Fc.
Table 14. Features of the vector in the E. coli and mammalian toolboxes. The bp within the primer that is complementary to the vector is indicated in italics.
Expression system Vector Tag Application Restriction enzymes Forward primer (5′–3′) Reverse primer (5′–3′, reverse complement)
E. coli pOPINVHH_his His Crystallography NcoI and Bsu36I GCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAG GTGATGGTGGCCTGAGGAGACGGTGACCTGGGTC
pOPINVHH_flag_his His and FLAG Epitope detection
pOPINVHH_myc_his His and myc Epitope detection
pOPINVHH_BAP_his Avi-tagTM and His Biotinylation ATCATTCAAGCCTGAGGAGACGGTGACCTGGGTC
pOPINVHH_cys_his Cys and His Chemical conjugation ATGGTGACAGCCTGAGGAGACGGTGACCTGGGTC
pOPINVHH_sort_his Sortase Enzymatic labelling CGGCAGGCCGCCTGAGGAGACGGTGACCTGGGTC
pOPINVHH_snap_his SNAP-tag® Enzymatic labelling GTCCTTGTCGCCTGAGGAGACGGTGACCTGGGTC
Mammalian pOPINTTGneo-huIg1Fc huIg1Fc and His Fc fusions KpnI and PmeI GCGTAGCTGAAACCGGCCAGGTGCAGCTGGTGGAG CAGAACTTCCAGTTTAGGGGAGACGGTGACCTGGGTC
pOPINTTGneo
huIg1FcA
(Y407T)
huIg1Fc and His Bispecific nanobody generation
pOPINTTGneo huIg1FcB
(T334Y)
huIg1Fc and His Bispecific nanobody generation
pOPINE-eGFP GFP and His Fluorescent fusions NcoI and PmeI AGGAGATATACCATGCAGGTGCAGCTGGTGGAG
pOPINE-eYFP YFP and His Fluorescent fusions
pOPINE-mCherry mCherry and His Fluorescent fusions
Depending on the desired application, the selected nanobody is subcloned into a suitable expression vector. Below we detail the subcloning of a hit nanobody into the pOPINVHH_His vector as an example. The same process is followed to subclone the same nanobody into a mammalian vector for mammalian cell expression.
Restriction digestion of the pOPINVHH_His vector
Note: Work at a lab bench.
Prepare the following digestion mixture in a PCR tube (1 × 100 µL) (Table 15).
Table 15. Restriction digestion reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 80.5 µL
10× rCutSmartTM buffer 1× 10 µL
NcoI 2.5 units 2.5 µL
Bsu36I 2.5 units 2.5 µL
400 ng/µL vector DNA 18 µg 4.5 µL
Total n/a 100 µL
Briefly vortex and centrifuge the reaction tube.
Incubate at 37 °C for 1 h and then at 80 °C for 20 min.
Add 4× volume of B3 buffer (400 µL) and purify using 1× Purelink column from the Purelink PCR purification kit according to the manufacturer’s instructions. Elute using 50 µL of E1 buffer. Measure the A280 and expect a concentration between 60 and 100 ng/µL.
Preparation of nanobody insert
Note: Work in a safety cabinet.
From the master plate (step D7d), add 10 µL of the selected nanobody clones to a 24-well plate already filled with 4 mL/well of 2× YT containing 100 µg/mL ampicillin, cover with cell culture adhesive seal, and incubate overnight at 37 °C, shaking at 600 rpm.
Note: Work at a lab bench.
Use 2 mL of the overnight culture to isolate the plasmid using the QIAprep spin miniprep kit according to the manufacturer’s protocol. Elute in a final volume of 50 µL of EB. Measure the A280 and expect a concentration between 100 and 500 ng/µL.
Send an aliquot for sequencing along with the PhD seq Fwd primer. If it matches the sequence obtained by PCR (step D9g), continue to the next step. See General note 18.
Prepare the following reaction in a PCR tube (1 × 25 µL) (Table 16).
Table 16. PCR reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 9 µL
10 µM Common Fwd primer 0.5 µM 1.25 µL
10 µM His FLAG Rev primer 0.5 µM 1.25 µL
2× Phusion flash PCR master mix 1× 12.5 µL
10 ng/µL vector template (step F2b) 10 ng 1 µL
Total n/a 25 µL
Briefly vortex and centrifuge the reaction tube.
Set up the thermal cycler as below and perform the PCR (Table 17).
Table 17. PCR cycling conditions
Step Temperature (°C) Duration Number of cycles
Denaturation 98 10 s 1
Annealing 98 1 s 30
Extension 60 1 s
Final extension 72 5 s
Hold 72 1 min 1
Denaturation 12 Infinite hold -
Add 5 µL of 6× DNA gel loading buffer to the reaction tube and run alongside a well of 1× GeneRuler 1 kb DNA ladder on a 1% agarose gel containing 1× SYBRTM safe DNA gel stain. Electrophorese in 1× TBE buffer at 80 V for 40 min.
Visualise the ~500 bp amplified nanobody band using a LED Blue transilluminator, excise from the gel using a scalpel knife, and gel extract using one spin column from the Nucleospin Gel and PCR clean-up kit according to the manufacturer’s protocol. Elute using 50 µL of NE, measure the A280, and expect a concentration between 30 and 100 ng/µL.
Caution: Use Safe ImagerTM viewing glasses when visualising the DNA during excision of the band from the agarose gel and use caution when using scalpel knives.
Ligation and transformation
Note: Work at a lab bench.
Prepare the following reaction in a PCR tube (1 × 10 µL) (Table 18).
Table 18. In-Fusion reaction composition
Reagent Final concentration Volume
Nuclease-free water n/a 5 µL
5× CEII buffer 1× 2 µL
4 ng/µL insert (step F2h) 4 ng 1 µL
19.83 ng/µL vector DNA (step F1d) 19.83 ng 1 µL
Exnase II 1 µL
Total n/a 10 µL
Briefly vortex and centrifuge the reaction tube.
Incubate at 42 °C for 30 min using a thermal cycler. Add 40 µL of TE buffer to terminate the reaction.
Note: Work in a safety cabinet.
Gently mix 5 µL of in-fusion reaction with 25 µL of StellarTM competent cells in a sterile 2 mL microcentrifuge tube. Incubate on ice for 10 min.
Heat shock at 42 °C for 45 s and incubate on ice for 2 min. Add 200 µL of LB medium and incubate for 1 h at 37 °C agitating at 600 rpm.
Spread 50 µL of culture on a 1% agar LB plate containing 100 µg/mL ampicillin, 2 mM IPTG, and 40 µg/mL X-gal until dry. Incubate overnight at 37 °C. Expect many white colonies and few-to-no blue colonies.
Transfer two white colonies from the transformation plate to two wells of a 24-well plate already filled with 4 mL/well of 2× YT containing 100 µg/mL ampicillin. Cover with cell culture adhesive seal and incubate overnight at 37 °C, shaking at 600 rpm.
Note: Work at a lab bench.
Use 2 mL of the overnight culture to isolate the plasmid using the QIAprep spin miniprep kit according to the manufacturer’s protocol. Elute in a final volume of 50 µL of EB. Measure the A280 and expect a concentration between 100 and 500 ng/µL.
Send an aliquot for sequencing along with the PhD seq Fwd primer. If it matches the sequence obtained by PCR (step D9g), continue to the next step. See General note 18.
Proceed to large-scale expression using WK6 E. coli or Expi293TM as detailed in Le Bas et al. [12] or use in imaging experiments using other mammalian cell lines (see section G) depending on the expression vector that was selected.
Nanobody functionalisation and application
By using a selection of vectors from the E. coli and mammalian toolboxes, we demonstrate how nanobodies can be used as a reagent for pull downs using streptavidin and the pOPINVHH_BAP vector, for imaging by chemical conjugation using the pOPINVHH_Cys vector and by transfection with the pOPINE-GFP and pOPINE-YFP for in vivo and live cell imaging.
Confirmation of biotinylation of anti-GFP nanobody
The anti-GFP nanobody was cloned into the pOPINVHH_BAP vector, expressed at large scale in BL21(DE3)-R3-pRARE2-BirA to allow for in vivo biotinylation, and purified using Ni-NTA and size exclusion chromatography as detailed in section F and in Le Bas et al. [12] with some modifications. Briefly, LB agar plates contained 34 µg/mL chloramphenicol, 50 µg/mL spectinomycin, and 50 µg/mL carbenicillin, cultures of terrific broth were supplemented with 50 µg/mL spectinomycin and 50 µg/mL carbenicillin, and a final concentration of 0.2 mM biotin was added alongside 0.1 mM IPTG. Below, we detail how we confirm that the anti-GFP nanobody has been biotinylated.
Note: Work at a lab bench.
Add 2 µg of anti-GFP expressed in pOPINVHH_BAP or pOPINVHH_His and 1.5 µL of 50 µg/mL Streptavidin Alexa FluorTM 488 conjugate to a 1.5 mL microcentrifuge tube and make up the volume to 10 µL with PBS. Incubate for 30 min at 37 °C.
Add 10 µL of 2× Laemmli sample buffer, incubate at 95 °C for 5 min, and run on a NuPage 4%–12% bis tris precast gel in 1× MES buffer alongside Mark12TM unstained standard and BenchMarkTM fluorescent protein standard at 200 V for 40 min.
Image the in-gel fluorescence using the ProQ emerald 488 image acquisition preset on the ChemiDocTM imaging system (blue Epi excitation, 532/28 emission filter). Stain with InstantBlue® Coomassie protein stain (Figure 9).
Figure 9. Confirmation of nanobody biotinylation after expression in pOPINVHH_BAP and co-expression with BirA by analysis on SDS-PAGE. Samples were visualised by in-gel fluorescence and stained with InstantBlue® (Coomassie). Protein molecular weight markers in kDa were run in parallel.
Biotin-mediated pull downs
Below, we detail the utilisation of purified biotinylated anti-GFP nanobody in the purification of GFP from spiked E. coli lysate.
Note: Work at a lab bench.
Wash 100 µL of streptavidin DynabeadsTM M-280 twice with 1 mL of PBS.
Add 100 µL of 10 µg biotinylated anti-GFP nanobody diluted in PBS to the prepared beads and incubate for 30 min at RT using a sample mixer.
Collect the unbound sample and wash the beads three times with 1 mL of PBS.
Add 10 µg of pure GFP to E. coli lysate and make up to a final volume of 100 µL using PBS. Add this to the washed beads and incubate for 30 min at RT using a sample mixer.
Collect the unbound sample and wash the beads three times with 1 mL of PBS.
Add 20 µL of 2× Laemmli sample buffer to the beads.
To 10 µL of each loaded and unbound sample, add 10 µL of 2× Laemmli sample buffer.
Incubate all Laemmli-treated samples at 95 °C for 5 min and run on a NuPage 4%–12% bis tris precast gel in 1× MES buffer alongside Mark12TM unstained standard at 200 V for 40 min.
Stain with InstantBlue® Coomassie protein stain (Figure 12A).
Fluorophore conjugation
The anti-vimentin (VB3) nanobody was cloned into the pOPINVHH_Cys vector, expressed at large scale in WK 6 E. coli, and purified using Ni-NTA and size exclusion chromatography as detailed in section F. Below, we detail how to prepare fluorescently labelled VB3 nanobody (VB3-AF647) and its application in in vivo imaging.
Note: Work at a lab bench.
To 300 µg of purified VB3 nanobody, add 1 mM TCEP pH 8.0 to make up to a final volume of 100 µL and incubate for 20 min at 4 °C using a sample mixer.
Add 1 µL of Alexa FluorTM 647 C2-maleimide to the VB3 nanobody solution and incubate for 1 h at 4 °C using a sample mixer.
Remove any unbound Alexa FluorTM 647 from the fluorescently labelled VB3 nanobody using a ZebaTM dye and biotin removal spin column as per the manufacturer’s protocol. This sample is stored at 4 °C for two weeks protected from light but can be flash frozen and stored at -80 °C for an extended period.
Mix 10 µL of the VB3-AF647 with 10 µL of 2× Laemmli sample buffer, incubate at 95 °C for 5 min, and run on a NuPage 4%–12% bis tris precast gel in 1× MES buffer alongside PageRulerTM prestained protein ladder at 200 V for 40 min.
Image the in-gel fluorescence using the Oriele image acquisition preset on the ChemiDocTM imaging system (UV excitation, 590/110 emission filter). Stain with InstantBlue® Coomassie protein stain (Figure 10).
Measure the absorbance of the VB3-AF647 from 200 to 800 nm with baseline correction at 750 nm using the Nanodrop. Expect a peak at 280 and 651 nm, which correspond to VB3 nanobody and to Alexa FluorTM 647, respectively (Figure 10).
Figure 10. Conjugation of Alexa FluorTM 647 to VB3 nanobody (VB3-AF647) expressed in pOPINVHH_Cys as determined by in-gel fluorescence (left), InstantBlue® staining (middle), and absorbance profile (right)
In vivo visualisation
Note: Work in a safety cabinet.
Place a single coverslip into the wells of a 6-well culture plate. Seed 1 mL of 10,000 HeLa cells/well and culture in phenol red–free DMEM medium supplemented with 10% FBS, 1× GlutaMAXTM, and 1× penicillin-streptomycin at 37 °C and 5% CO2 in a CO2 incubator.
Note: Work at a lab bench.
After 24 h, wash the cells three times with 2 mL of PBS and fix with 2 mL of 4% paraformaldehyde for 20 min at RT.
Wash the cells three times with 2 mL of PBS and block the cells with 2 mL of confocal blocking buffer for 1 h at RT.
During the blocking step, prepare the fluorescently labelled VB3 nanobody solution for labelling vimentin in HeLa cells. In a 1.5 mL microcentrifuge tube, add 5 µL of VB3-AF647 (step G3c) to 1 mL of confocal dilution buffer PBS and mix gently by inversion.
Remove the blocking buffer from the cells, add the prepared VB3-AF647 (step G4d), and incubate for 2 h at 4 °C using a gyratory rocker.
Remove the VB3-AF647 solution and wash the cells three times with 2 mL of PBS.
For each coverslip, add a small drop of Fluoroshield mounting media with DAPI onto a microscope slide. Using Dumont tweezers, gently remove the coverslip from the well and place cell side down onto the mounting media. Remove excess liquid by gently pressing the coverslip slide down towards the microscope slide and secure the coverslip by adding nail varnish around the perimeter of the coverslip.
The stained cells on the prepared slides are imaged using a Leica SP8 confocal microscope at 63× objective with type F immersion liquid. Use Ex405 and Em430–550nm for DAPI detection and Ex633 and Em650 for Alexa FluorTM 647 detection.
Live cell imaging
The VB3 nanobody was cloned into the pOPINE-eGFP and -eYFP vectors as detailed in section F. The produced vectors are referred to as pOPINE-VB3-eGFP and pOPINE-VB3-eYFP. Below, we detail how to transiently transfect HeLa cells with the pOPINE-VB3-eGFP and pOPINE-VB3-eYFP vectors for live-cell imaging of vimentin filaments.
Note: Work in a safety cabinet.
In an 8-well chambered coverslip, seed 200 µL containing 25,000 HeLa cells/well and culture in phenol red–free DMEM medium supplemented with 10% FBS, 1× GlutaMAXTM, and 1× penicillin-streptomycin at 37 °C and 5% CO2 in a CO2 incubator.
After 24 h, prepare the transfection mixture as follows. In a 500 µL microcentrifuge tube, add 10 µL of serum-free DMEM medium and gently mix in 2 µL of 100 ng/µL pOPINE-VB3-eGFP and pOPINE-VB3-eYFP vectors. Add 0.5 µL of Fugene transfection reagent and incubate for 10 min at RT.
Remove the old media from each of the wells in the 8-well microscope chambered coverslip and add 270 µL/well of DMEM medium supplemented with 10% FBS. Add each DNA-Fugene complex dropwise, by gently depressing the plunger of the pipette, into each well and incubate the chambered coverslip at 37 °C and 5% CO2 in a CO2 incubator for 48 h.
Live-cell imaging is captured using a Leica SP8 confocal microscope at 63× objective with immersion liquid. Use Ex405 and Em430–550nm for DAPI detection and Ex488 and Em518-558 for GFP and YFP detection.
Data analysis
Analysis of sequencing data (section D9)
Drag and drop the .seq files into IMGT/V-QUEST. Make the following selections on the site: Species: Vicugna Pacos (alpaca); Receptor type or locus: Ig; choose to get the results in excel format. Submit. See General note 21.
There are multiple tabs in the produced Excel file. The fifth tab (AA-sequences) is the one that we will work in. Sort the sequences by productive, non-productive, and no results.
Then, sort the CDR3-IMGT of the productive sequences to visualise the CDR3 clusters. Highlighting the different CDR3 clusters in different colour aids in the selection of representative clones, which will be used for small-scale expression.
Open the .seq files of the selected clones in SnapGene to obtain the amino acid sequences. Copy the single letter amino acid sequence and paste into Expasy - ProtParam tool. Delete the pelB sequence (MKYLLPTAAAGLLLLAAQPAMA), submit, and note the expected kDa, pI, and extinction coefficient (ϵ), assuming that all Cys residues are reduced.
Analysis of BLI data for off-rate determination (section E5)
In the Preprocess data tab: Subtract the background of the no nanobody well to all the other tested nanobody wells. Correct the Y axis to the baseline step and the interstep to the dissociation step. Apply the Savitzky-Golay filtering.
In the Kinetic analysis tab: Analyse both the association and dissociation steps. Use a 1:1 binding model. Use a local (individual) fitting. Fit the full association and dissociation steps and note the calculated off-rate (kdis).
Validation of protocol
Identification of nanobodies to the G protein of Nipah virus
The improved workflow for nanobody generation was exemplified by the identification of nanobodies to the receptor-binding G protein of the Nipah virus (NivG). The extracellular region of the NivG protein (residues 183–602) with N-terminal hexahistidine tag was expressed in Expi293TM cells and purified by a combination of immobilised metal affinity chromatography (IMAC) and size exclusion (Figure 11A). Comparison of pre- and post-immune sera in a seroconversion ELISA of biotin-tagged NivG confirmed that an immune response had been generated to the antigen (Figure 11B). Following two rounds of panning of the VHH library 13, 93 phage clones were picked and tested in a phage ELISA. The sequences of phage binders were amplified by PCR and sequenced and the translated VHHs were clustered by CDR3 sequence identity. A representative clone from each of the three major clusters was selected, expressed at small scale (4 mL cultures), and purified by IMAC (panel C). Binding to NivG was confirmed by titration ELISA (panel D). Analysis of binding off-rates by BLI confirmed that the three clones (A8, B7, and D5) bound to NivG (panel E) and were prioritised for large-scale expression.
Figure 11. Generation of NivG-binding nanobodies from an immunised llama. (A) SDS-PAGE of fractions from gel filtration of purified NivG, (B) seroconversion ELISA of pre- and post-immune sera following immunisation with NivG, (C) SDS-PAGE of purified anti-NiVG nanobodies from small-scale expression, (D) binding of purified nanobodies to NivG 183-602 by titration ELISA, (E) BLI traces of nanobody association to and dissociation from NivG 183-602; off-rates for the three nanobodies are shown.
Functionalisation of nanobodies for purification and imaging
The functionalisation of nanobodies using three of the vectors presented in Table 1 and their application was exemplified using an anti-GFP [9] and an anti-vimentin (VB3) [10] nanobody. The anti-GFP nanobody was expressed in the pOPINVHH_BAP vector, biotinylated in vivo, and used for pull-down of GFP from spiked E. coli lysate with magnetic-coupled streptavidin beads (Figure 12A). The anti-VB3 nanobody was expressed in E. coli using the pOPINVHH-cys vector, purified, and coupled to AlexaFluorTM 647 via a maleimide linker. HeLa cells were fixed and permeabilised and the vimentin filaments were stained with the anti-VB3 nanobody conjugate (Figure 12B). The same nanobody was genetically fused to either YFP or GFP by cloning into pOPINE-YFP and pOPINE-GFP vectors, respectively. Live HeLa cells were transfected with these vectors and imaged by confocal microscopy to visualise vimentin filaments (Figure 12C).
Figure 12. Functionalisation of nanobodies. (A) Pull-down of GFP spiked into an E. coli lysate by biotinylated anti-GFP nanobody coupled to streptavidin magnetic beads. Detection of vimentin in HeLa cells using (B) fluorescently labelled anti-vimentin nanobody (VB3-AF647) and (C) transiently transfected with VB3-eGFP and VB3-eYFP.
General notes and troubleshooting
General notes
Preparation of biotinylated proteins can be achieved in two ways: chemical conjugation or by in vitro biotinylation of a protein with the Avi-tag® sequence and co-expression with BirA.
Expect colour development within 5–15 min of substrate addition. If signals are low, the longest incubation before absorbance measurement is 1 h.
If we receive 170 mL of llama blood in 17 × 10 mL blood tubes without anticoagulant, the addition of an equal volume of PBS will result in a total of 340 mL of diluted blood. Fifteen millilitres of diluted blood are required for each 50 mL tube with 15 mL Histopaque®-1007 already added; therefore, a total of 22.6 tubes are required. As such, the 23rd tube will have less than 15 mL of diluted blood.
Holding the 50 mL conical centrifuge tube at a 45° angle allows for the gentle addition of the diluted blood over the Histopaque®-1007 to prevent the disturbance of the Histopaque®-1007 layer. During the process of gently layering the blood on top of the Histopaque®-1007 over the many 50 mL conical centrifuge tubes, the lower layer will turn red as the heavier red blood cells fall to the bottom of the tube. This is to be expected.
Mixing the trypan blue with the PMNCs too vigorously may damage the PMNCs, resulting in low viability values that may not be a true representation of the actual viability.
If no PCR workstation or dedicated RNase free space is available, keeping boxes of tips and bags microcentrifuge tubes that are used only for RNA work will be sufficient if gloves are changed regularly and RNaseZapTM is used.
RNA should be stored at -20 °C under ethanol where it is stable for extended periods of time. Once it is resuspended in water, the stability is reduced and, as such, for the best possible results, we recommend that cDNA synthesis is carried as soon as RNA has been resuspended in water (step C3i).
For the electroporations, expect ms values greater than 4.5.
Note that the estimated library sizes for the small-scale library will vary slightly from that of the scaled library.
An example on how to calculate how many microlitres of in-fusion reactions would be needed to make a library with a size of 1 × 106: We purify the 10 µL in-fusion reaction using a Purelink column, eluting in 20 µL nuclease-free water. We use 5 µL of this for the electroporation, which translates to 2.5 µL of the original in-fusion reaction being added to 30 µL of competent TG1. Hence, if 2.5 µL of in-fusion library yielded 373 CFU at 1:100 dilution, the small-scale library size would be 1.492 × 105 CFU/mL. To create a 1 × 106 CFU/mL library, 6.7 × 2.5 µL of in-fusion reactions would be required, which is just over 1 × 10 µL of in-fusion reaction. If enough VHH2 and digested pADL23c backbone has been prepared, we routinely use more in-fusion reactions than necessary, i.e., 10 × 10 µL, to ensure that we have the best chance of producing a 1 × 106 CFU/mL library.
If there is a large discrepancy between the library size as calculated by titre and by plate density, use the plate density to guide your decision if the library is big enough. See Figure 7.
Begin by using 200 µL of the TG1 stock when preparing the starting culture.
Begin by using 20 µL of the isolated library phage and pan 1 sub-library stock when preparing the starting culture.
In some cases, it might take longer than 2 h for the culture to reach an OD600 of 0.5. Continue culturing until the required OD600 is reached. As long as the TG1 control plate in either step D3k or D6i are negative, you can be assured that this might just be variance in the library and/or sub-library.
A TG1-only control functions to show that the TG1s used have not been infected with phage prior to this titration step. See Troubleshooting 1 and 2.
Steps C8h–i show how diverse the library is. The titration of the amplified and recovered library (step D3l) gives an indication of how many phage particles we can make using our diverse library.
Adjust the volume of the prepared PCR master mixture according to the number of actual ELISA positive clones.
Volumes and concentrations of the DNA and primers might be supplier specific. Check the requirements of your local sequencing company.
Use enough antigen to load the biosensors to result in a 1 nm signal increase.
Polypropylene plates must be used as they do not bind protein.
The IMGT site can only process 48 sequences at a time. If more than 48 clones have been sequenced, combine the two generated IMGT Excel files together before sorting by V-DOMAIN functionality and CDR3-IMGT.
Troubleshooting
Problem 1: Growth on TG1 control plates.
Possible cause: Indication of phage contamination of the E. coli before our isolated/eluted phage was added.
Solution: Prepare fresh log-phase TG1 from fresh aliquot of TG1 in the -80 °C. Repeat the titration of the phage in question.
Problem 2: Growth on TG1 control plates.
Possible cause: Ampicillin stock is old.
Solution: Using a new ampicillin aliquot, prepare fresh 1% agar LB plates with ampicillin. Take a colony from the “contaminated” TG1 control plate as well as the glycerol stock used to grow the log-phase TG1 and streak it on the new plates. If no growth is observed, this indicates that the ampicillin stock is old and that the TG1 was not infected with phage prior to this.
Problem 3: Only a single hit is found on the anti-M13 ELISA plate.
Possible cause: Only a few binders are present after the rounds of panning.
Solution: Another 93 colonies can be picked to prepare a second master plate to test in an anti-M13 ELISA.
Problem 4: None or very few anti-M13 ELISA hits present after the second round of panning using 5 nM of target protein.
Possible cause: Might not have enrichment.
Solution: Go back to 50 nM phage and pan using 10 nM antigen. If there are still not hits, try Troubleshooting 3. If there are still no hits, consider looking at another library. Use the seroconversion ELISA data to guide this decision.
Acknowledgments
This work was supported by the Rosalind Franklin Institute, with funding delivery partner the Engineering and Physical Sciences Research Council UK (EPSRC) and grants from the Biotechnology and Biological Sciences Research Council UK (BBSRC) for nanobody discovery (ref. BB/V018523/1) and the Wellcome Trust for technology development (ref. 223733/Z/21/Z). Graphical overview created with BioRender.
Competing interests
The authors declare that they have no competing interests with respect to the work described.
Ethical considerations
Immunisations and handling of the llamas were performed under the authority of the UK Home Office project license PA1FB163A.
References
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Bao, G., Tang, M., Zhao, J. and Zhu, X. (2021). Nanobody: a promising toolkit for molecular imaging and disease therapy. EJNMMI Res. 11(1): 6.
Muyldermans, S. (2020). A guide to: generation and design of nanobodies. FEBS J. 288(7): 2084–2102.
Parker, J. L., Deme, J. C., Wu, Z., Kuteyi, G., Huo, J., Owens, R. J., Biggin, P. C., Lea, S. M. and Newstead, S. (2021). Cryo-EM structure of PepT2 reveals structural basis for proton-coupled peptide and prodrug transport in mammals. Sci. Adv. 7(35): eabh3355.
Girt, G. C., Lakshminarayanan, A., Huo, J., Dormon, J., Norman, C., Afrough, B., Harding, A., James, W., Owens, R. J., Naismith, J. H., et al. (2021). The use of nanobodies in a sensitive ELISA test for SARS-CoV-2 Spike 1 protein. R. Soc. Open Sci. 8(9): e211016.
Huo, J., Mikolajek, H., Le Bas, A., Clark, J. J., Sharma, P., Kipar, A., Dormon, J., Norman, C., Weckener, M., Clare, D. K., et al. (2021). A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19. Nat. Commun. 12(1): 5469.
Akkermans, O., Delloye-Bourgeois, C., Peregrina, C., Carrasquero-Ordaz, M., Kokolaki, M., Berbeira-Santana, M., Chavent, M., Reynaud, F., Raj, R., Agirre, J., et al. (2022). GPC3-Unc5 receptor complex structure and role in cell migration. Cell 185(21): 3931–3949.e26.
Pardon, E., Laeremans, T., Triest, S., Rasmussen, S. G. F., Wohlkönig, A., Ruf, A., Muyldermans, S., Hol, W. G. J., Kobilka, B. K., Steyaert, J., et al. (2014). A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. 9(3): 674–693.
Kirchhofer, A., Helma, J., Schmidthals, K., Frauer, C., Cui, S., Karcher, A., Pellis, M., Muyldermans, S., Casas-Delucchi, C. S., Cardoso, M. C., et al. (2010). Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17(1): 133–138.
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Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin
EN Eider Nuñez
AM Arantza Muguruza-Montero
SA Sara M. Alicante
AV Alvaro Villarroel
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4963 Views: 735
Reviewed by: Olga KopachNeha Nandwani Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in eLIFE Feb 2023
Abstract
The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions.
Key features
• Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding.
• Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts.
• Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development.
• Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.
Graphical overview
Fluorescence emission profiles of dansylated-calmodulin (D-CaM) in different states. Fluorescence emission spectra of D-CaM upon excitation at 320 nm are depicted. Conditions include apo-D-CaM (gray), holo-D-CaM (red), apo-D-CaM bound to peptide (blue), and holo-D-CaM bound to peptide (purple). Corresponding structural representations of D-CaM next to each condition are superimposed on the respective spectra along with the hydrophobicity of the dansyl environment, which increases upon binding of peptide or Ca2+ to D-CaM. Upon peptide binding to D-CaM, there is an enhancement in the fluorescent intensity of the spectra; upon Ca2+ binding, there is an enhancement of the intensity and a leftward shift of the spectra.
Keywords: Dansyl-Calmodulin CaM-target Steady-state fluorescence spectroscopy Calcium dependency Dansyl-chloride Peptide Protein
Background
Calmodulin (CaM), a pivotal Ca2+-binding protein, intricately regulates essential biological functions by binding to the cation, thereby exerting meticulous control over an array of effector proteins. This interaction induces conformational changes in CaM, critically influencing cellular processes such as muscle contraction and neurotransmitter release, as exhaustively elucidated by Chin and Means [1] and Rhoads and Friedberg [2].
The trajectory of unraveling CaM's multifaceted role spans decades, commencing in the 1970s with the identification of cyclic nucleotide phosphodiesterase as one of the initial proteins binding to CaM, as underscored by Rasmussen et al. [3]. Subsequently, Klee and Vanaman's seminal work in 1982 laid the foundational understanding of CaM's centrality in cellular signal transduction. The ongoing delineation of over 300 target peptides for CaM, meticulously documented by Klee and Vanaman [4], accentuates its indispensability in diverse cellular processes.
Technological advancements, such as dansylation [5] and fluorogenesis [6], have significantly contributed to the precise identification and characterization of CaM targets, thereby enhancing our understanding of fundamental cellular processes. Among these tools, dansyl-CaM (D-CaM), a derivative of CaM conjugated with dansyl chloride (DNSC) [5-(dimethylamino)naphthalene-1-sulfonyl chloride], emerges as a distinctive and powerful instrument for analyzing interactions with peptides and proteins. DNSC specifically interacts with primary amino groups, forming stable blue or blue-green fluorescent sulfonamide adducts with aliphatic and aromatic amines (Tyr, Phe, Trp, etc.). The incorporation of dansyl into CaM enables the sensitive detection and thorough examination of this modified protein using fluorescence-based techniques.
High-resolution structural scrutiny of apo-CaM and holo-CaM has unveiled intricate Ca2+-induced structural changes, laying bare hydrophobic interfaces, aligning with the observations of Chin and Means [1] and Rhoads and Friedberg [2]. Structural analyses of CaM–peptide complexes reveal a commonality in interaction patterns, particularly in the hydrophobic domains of the CaM protein, resembling interactions observed with classical proteins and inhibitors [2]. Significantly, these peptides manifest a positively charged amphiphilic alpha-helical structure, irrespective of their amino acid sequences, as expounded upon by O’Neil and DeGrado [7]. The observed high affinity (Kd) of these peptides, falling in the range of 10-9–10-12 M, positions them as putative bioactive entities, exceeding the affinity of traditional organic CaM inhibitors (Kd ~10-3 M), as demonstrated by Chen et al. [8] and Peersen et al. [9].
Nevertheless, the conventional understanding of CaM function encounters challenges with the discovery of proteins preferentially binding to apo-CaM, influencing their Ca2+ affinity, as exemplified by Smith et al. [10]. Additionally, CaM-binding partners exhibit minimal or no sequence similarity, posing a conundrum for attempts at structural categorization.
In navigating this intricate terrain, the elucidated method emerges as a potent tool, facilitating expeditious, cost-effective, and reliable identification of CaM targets even in instances of deviation from canonical binding patterns. This approach not only sheds light on diverse interactions involving D-CaM but also imparts invaluable insights into the nuanced relationships between proteins within the dynamic realm of CaM.
Materials and reagents
Biological materials
Calmodulin in pET14b plasmid or other bacterial protein expression vector
Peptides from Proteogenix (https://www.proteogenix.science/) or another source
Peptides exhibiting sequences, structures, or functions favorable for CaM binding are recommended, with an advised minimum length of 5 and a maximum of 50 amino acids to avoid potential challenges in purity and yield. See Note 1.
E. coli BL21 DE3 (Sigma-Aldrich, catalog number: 69450-M) (other strains may work as well)
Reagents
Dansyl chloride (DNSC) (Merck, Supelco, catalog number: 03641)
Sephadex G-25 (Merck, Sigma-Aldrich, catalog number: S5772)
Sepharose CL-4B (Merck, Sigma-Aldrich, catalog number: 4B200)
Base Trizma (Tris) (Merck, Sigma-Aldrich, catalog number: T1503)
HEPES (Thermo Fisher, Thermo Scientific Chemicals, catalog number: J16926.A1)
Potassium chloride (KCl) (Merck, Sigma-Aldrich, catalog number: P9541)
Ethyleneglycol-bis(β-aminoethyl)-N,N,N,N-tetraacetic acid (EGTA) (Merck, Millipore, catalog number: 324626)
Calcium chloride dihydrate (CaCl2) (Merck, Millipore, catalog number: 208291)
Sodium chloride (NaCl) (Merck, Sigma-Aldrich, catalog number: S3014)
30% Acrylamide/Bis solution, 37.5:1 (Bio-Rad, catalog number: 1610158)
Sodium dodecyl sulfate (SDS) (Merck, Sigma-Aldrich, catalog number: 436143)
Ammonium persulfate (APS) (Merck, Sigma-Aldrich, catalog number: A3678)
Tetrametiletilendiamina (TEMED) (Thermo Fisher, catalog number: 17919)
Isopropanol (Merck, Sigma-Aldrich, catalog number: I9516)
Coomassie Brilliant Blue R-250 powder (CBB R-250) (Bio-Rad, catalog number: 1610400)
Methanol (Merck, Sigma-Aldrich, catalog number: 1060351000)
Acetic acid (glacial) 100% (Merck, Supelco, catalog number: 100066)
Phenyl-Sepharose CL-4B (Merck, Cytiva, catalog number: GE17-0150-01)
Dimethyl Sulfoxide (DMSO) (Merck, Sigma-Aldrich, catalog number: D2650)
N,N-Dimethylformamide (DMF) (Merck, Supelco, catalog number: DX1730)
Acetone (Merck, Sigma-Aldrich, catalog number: 179124)
Ethanol (Merck, Sigma-Aldrich, catalog number: 34852-M)
Glycerol (Merck, Sigma-Aldrich, catalog number: G5516)
Nitrocellulose membrane (Merck, Cytiva, catalog number: GE10600001)
Bradford (Merck, Supelco, catalog number: B6916)
Ponceau Red (Ponceau 4R) (Merck, Supelco, catalog number: 18137)
Trifluoroacetic acid (TFA) (Merck, Sigma-Aldrich, catalog number: 302031)
Ammonium hydroxide solution (NH4OH) (Merck, Sigma-Aldrich, catalog number: 221228)
Acetonitrile (Merck, Sigma-Aldrich, catalog number: 34851)
LB broth (Lennox) (Merck, Sigma-Aldrich, catalog number: L3022)
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Merck, Sigma-Aldrich, catalog number: I5502)
Phenylmethanesulfonyl fluoride (PMSF) (Merck, Roche, catalog number: 10837091001)
Solutions
Dansylation buffer (D buffer) (see Recipes)
Lysis buffer (L buffer) (see Recipes)
Equilibration buffer (CQ buffer) (see Recipes)
Wash buffer (CW buffer) (see Recipes)
High salt wash buffer (CHSW buffer) (see Recipes)
Elution buffer (CE buffer) (see Recipes)
Fluorescence buffer (F buffer) (see Recipes)
Calcium buffer (Ca buffer) (see Recipes)
APS 10% (see Recipes)
SDS 10% (see Recipes)
Tris-HCl 1.5 M, pH 8.8 (see Recipes)
Tris-HCl 1 M, pH 6.8 (see Recipes)
15% Acrylamide electrophoresis gel RESOLVING (see Recipes)
Stacking acrylamide gel (see Recipes)
Running buffer 10× (see Recipes)
Loading buffer 5× (see Recipes)
Coomassie Blue (see Recipes)
Fast De-staining solution (see Recipes)
De-staining solution (see Recipes)
Ampicillin 100 μg/mL (see Recipes)
Ponceau Red (see Recipes)
Recipes
Dansylation buffer (D buffer)
Reagent Final concentration Quantity
CaCl2 (1 M) 20 mM 2 mL
Tris-HCl (1 M, pH 8.5) 100 mM 10 mL
H2O n/a 88 mL
Total n/a 100 mL
Lysis buffer (L buffer)
Reagent Final concentration Quantity
EDTA (1 M) 2 mM 0.2 mL
Tris-HCl (1 M, pH 7.5) 50 mM 5 mL
PMSF (0.1 M) 2 mM 2 mL
H2O n/a 83.8 mL
Total n/a 100 mL
Equilibration buffer (Q buffer)
Reagent Final concentration Quantity
CaCl2 (1 M) 5 mM 0.5 mL
Tris-HCl (1 M, pH 7.5) 50 mM 5 mL
NaCl (1 M) 100 mM 10 mL
H2O n/a 84.5 mL
Total n/a 100 mL
Wash buffer (W buffer)
Reagent Final concentration Quantity
CaCl2 (1 M) 0.1 mM 10 µL
Tris-HCl (1 M, pH 7.5) 50 mM 5 mL
NaCl (1 M) 100 mM 10 mL
H2O n/a 84.90 mL
Total n/a 100 mL
High salt wash buffer (CHSW buffer)
Reagent Final concentration Quantity
CaCl2 (1 M) 0.1 mM 10 µL
Tris-HCl (1 M, pH 7.5) 50 mM 5 mL
NaCl (1 M) 500 mM 50 mL
H2O n/a 44.90 mL
Total n/a 100 mL
Elution buffer (E buffer)
Reagent Final concentration Quantity
EGTA (1 M) 1 mM 100 µL
Tris-HCl (1 M, pH 7.5) 50 mM 5 mL
Total n/a 94.9 mL
Fluorescence buffer (F buffer)
Reagent Final concentration Quantity
KCl (1 M) 120 mM 12 mL
HEPES (1 M, pH 7.4) 50 mM 5 mL
NaCl (1 M) 5 mM 0.5 mL
EGTA (1 M) 5 mM 0.5 mL
H2O n/a 82 mL
Total n/a 100 mL
See Note 2.
Calcium buffer (Ca buffer)
Reagent Final concentration Quantity
KCl (1 M) 120 mM 12 mL
HEPES (1 M, pH 7.4) 50 mM 5 mL
NaCl (1 M) 5 mM 0.5 mL
EGTA (1 M) 5 mM 0.5 mL
CaCl2 (1 M) 20 mM 2 mL
H2O n/a 80 mL
Total n/a 100 mL
See Note 3.
APS 10%
Reagent Final concentration Quantity
APS 10% 1 g
H2O n/a 10 mL
SDS 10%
Reagent Final concentration Quantity
SDS 10% 1 g
H2O n/a 10 mL
Tris-HCl pH 8.8 1.5 M
Reagent Final concentration Quantity
Tris 1.5 M 36.3 g
H2O n/a 200 mL
*Note 4
Tris-HCl 1 M pH 6.8
Reagent Final concentration Quantity
Tris 1 M 12.1 g
H2O n/a 100 mL
See Note 5.
15% Acrylamide electrophoresis gel RESOLVING (for one gel)
Reagent Final concentration Quantity
Acrylamide 15% 2.5 mL
Tris 1.5 M, pH 8.8 390 mM 1.3 mL
10% SDS 0.1% 50 µL
10% APS 0.1% 50 µL
TEMED 0.004% 2 µL
H2O n/a 1.2 mL
Total n/a 5 mL
Electrophoresis gel STACKING (for one gel)
Reagent Final concentration Quantity
Acrylamide 4.95% 0.33 mL
Tris 1 M, pH 6.8 125 mM 0.250 mL
10% SDS 0.1% 20 µL
10% APS 0.1% 20 µL
TEMED 0.01% 2 µL
H2O n/a 1.2 mL
Total n/a 2 mL
Running buffer 10×
Reagent Final concentration Quantity
Tris 25 mM 33 g
Glycine 1.92 M 144 g
SDS 1% 10 g
H2O n/a 1,000 mL
Total n/a 1,000 mL
Loading buffer 5×
Reagent Final concentration Quantity
Tris HCl 1 M pH 6.8 250 mM 5 mL
Glycerol 50% 10 mL
SDS 10% 2 g
Bromophenol blue 1% 0.125% 250 µL
H2O n/a 4.75 mL
Total n/a 10 mL
Coomassie Blue
Reagent Final concentration Quantity
Ethanol 50% 125 mL
Acetic acid 10% 25 mL
CBB R-250 0.25% 625 g
H2O n/a 100 mL
Total n/a 200 mL
*Note 6
Fast Coomassie de-staining
Reagent Final concentration Quantity
Ethanol 10% 10 mL
Acetic acid 20% 20 mL
H2O n/a 70 mL
Total n/a 100 mL
Coomassie de-staining
Reagent Final concentration Quantity
Acetic acid 10% 10 mL
H2O n/a 90 mL
Total n/a 100 mL
Ampicillin 100 µg/mL
Reagent Final concentration Quantity
Ampicillin 0.1 g/mL 1 g
H2O n/a 10 mL
Laboratory supplies
Standard dialysis tubing with 2000 MW cut off (e.g., Membra-Cel, Viskase, catalog number: 300911011)
Filters, 0.2 µm diameter (SARSTEDT, catalog number: 83.1826.001)
Protein concentrator (Amicon R-Ultra, 15 mL, 3 KDa) (Millipore, Merck, catalog number: UFC9003)
Magnetic stirrer (Fisherbrand, Fisher Scientific, catalog number: 11808892)
Small columns (1–5 mL bed volume) (empty PD-10 column for gravity flow purification) (Cytiva, catalog number: 17043501)
Electrophoresis chamber (Mini-PROTEAN Tetra Vertical Electrophoresis Cell) (Bio-Rad, catalog number: 1658004)
Handcast electrophoresis gel accessories (for 1.00 mm thickness gels) (Bio-Rad, catalog number: 1658001FC)
Microtubes, 1.5 mL (Eppendorf, catalog number: 0030120086)
Centrifuge tube, 50 mL (Avantor, VWR, catalog number: 525-0634)
Equipment
Orbital incubator (Sartorius, model: Certomat BS-1, catalog number: 20444445202)
Spectrophotometer (VWR, model: V-1200, catalog number: 634-60009
Balance (Fisher Scientific, model: FPOS622, catalog number: 8344272595)
Hot plate stirrer (Fisher Scientific, model: AREX, catalog number: 15369664)
pH meter (pH and ORP table-top bench meter laboratory Tester Hanna) (Servovendi, catalog number: 1965)
Centrifuge with fixed-angle rotor (Beckman, model: Avanti J-20 XP, catalog number: 8043-30-1171)
Rotor JA14 (Beckman, catalog number: 339247)
Microcentrifuge (Eppendorf, model: 5430R, catalog number: 5428000210)
Eppendorf® rotor F-35-6-30 (Eppendorf, Merck, catalog number: EP5427716009)
Eppendorf® rotor F-45-48-11 (Eppendorf, Merck, catalog number: EP5427755004)
Spectro fluorimeter (we used an SLM-Aminco 8100 Series 2, not commercially available)
Quartz cuvette with two transparent faces (we used 3 mm light path, 100 μL volume) (Hellma, catalog number: 105-251-15-40)
Software and datasets
Sigmaplot 11.0 (Systat Software, Inc; 2008) (any other scientific data analysis and graphing software can be used)
Maxchelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-TS.htm)
Procedure
CaM expression and purification
The human CaM gene, inserted into the pET-14b expression vector, is introduced into BL21-DE3 E. coli (other bacteria strain to express non-toxic heterologous genes can also be used). The purification procedure for CaM has been adapted from existing literature [11] and results in substantial yields of soluble protein, as outlined below.
Protein expression
Cultivate BL21-DE3 cells from glycerol stock in 1 L of LB medium at 37 °C, supplemented with 100 μg/mL ampicillin, until the optical density (A600) reaches 0.8–1.
Induce protein expression by adding 0.4 mM IPTG and continue cultivation for 4–6 h at 37 °C or overnight at 20 °C.
Cell harvesting and resuspension
Centrifuge the cells to collect them (9,000× g for 9 min at 4 °C) and wash the cell pellet twice with 50 mL of fresh lysis buffer.
Resuspend the cell pellet in 30 mL of lysis buffer and store the sample in 10 mL aliquots at -20 °C.
Sample preparation
Thaw an aliquot on ice and perform sonication (three cycles of 10 s at 50 kHz; keep the sample on ice).
Subject the sample to three freeze–thaw cycles by alternating between a dry ice ethanol bath and a 37 °C water bath.
Centrifuge the sample in a microcentrifuge at 14,000× g for 15 min.
Heat the supernatant to 95 °C for 5 min, followed by centrifugation as previously described. This step leverages CaM's enhanced thermal stability.
Chromatography
Introduce CaCl2 to the supernatant (final concentration: 5 mM). Load the sample at room temperature onto a 5 mL Phenyl–Sepharose column pre-equilibrated with CQ buffer. The chromatography can be achieved through gravity flow or by utilizing a peristaltic pump. Wash the column with 20 column volumes of CW buffer followed by 10 column volumes of CHSW buffer.
Elute CaM with 20 column volumes of CE buffer, taking fractions of 500 µL.
Analysis and storage
Mix 20 µL of CaM with 5 µL of 5× loading buffer. Load the mixture onto a 15% acrylamide gel and perform electrophoresis using a 1× dilution of the running buffer. Run the gel at a voltage of approximately 120–150 V for 90 min. Stain the gel with Coomassie Blue for 10 min and destain first using fast destaining for 15 min followed by regular destaining for 30 min. Concentrate the sample to have a final concentration of at least 1 mg/mL using an Amicon centrifugal filter of 3 kDa. Dialyze the fractions against MilliQ water,
Store the purified CaM at -20 °C at 1 mg/mL in fractions of 1 mL or lyophilize it in fractions of 1 mL.
CaM concentration estimation can be done via absorbance at 276 nm, with ϵ276 = 3,030 M-1·cm-1. Alternatively, employ the Bradford method for quantification.
Dansylation of calmodulin
CaM preparation
Begin by diluting CaM in D buffer to achieve a final concentration of 1 mg/mL.
Dansyl chloride preparation
Dissolve dansyl chloride in acetone at a concentration of 2.17 mg/mL.
Store this dansyl chloride solution at either 4 °C or -20 °C in a dark environment. It remains stable for an extended period, often several months.
Dansylation process
Add 12.5 μL of the prepared dansyl chloride solution to 1 mL of the CaM solution at 1 mg/mL. This results in a final concentration of dansyl chloride of approximately 100 μM.
Incubate the mixture at room temperature, in darkness, for 2 h. During this time, gently vortex the mixture every 20 min.
Separation of dansylated CaM (D-CaM)
To separate D-CaM from any unreacted dansyl chloride, you will need a disposable column packed with approximately 1 mL of Sephadex G-25.
Equilibrate approximately 250 mg of dry resin with distilled water.
Load the D-CaM mixture onto the column and collect fractions of 50–100 μL each.
The initial fractions eluted, known as the "excluded fraction," contain the D-CaM conjugate.
See Note 7.
To determine the specific dansylated residues in CaM, tryptic digestion coupled with mass spectroscopy or gas-phase protein sequencers have been employed, reporting dansylation at either Lys75 or Lys 115 [12,13]. The mass spectrometry analysis revealed the binding of up to four dansyl molecules per CaM. Furthermore, tandem mass spectrometry of tryptic peptides strongly indicates dansylation at Ala1 and Lys148 [14]. The identification of the remaining two dansylated residues is pending further investigation; however, the data are consistent with the possibility of them being Lys75 and Lys115 [14].
Fraction analysis
Swiftly verify the presence of the protein in the collected fractions by performing dot blotting on nitrocellulose and staining it with Ponceau Red. Additionally, analyze the fractions using 15% SDS-PAGE gels. For a more detailed examination, record the emission spectra of each sample (as further explained below).
D-CaM storage
Gather the fractions containing D-CaM and concentrate them if needed. We recommend stocks between 0.5 and 5 µM.
Store the resulting D-CaM aliquots in a dark environment at -20 °C or, alternatively, as lyophilized samples. Typically, the conjugate retains its properties when stored at -20 °C for several months or more.
See Note 8.
Protein concentration determination
Utilize the Bradford assay to determine the protein concentrations of D-CaM, using unlabeled CaM as a standard. Alternatively, measure the concentration of D-CaM by UV absorption at 320 nm (ϵ320 = 3,400 M-1·cm-1).
Dansyl moiety concentration:
Determine the concentration of the incorporated dansyl moiety via spectroscopy.
When possible, calculate the number of specific dansylated residues within a D-CaM molecule.
This process allows for the efficient dansylation of CaM, making it ready for various downstream applications and analyses.
Peptide resuspension
When working with peptides, start by dissolving them in distilled, sterile water. This is especially suitable for short peptides (<5 residues). For each specific peptide, choose the most appropriate conditions to ensure optimum solubility based on its sequence.
Calculate overall charge; begin by assessing the overall charge of the peptide:
Assign a value of -1 for each acidic residue, including aspartic acid (Asp or D), glutamic acid (Glu or E), and the C-terminal -COOH.
Assign a value of +1 for each basic residue, including arginine (Arg or R), lysine (Lys or K), histidine (His or H), and the N-terminal -NH2. Calculate the net charge of the peptide.
Positive charge peptides; if the overall charge of the peptide is positive:
Attempt to dissolve the peptide in water initially. If water does not work, try a 10%–30% acetic acid solution. If the peptide still does not dissolve, add a small amount of TFA (<50 μL) to solubilize it and then dilute to the desired concentration.
Negative charge peptides; if the overall charge of the peptide is negative:
Start by attempting to dissolve the peptide in water. If water is ineffective to dissolve the peptide, add a small amount of NH4OH (<50 μL) and dilute to the desired concentration. See Note 9.
Neutral charge peptides; for peptides with a net charge of zero:
Introduce organic solvents as follows: First, try adding acetonitrile, methanol, or isopropanol. For highly hydrophobic peptides, start with a small amount of DMSO (30–50 μL, 100%). Gradually add this solution drop by drop to a stirring aqueous buffered solution like PBS or your preferred buffer until the desired peptide concentration is achieved. If turbidity appears in the peptide solution, it indicates that you have reached the limit of solubility. In such cases, sonication can help to dissolve the peptides. If the peptide includes cysteine residues, use DMF instead of DMSO. See Note 10.
In cases where peptides tend to aggregate, incorporate 6 M guanidine•HCl or 8 M urea before proceeding with necessary dilutions.
Peptide interaction with Apo-CaM
Dilute the peptide stock in fluorescence buffer to have a 20 μM peptide solution.
Calculate the volumes of peptide required for each titration step using the formula: C1·V1 = C2·V2, where C1 is the initial peptide concentration in the stock (20 µM), V1 is the volume of peptide to add in each step, C2 is the desired final peptide concentration in each step, and V2 is the total sample volume (100 µL).
Prepare a table with the calculated volumes (Table 1):
Table 1. Sample composition for peptide titration in absence of CaCl2
Peptide final concentration (µM) D-CaM 1 µM (µL) Peptide 20 µM (µL) Fluorescence buffer volume (µL)
0 5 0 95
0.1 5 0.5 94.5
0.2 5 1 94
0.5 5 2.5 92.5
1 5 5 90
1.5 5 7.5 87.5
2 5 10 85
4 5 20 75
8 5 40 55
16 5 80 15
Following sample preparation, centrifuge them at 185,494× g (radius 98 mm) for 10 min and carefully transfer the resulting supernatants to fresh 500 µL tubes to remove any potential aggregates.
Ensure the absence of air bubbles as they can distort fluorescence readings. Insert the cuvette into the fluorimeter and proceed to acquire emission spectra. Employ an excitation wavelength of 340 nm, recording emissions across the 400–660 nm range. All measurements are conducted at a temperature of 25 °C, with necessary corrections applied to account for any buffer-related interference. These conditions should yield a prominent fluorescence peak around 500 nm in the D-CaM spectrum. Record the fluorescence values for each peptide concentration.
Analyze data to determine the interaction between the peptide and D-CaM at different concentrations.
Calcium titration:
Start the process by gradually adding concentrated CaCl2 aliquots into a cuvette containing the peptide saturation sample once the peptide concentration for signal saturation is achieved.
In a 100 μL sample of the peptide saturation sample, add 1 μL of Ca buffer solution sequentially. It is important to note that all experiments will be conducted under constant conditions of pH 7.4 and 25 °C. Remember that EGTA buffering is pH dependent.
It is worth emphasizing that the experimental design incorporates a 10 mM Ca buffer, affording the evaluation of free calcium concentrations spanning from nM to µM ranges. Notably, adjustments in CaCl2 concentration are permissible, and alternative buffers may be employed to facilitate analyses across diverse calcium concentration ranges, whether elevated or diminished.
Utilize the Ca-EGTA Calculator v1.3, incorporating constants sourced from Theo Schoenmakers' Chelator, particularly Table 2, which corresponds to the 10 mM CaCl2 calcium buffer. After each Ca2+ addition, ensure thorough mixing to attain homogeneity.
See Note 11.
Table 2. Free calcium concentration calculation for 10 mM CaCl2 solution
Volume of Ca Buffer (μL) Total volume (μL) [Ca2+] total (mM) [Ca2+] free (µM)
0 100 0 0
1 101 0.1 0.0030
2 102 0.2 0.0079
3 103 0.3 0.177
4 104 0.4 0.471
5 105 0.5 2.45
6 106 0.6 10.01
7 107 0.7 20
8 108 0.8 30
9 109 0.9 40
10 110 1.0 50
Record the fluorescence spectra approximately 20–30 s after adding each sample to the cuvette. Note that extended equilibration times do not yield improved data.
Continue the titration of Ca2+ until saturation is reached, signified by the absence of further observable changes in the spectra.
Peptide interaction with Holo-CaM
To evaluate the peptide's interaction with holo-D-CaM, first saturate CaM with 1 mM free CaCl2.
Calculate the necessary peptide volumes for each titration step using the equation: C1·V1 = C2·V2, where C1 represents the initial peptide concentration in the stock (20 μM), V1 is the volume of peptide to be added in each step, C2 is the desired final peptide concentration for each step, and V2 is the total sample volume (100 μL).
Create a table to document the calculated volumes (Table 3):
Table 3. Sample composition for peptide titration in the presence of 1 mM CaCl2
Final peptide concentration (µM) Peptide 20 µM volume (µL) F buffer (µL) Ca buffer (µL) Total V (µL)
0 0 90 10 100
0.2 1 89 10 100
0.5 2.5 87.5 10 100
1 5 85 10 100
2 10 90 10 100
4 20 80 10 100
8 40 50 10 100
16 80 10 10 100
After preparing the samples, centrifuge them at 185,494× g (radius 98 mm) for 10 min and carefully transfer the resulting supernatants to fresh 500 μL tubes to eliminate any potential aggregates.
Ensure the absence of air bubbles, as they can distort fluorescence measurements. Insert the cuvette into the fluorimeter and proceed to acquire emission spectra. Use an excitation wavelength of 340 nm, recording emissions across the 400–660 nm range. All measurements are conducted at a temperature of 25 °C, with appropriate corrections made to account for any buffer-related interference. These conditions should yield a prominent fluorescence peak around 500 nm in the D-CaM spectrum. Record the fluorescence values for each peptide concentration.
Data analysis
In evaluating the affinity of a specific peptide for D-CaM, our approach involves the analysis of emission spectra obtained at various peptide concentrations, depicted in Figure 1A. Notably, a correlation is observed between peptide concentration and the intensity of the emission spectrum. The emission spectra exhibit a discernible trend until reaching a saturation point, wherein further increments in peptide concentration no longer elicit changes in the maximal emission intensity.
To better assess changes in intensity, we focus on the wavelength range of 490–500 nm (Table 4). We quantify intensity increases by summing up the values within this range. For normalization, we use the apo-CaM condition as the baseline, ensuring that D-CaM with 0 μM peptide corresponds to an intensity of 0. To normalize, we divide the sum of intensities by the corresponding value obtained in the apo-CaM condition (Table 4).
The experimental protocol is subsequently reiterated under Holo-CaM conditions, wherein a saturating concentration of unbound Ca2+ (e.g., 10 mM) is initially introduced, followed by the repetition of the peptide titration process.
For a more lucid depiction of intensity changes, we present the normalized values as percentages, setting the maximum intensity as 100. This normalization entails dividing each value by the maximum intensity obtained and then multiplying the result by 100 (Table 1).
Figure 1. Data analysis from emission spectra. A. Effect of incremental concentrations of a peptide in the dansyl-CaM (D-CaM) fluorescence emission spectrum in absence (left) and presence (right) of Ca2+. The wavelength at which the maximal emission is achieved is indicated by the vertical discontinuous line (adapted from Alaimo et al. [15]). B. Relative concentration-dependent enhancement of fluorescence at varying peptide concentrations using fixed 12.5, 100, and 400 nM D-CaM concentrations, in absence (left) and presence (right) of Ca2+. EC50 values are indicated in blue for each condition. C. Extrapolation to the true affinity. Plot of the concentration of the peptide at which 50% of maximal response is achieved (EC50) at different D-CaM concentrations (adapted from Bonache et al. [16]).
Table 4. Experimental data summary. The values in the Normalized data column are obtained by dividing each intensity value by the corresponding value obtained in apoD-CaM and subtracting 1. The Percentage column represents the percentages, with the maximum taken as 100%, calculated by dividing all values by the maximum normalized value.
Peptide [μM] Sum of intensity (490–500 nm) Normalized data (relative to apoD-CaM) Percentage
0 x1 (x1/x1_apoD-CaM) - 1 = y1 (y1/ymax) * 100
2 x2 (x2/x1_apoD-CaM) - 1 = y2 (y2/ymax) * 100
4 x3 (x3/x1_apoD-CaM) - 1 = y3 (y3/ymax) * 100
... ... ... ...
n x_max (x_max/x_1_apoD-CaM) – 1 = ymax (ymax/ymax)*100 = 100
If we plot the obtained values against the peptide concentration, a dose-response curve in percentage is generated. This approach facilitates a robust comparison of intensity changes across various peptide concentrations, culminating in a percentage-based representation of the specified peptide's affinity for D-CaM.
To generate concentration-response curves, plot fluorescence enhancement against the peptide-D-CaM ratio or [peptide] and fit the data using the three-parameter Hill equation through curvilinear regression.
EC50 values vary with D-CaM concentration due to ligand depletion, especially at low concentrations. Correct for depletion by determining EC50 values across a range of D-CaM concentrations. At infinitely low D-CaM concentrations, depletion should be negligible, making EC50 a true affinity value [1]. For accurate dissociation constant (Kd) determination, perform titrations with varying initial concentrations of D-CaM (6.25–200 nM) and create concentration-response curves at each D-CaM concentration (Figure 1B). Calculate apparent dissociation constants (EC50) from Figure 1B and plot them against D-CaM concentrations (Figure 1C). Obtain true dissociation constants through linear fitting and extrapolation to D-CaM concentrations equal to zero.Principio del formulario
If stoichiometry is known, estimate Kd values using a Scatchard plot analysis. However, we recommend the previous method for its accuracy and reliability in Kd determination, particularly in complex binding interactions.
To estimate Kd, assuming a 1:1 stoichiometry, apply the equation:
Here, F represents the increase in fluorescence, Fmax is the maximal fluorescence (variable), [peptide] is the known total peptide concentration, [CaM] is the known concentration of total D-CaM, and Kd is the variable for the affinity constant.
Express results as means ± S.E.M from three or more experiments. For statistical analysis, use the unpaired Student t-test, with P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) considered statistically significant. This protocol provides a comprehensive method for estimating the Kd in a fluorescence-based ligand displacement assay with D-CaM, considering the impact of D-CaM concentration and correcting for ligand depletion at low concentrations. It offers a robust approach for characterizing the binding affinity and cooperativity between D-CaM and the ligand.
Validation of protocol
This methodology, initially validated by Kincaid and Vaughan in 1986 [17], has exhibited consistent efficacy in discerning conformational changes resulting from interactions with Ca2+, peptides, or proteins. Subsequent studies Yuan by and Graves in 1989 [18] investigated the interaction of CaM with the γ subunit of phosphorylase kinase, while Munier et al. in 1991 [19] characterized the catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase (refer to Figures 3 and 4). Filoteo et al. [20] delved into the binding between the lipid-binding region (G region) of the erythrocyte Ca2+ pump and CaM (see Figures 4 and 5). In 2013, Alaimo et al. [14] explored the interaction of CaM with different components of the Kv7.2 channel (refer to Figures 5, 6, and 7). Zhang et al. [21] identified two distinct CaM binding sites in the angiotensin II (AT1A) receptor (refer to Figures 4 and 6). Alcalde et al. [22] demonstrated that non-myristoylated peptides derived from the CaM binding site of Grb7 exhibit higher efficiency in binding dansyl-CaM in the presence of Ca2+ compared to its absence (see Supplementary Figure 2). More recently, Nuñez et al. [23] showcased that the oxidation of peptides derived from the S2S3 linker of Kv7 channels inhibits their binding to CaM (refer to Figure 3), and numerous other studies contribute to the comprehensive utility of this method.
General notes and troubleshooting
We request peptides with a purity level exceeding 90%, which, alongside maintaining the correct sequence, should also possess an amino group (-NH3) at the N-terminus and a carboxyl group (-COOH) at the C-terminus. These terminal groups are instrumental in promoting the stability and suitability of the peptide for subsequent experimental procedures, such as binding assays and functional studies.
The initial Ca2+ concentration within the D-CaM sample plays a pivotal role in these assays. Consequently, it is imperative to minimize any residual Ca2+ presence. To achieve this, we use 5 mM EGTA to ensure that the minimal endogenous Ca2+ bound to the protein under titration does not interfere with free Ca2+ levels.
Ensure the pH is promptly readjusted to 7.4 after buffer preparation, as CaCl2 addition can lead to pH reduction.
We suggest dissolving Tris in 150 mL of solution, carefully adjusting the pH to 8.8 with HCl, and then precisely adding the necessary volume to reach a final volume of 200 mL.
We suggest dissolving Tris in 75 mL of solution, carefully adjusting the pH to 6.8 with HCl, and then precisely adding the necessary volume to reach a final volume of 100 mL.
Dissolve for 2 h with continuous stirring and filter using filter paper. Store at room temperature, protected from light.
To eliminate the excess dansyl chloride, we recommend dialyzing the D-CaM samples in conjunction with the gel filtration process outlined earlier.
Collect the D-CaM fractions, concentrate if required, and safeguard them by storing in light-protected aliquots at -20 °C or by lyophilization. Our thorough investigations have demonstrated that the conjugate's properties remain largely unchanged when stored at -20 °C for months, if not longer [14].
Ensuring that the protein or peptide samples remain uniform without aggregation is of utmost importance. We routinely employ dynamic light scattering (DLS) with a Zetasizer Nano instrument (Malvern Instruments Ltd.) to assess sample dispersion. It is essential to use samples exhibiting monodispersity, where both the correlation function and polydispersity index are below 0.2.
When dealing with peptides that contain cysteine (Cys) residues, it is advisable to abstain from employing basic solutions for dissolution and explore alternative methods. In such cases, ideal solvents include degassed mediums like low-pH buffers, diluted acetic acid, or a solution consisting of 0.1% trifluoroacetic acid in aqueous acetonitrile. Particular caution should be exercised to avoid the use of DMSO, especially when working with peptide trifluoroacetates.
The key to achieving accurate Ca2+ titrations is the precise control of free Ca2+ levels using chelators like EGTA or EDTA. We prefer EGTA for experiments conducted at pH 7.5 to replicate intracellular conditions and physiological magnesium concentrations. Maintaining strict pH control is vital because chelating efficiency is highly sensitive to proton concentration. To calculate free Ca2+ concentrations, we employ the Maxchelator program (http://maxchelator.stanford.es) and custom software. Various other programs are available to determine free Ca2+ concentrations based on the total added Ca2+, given EGTA levels, and considering different temperature, ionic strength, and pH conditions.
Acknowledgments
This article has been funded through the following research projects: the Ministry of Science and Innovation under the project PID2021-128286NB-100 funded by MCIN/AEI/10.13039/501100011033/FEDER, UE; support from the Basque Government under the project IT1707-22. S.M-A and E.N. received support from predoctoral (PRE_2021_1_0101) and postdoctoral (POS_2021_1_0017) contracts, respectively, provided by the Basque Government.
This article has been previously described and validated by Alaimo et al. [14] and Nuñez et al. [23]. Subsequent verification has been conducted in various studies, as documented in the validation of the protocol section.
Competing interests
The authors declare that they have no competing interests.
References
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Agrobacterium-Mediated Transient Gene Expression Optimized for the Bioenergy Crop Camelina sativa
PK Pawan Kumar
ZB Zeeshan Z. Banday
JR John L. Riley
JG Jean T. Greenberg
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4964 Views: 735
Reviewed by: Wenrong HeJianyan Huang Anonymous reviewer(s)
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Abstract
Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1–32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system.
Key features
• This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop Camelina species.
• A constitutive and inducible transient gene expression in the hexaploid species Camelina sativa cultivar DH55.
• Requires only 16–18 days to complete with high efficacy.
Graphical overview
Agrobacterium-mediated transient gene expression optimized for Camelina sativa
Keywords: Camelina sativa Transient gene expression Brassicaceae Subcellular organelle localization Agrobacterium
Background
Camelina sativa, an important Brassicaceae family crop and crop model, is used to make fodder, human food, industrial chemical raw materials, biofuels, and compost to enhance soil properties [2]. It is planted worldwide, particularly in North America, Europe, and Asia, and is also known as large-seeded False Flax seed due to its high level of polyunsaturated fatty acids [3]. Camelina has lately become noted for its potential to be a high-value, climate-resilient oilseed crop owing to its resistance to a variety of abiotic and biotic stresses [4].
Using different technologies like classical breeding, transgenics, and CRISPR/Cas9 genome editing, various genetic improvements in Camelina have been made to increase its seed yield and oil contents and reduce its anti-nutritional glucosinolate content. Stable transformation systems that employ the floral-dip method and in vitro leaf explants are available in Camelina sativa [5]. Nevertheless, the transformation efficiency is very low (i.e., 0.8%–1%) and the method is laborious [6,7]. While an Agrobacteria-mediated infiltration system for rapid and transient gene expression for studying protein localization, protein–protein interaction, etc., has been well established in Nicotiana benthamiana, this technique has had limited applications for other crop species [8]. Hence, with regards to time and labor efficiency, the development of a transient expression system will provide an alternative and convenient way to study genes in Camelina.
Previously, Zhang et al. [1] developed a transient gene expression protocol using different strains of Agrobacteria in the model plant Arabidopsis thaliana, along with seven other plant species: Brassica oleracea, Capsella rubella, Thellungiella salsuginea, T. halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Herein, we modified this protocol for Camelina sativa cultivar DH55. The major modification is the age of the plants, which is younger than that described by Zhang et al. [1], enabling more rapid acquisition of data. In addition, this optimized protocol functions with constitutive and inducible promoters and is suitable for both subcellular localization and colocalization of proteins in Camelina. Using the leaves of 10–12-days-old plants infected with Agrobacterium strain C58C1, we show the subcellular localization of the organelle markers to chloroplasts, peroxisomes, Golgi bodies, and ER [9]. Leaves of 14-day-old plants showed a significantly decreased transformation efficiency, highlighting the benefit of using younger plants for these experiments. We also report the intracellular localization of the N-terminal ER signal (1–32 aa) region of the protein encoded by the At3G28580 gene in intact leaves. This study demonstrates an efficient transient gene expression protocol for Camelina.
Materials and reagents
Biological materials
Camelina sativa cultivar DH55 seeds
Agrobacterium strain C58C1 containing constitutive or chemically inducible plasmid expression constructs.
Plasmids used in this study harbor constructs encoding chloroplast-GFP (CD3-995), peroxisomes-mCherry (CD3-983), Golgi body-mCherry (CD3-967), ER-mCherry (CD3-959) as described in Nelson et al. [9], and Dex-inducible pBAV150 [10] plasmid with DNA encoding the N-terminal (1–32 aa) region of a protein coded by the At3G28580 gene
Reagents
Plastic domes (Hummert International, catalog number: 11-33480) and trays (Hummert International, catalog
number: 11-33010)
Pots 8 cells; width × length × depth: 12.34 × 12.34 × 5.77 cm (T.O. PLASTICS, catalog number: 715352C)
Soil (Berger 60, catalog number: BM2; BM6, mixed in 1:1 ratio)
Yeast extract (Fisher Scientific, catalog number: BP1422-500)
BactoTM peptone (Thermo Fisher Scientific, catalog number: 211677)
Sucrose (Fisher Scientific, catalog number: S5-500)
MgSO4·7H2O (Fisher Scientific, catalog number: 10034-99-8)
NH4Cl (Sigma, catalog number: 12125-02-9)
KCl (Fisher Scientific, catalog number: P217-500)
CaCl2 (Sigma, catalog number: C2661)
FeSO4·7H2O (Sigma, catalog number: F-7002)
NaH2PO4·H2O (JT Baker, catalog number: JTB-3818-05)
Na2HPO4 (Fisher Scientific, catalog number: 7558-79-4)
Glucose (Sigma, catalog number: G7021)
MES (Sigma, catalog number: M8250)
MgCl2·6H2O (Fisher Scientific, catalog number: BP214-500)
Murashige and Skoog medium (Sigma, catalog number: M5519)
Bleach (Clorox Concentrated Germicidal Bleach)
Ethanol (Decon Labs Inc., catalog number: 2701)
Agarose (GOLDBIO, catalog number: 9012-36-6)
Silwet L-77 (Vac-In-Stuff, catalog number: VIS-30)
Tween® 20 (Hoefer, catalog number: 9005-64-5, GR128-500)
Antibiotics: kanamycin sulfate (Kan) and rifampicin (Rif) (Fisher Scientific, catalog number: BP906-5, 13292-46-1, respectively)
NalgeneTM Rapid-FlowTM sterile disposable filter units (Thermo Fisher Scientific, catalog number: 568-0020)
Dexamethasone (Sigma, catalog number: D1756)
Perfluorodecalin (Strem Chemicals, catalog number: 09-5960)
Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418)
Agar (Fisher Scientific, catalog number: BP1423-500)
Acetosyringone (Sigma, catalog number: D134406)
Solutions
Washing solution (see Recipes)
Infiltration solution (see Recipes)
Agrobacterium growth media (see Recipes)
30 μM Dexamethasone (Dex) solution with 0.04% Tween-20 (see Recipes)
70% ethanol (see Recipes)
0.1% agar media (see Recipes)
Phosphate buffer (50 mM, 25×, pH 5.5) (see Recipes)
Recipes
Recipes 1, 2, and 3 are adapted from Zhang et al. [1].
Washing solution (1 L)
Reagent Final Concentration Volume
MgCl2 (1 M) 10 mM 10 mL
Acetosyringone (0.1 M) 100 μM 1 mL
ddH2O n/a 989 mL
Total n/a 1 L
Infiltration solution (1 L)
Note: Water should be added in increments of 100 mL. Allow the solution to mix completely before adding any more water. Murashige and Skoog medium and sucrose will take a substantial volume when fully dissolved. Adjust pH to 6.0 using 1 M KOH; use freshly prepared.
Reagent Final Concentration Quantity or Volume
Murashige and Skoog medium 1/4 × (w/v) 1.1 g
Sucrose 1% (w/v) 10 g
Acetosyringone (0.1 M) 100 µM 1 mL
Silwet L-77 0.01% (v/v) 100 μL
ddH2O n/a see note
Total n/a 1 L
Agrobacterium growth media (1 L)
Note: Adjust the pH to 5.5 using 1 M KOH and autoclave the media. After autoclaving, add the filtered induce buffer components. Acetosyringone is prepared in DMSO.
Reagent Final Concentration Quantity or Volume
Yeast extract 0.1% (w/v) 1.0 g
BactoTM peptone 0.5% (w/v) 5.0 g
Sucrose 0.5% (w/v) 5.0 g
MgSO4·7H2O 2.03 mM 0.5 g
Agarose 1% (w/v) 10.0 g
NH4Cl 18.70 mM 1.0 g
KCl 2.01 mM 0.15 g
CaCl2 90.10 μM 0.01 g
FeSO4·7H2O 8.99 μM 0.0025 g
ddH2O n/a 889 mL (see note)
Induce buffer composition:
Phosphate buffer (50 mM, 25×, pH 5.5) 1× 40 mL
Glucose (20%, 20×) 1× 50 mL
MES (1 M, 50×, pH 5.5) 1× 20 mL
Acetosyringone (200 mM, 1,000×) 1× 1 mL
Total n/a 1 L
30 μM Dexamethasone (Dex) solution with 0.04% Tween-20 (100 mL)
Note: Dex is prepared in DMSO.
Reagent Final Concentration Volume
Dex (30 mM) 30 μM 100 μL (see note)
Tween-20 0.04% 40 μL
ddH2O n/a 99.86 mL
Total n/a 100 mL
70% ethanol (100 mL)
Reagent Final Concentration Volume
Ethanol (95%) 70% 73.7 mL
ddH2O n/a 26.3 mL
Total n/a 100 mL
0.1% agar media (100 mL)
Note: Autoclave the media and stir before it cools.
Reagent Final Concentration Volume
Agar 0.1% 100 mg
ddH2O n/a 100 mL
Total n/a 100 mL (see note)
Phosphate buffer (50 mM, 25×, pH 5.5; 100 mL)
Note: Adjust the pH to 5.5 using 1 M KOH.
Reagent Final Concentration Volume
NaHPO.HO 48.02 mM 662.7 mg
NaHPO 1.98 mM 28.05 mg
ddH2O n/a 100 mL
Total n/a 100 mL (see note)
Laboratory supplies
Laboratory glassware
Pipette tips: 10 μL, 200 μL, and 1,000 μL (USA Scientific, catalog number: 1111-3700, 1111-1700, 1112-1720)
Black marker (Sharpie Permanent Markers, catalog number: 33861PP)
Kimwipes (Fisher Scientific, catalog number: 06-666A)
15 mL and 50 mL centrifuge tubes (Fisher Scientific, catalog numbers: 14-959-53A, 14-432-22)
9 cm round Petri dishes (Fisher Scientific, catalog number: FB0875712)
1.5 mL microcentrifuge tubes (Fisher Scientific, catalog number: 01-549-746)
1 mL tuberculin syringes without needle (BD Biosciences, catalog number: 309659)
Damp paper towel (Georgia-Pacific, catalog number: P200)
Sterile double-distilled water
Micropipettes 20 μL, 200 μL, and 1 mL (Gilson, catalog number: F144056M, F144058M, F144059M, respectively)
1 L beaker (Pyrex, catalog number: 1000)
Equipment
Cork borer 4 mm diameter
Autoclave (Primus Sterilizer Co. Inc. 1317, catalog number: 09415.1256)
Fisher vortex Genie 2 (Fisher Scientific, catalog number: 12-812)
Plant growth chamber or walk-in growth room with humidity maintained at ≥ 50%. Light intensity should be 135–145 μmols-1·m-2 at soil level
Freezer (-80 °C) (Panasonic VIP Plus, model: MDF-V76VC-PA)
Balance (Mettler Toledo, model: PB1501)
Spectrophotometer (Bio-Mini, model: SHIMADZU)
Laminar flow hood (SterilGARD, model: 3 Advance)
Incubator at 28 °C (VWR, model: 3020)
Analytical balance for weighing chemicals
pH meter (SevenCompact, serial number: B408309625)
Confocal microscope (Zeiss, model: LSM 800)
Software and datasets
Fiji (ImageJ, version 2.15.0, 10/12/2023)
Photoshop (Adobe PS, 22.4.3, 07/19/2021)
Prism v10.1 (GraphPad, 07/01/2023)
BioRender.com online tool
Procedure
Plant growth and maintenance
Soak non-sterilized, dry Camelina seeds (~100–150) in 4–6 mL of 0.1% sterile agar in 15 mL falcon tubes and keep at 4 °C in the dark for 3–7 days. The Camelina seeds utilized in this study were multiplied and bulked inside the greenhouse chamber.
Sow approximately 18–20 seeds in individual soil mix–containing pots (12.34 × 12.34 × 5.77 cm) with the help of a Pasteur pipette and cover with a transparent plastic lid to maintain moisture. Keep pots in a plant growth chamber or growth room chamber under 12 h light and 12 h dark conditions at 22 ± 2 °C day/night temperature regime and light intensity of ~135–145 μmols-1·m-2. We use a mix of 50/50 sodium and metal halide light (but other lights might be suitable) at 50%–70% relative humidity.
After 4–5 days, thin seedlings to nine plants per pot and remove the lid. Select the seedlings that look visibly similar and discard the rest (Figure S1).
Allow seedlings to establish for the next 5–7 days until the first true leaves are completely opened (Figure 1).
Figure 1. Representative image depicting Agrobacterium-infiltrated plants of Camelina sativa DH55 cultivar. Ten-to-twelve-day-old plants with opened true leaves were infiltrated with Agrobacterium strain C58C1 carrying desired constructs. White arrows indicate representative leaves imaged immediately after infiltration. The abaxial part of the leaves will have a wet appearance until the infused liquid containing Agrobacteria evaporates. Scale bar is in centimeters.
Agrobacterium strain suspension preparation for infiltration
Agrobacterium suspension preparation
Streak an Agrobacterium strain C58C1 stock with the desired constructs on a sterile plate of Agrobacterium growth media containing appropriate antibiotics and 200 μM acetosyringone inside a laminar airflow cabinet.
Keep plates with bacteria at 28 °C for 24–36 h. In this work, marker plasmids of plastid-GFP (CD3-995), peroxisomes-mCherry (CD3-983), Golgi body-mCherry (CD3-967), ER-mCherry (CD3-959), and the N-terminal ER signal (1–32 aa) region of the At3G28580-encoded protein were used, and plates contained Rif (34 mg/mL) + Kan (50 mg/mL).
Scrape a thin layer of Agrobacterial cells from each plate using a 200 μL pipette tip and resuspend bacteria in 1 mL of washing solution (see Recipes) in a 1.5 mL microcentrifuge tube by vortexing.
Dilute an aliquot of 100 μL of resuspended Agrobacteria 10× in order to measure the OD600. A value equal to or greater than 0.6 is desired in the final infiltration solution. Multiply this OD value by 10 to determine the OD600 of the original undiluted resuspended Agrobacteria. This is done to obtain enough inoculum, sufficient to infiltrate all the true leaves per pot for each construct. After that, adjust the original suspension of Agrobacteria to an OD600 of 0.6 using the infiltration solution (see Recipes). For colocalization studies, mix strains in a 1:1 ratio using an OD600 of 0.6 for each strain and immediately infiltrate the leaves.
Agrobacterium infiltration in Camelina
Infiltrate the Agrobacterium in the infiltration solution into the abaxial surface of Camelina leaves (2 true leaves per plant) with the help of a 1 mL needleless plastic syringe. Use four or more plants per construct for infiltrations. Keep plants in the light for 1 h to allow the water-soaked leaves to dry before placing plants in the dark for 24 h at room temperature using plastic trays.
Return the Agrobacterium-infiltrated plants to a growth chamber or growth room for another 3–5 days before observing leaves using confocal microscopy. If using an inducible promoter, spray the inducer over leaves 21 h before examination, as described in Banday et al. [11], using a confocal microscope (Figure 2A and 2B). In the example in Figure 2A, 30 μM Dex with 0.04% Tween-20 was applied.
Figure 2. Agrobacterium-mediated transient subcellular localization in Camelina sativa DH55 cultivar. A. Upper panel: Confocal micrographs showing localization of Chloroplast-GFP (CD3-995), Peroxisome-mCherry (CD3-983), and Golgi-mCherry (CD3-967). Lower panel: Confocal micrographs of ER-mCherry (CD3-959), N-Term-ER-GFP (Dex: At3G28580; 1-32 aa region), and colocalization of N-Term-ER-GFP (Dex: At3G28580) with ER-mCherry (CD3-959; shown in magenta color). For localization and colocalization studies, images were taken 3 and 5 days after transfer to the light conditions, respectively. Chloroplast autofluorescence is shown in blue. Yellow arrowheads indicate chloroplast autofluorescence (shown in insets), magenta arrowheads indicate chloroplast-GFP overlap with chloroplast autofluorescence, and white arrows indicate colocalization of N-Term-ER-GFP (Dex: At3G28580) and ER-mCherry (CD3-959). Scale bars, 100 μm. B. Details of the separate channels of individual confocal micrographs: chloroplast autofluorescence (Blue), GFP signal (Green), and mCherry signal (Red). For colocalization, the mCherry signal is shown in magenta color. Scale bars, 100 μm. C. Efficiency of each transiently expressed construct. Data represent the average ± SE (n = 8) in percentage (%) obtained from experiments performed on two different days. The four images from each independent experiment were used for the calculations. See Table S1 for details.
Data analysis
The confocal microscope method was adapted from Banday et al. [11]. Zeiss LSM 800 was used to visualize GFP and mCherry fluorescence (Argon laser/excitation: 488 nm; emission collection: 505–530 nm for GFP; Argon laser/excitation: 594 nm; emission collection: 610–630 nm for mCherry) and chlorophyll autofluorescence (He-Ne laser/excitation: 633nm; emission collection: 650–750 nm or 645–700 nm). Images of abaxial leaf surface were captured using EC Plan-Neofluar 40/1.3 Oil DIC M27 objectives, pinhole at 1 AU for each channel, and photomultipliers master gain between 450 and 700. The images were optical sections captured at 1,024 × 1,024 pixels scanning resolution in maximum speed mode. Fluorescence of GFP, mCherry, and chlorophyll was acquired in sequential acquisition mode. Plant leaves were mounted in perfluorodecalin for optical enhancement [12]. ImageJ (Fiji) and Adobe PS were used to process the images. Each C. sativa imaging experiment included four independent samples per construct, and the experiments were done twice. Four images from different plant leaves from each experiment were used to calculate the efficiency of the transformation of cells per image. Using this protocol, all infiltrated leaves showed expression of the respective constructs. However, the efficiency of cells transformed/image was lower in the leaves of older plants (Table S1).
For calculation of efficiency analysis, the formula was adapted from Zhang et al. [1]. The graph was plotted using Prism software (Figure 2C and Table S1).
Efficiency (%) = Number of transiently transformed cells *100/Total number of cells
Validation of protocol
This protocol was adapted from Zhang et al. [1] and optimized for the Camelina crop. The protocol was validated using five different constitutive or chemically inducible plasmid expression constructs.
Each Camelina imaging experiment included four independent samples per construct, and the experiments were done twice. Four images from different plant leaves from each experiment were used to calculate the efficiency of the transformation of cells per image.
The student’s t-test shows that the efficiency of cells transformed/image was significantly lower in leaves of older plants (Table S1).
General notes and troubleshooting
Plants' age is very important. Leaves of older Camelina plants require needle punctures for successful infiltration and the efficiency of transformation declines (Table S1).
Pots must be well watered during the experiment to maintain humidity.
Keep only nine plants in one pot; plant density affects the growth of plants. A higher number of plants results in slower growth. Conversely, a lower number of plants per pot hastens the growth of plants.
Do not keep bacterial cultures for more than 36 h.
For better infiltration, the infiltration solution should be introduced into the abaxial surfaces of leaves.
Take confocal images of the abaxial surface to obtain a greater number of cells in the same focal plane.
Use perfluorodecalin to prevent the leaves from drying and for better imaging.
Clean the workbench before making an Agrobacterial suspension with the help of 70% ethanol. Later on, after completion of infiltration in plants, the remaining bacterial suspension must be treated with bleach for 24 h before being discarded into the sink.
Acknowledgments
This work was supported by a Dropkin Postdoctoral Fellowship awarded to PK and an Undergraduate fellowship from Arnold and Mabel Beckman Foundation to JLR. This protocol was adapted from Zhang et al. [1] and modified for the Camelina crop. We thank Dr. Isobel Parkin (Agriculture and Agri-Food Canada) for providing Camelina seeds. We thank Dr. Joanna Jelenska for valuable suggestions.
Competing interests
The authors declare that they have no conflicts of interest.
References
Zhang, Y., Chen, M., Siemiatkowska, B., Toleco, M. R., Jing, Y., Strotmann, V., Zhang, J., Stahl, Y. and Fernie, A. R. (2020). A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species. Plant Commun. 1(5): 100028. https://doi.org/10.1016/j.xplc.2020.100028
Sydor, M., Kurasiak-Popowska, D., Stuper-Szablewska, K. and Rogoziński, T. (2022). Camelina sativa. Status quo and future perspectives. Ind. Crops Prod. 187: 115531. https://doi.org/10.1016/j.indcrop.2022.115531
Waraich, E. A., Ahmed, Z. I., Ahmad, R., Ashraf, M. Y., Saifullah, Naeem, M. and Rengel, Z. (2013). Camelina sativa, a climate proof crop, has high nutritive value and multiple uses: A review. Aust. J. Crop Sci. 7(10):1551–1559. https://www.cropj.com/waraich_7_10_2013_1551_1559.pdf
Neupane, D., Lohaus, R. H., Solomon, J. K. Q. and Cushman, J. C. (2022). Realizing the Potential of Camelina sativa as a Bioenergy Crop for a Changing Global Climate. Plants 11(6): 772. https://doi.org/10.3390/plants11060772
Ghidoli, M., Ponzoni, E., Araniti, F., Miglio, D. and Pilu, R. (2023). Genetic Improvement of Camelina sativa (L.) Crantz: Opportunities and Challenges. Plants 12(3): 570. https://doi.org/10.3390/plants12030570
Lu, C. and Kang, J. (2007). Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep. 27(2): 273–278. https://doi.org/10.1007/s00299-007-0454-0
Liu, X., Brost, J., Hutcheon, C., Guilfoil, R., Wilson, A. K., Leung, S., Shewmaker, C. K., Rooke, S., Nguyen, T., Kiser, J., et al. (2012). Transformation of the oilseed crop Camelina sativa by Agrobacterium-mediated floral dip and simple large-scale screening of transformants. In Vitro Cell. Dev. Biol. Plant48(5): 462–468. https://doi.org/10.1007/s11627-012-9459-7
Krenek, P., Samajova, O., Luptovciak, I., Doskocilova, A., Komis, G. and Samaj, J. (2015). Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol. Adv. 33(6): 1024–1042. https://doi.org/10.1016/j.biotechadv.2015.03.012
Nelson, B. K., Cai, X. and Nebenführ, A. (2007). A multicolored set of in vivo organelle markers for co‐localization studies in Arabidopsis and other plants. Plant J. 51(6): 1126–1136. https://doi.org/10.1111/j.1365-313x.2007.03212.x
Vinatzer, B. A., Teitzel, G. M., Lee, M., Jelenska, J., Hotton, S., Fairfax, K., Jenrette, J. and Greenberg, J. T. (2006). The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non‐host plants. Mol. Microbiol. 62(1): 26–44. https://doi.org/10.1111/j.1365-2958.2006.05350.x
Banday, Z. Z., Cecchini, N. M., Speed, D. J., Scott, A. T., Parent, C., Hu, C. T., Filzen, R. C., Agbo, E. and Greenberg, J. T. (2022). Friend or foe: Hybrid proline-rich proteins determine how plants respond to beneficial and pathogenic microbes. Plant Physiol. 190(1): 860–881. https://doi.org/10.1093/plphys/kiac263
Littlejohn, G. R., Gouveia, J. D., Edner, C., Smirnoff, N. and Love, J. (2010). Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. New Phytol. 186(4): 1018–1025. https://doi.org/10.1111/j.1469-8137.2010.03244.x
Supplementary information
The following supporting information can be downloaded here:
Figure S1: Visible appearance of 5-days-old seedling.
Table S1: Details of efficiency analysis in percentage.
Article Information
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Method for Large-scale Production of hIPSC Spheroids
LL Lucas Lemarié
EC Edwin-Joffrey Courtial
JS Jérôme Sohier
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4965 Views: 711
Reviewed by: Alessandro DidonnaLionel Schiavolin Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Bioengineering Dec 2023
Abstract
Stem cell spheroids are rapidly becoming essential tools for a diverse array of applications ranging from tissue engineering to 3D cell models and fundamental biology. Given the increasing prominence of biotechnology, there is a pressing need to develop more accessible, efficient, and reproducible methods for producing these models. Various techniques such as hanging drop, rotating wall vessel, magnetic levitation, or microfluidics have been employed to generate spheroids. However, none of these methods facilitate the easy and efficient production of a large number of spheroids using a standard 6-well plate. Here, we present a novel method based on pellet culture (utilizing U-shaped microstructures) using a silicon mold produced through 3D printing, along with a detailed and illustrated manufacturing protocol. This technique enables the rapid production of reproducible and controlled spheroids (for 1 × 106 cells, spheroids = 130 ± 10 μm) from human induced pluripotent stem cells (hIPSCs) within a short time frame (24 h). Importantly, the method allows the production of large quantities (2 × 104 spheroids for 1 × 106 cells) in an accessible and cost-effective manner, thanks to the use of a reusable mold. The protocols outlined herein are easily implementable, and all the necessary files for the method replication are freely available.
Key features
• Provision of 3D mold files (STL) to produce silicone induction device of spheroids using 3D printing.
• Cost-effective, reusable, and autoclavable device capable of generating up to 1.2× 104 spheroids of tunable diameters in a 6-well plate.
• Spheroids induction with multiple hIPSC cell lines.
• Robust and reproducible production method suitable for routine laboratory use.
Graphical overview
Spheroid induction process following the pellet method on molded silicon discs
Keywords: Spheroids Induced pluripotent stem cell Large-scale Uniform Autoclavable Tissue engineering.
Background
Stem cell spheroids ideally mimic the physiological 3D environment of tissues, making them increasingly indispensable in tissue engineering [1]. Since the inception of cellular aggregates in the 1930s, spheroids have become a model of choice across various fields of fundamental biology. Their applications span from drug testing to disease modeling, encompassing oncology and regenerative medicine [2,3]. Despite the growing use of spheroids in the past two decades, the demand for spheroids as a raw material remains substantial [4]. This development has given rise to numerous spheroid production methods, tailored to specific applications.
Concurrently, the use of human induced stem cells (hiPSCs) has surged since their discovery in 2007 [5]. This model, based on the reprogramming of unipotent cells into pluripotent ones, proves valuable from practical and ethical perspectives, offering an excellent substitute for embryonic stem cells. Today, hIPSCs play a pivotal role in both basic research and applied biology [6,7]. Simultaneously, spheroids represent a highly promising tool for both tissue engineering [8] and the study of fundamental biological phenomena [9] and have been recently used to assess the impact of the viscoelastic properties of the 3D environment on their growth [10]. The most employed techniques to form spheroids from stem cells, including hiPSCs, are rotary cell culture, hanging drop, cell culture in non-adhesive hydrogel, pellet method, and microfluidics [11]. However, these methods share drawbacks related to low productivity, time inefficiency, high cost, and very limited reusability.
The technique presented here is built upon the pellet method (U-shaped), offering a compromise between existing methods. Optimized for rapid and cost-effective production of well-defined spheroids from hiPSCs, this solution is easily transferable for integration into a routine production pipeline. Exhibiting the ability to tune the diameter of spheroids (50–150 μm) by modulating the number of cells used (from 1.0 × 105 to 1.5 × 106), its adaptability extends to other cell lines, demonstrating robust aggregation capabilities.
Materials and reagents
Biological materials
hiPSCs AG08C5 cell line (European hPSCreg as PGNMi001-A) ( https://hpscreg.eu/cell-line/PGNMi001-A) [12]
Healthy Control Human iPSC Line, Female, SCTi003-A (STEMCELL, catalog number: 200-0511)
Reagents
Maintenance culture media mTeSRTM Plus kit, cGMP (STEMCELL, catalog number: 100-0276)
Vitronectin XFTM (STEMCELL, catalog number: 100-0763)
TrypLETM (Thermo Fisher, catalog number: 12604013)
Y-27632 (Dihydrochloride), RHO/ROCK pathway inhibitor (STEMCELL, catalog number: 72302)
Fetal bovine serum (FBS) (Sigma Aldrich, catalog number:341506)
Gibco Dulbecco’s phosphate buffered saline (DPBS) (Thermo Scientific, catalog number: 14190)
Soap (e.g., Enzypin, Distrimed, catalog number: 201261)
Ethanol 70% (e.g., Fisher Scientific, catalog number: 16320638)
Solutions
hiPSC spheroids induction medium (see Recipes)
TrypLETM inhibition medium (see Recipes)
Recipes
hiPSC spheroids induction medium
Composition Final concentration Volume
mTeSRTM Plus - 50 mL
Y-27632 (10 mM) 10 μM 50 μL
TrypLETM inhibition medium
Composition Final concentration Volume
mTeSRTM Plus - 9.8 mL
FBS 2% 200 μL
The solutions should be freshly prepared immediately before each experiment, with volumes adjusted according to the specific requirements. Prior to contact with the cells, these solutions must be prewarmed to 37 °C. Antibiotics can be supplemented to the medium according to your culture protocol.
Laboratory supplies
P1,000 pipette (e.g., Pipetman 100–1,000 µL, Gilson, catalog number: F144059M).
Tips 1,000 µL, (e.g., AmpliPur Expert Tips 100–1,000 µL, Gilson, catalog number: F174401)
Acrylic resin (Acrylate-like) (e.g., Stratasys, model: Vero ClearTM )
Silicone (e.g., Elkem Silicones, model: BLUESILTM RTV 3503)
Disposable autoclave bag (Sigma-Aldrich, catalog number: Z692212)
6-well plate, round (Thermo Fischer, catalog number: 140675)
Falcon 15 mL centrifuge tubes, PET, conical bottom w/plug seal cap (Sigma-Aldrich, catalog number: CLS430055)
Falcon 50 mL centrifuge tubes, PET, conical bottom w/plug seal cap (Sigma-Aldrich, catalog number: CLS4558)
Petri dish B60 (e.g., Corning, catalog number: BP53-03)
Tweezer (e.g., EMS 96, Sigma Aldrich, catalog number: 932922)
Disposable hemocytometer (e.g., Millicell®, Sigma-Aldrich, catalog number: MDH-2N1-50PK)
Positive displacement pipette (e.g., Microman 100–1,000 µL, Gilson, catalog number: FD10006)
Capillary pistons (e.g., CP1,000ST 2 × 91 TIPACK, Gilson, catalog number: F148180)
Microtube Eppendorf 2 mL (Dutscher, catalog number: 033297)
Equipment
Inkjet 3D printer (e.g., Stratasys, model: Objects 30 pro)
Autoclave (e.g., Sigma-Aldrich, model: BioCLAVETM mini digital autoclave, catalog number: Z680109)
Centrifuge (e.g., Sigma Aldrich, catalog number: C166500)
CO2 incubator (e.g., MEMMERT, catalog number: I227880)
Vacuum drying oven (e.g., Goldbrunn, model: 450)
Fluid aspiration systems (e.g., BVC control, Vaccubrand, catalog number: 20727200)
6 well-plate rotor (e.g., Eppendorf A-4-81 Rotor, Marshall Scientific, catalog number: EP-WPB)
Software and datasets
MATLAB (2022b, September 20, 2022)
Prism v9.0 (GraphPad, October 27, 2020)
Procedure
The aim of the procedure is to generate a biocompatible and autoclavable device, simplifying the induction of spheroids. As illustrated in Figure 1, the experimenter needs only to 3D-print the provided molds using the supplied 3D files (STL) to create an unlimited number of devices.
Figure 1. Silicone disc fabrication process. Scale bar = 1 mm. SEM: TM4000 (Itachi, Japan).
Mold 3D printing
Use an inkjet 3D printer to produce the acrylic resin molds from the “EBs-mold-base.STL,” “EBs-mold-intern.STL,” and “EBs-mold-extern.STL” STL files (available at https://github.com/Klux1rst/Spheroids-Inducer-Mold-Bioprotocol/tree/main). It is recommended to use the most resolution-enhancing default parameters to achieve a high-quality mold (See General Notes 1).
Perform post-printing cleaning on the 3D printed molds (usually by soaking in 50 mL of EtOH 70% in an open container for 12 h at room temperature but consult your material instructions because the post-printing process is resin dependent). Allow the acrylate mold to dry at room temperature for 30 min before use.
Silicone disc production
Note: The following steps are illustrated in Figure S1.
Handle the basal piece (EBs-mold-base).
Snap the central piece into the basal piece (EBs-mold-intern).
To complete the assembly, snap the external part into the basal piece (EBs-mold-extern).
Confirm proper assembly, ensuring all pieces fit securely. Place the mold in the upper lid of a B60 Petri dish.
Deposit 1 mL of silicone, starting with the peripheral groove between the central and external pieces. A positive displacement pipette is recommended to ensure an accurate volume.
Deposit another 1 mL a second time at the center of the mold.
Note: If only classic pipettes are available, approximately 2 mL of silicone can be deposited directly. Use a balance for accuracy (approximately 2 g).
Place the assembly in a sealed chamber and create a vacuum for 5 min. This step is crucial to eliminate bubbles resulting from the mixing of the two silicone phases (if RTV silicon) and to ensure silicone penetration into the peripheral groove.
Gently place the lower part of the B60 to smooth the silicone on the mold surface. Starting from one corner, gradually deposit to avoid trapping air. A weight can be placed on top to maintain pressure.
Incubate the assembly at 37–55 °C for 20 min to catalyze crosslinking. Alternatively, incubate for 1.5 h at room temperature (depending on the silicone used).
Retrieve the assembly after complete silicone crosslinking.
Remove the upper part of the B60 (no need to force).
Gently remove (lever from an angle) the lower part of the Petri dish while manually holding the mold.
Using a spatula, lever at the notches located at the base of the external part (EBs-mold-extern) of the mold. Gradually proceed from notch to notch until complete separation from the lower part (EBs-mold-base).
The base (EBs-mold-base) of the mold is completely removed.
Manually remove the external part (EBs-mold-extern) by applying gentle pressure.
The external part (EBs-mold-extern) of the mold is completely removed.
Finally, gently remove the silicone disk from the internal part ( EBs-mold-intern) with your fingers.
The internal part (EBs-mold-intern) of the mold is completely removed.
The molding process is complete. After a soap washing step (optional: use a toothbrush with soft bristle), the resulting disk can be autoclaved (liquid or dry) and used.
hiPSCs aggregation
Note: Perform the culture operation in a level 1 or 2 laboratory in a dedicated laminar airflow cabinet. Use autoclaved tweezers to ensure sterility. After use, store the forceps in a 15 mL Falcon filled with 70% EtOH until the next autoclaving.
Using tweezers, place the silicon discs at the bottom of 6-well plates.
Prepare the hiPSC spheroids induction medium (see Recipe 1) and TrypLETM inhibition medium (see Recipe 2).
Culture hiPSCs in mTeSRTM Plus containing medium on plates coated with Vitronectin XFTM until cells reach 80%–90% of confluency (Figure 2A).
Figure 2. Different steps of the human induced pluripotent stem cells (hiPSC) spheroids induction protocol. A. Colonies of hIPSCs before trypsination. B. Appearance of cells in wells after plating and centrifugation. C. Aspect of spheroids after 24 h aggregation. Scale bar = 200 µm.
Aspirate the medium from the cells and wash with 2.5 mL of room temperature DPBS. For aspiration, use a vacuum pump with a pipette and appropriate tubing.
Add 2 mL of TrypLETM and incubate for 5 min at 37 °C.
After cell detachment, add 2.5 mL of TrypLETM inhibition medium. Transfer the total volume to a 15 mL Falcon tube and centrifuge at 200× g for 4 min at room temperature.
Resuspend the cell pellet in 1 mL of hiPSC spheroids induction medium and count the cells (using a hemocytometer).
Transfer the desired cell quantity to a 2 mL Eppendorf tube. Gently mix.
Note: The diameter of the resulting hiPSC spheroids is directly related to the number of cells deposited on the disc, following linear Equation 1 (example: with 1 × 106 cells, D = 79.4 + 42.6 = 122 μm).
Equation 1: Spheroid diameter (D) as a function of the number of hIPSCs deposited on the disc.
D = 79.4x + 42.6
with D in µm and x the number of cells per disc in 10 5
Distribute the cell solution evenly on the molded disc by depositing drops in a circular motion. To ensure homogeneity, deposit the cell solution across the entire disc rather than locally, preventing strong size variations of the obtained spheroids.
Wait for 1 h in a 37 °C, 5% CO2 incubator for sedimentation. This step is crucial for ensuring good cell distribution and, consequently, uniformity of the spheroids (Figure 2B ).
Centrifuge at 500× g for 2 min at 20 °C to enhance cell aggregation.
Place the 6-well plate in a 37 °C, 5% CO2 incubator for 24 h (Figure 2C).
Spheroids extraction
Note: Perform the culture operation in a level 1 or 2 laboratory in a dedicated laminar airflow cabinet. Use autoclaved tweezers to ensure sterility. After use, store the forceps in a 15 mL Falcon filled with 70% EtOH until the next autoclaving.
After 24 h, use a P1000 pipette and 1 mL of DPBS warmed at 37 °C to pipette. Mix by aspiration, dislodging spheroids from the microwells.
Gently invert the silicone discs using fine forceps.
Centrifuge at 200× g for 4 min to extract the spheroids from the microwells.
Collect the culture medium containing spheroids in a 50 mL collection tube for each well.
Flush each disc with 5 mL of DPBS in order to recover any remaining spheroids at the well bottom and add this volume to the collection tube.
Rinse each silicone disc with 2 mL of DPBS. Submerge the discs in EtOH 70% for 30 min. Wash with water and soap, rinse in dezionized water, and autoclave in an autoclave bag for the next experiment.
Check spheroids integrity (refer to Figure 3A and Figure 3B) and gently centrifuge the collection tubes at 50× g for 4 min at 20 °C to obtain a pellet containing the spheroids.
Figure 3. Spheroids of two different human induced pluripotent stem cells (hiPSC) cell lines 24 h after deposition of 1 × 106 cells per disc. A. Cell line AG08C5. B. Cell line SCTi003-A. Scale bar = 200 µm.
Proceed with your experiment using the hiPSC spheroids.
Data analysis
To demonstrate the robustness of the methodology, varying quantities of hiPSCs were applied onto the discs following the protocol described above (Figure 4).
Figure 4. Human induced pluripotent stem cells (hIPSC) spheroids diameter depending on the number of cells deposited. A. 1 × 105. B. 2.5 × 105. C. 5 × 10 5. D. 7.5 × 105. E. 1 × 106. F. 1.5 × 106. Scale bar = 100 µm.
The obtained results were analyzed using a custom image analysis algorithm developed in MATLAB, leveraging the ImFindCircle function, which is relevant for the measurement of spheroid diameters (https://www.mathworks.com/help/images/ref/imfindcircles.html). Although not perfect due to occasional spheroid escape detection, the large number of events analyzed helps mitigate data variability. This analysis reveals a direct relationship between the quantity of cells deposited on a disc (each disc contains approximately 20,00 wells) and the resulting spheroid diameter. Spheroid polydispersity analysis based on each cell’s quantity is depicted in Figure 5.
Figure 5. Human induced pluripotent stem cells (hIPSC) spheroids diameter distribution depending on the number of cells deposited (n = 300, D = trend equation, R = 0.87).
These box plots demonstrate the technique's versatility, generating spheroids ranging from 50 to 150 μm in diameter with the same mold by varying only the initial number of cells deposited. Notably, deviation is important for small cell numbers (<1 × 106), reaching a minimum value of 1 × 106 cells per disc. With the designed microwell diameter (approximately 400 µm), this condition appears most suitable for the production of homogeneous spheroids (Figure 4E). This observation can be attributed to a relation between microwell volume and the optimal volume occupied by cells (and hence cell quantity) for reproducible filling.
Moreover, the method demonstrates good reproducibility, with triplicate independent experiments using 1 × 106 cells per disc (approximately 5,000 cells per microwell) showing a deviation of spheroid median diameters of only 10.2 µm (run 1: median 130.2 ± 10.2 μm; run 2: 127.9 ± 9.9 μm; run 3: 128 ± 10.4 μm).
The integrity of hiPSC spheroids produced by the method was observed after extraction (Figure 6A). In all cases, a clear peripheral membrane was observed, indicating good aggregation. After 5 days in culture medium (mTeSR™ Plus), significant growth was observed (Figure 6B), validating the viability and quality of the produced spheroids.
Figure 6. Spheroid diameter for the condition 1 × 106 cells per disc (≃5000 cells per well) at day 1 (A) and day 5 (B). Scale bar = 100 µm.
Validation of protocol
The protocol has been successfully employed in various studies in the lab, as well as in our most recent publication (Lemarié et al, DOI: 10.3390/bioengineering10121418). In this particular study, hiPSC-induced spheroids were incorporated into different alginate-gelatine hydrogels to investigate the impact of viscoelastic properties on the spheroid’s fate in the absence of growth factor.
General notes and troubleshooting
General notes
When 3D-printing molds, the nature of the resin is relatively unimportant as long as the post-process is executed correctly. Notably, non-polymerized resins can hinder silicone cross-linking [13]. Regarding printing parameters, it is strongly recommended to employ the maximum resolution (layer height < 50 μm) for the central part (EBs-mold-intern). Low resolution may result in a staircase effect in the microwells, negatively impacting cell aggregation and, consequently, spheroid quality.
The extraction of spheroids from the well is a critical step. Turning the disc over followed by centrifugation removes most spheroids, but some may remain in the microwells. The rinsing step with DPBS allows for their recovery. It is recommended to vigorously flush (employ an important backflow rate) the disc during this step. To standardize spheroid diameter more rigorously, a strainer with defined mesh size (usually < 200 μm) can be used to set aside spheroids of excessively large diameter, particularly at the disc’s edge, which can impact spheroid diameter distribution (see Figure S2a and Figure S2b). Silicone discs are, in principle, infinitely reusable if not damaged by handling with fine forceps or during washing steps.
The reduced dispersion observed in spheroids induced with a 106 cell deposit aligns with expectations. This deposition corresponds to approximately 500 cells per microwell, an optimal quantity considering the microwell's conical geometry with a depth of 400 μm and a base of 600 μm. The adaptability of this chosen format is elucidated in Figure 4. Note that the suitability of dimensions depends on cell type and quantity, highlighting the versatility of the selected configuration for this study.
After a 24 h incubation, hIPSC spheroids show optimal aggregation and structural integrity and do not disaggregate. It is recommended to use them within the first hour post-extraction to prevent fusion. They can be reintegrated into standard cell culture, adhering to the substrate coating and proliferating. Alternatively, they can be incorporated into viscous solutions like Matrigel® while maintaining integrity. 100 μm hIPSC spheroids were successfully embedded in alginate-gelatin hydrogels with preserved viability (live-dead staining, [10]). Note the increased risk of necrotic core formation with larger spheroid diameters due to diffusion constraints.
The 3D model presented here (designed using Autodesk Fusion 360) is the result of several iterations and is considered a very good compromise between the average resolution of inkjet printers currently on the market and the number of spheroids obtained in each experiment.
Troubleshooting
Problem 1: No aggregation.
Possible cause: Quantity of cells.
Solution: Deposit more cells (a minimum quantity is required to allow aggregation depending on cell type).
Problem 2: Spheroids’ diameter homogeneity.
Possible cause: Deposition of cells.
Solution: Achieving a uniform deposition across the entire disc structure, for example through circular motion, is crucial to prevent local cell concentration.
Problem 3: Several spheroids per well.
Possible cause: Bad centrifugation.
Solution: Double the centrifugation time.
Problem 4: Low spheroids yield.
Possible cause: Spheroids stuck in the silicone disc microwells.
Solution: Vigorously flush the disc before flipping and centrifuge the disc.
Acknowledgments
This research received support from SEGULA Technologies and the ANRT (National Association for Research and Technology). The authors express their gratitude to the IPS-PGNM core facility (https://pgnm.inmg.fr/plateformes-inmg/, Lyon, FRANCE) for providing the AG08C5 and SCTi003-A (Stem Cell Technologies) control IPS lines. The AG08C5 line is officially registered in the European hPSCreg as PGNMi001-A (https://hpscreg.eu/cell-line/PGNMi001-A) [12]. The SCTi003-A is a commercial cell line, female, and derived from blood cells (STEMCELL, Vancouver, Canada). The authors also acknowledge that all human cells utilized at the iPS-PGNM platform are duly declared to the French Ministry of Health (CODECOH DC-2022-5055).
The experiments were conducted with the invaluable assistance of the LBTI (Laboratory for Tissue Biology and Therapeutic Engineering, Lyon, France) and the academic platform 3d.FAB (Villeurbanne, France). Special thanks to Rania Hadji for her assistance in drafting the pictorial protocol. This protocol was applied in [10].
Competing interests
The authors declare no conflict of interest or competing interests.
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Nguyen, D., Hägg, D. A., Forsman, A., Ekholm, J., Nimkingratana, P., Brantsing, C., Kalogeropoulos, T., Zaunz, S., Concaro, S., Brittberg, M., et al. (2017). Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink. Sci. Rep. 7(1): e1038/s41598–017–00690–y. https://doi.org/10.1038/s41598-017-00690-y
Baptista, L., Kronemberger, G., Côrtes, I., Charelli, L., Matsui, R., Palhares, T., Sohier, J., Rossi, A. and Granjeiro, J. (2018). Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering. Int. J. Mol. Sci. 19(5): 1285. https://doi.org/10.3390/ijms19051285
Boot, R. C., Koenderink, G. H. and Boukany, P. E. (2021). Spheroid mechanics and implications for cell invasion. Adv. Phys.: X 6(1): e1978316. https://doi.org/10.1080/23746149.2021.1978316
Lemarié, L., Dargar, T., Grosjean, I., Gache, V., Courtial, E. J. and Sohier, J. (2023). Human Induced Pluripotent Spheroids’ Growth Is Driven by Viscoelastic Properties and Macrostructure of 3D Hydrogel Environment. Bioengineering 10(12): 1418. https://doi.org/10.3390/bioengineering10121418
Białkowska, K., Komorowski, P., Bryszewska, M. and Miłowska, K. (2020). Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application. Int. J. Mol. Sci. 21(17): 6225. https://doi.org/10.3390/ijms21176225
Badja, C., Maleeva, G., El-Yazidi, C., Barruet, E., Lasserre, M., Tropel, P., Binetruy, B., Bregestovski, P. and Magdinier, F. (2014). Efficient and Cost-Effective Generation of Mature Neurons From Human Induced Pluripotent Stem Cells. Stem Cells Transl. Med. 3(12): 1467–1472. https://doi.org/10.5966/sctm.2014-0024
Venzac, B., Deng, S., Mahmoud, Z., Lenferink, A., Costa, A., Bray, F., Otto, C., Rolando, C. and Le Gac, S. (2021). PDMS Curing Inhibition on 3D-Printed Molds: Why? Also, How to Avoid It?. Anal. Chem. 93(19): 7180–7187. https://doi.org/10.1021/acs.analchem.0c04944
Supplementary information
The following supporting information can be downloaded here
Figure S1: Illustrated protocol of the different steps for producing silicone discs from preprinted molds.
Figure S2: Diameter distribution of AG08C5 spheroids for 1 × 106 cells per disc.
Article Information
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Stem Cell > Organoid culture
Cell Biology > Cell isolation and culture > 3D cell culture
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Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins
YW Yueying Wang *
XZ Xinting Zhang *
XF Xiao Feng
XW Xia Wang
QJ Qi Jin
FJ Feng Jiang
(*contributed equally to this work)
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4966 Views: 627
Reviewed by: David A. CisnerosBhanu JagilinkiJoyce ChiuGuillaume Lenoir
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Original Research Article:
The authors used this protocol in Science Advances Apr 2022
Abstract
Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.
Keywords: Contractile injection system Photorhabdus asymbiotica PVC T6SS Phage tail Protein complex Purification
Background
Typical contractile injection systems (CISs) have been studied for decades for their structure, assembly, and mechanism. They can take part in multiple interactions between microorganisms and hosts by delivering nucleic acids and protein substrates using contractile sheath–tube assemblies, such as the contractile tails of bacteriophage T4, P2, and Mu [1]. The type VI secretion system, which spans both the inner and outer membranes of Gram-negative bacteria and transports substrates from the cytoplasm, is a well-studied example of a contractile apparatus [2]. Extracellular CIS (eCISs), found in both prokaryotes and archaea, such as Photorhabdus virulence cassette (PVC), the antifeeding prophage [3], and the metamorphosis-associated contractile structure [4], have also been studied extensively in recent years.
Although the species from the Photorhabdus genus are generally considered pathogens infecting insects, P. asymbiotica has been reported to infect humans in the United States and Australia [5]. Five PVC clusters have been identified in the genome of P. asymbiotica, all phylogenetically related to the tail of T4 bacteriophage. PVC could translocate toxins into eukaryotic cells [6], which means PVC is different from T4 phage or R-type pyocins, which can only target prokaryotic cells. Moreover, a group of N-terminal signal peptides are identified within the PVC effectors to facilitate the cargo loading process [7]. Taking into consideration that PVC complexes could be released outside the bacterial cells for function, this apparatus would be an ideal model for manipulation as a potent delivery vector in precision medicine [8].
The purification workflow of PVC can be divided into four steps: bacterial collection, cell rupture, cell debris removal, and protein extraction. Centrifugation and ultracentrifugation are the primary methods for bacterial collection, cell debris removal, and protein extraction. As for the cell rupture, chemical and biological methods are applied to break the cell membrane and release the PVC protein complexes. This protocol has been used several times to purify PVC complexes for structural and functional characterization experiments, demonstrating the effectiveness of this method.
Materials and reagents
15 mL centrifuge tubes (Corning, catalog number: 430791)
50 mL centrifuge tubes (Corning, catalog number: 430829)
500 mL triangular culture flask (Corning, catalog number: 431145)
1.5 mL microtubes (Axygen, catalog number: MCT-150-C-S)
Sterile syringe filter (Millipore, catalog number: SLGVR33RB)
500 mL centrifuge bottle (Nalgene, catalog number: 3120-0500)
50 mL round bottom centrifuge tubes (Biosharp, catalog number: BS-500-MC)
13.2 mL centrifuge tubes (Beckman Coulter, catalog number: 344059)
Escherichia coli strain EPI300 (Lucigen, catalog number: EC300110)
Plasmids pCNM3, pBR60, and pBBRN Pnf N50-TkTcsT, which were used previously [4,7] (Figure 1)
Figure 1. Schematic illustration of the Photorhabdus virulence cassette (PVC) locus and the map of plasmids used for PVC complex and cargo protein production. A. The 16 PVC structural genes (annotated Pvc1–Pvc16, shown in blue) involved in protein complex production are encoded consecutively within the P. asymbiotica genome. B–D. The plasmids pCNM3 (B), pBR60 (C), and pBBRN Pnf N50-TkTcsT (D) were constructed by our lab and the maps were generated using SnapGene. pCNM3 was constructed by ligating the complete PVC operon into pRK404 vector for constitutive expression under their own promoters [9]. pBR60 was created by ligating the DNA fragment containing the PAU_RS16560 (LysR regulator gene, shown in blue) into pBR322 plasmid (BamHI-SalI insertion) for constitutive expression of the regulator gene under its own promoter. pBBRN Pnf N50-TkTcsT was a plasmid used for cargo production. This plasmid was created by inserting the open reading frame of the toxic TkTcsT protein (shown in blue) from Trichosanthes kirilowii into a broad host range vector pBBR1MCS5, with a PVC signal peptide sequence (shown in red) fused at the N-terminus of the TkTcsT protein. This allows the production of signal peptide–fused proteins in E. coli.
LB broth, powder (Solarbio, catalog number: L1010)
Phosphate-buffered saline (PBS) (Gibco, catalog number: C10010500BT)
Tetracycline HCl (Solarbio, catalog number: T8180)
Ampicillin, sodium salt (Solarbio, catalog number: A8180)
Gentamycin sulfate (Solarbio, catalog number: G8170)
Anhydrous ethanol (Beijing Chemical Works, catalog number: C320080)
TBS tablets (VWR, catalog number: K859-100TABS)
Triton X-100 solution (Sigma, catalog number: X-100)
Protease inhibitor cocktail (MCE, catalog number: HY-K0010)
Lysozyme (Sigma, catalog number: L6876)
DNase I (MCE, catalog number: HY-108882)
Sodium chloride (Solarbio, catalog number: S8210)
Magnesium chloride hexahydrate (Solarbio, catalog number: M8161)
Solution endotoxin erasol (TianDZ, catalog number: 60607-25)
Solutions
LB broth medium (see Recipes)
10 mg/mL tetracycline in solution (see Recipes)
100 mg/mL ampicillin in solution (see Recipes)
50 mg/mL gentamycin in solution (see Recipes)
5 mg/mL NaCl (see Recipes)
2 M MgCl2 (see Recipes)
20 mg/mL lysozyme (see Recipes)
5 mg/mL DNase I (see Recipes)
P buffer (see Recipes)
Recipes
LB broth medium
Add 25 g of LB broth powder to 1,000 mL of distilled water. Sterilize using a high-pressure steam sterilization pot at 121 °C for 15 min.
10 mg/mL tetracycline in solution
Add 0.5 g of tetracycline HCl to 50 mL of anhydrous ethanol. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
100 mg/mL ampicillin in solution
Add 1 g of ampicillin to 10 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
50 mg/mL gentamycin in solution
Add 1 g of gentamycin sulfate to 20 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
5 mg/mL NaCl
Dissolve 1 g of sodium chloride in 200 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
2 M MgCl2·6H2O
Dissolve 406 g of magnesium chloride hexahydrate in 800 mL of distilled water and adjust volume to 1 L. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
20 mg/mL lysozyme
Dissolve 1 g of lysozyme in 50 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
5 mg/mL DNase I
Dissolve a bottle of DNase I (100 mg) in 20 mL of NaCl solution (5 mg/mL). Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.
P buffer
Take 50 mL of distilled water into a 50 mL centrifuge tube and add a piece of TBS tablet. Divide the solution into two 50 mL centrifuge tubes equally after the TBS tablet is completely dissolved.
Add 2.5 mL of 10% Triton X-100 solution, 500 μL of 20 mg/mL lysozyme, 500 μL of 5 mg/mL DNase I, 125 μL of 2 M MgCl2, and 500 μL of protease inhibitor cocktail to each tube. Add distilled water to fill 50 mL.
Notes:
P buffer should be freshly prepared.
TBS tablets could cause skin and eye irritation; avoid breathing dust, fumes, gas, mist, vapors, or spray.
Triton X-100 solution could cause serious eye damage; wear protection when using it.
The final concentration of lysozyme and DNase I are 200 µg/mL and 50 µg/mL in P buffer, respectively. Triton X-100 and MgCl2 are 50 mg/mL, 0.5%, and 5 mM, respectively.
Equipment
Innova44 Incubator Shaker Series (NBS, model: SHA1104)
Biological safety cabinet (Shanghai Lishen Scientific Equipment Co, model: Hfsafe 1200LC)
Milli-Q (Millipore, catalog number: IX 70XX)
High-pressure steam sterilization pot (YAMATO, model: SM830)
High-speed refrigerated centrifuge (Eppendorf, model: 5424 R)
Ultracentrifuge (Beckman, model: Optima L-100 XP)
-80 °C ultra-low temperature freezer (Haier, model: DW-86L728J)
Thermostatic water bath (GFL, model: GFL1002)
High-speed refrigerated centrifuge (Hitachi, model: CR22G)
Ice machine (Panasonic, model: SIM-F140BDL)
NanoDrop 1000 spectrophotometer
Procedure
Production of empty and cargo-loaded PVC complexes
The heterologous expression of the empty PVC complexes in E. coli requires two plasmids, pCNM3 and pBR60. Plasmid pCNM3 carries 16 structural genes required for PVC complexes assembly based on a tetracycline-resistant broad host range vector pRK404 (Figure 1A). Plasmid pBR60 harbors the LysR regulator gene (PAU_RS16560) fragment, which is indispensable for the maturation of PVC complexes (Figure 1C). The backbone vector for pBR60 is ampicillin-resistant pBR322 (the original tetracycline-resistance gene of pBR322 is disrupted by gene insertion). Both genes are expressed constitutively under their own promoters. The E. coli strain EPI300 co-expressing these two plasmids can be used to produce the empty PVC complexes accordingly (two plasmids in E. coli for empty PVC).
For the cargo-loaded PVC complex to be produced, a third plasmid based on pBBRN (a derivative of broad host range vector pBBR1MCS5, which introduces a NdeI site to generate a start codon at the beginning of LacZα gene) with gentamycin resistance can be used. In this protocol, the DNA fragment producing fusion protein of toxic TkTcsT and PVC signal peptide at the N-terminus is inserted into multiple cloning sites of pBBRN, whose expression is controlled by the lac promoter (Figure 1D). Similar operations can be applied to generate cargo-loaded PVC complexes using the empty PVC-producing E. coli strain mentioned above, co-transformed with the third cargo-expressing plasmid (three plasmids in E. coli for cargo-loaded PVC).
Bacterial collection
Prepare LB broth medium according to Recipe 1. Add antibiotics to the LB broth medium if needed. Prepare 10 mg/mL tetracycline solution (Recipe 2) and dilute 100 times to bring the final concentration to 10 μg/mL at the time of use. Prepare 100 mg/mL ampicillin solution (Recipe 3) and dilute 100 times to bring the final concentration to 100 μg/mL at the time of use. Prepare 50 mg/mL gentamycin solution (Recipe 4) and dilute 500 times to bring the final concentration to 10 μg/mL at the time of use.
Load 3–4 mL of LB broth medium supplemented with 100 μg/mL ampicillin and 10 μg/mL tetracycline (additional 10 μg/mL gentamycin for cargo-loaded PVC production) into 15 mL centrifuge tubes. The same type and concentration of antibiotics are used throughout step B.
Pick one single colony of E. coli EPI300 stain harboring corresponding plasmids on LB solid medium or frozen bacteria (in a -80 °C ultra-low temperature refrigerator) in LB broth medium with antibiotics and incubate overnight on a shaker at 220 rpm and 37 °C.
Inoculate 2 mL of bacteria incubated overnight (from steps B2 and B3) in 200 mL of LB broth medium with antibiotics and incubate for 24 h at 220 rpm and 30 °C in a shaker.
Load the 200 mL bacterial cultures into 500 mL centrifuge bottles and centrifuge using a high-speed refrigerated centrifuge at 12,000× g for 10 min at 4 °C.
Discard the supernatant and resuspend in 30 mL of sterile PBS.
Transfer the suspension to 50 mL centrifuge tubes and centrifuge at 3,500× g for 30 min at 4 °C.
Discard the supernatant and store the bacterial pellet in a -80 °C ultra-low temperature refrigerator overnight until use.
Cell rupture
Prepare P buffer (see Recipes).
Take out the frozen bacteria from the -80 °C ultra-low temperature refrigerator, add 10 mL of P buffer to each tube, resuspend, and incubate in a water bath at 37 °C for 30 min.
Cell debris removal
Transfer the lysate to round bottom centrifuge tubes and spin at 27,000× g for 10 min at 4 °C using a high-speed refrigerated centrifuge.
Protein extraction
Transfer the supernatant to ultracentrifuge tubes, balance with P buffer precooled at 4 °C, and centrifuge at 150,000–250,000× g for 1 h at 4 °C using an ultracentrifuge.
Discard the supernatant and resuspend sediment with 1 mL of sterile PBS precooled at 4 °C. Transfer the suspension into 1.5 mL microtubes and centrifuge at 18,400× g for 10 min at 4 °C using a high-speed refrigerated centrifuge.
Transfer the supernatant into ultracentrifuge tubes, balance with PBS precooled at 4 °C, and centrifuge at 150,000–250,000× g for 1 h at 4 °C using an ultracentrifuge.
Discard the supernatant and resuspend sediment with 200 μL of sterile PBS precooled at 4 °C. Transfer the suspension into 1.5 mL microtubes and centrifuge at 18,400× g for 10 min at 4 °C using a high-speed refrigerated centrifuge.
Collect supernatant and measure the concentration of PVC at 280 nm using a NanoDrop 1000 spectrophotometer. Store the supernatant containing the PVC particles at 4 °C (Figure 2).
Figure 2. SDS-PAGE analysis of protein samples from major steps during Photorhabdus virulence cassette (PVC) purification. Lane 1: Whole-cell lysate from step D1. Lane 2: The supernatant from step E1. Lane 3: The supernatant from step E3. Lane 4: Final purified PVC from step E4, as used for negative staining analysis. M: Marker.
Endotoxin removal (optional)
Precool the solution endotoxin erasol reagent and the protein sample.
Add 50 μL of solution endotoxin erasol reagent to 500 μL of protein sample, mix, leave it on an ice bath for 10 min, and then incubate in a water bath at 65 °C for 20 min.
Centrifuge at 18,400× g for 10 min using a high-speed refrigerated centrifuge. Collect the supernatant.
Validation of protocol
This protocol has been used and validated in the following research articles:
Jiang et al. [7]. N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system. Science Advances (Figure 1, panel D; Figure 2, panel B; Figure 3).
Wang et al. [6]. Characterization of Photorhabdus Virulence Cassette as a causative agent in the emerging pathogen Photorhabdus asymbiotica. Science China Life Sciences (Figure 1, panel B; Figure 2, panel F).
Jiang et al. [9]. Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System. Cell (Figure 1; Figure 6, panel D; Figure 7, panel A; Figure S1, panel C–D).
General notes and troubleshooting
In steps B4 and B5, the OD600 of bacteria incubated overnight should be higher than 0.6. If the OD600 of bacteria is lower than 0.6, it generally indicates some problems in the culture process or that the cargo protein may be toxic to bacteria.
In step B4, incubate at 30 °C, because the protein expression and assembly at 30 °C is better than at 37 °C.
In step B8, the collected bacterial pellet can be stored at -80 °C overnight to facilitate complete rupture of the bacterial cells, which is helpful for a greater production of the PVC complexes.
In steps E1 and E3, any centrifugal force between the ranges of 150,000× g and 250,000× g can efficiently sediment the particles. For example, spinning at a higher speed produces a greater number of PVC particles but with many undesirable immature components.
In step E5, a NanoDrop 1000 spectrophotometer is used to measure the concentration of PVC protein samples. NanoDrop 1000 spectrophotometer is a convenient equipment for protein and nucleic acid concentration determination; samples as low as 2 μL can be accurately quantified. Here, the concentration of protein samples is estimated by measuring UV absorption at 280 nm. Although this method is not strictly quantitative, it is quick, convenient, and suitable for protein concentration determination in this protocol.
In step E5, the concentration of protein extracted under this protocol should generally be in the range of 1–5 mg/mL. If the concentration is higher than 10 mg/mL, it is recommended to repeat steps E4–E5 to exclude any remaining bacterial debris or other protein components.
Step F is optional; however, it is an essential step if the protein sample is to be tested on animals.
Data analysis
Negative staining is the best method to assess the quality of PVC samples in the solution [9]. Aliquots of 1–5 μL of PVC samples (depending on the protein concentration) are applied onto glow-discharged holey-carbon copper grids (300 mesh, Zhongjingkeyi, Beijing), washed, and stained with 2% uranyl acetate. The negative stain grids are dried at room temperature and checked using an electron microscope. PVC complexes, if present, are seen as bullet-shaped (Figure 3).
Figure 3. Representative negative-staining electron microscopy figure of Photorhabdus virulence cassette (PVC) particles purified from E. coli. Empty PVC (A) and PVC complexes loaded with TkTcsT cargos (B) share similar T4-tail-like morphology by negative staining electron microscopy observation. Mature (black arrows) and immature (white arrow) components of PVC are shown.
Acknowledgments
We thank Prof. Ning Gao and the Electron Microscopy Laboratory of Peking University for help with negative staining. We also thank all the lab members of Qi Jin and Feng Jiang for beneficial advice. The work was funded by the National Key Research and Development Program of China (2022YFC2303200), National Natural Science Foundation of China (NSFC) (32070081), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-037) and the Special Research Fund for Central Universities, Peking Union Medical College (3332023055). The original protocol was developed and used in previous works [6,7,9].
Competing interests
No competing interest is declared.
References
Taylor, N. M. I., van Raaij, M. J. and Leiman, P. G. (2018). Contractile injection systems of bacteriophages and related systems. Mol. Microbiol. 108(1): 6–15.
Wang, J., Brodmann, M. and Basler, M. (2019). Assembly and Subcellular Localization of Bacterial Type VI Secretion Systems. Annu. Rev. Microbiol. 73: 621–638.
Desfosses, A., Venugopal, H., Joshi, T., Felix, J., Jessop, M., Jeong, H., Hyun, J., Heymann, J. B., Hurst, M. R. H., Gutsche, I., et al. (2019). Atomic structures of an entire contractile injection system in both the extended and contracted states. Nat. Microbiol. 4(11): 1885–1894.
Rocchi, I., Ericson, C. F., Malter, K. E., Zargar, S., Eisenstein, F., Pilhofer, M., Beyhan, S. and Shikuma, N. J. (2019). A Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector. Cell Rep. 28(2): 295–301 e294.
Costa, S. C., Girard, P. A., Brehelin, M. and Zumbihl, R. (2009). The emerging human pathogen Photorhabdus asymbiotica is a facultative intracellular bacterium and induces apoptosis of macrophage-like cells. Infect. Immun. 77(3): 1022–1030.
Wang, X., Cheng, J., Shen, J., Liu, L., Li, N., Gao, N., Jiang, F. and Jin, Q. (2022). Characterization of Photorhabdus Virulence Cassette as a causative agent in the emerging pathogen Photorhabdus asymbiotica. Sci. China Life Sci. 65(3): 618–630.
Jiang, F., Shen, J., Cheng, J., Wang, X., Yang, J., Li, N., Gao, N. and Jin, Q. (2022). N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system. Sci. Adv. 8(17): eabm2343.
Kreitz, J., Friedrich, M. J., Guru, A., Lash, B., Saito, M., Macrae, R. K. and Zhang, F. (2023). Programmable protein delivery with a bacterial contractile injection system. Nature 616(7956): 357–364.
Jiang, F., Li, N., Wang, X., Cheng, J., Huang, Y., Yang, Y., Yang, J., Cai, B., Wang, Y. P., Jin, Q. and Gao, N. (2019). Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System. Cell 177(2): 370–383 e315.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Microbiology > Heterologous expression system > Escherichia coli
Biochemistry > Protein > Expression
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Peer-reviewed
In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase
AM Aisha Mahboob *
NF Nishat Fatma *
AH Afzal Husain
(*contributed equally to this work)
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4967 Views: 913
Reviewed by: Marcelo S. da SilvaRitu Gupta Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Protein Expression and Purification Aug 2023
Abstract
The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase.
Key features
• We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d.
• Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance.
• Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers.
• The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.
Keywords: Fusion DNA polymerase Pfu-Sso7d PCR Precipitation Processivity
Background
DNA polymerases are extensively used in PCR to exponentially amplify DNA and generate a substantial quantity from a minimal initial DNA template. An efficient PCR amplification necessitates a DNA polymerase that is not only thermostable but also has excellent fidelity and processivity. These essential DNA polymerase characteristics shorten extension times and enable error-free amplification of lengthy DNA templates. A variety of approaches are utilized to enhance the processivity of the DNA polymerase. One such approach is using fusion DNA polymerases, created by the covalent fusion of a tiny DNA-binding protein to the polymerase domain of the enzyme. Pfu-Sso7d fusion DNA polymerase, for instance, is produced by the fusion of Pfu DNA polymerase with Sso7d, a tiny 7 kDa protein derived from Sulfobulus solfataricus, and binds to dsDNA in a sequence-independent manner [1,2]. This fusion significantly increases processivity by preventing the Pfu-Sso7d from frequently dissociating from the template.
Recombinant DNA polymerases are typically purified through a time-consuming, cost-intensive, two-step affinity purification followed by dialysis [3,4]. We recently reported a straightforward, economical, and time-saving method for expressing and purifying the Pfu-Sso7d fusion DNA polymerase. This involves heat denaturation and DNase I treatment of bacterial lysate to recover thermostable DNA polymerase (Figure 1). The heat-cleared and DNase I–treated lysates are then precipitated using ethanol or acetone [5]. We also reported an in-house PCR buffer system that outperforms commercially available alternatives for PCR amplification of various DNA templates. Laboratories dealing with a large number of PCRs and constrained resources can greatly benefit from the in-house purification of thermostable polymerases and the preparation of in-house buffer solutions.
Figure 1. Precipitation-based protocol for the purification of Pfu-Sso7d fusion DNA polymerase. A. Schematic representation showing the extraction and purification of Pfu-Sso7d from the IPTG-induced bacterial culture. Briefly, a colony of BL21 (DE3) pLysS transformed with pET-28b-Pfu-Sso7d expression plasmid was cultured and induced with IPTG. Bacterial cells were harvested and lysed using a lysis buffer containing lysozyme. Heat-cleared and DNase I–treated lysates were then precipitated using acetone or ethanol and analyzed by SDS-PAGE and PCR. B. SDS-PAGE analysis of the Pfu-Sso7d in heat-cleared and DNase I–treated cell lysate and of the precipitates obtained with 67% (v/v) ethanol or 50% (v/v) acetone. C. The precipitated Pfu-Sso7d was tested in the PCR amplification of a 2.4 kb fragment from the plasmid template.
Materials and reagents
Biological materials
BL21 (DE3) pLysS cells for protein expression (Thermo Fisher Scientific, catalog number: C606010)
pET-28b-Pfu-Sso7d plasmid (a gift from Dr. Alexander Klenov, York University, Canada) (Sequence can be downloaded from https://barricklab.org/twiki/pub/Lab/ProtocolsReagentsPfuSso7d/6his-pfu-sso7d-pET28.gbk)
Reagents
Tris (hydroxymethyl) aminomethane, Tromethamine, Tris base (Sisco Research Laboratory, catalog number: 71033)
Luria broth (LB) (Himedia, catalog number: M575)
Agar (Himedia, catalog number: GRM026)
Super optimal catabolite (SOC) (Himedia, catalog number: G015)
Kanamycin (Himedia, catalog number: A008)
Chloramphenicol (Himedia, catalog number: CMS218)
Deoxyribonucleotides (dNTPs) (Sisco Research Laboratory, catalog number: 14464)
Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Himedia, catalog number: MB072)
Phenylmethylsulphonyl fluoride (PMSF) (Sisco Research Laboratory, catalog number: 87606)
Lysozyme (Sisco Research Laboratory, catalog number: 45822)
Sodium dodecyl sulfate (SDS) (Sisco Research Laboratory, catalog number: 1948101)
Acrylamide 1× crystal (Sisco Research Laboratory, catalog number: 89314)
Bis-acrylamide (Sisco Research Laboratory, catalog number: 38516)
Ammonium persulfate (APS) (Himedia, catalog number: MB003)
N,N,N′,N′-Tetramethyl ethylenediamine (TEMED) (Sisco Research Laboratory, catalog number: 84666)
β-mercaptoethanol (Sisco Research Laboratory, catalog number: 83759)
Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: 71505-250)
Glycerol (Sisco Research Laboratory, catalog number: 59991)
Dithiothreitol (DTT) (Sisco Research Laboratory, catalog number: 17315)
Bromophenol blue (Himedia, catalog number: GRM914)
Pair of specific primers (Integrated DNA Technology)
Deoxyribonuclease I (DNase I) (Invitrogen, catalog number: AM2222)
Agarose (Himedia, catalog number: MB002)
Betaine solution (Sigma-Aldrich, catalog number: B0300)
Ethylenediaminetetraacetic acid (EDTA) (Qualigens, catalog number: Q18455)
Ethanol, absolute 99.9% (any brand)
Acetone (Sisco Research Laboratory, catalog number: 31566)
Tween 20 (Merck, catalog number: SB3S630097)
Triton X-100 (Sisco Research Laboratory, catalog number: 64518)
Sodium chloride (NaCl) (Sisco Research Laboratory, catalog number: 76945)
Glycine (Merck, catalog number: MA7M562461)
Coomassie brilliant blue R-250 (Sisco Research Laboratory, catalog number: 93473)
Sodium hydrogen phosphate dodecahydrate (Na2HPO4·2H2O) (Sisco Research Laboratory, catalog number: 83417)
Potassium dihydrogen orthophosphate extra pure AR, 99.5% (KH2PO4) (Sisco Research Laboratory, catalog number: 50451)
Nonidet P-40 (NP-40) (Thermo Fischer Scientific, catalog number: 28324)
Bovine serum albumin solution (Sigma-Aldrich, catalog number: A8412)
Acetic acid (Sisco Research Laboratory, catalog number: 85801)
Ammonium sulphate [(NH4)2SO4] (Sisco Research Laboratory, catalog number: 88064)
Magnesium sulphate (MgSO4) (Sisco Research Laboratory, catalog number: 50014)
Potassium chloride extra pure AR, 99.5% (KCl) (Sisco Research Laboratory, catalog number: 38630)
Hydrochloric acid, 6N aqueous solution (HCl) (Sisco Research Laboratory, catalog number: 17560)
Ethidium bromide (Sigma-Aldrich, catalog number: E7637)
Magnesium chloride anhydrous extra pure, 98% (MgCl2) (Sisco Research Laboratory, catalog number: 31196)
Solutions
Phosphate buffer saline (PBS) (see Recipes)
Lysis buffer (see Recipes)
Storage buffer (see Recipes)
100 mM IPTG (see Recipes)
1 M Tris-HCl, pH 6.8 (see Recipes)
1.5 M Tris-HCl, pH 8.8 (see Recipes)
4× SDS dye (see Recipes)
10× Tris-Glycine-SDS buffer (see Recipes)
Staining solution (see Recipes)
De-staining solution (see Recipes)
10× PCR buffer (see Recipes)
Recipes
PBS (100 mL)
Reagent Final concentration Quantity
Na2HPO4·2H2O 100 mM 1.779 g
KH2PO4 18 mM 0.244 g
NaCl 137 mM 0.8 g
KCl 2.7 mM 0.02 g
Adjust pH to 7.4 with HCl/NaOH
Double-distilled water n/a up to 100 mL
Autoclave and store at 4 °C.
Lysis buffer (50 mL)
Reagent Final concentration Quantity
NaCl (5 M) 300 mM 3 mL
NaH2PO4 (1 M, pH 8.0) 50 mM 2.5 mL
Glycerol (100%) 10% (v/v) 5 mL
Triton-X 100 (10%) 0.1% (v/v) 0.5 mL
Double-distilled water n/a up to 50 mL
Store at 4 °C. Add 0.5 mM PMSF and 2 mg/mL lysozyme just before use.
Storage buffer (50 mL)
Reagent Final concentration Quantity
EDTA (0.5 M) 0.1 mM 0.01 mL
Tris-HCl (1 M, pH 8.0) 25 mM 1.25 mL
NaCl (5 M) 250 mM 2.5 mL
NP-40 (100%) 0.2% (v/v) 0.1 mL
Glycerol (100%) 50% (v/v) 25 mL
Tween 20 (100%) 0.2% (v/v) 0.1 mL
Double-distilled water n/a up to 50 mL
Filter sterilize, aliquot, and store at -20 °C. Add 2 mM DTT just before use.
100 mM IPTG (10 mL)
Reagent Final concentration Quantity
IPTG 100 mM 0.238 g
Double-distilled water n/a up to 10 mL
Filter sterilize, aliquot, and store at -20 °C.
1 M Tris-HCl, pH 6.8 (100 mL)
Reagent Final concentration Quantity
Tris base 1 M 12.1 g
Adjust pH to 8.8 with HCl/NaOH
Double-distilled water n/a up to 100 mL
Autoclave and store at 4 °C.
1.5 M Tris-HCl, pH 8.8 (100 mL)
Reagent Final concentration Quantity
Tris base 1.5 M 18.1 g
Adjust pH to 8.8 with HCl/NaOH
Double-distilled water n/a up to 100 mL
Autoclave and store at 4 °C.
4× SDS dye (5 mL)
Reagent Final concentration Quantity
Tris-HCl (1 M, pH 6.8) 200 mM 1 mL
Glycerol (100%) 40% (v/v) 2 mL
SDS (10%) 4% (w/v) 2 mL
Bromophenol blue 0.08% (w/v) 0.004 mL
Double-distilled water n/a up to 5 mL
Aliquot and store at -20 °C. Add β-mercaptoethanol to 5% (v/v) just before use.
10× Tris-Glycine-SDS buffer (100 mL)
Reagent Final concentration Quantity
Tris base 250 mM 3.03 g
SDS 35 mM 1 g
Glycine 1.92 M 14.4 g
Double-distilled water n/a up to 100 mL
Store at room temperature.
Staining solution (500 mL)
Reagent Final concentration Quantity
Methanol (100%) 40% (v/v) 200 mL
Acetic acid (100%) 8% (v/v) 40 mL
Coomassie brilliant blue R-250 0.1% (w/v) 0.5 g
Double-distilled water n/a up to 500 mL
Store at room temperature.
De-staining solution (500 mL)
Reagent Final concentration Quantity
Methanol (100%) 40% (v/v) 200 mL
Acetic acid (100%) 8% (v/v) 40 mL
Double-distilled water n/a up to 500 mL
Store at room temperature.
10× PCR buffer (25 mL)
Reagent Final concentration Quantity
Tris-HCl (1.5 M, pH 8.8) 200 mM 3.3 mL
KCl (1 M) 100 mM 2.5 mL
(NH4)2SO4 (1 M) 100 mM 2.5 mL
MgSO4 (1 M) 20 mM 0.5 mL
Triton X-100 (10%) 1% (v/v) 2.5 mL
Nuclease-free BSA (100 mg/mL) 1 mg/mL 0.25 mL
Double-distilled water n/a up to 25 mL
Filter sterilize, aliquot, and store at -20 °C.
Laboratory supplies
100 mm cell culture dishes (Sigma-Aldrich, catalog number: Z755923-150EA)
50 mL tubes (Abdos, catalog number: P10424)
1.5 mL tubes (Abdos, catalog number: P10202)
Equipment
Micropipettes 10, 100, 200, and 1,000 μL (any brand)
Orbital shaker (any brand)
Vortex mixer (Thermo Fisher Scientific, catalog number: 128101)
Water bath (MAC Serological water bath, catalog number: MSW-273)
Incubator (any brand)
Centrifuge (any brand)
Biosafety cabinet (MAC Horizontal laminar flow bench, catalog number: MSW-161)
PCR machine (Thermo Fisher Scientific, model: VeritiTM 96-well fast thermal cycler)
Agarose gel apparatus (Bio-Rad, catalog number: 1703940)
Protein electrophoresis system (Bio-Rad, model: Mini-PROTEAN® Tetra cell, catalog number: 1658005EDU)
Visible spectrophotometer (Labman, model: LMSP-V320)
Autoclave (any brand)
UV transilluminator (any brand)
Quantus fluorometer (Promega, catalog number: E6150)
Procedure
Transformation of BL21 (DE3) pLysS cells
Mix 1 μL (10–25 ng) of pET-28b-Pfu-Sso7d plasmid with 50 μL of BL21 (DE3) pLysS competent cells in a microcentrifuge tube and incubate the mixture on ice for 20–30 min.
Heat-shock the transformation tube at 42 °C for 45 s.
Put the tubes back on ice for 2 min.
Add 500 μL of prewarmed super optimal catabolite (SOC) media (without antibiotic) to the competent cells and grow at a 37 °C shaking incubator for 30–60 min.
Plate 100–200 μL of the transformation onto an LB agar plate containing kanamycin (50 μg/mL) and chloramphenicol (35 μg/mL).
Incubate the plate overnight at 37 °C.
Expression of Pfu-Sso7d in BL21 (DE3) pLysS cells
Culture a single transformed colony in 2 mL of LB media containing kanamycin (50 μg/mL) and chloramphenicol (35 μg/mL) with constant shaking at 200–250 rpm overnight at 37 °C.
Note: Glycerol stock of the culture can be stored at -80 °C.
Inoculate a 1 mL aliquot of this overnight starter culture in 100 mL of LB media containing kanamycin (50 μg/mL) and chloramphenicol (35 μg/mL) with constant shaking at 200–250 rpm at 37 °C.
When the culture's optical density at 600 nm (OD600) reaches 0.4–0.5, add 0.5 mM of IPTG and incubate the culture overnight at 18 °C with constant shaking.
Preparation of heat-cleared lysate
Harvest the bacterial cells by centrifugation at 2,000× g for 15 min at 4 °C.
Wash the cell pellet with 5 mL of PBS buffer and then resuspend it in 4.5 mL of lysis buffer freshly supplemented with 0.5 mM PMSF and 2 mg/mL lysozyme.
Incubate at 37 °C for 30 min with occasional mixing.
Spin the sample tubes briefly and then heat the tubes at 70 °C for 30 min.
Place it on ice for 15 min, centrifuge the lysate at 13,500× g for 10 min, and collect the supernatant in a fresh tube.
Add DNase I and MgCl2 to a final concentration of 40 U/mL and 2 mM, respectively.
Incubate the tubes at 37 °C for 30 min.
Spin the sample tubes briefly and heat the tubes at 70 °C for 30 min.
Centrifuge at 13,500× g for 10 min and transfer the supernatant to a fresh tube.
Save a portion of this heat-cleared lysate to check the purity and efficiency of heat denaturation on SDS-PAGE.
Acetone precipitation method
Add ice-cold acetone in a 1:1 (v/v) ratio (50% final) and keep the tubes at -20 °C for 20 min.
Centrifuge at 13,500× g for 20 min and carefully discard the supernatant. Tap the tube on a paper towel to eliminate any remaining supernatant. If required, save a portion of this supernatant to check acetone precipitation efficiency.
Add 2–3 mL of storage buffer freshly supplemented with 2 mM DTT.
Vigorously vortex the tube and leave it overnight at -20 °C.
Centrifuge the sample at 2,500× g for 5 min. Collect the supernatant, aliquot, and store at -20 °C or -80 °C.
If necessary, the pellets can be extracted once more with 1–2 mL of storage buffer and repeating steps D3 and D5.
Save a portion of this precipitated and solubilized polymerase to check the purity and acetone precipitation efficiency on SDS-PAGE.
Ethanol precipitation method
Add ethanol in 1:2 (v/v) ratios (67% final) and keep the tubes at room temperature for 20 min.
Centrifuge at 13,500× g for 20 min and carefully discard the supernatant. Tap the tube on a paper towel to eliminate any remaining supernatant. If required, save a portion of this supernatant to check ethanol precipitation efficiency.
Add 2–3 mL of storage buffer freshly supplemented with 2 mM DTT.
Vigorously vortex the tube and leave it overnight at -20 °C.
Centrifuge the sample at 2,500× g for 5 min. Collect the supernatant, aliquot, and store at -20 °C or -80 °C.
If necessary, the pellet can be extracted once more with 1–2 mL of storage buffer and repeating steps E3 and E5.
Save a portion of this precipitated and solubilized polymerase to check the purity and ethanol precipitation efficiency on SDS-PAGE.
Analysis of the purified Pfu-Sso7d
Run 10–20 μL of the various samples saved above on SDS-PAGE (10% separating gel, 5% stacking) and stain with Coomassie Brilliant Blue R-250.
To conduct PCR, combine 1× homemade or commercial PCR buffer, 200 nM primers, 0.2 mM dNTPs, 0.1–0.5 μL of polymerase, 0.7–1.5 M betaine, and an appropriate quantity of DNA template (genomic DNA: 50–250 ng; plasmid or viral DNA: 10 pg–20 ng; cDNA: up to 5 μL) in a total reaction volume of 25–50 μL for 30–35 cycles. Execute PCR with an initial denaturation at 95 °C for 30 s, followed by 25–35 cycles of denaturation at 95 °C for 10 s, primer annealing at 45–72 °C for 10–20 s, extension at 72 °C (2 kb/min), and a final extension of 5 min at 72 °C.
Subject the PCR products to electrophoresis on a 0.8%–2.0% agarose gel containing 1 μg/mL ethidium bromide and visualize the separated PCR products under UV illumination.
Fractions of acetone- and ethanol-precipitated and solubilized polymerase were diluted, and proteins were estimated using Bicinchoninic acid (BCA) method. The protein concentration was generally in the range of 1.0–4.0 μg/μL.
Validation of protocol
This protocol has been used and validated in the following research article:
Farooqui et al. [5]. Quick and easy method for extraction and purification of Pfu-Sso7d, a high processivity DNA polymerase. Protein Expression and Purification.
General notes and troubleshooting
General notes
Acetone and ethanol can both efficiently precipitate Pfu-Sso-7d, but we suggest acetone over ethanol because the former showed a relatively lesser amount of contaminating DNA and requires a lesser amount of acetone (50%) compared to ethanol (67%).
Although we have performed the heat-shock method to transform plasmid DNA into E. coli, other methods of transformation could also be followed.
When prepared and stored as advised, all the buffers are stable for up to one year; polymerases prepared through this protocol and stored as described remain stable for up to three years. We have not tested them beyond the mentioned time.
Troubleshooting
If activity loss over long-term storage at -20 °C or -80 °C is noticed, it may be recovered by supplementing Pfu-Ss07d solution with 2 mM fresh DTT.
Acknowledgments
We are grateful for the financial support from the Department of Science and Technology-Science and Engineering Research Board (SRG-2020-000819) and the University Grants Commission [F.30-564/2021 (BSR)], Government of India, through Start-Up Research Grants. The protocol is adapted from Farooqui et al. [5].
Competing interests
The authors declare no competing financial interests.
References
Wang, Y. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res. 32(3): 1197–1207.
Gera, N., Hussain, M., Wright, R. C. and Rao, B. M. (2011). Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold. J. Mol. Biol. 409(4): 601–616.
Dabrowski, S. and Kur, J. (1998). Recombinant His-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase. Acta Biochim. Pol. 45(3): 653–660.
Rivera, M., Reyes, J., Blazquez-Sanchez, P. and A Ramirez-Sarmiento, C. (2021). Recombinant protein expression and purification of codon-optimized Pfu-Sso7d v2. Doi: dx.doi.org/10.17504/protocols.io.bzusp6we.
Farooqui, A. K., Ahmad, H., Rehmani, M. U. and Husain, A. (2023). Quick and easy method for extraction and purification of Pfu-Sso7d, a high processivity DNA polymerase. Protein Expr. Purif. 208–209: 106276.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Transient Expression Assay and Microscopic Observation in Kumquat Fruit
JG Jinli Gong
XS Xuepeng Sun
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4968 Views: 522
Reviewed by: Wenrong HeXiongjie ZhengMin Cao
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Cited by
Original Research Article:
The authors used this protocol in Horticulture Research Aug 2021
Abstract
Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, Agrobacterium tumefaciens–mediated genetic transformation of Hongkong kumquat (Fortunella hindsii Swingle) has been widely employed for gene function analysis. However, the perennial nature of woody plants results in the generation of transgenic fruits taking several years. Here, we show the procedures of Agrobacterium-mediated transient transformation and live-cell imaging in kumquat (F. crassifolia Swingle) fruit, using the actin filament marker GFP-Lifeact as an example. Fluorescence detection, western blot analysis, and live-cell imaging with confocal microscopy demonstrate the high transformation efficiency and an extended expression window of this system. Overall, Agrobacterium-mediated transient transformation of kumquat fruits provides a rapid and effective method for studying gene function in fruit, enabling the effective observation of diverse cellular processes in fruit biology.
Keywords: Transient expression Agrobacterium tumefaciens–mediated transformation Fluorescent protein Confocal microscopy Actin cytoskeleton Citrus
Graphical overview
Schematic illustration of instantaneous transformation system of citrus fruit
Background
Citrus, one of the most important fruit crops in the world, is subjected to a variety of environmental stimuli and developmental signals during its growth. Observing the dynamics of organelles may provide new insights into the vital behavior of fruits in response to these developmental and environmental signals. The use of green fluorescent protein (GFP) and its derivatives is a great improvement for cell biological studies, as it can be fused to genes of interest to study their subcellular localization, dynamics, and protein–protein interactions [1]. However, cell structures and subcellular activities from citrus fruits can hardly be observed by light microscopy due to their 3D structure and little transparency. The lack of efficient transformation techniques and the long juvenile phase of citrus plants [2] make the study of cell biology even more difficult. Therefore, developing an effective method for transient transformation of citrus species is important. Agrobacterium-mediated infiltration of tobacco leaves was an early-developed method for transient expression [3] and has been widely used to study cell morphology and dynamics. Subsequently, Agrobacterium-mediated transient expression methods have been developed for a wide range of leaves and fruits including Arabidopsis [4], tomato [5], strawberry [6], apple [7,8], and also citrus fruits [9,10]. Here, the kumquat (Fortunella crassifolia Swingle) fruit has been selected for Agrobacterium-mediated transient expression. It is a major citrus cultivar in southern China, bearing smaller fruits in the Citrus genus compared to pomelo, oranges, and tangerines [11]. It is also characterized by its high sugar content, thin skin, and fewer juice cells, which provides a good environment for infiltration and infection by Agrobacterium [12].
In eukaryotic cells, the cytoskeleton is a network of protein–fiber reticulum consisting of microtubules, microfilaments, and intermediate filaments, which not only plays an important role in maintaining cell morphology, withstanding external forces, and maintaining the orderliness of the internal cell structure, but also participates in many important biological processes. Hence, imaging this network in its native and mobile states is of great importance. Here, using the GFP-Lifeact for actin cytoskeleton as an example, we describe methods for Agrobacterium--mediated transient transformation of citrus fruit and live-cell imaging in citrus fruit. The high expression of fluorescent protein in kumquat fruit, combined with subsequent live cell imaging, provides a method for studying gene function, subcellular localization, and cellular activity in fruit.
Materials and reagents
Biological materials
Kumquat (F. crassifolia Swingle) fruit (collected from Guangxi Institute of Citrus Research, located at Guilin city, Guangxi, China)
Reagents
Actin cytoskeleton marker GFP-Lifeact containing a 35S::GFP cassette as a visual reporter [13]
P19 Agrobacterium
Yeast extract (OXOID, catalog number: LP0021)
Tryptone (OXOID, catalog number: LP0042)
NaCl (HUSHI, catalog number: 10019318, CAS: 7647-14-5)
D-glucose (HUSHI, catalog number: 10010518, CAS: 14431-43-7)
MES (Sigma-Aldrich, catalog number: M8250)
Na3PO4 (HUSHI, catalog number: 20040928, CAS: 7601-54-9)
Acetosyringone (Sigma-Aldrich, catalog number: D134406)
GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology, catalog number: AC1001)
Antibiotics: Kanamycin (Kan) (Genview, catalog number: AK177-10G) and Rifampicin (Rif) (Genview, catalog number: AR280-1G)
Antibodies: Polyclonal mouse antibody against GFP (Biorbyt, catalog number: orb688437) as the primary antibody and goat anti-mouse HRP (Biorbyt, catalog number: orb670233) as the secondary antibody
Dimethyl sulfoxide (DMSO) (MACKLIN, catalog number: C13178602)
Mannitol (Solarbio, catalog number: M8141)
KCl (Rhawn, catalog number: R051965)
CaCl2 (MACKLIN, catalog number: C832203)
BSA (MACKLIN, catalog number: B928042)
Cellulase R10 (Yakult, catalog number: 220908-02)
Sodium hypochlorite (MACKLIN, catalog number: S935360)
Solutions
Luria Broth (LB) medium (see Recipes)
Infiltration buffer (see Recipes)
Enzymatic solution (see Recipes)
Antibiotics (see Recipes)
Recipes
LB media
10 g of tryptone, 10 g of NaCl, 5 g of yeast extract; make up to 1 L with dH 2O, following autoclave sterilization.
Infiltration buffer
Infiltration medium stock solutions:
500 mM MES: 4.88 g of MES in 50 mL of dH2O. Store at 4 °C.
20 mM Na3PO4: 0.17 g of Na3PO4 in 50 mL of dH2O. Store at 4 °C.
1 M Acetosyringone (3’,5’-dimethoxy-4’-hydroxyacetophenone): 0.196 g of Acetosyringone in 1 mL of DMSO. Divide into single-use aliquots and store at -20 °C.
50 mL infiltration buffer
250 mg of D-glucose, 5 mL of MES stock solution, 5 mL of Na3PO 4 stock solution, 5 μL of Acetosyringone stock solution; make up to 50 mL with dH2O.
Enzymatic solution
Enzymatic solution stock solutions:
0.8 M mannitol stock: 7.29 g of mannitol in 50 mL of dH2O.
2 M KCl stock: 7.455 g of KCl in 50 mL of dH2O.
0.2 M MES stock: 1.952 g of MES in 50 mL of dH2O. Store at 4 °C.
1 M CaCl2 stock: 1.11 g of CaCl2 in 10 mL of dH2 O.
10% BSA stock: 0.1 g of BSA in 1 mL of dH2O.
10 mL enzymatic solution: 0.15 g of cellulase R10, 0.04 g of macerozyme R1, 5 mL of 0.8 M mannitol stock, 100 μL of 2 M KCl stock, and 1 mL of 0.2 M MES (pH 5.7) stock. Heat solution in heat block or oven set to 55 °C (maximum) for 10 min to help enzymes dissolve (or place in 55 °C water) and then cool to room temperature (RT). Next, add 100 μL of 1 M CaCl 2 stock, 100 μL of 10% BSA stock, and dH2O up to 10 mL. Sterilize by filtration (0.45 μm).
Antibiotics
50 mg/L Rif
50 mg/L Kan
Laboratory supplies
Tips (Axygen, catalog number: AXYT1000B)
1 mL syringe with needle (GEMTIER, catalog number: 0.45X16 RW LB)
Glass microscope slides, coverslips (Corning, catalog number: CLS294875X25, CLS285522), and Vectashield (Vectorlabs, catalog number: H-1000) for microscopy
Test tubes (Eppendorf, catalog number: EP0030122178) and flask (Corning, catalog number: CLS4444250) for culturing Agrobacterium
Fine forceps and razor blades (Artis Tweezer, catalog number: Z742671; smartSlicer, catalog number: Z740503)
Marker pen (STATMARK, catalog number: Z648191)
Tray (PureSolv, catalog number: Z681687) and Falcon tube cap (Corning, catalog number: 352070) for placing fruit
Petri dishes (60 mm, optional, for enzymatic digestion of protoplasts) (Nalgene, catalog number: TMO5921-0060)
Pipette (Eppendorf, catalog number: GN686271)
Rubber gloves (Microflex, catalog number: Z265179)
Bibulous paper (for uptaking the extravasated Agrobacterium)
50 mL Falcon tubes (Corning, catalog number: 352070)
Equipment
Shaking incubator (Radobio, model: Stab M1T) used for the growth of Agrobacterium at 28 °C
Centrifuge (Eppendorf, model: 5424)
Clean workbench (AIRTECH, model: SW-CJ-2FD)
Fluorescence dissecting stereomicroscope (Olympus, model: SZX7)
Confocal microscope (Leica, model: SP8)
Nanodrop spectrophotometer (or equivalent) to determine optical density of bacterial culture
Procedure
Preparation of kumquat fruit for infestation
Carry out transformation of citrus fruit on the kumquat (see Note 1). Harvest “Huapi” kumquats or “Rongan” kumquats 150–210 days after flowering (see Note 2). Sterilize with 2% sodium hypochlorite solution for 2 min and rinse thoroughly in water; then, drain on bibulous paper before Agrobacterium injection.
Choose the healthy and fresh fruit for infiltration.
Agrobacterium preparation
Incubate Agrobacterium strain GV3101 (see Note 3) carrying GFP-Lifeact and P19 in liquid LB medium with Kan and Rif antibiotics overnight at 28 °C with shaking at 220 rpm for 12–16 h, to an optical density (O.D.) over 1.0.
Centrifuge Agrobacterium culture in a 50 mL Falcon tube at 5,000× g for 5 min to pellet the cells.
Discard the supernatant and resuspend Agrobacterium pellet using infiltration buffer.
Spin down the culture at 5,000× g for 5 min and discard the supernatant. Suspend the culture with the infiltration buffer to reach a final OD600.
Measure the OD600 of Agrobacterium cells and add appropriate volumes of infiltration buffer to dilute the Agrobacteria to the desired O.D. (usually 0.8 O.D., see Note 4). If you want to co-express two or multiple proteins, mix different kinds of Agrobacteria with the desired O.D. for each one. Every construct needs to be mixed in equal amounts with P19 Agrobacterium (see Note 5).
Agrobacterium infiltration
Cut off part of the needle of the syringe, leaving a needle approximately 0.1–0.3 cm in length.
Hold the bottom of the fruit in one hand and gently inject the bacteria solution into the epicarp at a depth of 0.1–0.3 cm. With gentle pressure on the plunger, carefully inject bacteria mixture into the fruit tissues (Video 1). Try to be as gentle as possible when injecting to avoid damaging the fruit. Inject each fruit with 0.2–0.5 mL of infiltration solution (see Note 6). The mixture could potentially distribute to an area with a radius of 0.8–1.3 cm from the injection point (data not shown). The infiltrated area can easily be seen as a dark, water-soaked region.
Get rid of the overflowing Agrobacterium on the surface of the fruit with bibulous paper. Mark the injection site with a marker pen to facilitate sampling (Video 1).
Video 1. Procedure for fruit injection
Place the infiltrated fruit on the cap of 15/50 mL Falcon tubes and store at RT (25 °C) for approximately two days. Then, wrap with cling film to prevent dehydration (see Note 7) and continue to store at RT.
Observation of fluorescent signals
Approximately five days after infiltration (see Note 8), swiftly dissect the tissue infiltrated with Agrobacterium using a sharp blade (underscored area), with no particular concern for the size or thickness of tissue blocks. Subsequently, place these tissue blocks gently on a microscopy slide and immediately observe under a stereomicroscope equipped with a DP70 camera. Fluorescent signals near the injection site are distinctly visible (see Note 9). The purpose of this step is to preliminarily assess the expression, intensity, and tissue localization of the target protein through fluorescent signals, facilitating subsequent sampling. Detailed subcellular imaging, providing information on cellular localization, necessitates confocal microscopy.
Confocal microscopy
Hand-slice tissues with strong fluorescence signals into small pieces, as thin as possible, and transfer to a slide for confocal microscopy directly (see Note 10).
(Optional if you need to use protoplasts for microscopic observation) Incubate samples for confocal microscopy in sterilized enzymatic solution for 1–2 h in the dark (see Note 11) with shaking at 40 rpm at room temperature to partially digest the cell wall. The main purpose of this additional step is to release the cells from the tissue to facilitate observation. Prolonged digestion can result in the production of protoplasts, but it is normally unnecessary; ideally, it is good to keep any disruptions of this process to a minimum (Observation of Golgi apparatus movement using protoplasts is shown in Video 2).
Video 2. Golgi movement in citrus fruit protoplast
Mount samples in microscope slides and seal with Vectashield; then, press the coverslip gently from one side.
Carry out live-cell imaging using a Leica SP8 laser scanning confocal microscope with a 63× oil immersion lens. For wavelength settings, GFP was excited at 488 nm and detected at 505–550 nm.
Data analysis
“Huapi” or “Rongan” kumquat fruits (Figure 1A) were used for the transient overexpression of GFP-Lifeact. Five days after the injection, the injection site was incised, and obvious green fluorescence was visible under a stereomicroscope (Figure 1B). Fluorescent signals could be detected in different tissues of the kumquat, including the flavedo, albedo, juice sacs, and partition, into which the Agrobacterium solution penetrated [12]. Western blot analysis conducted on kumquat tissues five days post-injection revealed that the expression of GFP-Lifeact was discernible at all Agrobacterium OD values exceeding 0.2. Furthermore, the protein expression level of GFP-Lifeact exhibited enhancement with escalating concentrations of Agrobacterium injection (Figure 1C). Tissues exhibiting robust fluorescent signals were meticulously sectioned as thinly as possible, and the GFP-Lifeact-labeled actin cytoskeletons were clearly observed under confocal microscopy through manual sections and protoplasts (Figure 1D). However, the free GFP is ubiquitously distributed throughout the fruit cells, with signals of free GFP detectable in both the membrane and cytoplasm (Figure 2).
Figure 1. Transient expression of fluorescent protein GFP-Lifeact in kumquat fruit cells. A. Representative image of “Huapi” (left) and “Rongan” (right) kumquat fruit used for injection. B. Dissected sections expressing GFP fusion proteins. C. Western blotting analysis of GFP-Lifeact and free GFP (from pMDC43 binary vector) expression in kumquat fruit using different concentrations of Agrobacterium suspension; 20 μg of total protein from each lane were probed with anti-GFP antibodies with 1:2,000 dilution. D. Representative images of fluorescent protein fusions localized to actin cytoskeleton in fruit cells through manual sectioning (up) and protoplasts (down). The construct GFP-Lifeact was infiltrated at OD600 = 0.8 and images were taken five days after infiltration.
Figure 2. Transient expression of the free GFP in kumquat fruit cells
Notes
Fruit variety selection: The meticulous choice of fruit varieties holds paramount significance. Kumquat fruits were identified as our preferred material for transient transformation due to their characteristics of thin skin and tender flesh (Figure 1A); additionally, they contain high sugar content and low acid, which provides a suitable growth environment for Agrobacterium. Other citrus cultivars, such as orange, pomelo, and mandarin, could also be considered. Notably, kumquat exhibited sustained production of robust signals and high conversion efficiency (data not shown).
Determination of the fruit stage: The mature green stage of kumquat fruit (approximately 150–210 days after flowering) has proven to be optimal for transient transformation, owing to its firm texture, heightened metabolic activity, and finer peels. Additionally, post-harvest kumquat fruit remains suitable for experimentation, underscoring the importance of selecting fresh citrus fruits for injection in subsequent procedures. It is advised to avoid fruit at the end of the ripening stage, as it is susceptible to rot following Agrobacterium injection.
Strain selection: Various strains of Agrobacterium tumefaciens (e.g., EHA105 or GV3101) were selected for testing. The findings revealed that transformation mediated by GV3101 yielded the highest protein output.
Optical density value: Attaining the optimal Agrobacterium density (OD600) for each construct is crucial, and this requirement varies significantly from gene to gene. The OD600 of Agrobacterium also exerts an impact on the level of protein expression, necessitating the optimization of the ideal OD600 for a new construct prior to the commencement of the actual study. An OD600 concentration of Agrobacterium below 0.1 typically results in weak expression, while an OD600 exceeding 1.0 tends to induce yellowing and rotting of fruit. If mitigating potential overexpression artifacts is a primary concern, it is advisable to use the lowest O.D. that still yields sufficient signal. Otherwise, an OD600 within the range of 0.5–0.8 can be employed to achieve maximum expression.
P19: Augmented expression can be attained through the utilization of p19 constructs, effectively averting gene silencing.
Injection dose: Maintaining the injection volume within the range of 0.2–0.5 mL for each fruit is imperative, as excessive infiltration buffer may lead to unforeseen rotting.
Bagging: After two days of fruit injection, it is advisable to individually encase fruits with cling film to enhance water retention and ensure air permeability; bagging should not occur too early, otherwise it will cause fruit rot due to high humidity.
Time of expression: Different proteins may exhibit distinct expression timelines. Generally, protein expression becomes detectable 4–5 days after infiltration. If the fruit preservation conditions are favorable, protein expression can even be detected up to one month post-infiltration. The persistence and abundance of protein expression typically depend on the plasmid itself and the vitality of the fruits.
Fruit tissues for expression: Fluorescent signals can be observed in various tissues of kumquat, encompassing flavedo, albedo, and juice sacs, where the Agrobacterium solution can penetrate. The efficacy of expression is contingent upon the uptake of Agrobacterium by different tissues, with superior absorption correlating to enhanced expression strength. Typically, the expression of fluorescent proteins in albedo surpasses that in other tissues.
During the process of slicing fruit tissue, it is imperative to minimize external pressure applied to the sample. The use of a sharp razor blade is equally crucial as it mitigates mechanical damage and reduces the risk of fractures.
Enzymolysis: The primary objective of this supplementary step is to release protoplasts from tissues to facilitate observation. Although prolonged digestion could yield protoplasts, it is generally unnecessary, and the ideal approach is to minimize any disruptions.
Validation of protocol
This protocol has been used and validated in the following published articles:
Gong, J. L. et al. (2021). Illuminating the cells: transient transformation of citrus to study gene functions and organelle activities related to fruit quality. Hortic Res 8(1): e1038/s41438–021–00611–1, DOI: 10.1038/s41438-021-00611-1.
Gong, J. L. et al. (2021). Red light-induced kumquat fruit coloration is attributable to increased carotenoid metabolism regulated by FcrNAC22. Journal of experimental botany 72: 6274–6290, DOI: 10.1093/jxb/erab283.
Acknowledgments
We are grateful for the financial support from the Zhejiang Provincial Natural Science Foundation of China (LQ23C150004 and LR23C150001), the National Natural Science Foundation of China (32102318), and the Key Project for New Agricultural Cultivar Breeding in Zhejiang Province, China (2021C02066-1). This protocol was modified from our previously published work [12].
Competing interests
The authors declare no conflict of interest.
References
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Zhu, C., Zheng, X., Huang, Y., Ye, J., Chen, P., Zhang, C., Zhao, F., Xie, Z., Zhang, S., Wang, N., et al. (2019). Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii). Plant Biotechnol. J. 17(11): 2199–2210. https://doi.org/10.1111/pbi.13132
Sparkes, I. A., Runions, J., Kearns, A. and Hawes, C. (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1(4): 2019–2025. https://doi.org/10.1038/nprot.2006.286
Tsuda, K., Qi, Y., Nguyen, L. V., Bethke, G., Tsuda, Y., Glazebrook, J. and Katagiri, F. (2011). An efficient Agrobacterium‐mediated transient transformation of Arabidopsis. Plant J. 69(4): 713–719. https://doi.org/10.1111/j.1365-313x.2011.04819.x
Orzaez, D., Mirabel, S., Wieland, W. H. and Granell, A. (2006). Agroinjection of Tomato Fruits. A Tool for Rapid Functional Analysis of Transgenes Directly in Fruit. Plant Physiol. 140(1): 3–11. https://doi.org/10.1104/pp.105.068221
Zhao, Y., Mao, W., Chen, Y., Wang, W., Dai, Z., Dou, Z., Zhang, K., Wei, L., Li, T., Zeng, B., et al. (2019). Optimization and standardization of transient expression assays for gene functional analyses in strawberry fruits. Hortic. Res. 6(1): e1038/s41438–019–0135–5. https://doi.org/10.1038/s41438-019-0135-5
An, J. P., Wang, X. F., Li, Y. Y., Song, L. Q., Zhao, L. L., You, C. X. and Hao, Y. J. (2018). EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 Act in a Regulatory Loop That Synergistically Modulates Ethylene Biosynthesis and Anthocyanin Accumulation. Plant Physiol. 178(2): 808–823. https://doi.org/10.1104/pp.18.00068
Li, T., Jiang, Z., Zhang, L., Tan, D., Wei, Y., Yuan, H., Li, T. and Wang, A. (2016). Apple (Malus domestica) MdERF2 negatively affects ethylene biosynthesis during fruit ripening by suppressing MdACS1 transcription. Plant J. 88(5): 735–748. https://doi.org/10.1111/tpj.13289
Shen, S. l., Yin, X. r., Zhang, B., Xie, X. l., Jiang, Q., Grierson, D. and Chen, K. s. (2016). CitAP2.10activation of the terpene synthaseCsTPS1is associated with the synthesis of (+)-valencene in ‘Newhall’ orange. J. Exp. Bot. 67(14): 4105–4115. https://doi.org/10.1093/jxb/erw189
Yin, X., Xie, X., Xia, X., Yu, J., Ferguson, I. B., Giovannoni, J. J. and Chen, K. (2016). Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening. Plant J. 86(5): 403–412. https://doi.org/10.1111/tpj.13178
Zhu, M., Lin, J., Ye, J., Wang, R., Yang, C., Gong, J., Liu, Y., Deng, C., Liu, P., Chen, C., et al. (2018). A comprehensive proteomic analysis of elaioplasts from citrus fruits reveals insights into elaioplast biogenesis and function. Hortic. Res. 5(1): e1038/s41438–017–0014–x. https://doi.org/10.1038/s41438-017-0014-x
Gong, J., Tian, Z., Qu, X., Meng, Q., Guan, Y., Liu, P., Chen, C., Deng, X., Guo, W., Cheng, Y., et al. (2021). Illuminating the cells: transient transformation of citrus to study gene functions and organelle activities related to fruit quality. Hortic. Res. 8(1): e1038/s41438–021–00611–1. https://doi.org/10.1038/s41438-021-00611-1
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4,969 | https://bio-protocol.org/en/bpdetail?id=4969&type=0 | # Bio-Protocol Content
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Peer-reviewed
Nerve Preparation and Recordings for Pharmacological Tests of Sensory and Nociceptive Fiber Conduction Ex Vivo
VK Volodymyr Krotov
Olga Kopach
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4969 Views: 422
Reviewed by: Willy R Carrasquel-UrsulaezSalim GasmiMarco Pagliusi Jr.
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Original Research Article:
The authors used this protocol in Frontiers in Cellular Neuroscience Jan 2023
Abstract
Measuring signal propagation through nerves is a classical electrophysiological technique established decades ago to evaluate sensory and motor functions in the nervous system. The whole-nerve preparation provides a valuable model to investigate nerve function ex vivo; however, it requires specific knowledge to ensure successful and stable measurements. Although the methodology for sciatic nerve recordings has long existed, a method for reliable and long-lasting recordings from myelinated and non-myelinated (nociceptive) fibers still needs to be adapted for pharmacological testing. This protocol takes benefits from epineurium sheath removal for pharmacological tests and provides a detailed description of how to make accurate nerve preparations, from the dissection and handling of nerves to epineurium cleaning, fabrication of adaptable suction electrodes for appropriate fiber stimulation and recordings, setting of electrophysiological protocols for compound action potential (CAP) recordings to distinguish between myelinated and non-myelinated (nociceptive) fibers, and finally to the analysis of the datasets of CAP components. We also demonstrate the feasibility of CAP recordings from individual branches in epineurium-free nerve preparations and provide clues to help retain nerve viability and maintain stable recordings over time. Although a sciatic nerve preparation was used here, the methodology can be applied to other nerve-type preparations.
Key features
• Detailed and simplified protocol for peripheral nerve preparation for recording sensory inputs ex vivo.
• Recordings from myelinated and non-myelinated (nociceptive) fibers can be performed hours after nerve preparation.
• The protocol involves the epineurium removal to facilitate drug permeability into nerve tissue for pharmacological tests.
• The protocol allows physiological and pathological studies (pain/chronic pain conditions).
Graphical overview
Preparation and recordings from the sciatic nerve, including myelinated and non-myelinated (nociceptive) fibers
Keywords: Peripheral nerves Myelinated fibers Nociceptive fibers Sensory inputs Nerve conductance Pain
Background
It is estimated that more than 1.5 billion people worldwide suffer from pain, with hundreds of millions experiencing chronic pain that remains untreated. As a result, there is a need for new therapeutic options to be developed and new therapeutics are actively being searched [1]. Although there is consensus that a wide range of ex vivo nervous system models provide a valuable tool for studying pain and the mechanisms of nervous system pathologies, whole-nerve models are the only ones that can directly assess nerve conduction in both physiological and pathological conditions. The sciatic nerve is the largest and longest nerve in the body, making it the preferred nerve for many peripheral nerve models [2]. Despite the variety of rodent models that have been established in the past for in vivo and ex vivo recordings [3–7], the methodology for sciatic nerve recordings remains challenging and requires a detailed step-by-step description. Furthermore, detailed protocols for long-lasting readouts from both myelinated and non-myelinated (nociceptive) fibers, especially in the context of pharmacological testing, are still lacking. Stable and reliable recordings are critical to determine the effectiveness of any treatment being tested. This protocol provides a step-by-step description of how to accurately make reliable nerve preparations from rodents, which can be used to monitor nerve conduction over an extended period of time, in physiological conditions, and under pharmacological treatment. The distinctive feature of our protocol is nerve preparation with epineural sheath removal to facilitate pharmacological tests, including recordings from individual nerve branches. We describe electrophysiological protocols for recording compound action potential (CAP), which reflects action potentials (APs) generated by peripheral fibers, both afferents and efferents. In a bundle of nerve fibers, each fiber generates APs, resulting in a current flowing through the surface of the nerve bundle, which has high resistance and can be measured as a potential difference, i.e., CAP, using two electrodes placed on the nerve.
The protocol results in accurate measurements from myelinated and non-myelinated (nociceptive) fibers for a few hours. Additionally, we have demonstrated the effect of lidocaine, a widely used classical local anesthetic, on the nerve as a proof-of-concept to validate the model’s applicability for testing pharmacological treatment.
Materials and reagents
Biological materials
Sprague-Dowley rats 3–6 months old (Charles River, strain code: 001)
Reagents
Glucose (Sigma-Aldrich, catalog number: G8270)
Sodium chloride (Sigma-Aldrich, catalog number: S9888)
Sodium bicarbonate (Sigma-Aldrich, catalog number: S0751)
Sodium monophosphate (Sigma-Aldrich, catalog number: S6040)
Potassium chloride (Sigma-Aldrich, catalog number: P9333)
Magnesium chloride (Sigma-Aldrich, catalog number: M2670)
Calcium chloride (Sigma-Aldrich, catalog number: C7902)
Solutions
Krebs bicarbonate solution (see Recipes)
Recipes
Krebs bicarbonate solution (final volume 0.5 L in ddH2O)
Reagent Final concentration (mM) Quantity or Volume
Sodium chloride 125 3,653 mg
Glucose 10 901 mg
Sodium bicarbonate 26 1,092 mg
Sodium monophosphate 1.25 75 mg
Potassium chloride 2.5 93 mg
Magnesium chloride 1 102 mg
Calcium chloride 2 147 mg
Total n/a 500 mL
Note: To avoid precipitation, the solution should be bubbled with 95% O2 and 5% CO2 before adding calcium chloride.
Laboratory supplies
Isoflurane (Abbvie, catalog number: B506; also, Henry Schein, catalog number: 1182097 or any available)
Spray bottle with 70% ethanol (Fisher Scientific, catalog number: BP82031GAL)
Petri dish 100 mm × 15 mm (Corning, catalog number: 351029)
Glass capillaries with filament O.D. 1.5 mm, I.D. 0.86 mm (e.g., Sutter Instruments, catalog number: BF-150-86-10 or Harvard Apparatus, catalog number: GC150F-10)
Butane blow torch
95% O2 and 5% CO2 gas mixture
Equipment
Dissection and preparation
Anesthesia induction chamber (VetEquip, US) or any other available
Standard scissors (Fine Science Tools, catalog number: 14002-12)
Blunt surgical scissors (Fine Science Tools, product number: 14003-12). Alternatively, hemostat (Fine Science Tools, catalog number: 13003-10)
Coarse forceps (Fine Science Tools, catalog number: 11652-10)
Spring scissors (Fine Science Tools, catalog number: 15025-10)
Fine forceps (Fine Science Tools, catalog number: 11412-11)
Fine forceps (Fine Science Tools, catalog number: 11413-11)
Cordless trimmer for rodents (Bioseb, model: Bio-1584)
Electrophysiology and visualization
Stereomicroscope (Olympus, model: SZX7)
Light source (AmScope, model: 6 W LED Dual Gooseneck Illuminator or any available)
Upright microscope (Olympus, model: BX50WI)
4× objective (Olympus, PLN)
Camera (any equipped with the microscope)
Patch clamp amplifier with headstage (Molecular Devices, model: Multiclamp 700B)
Digitizer (Molecular Devices, model: Digidata 1440)
Micromanipulators (Scientifica, model: PatchStar)
Constant current stimulator (Digitimer, model: DS3)
Pipette holders (Molecular Devices, model: 1-HL-U)
Faraday cage (Sutter Instruments, model: AT-36FC)
Silver wire AWG 26 for electrodes (WPI, catalog number: AGW1510)
Ag/AgCl pellets (WPI, catalog number: EP1)
BNC cables
PC computer
Software and datasets
pClamp (version 10.7, Molecular Devices)
Clampfit (version 10.7, Molecular Devices)
Procedure
Initial preparations
Prior to beginning any procedures, warm up the Krebs bicarbonate solution to room temperature and bubble the solution with a mixture of 95% O2 and 5% CO2 gas. We recommend oxygenating the solution for 15 min.
Collect all surgical tools (Figure 1) and disinfect them and the working areas with 70% ethanol.
Figure 1. Preparation of surgical tools for nerve isolation
Before starting the dissection, prepare a large Petri dish for the initial collection of the dissected nerves and fill it with Krebs bicarbonate solution. Prepare another Petri dish with Krebs bicarbonate solution to where the cleaned nerves will be transferred. After dissection, the nerves can be kept in a Petri dish at room temperature.
Terminally anesthetize an animal with isoflurane (4%–5%) in a chamber for anesthesia. Ensure complete anesthesia by the loss of pedal reflection (pinch the hind paw) and/or corneal reflexes (gently touch the eye) and cull the animal (decapitation).
Place the animal in a designed tissue dissection area. Remove the fur from the outer surface of the leg using clippers. Repeat for the other leg if both sciatic nerves are to be harvested.
Nerve isolation
Before opening the tissues, spray the skin with 70% ethanol.
Position the animal on its side so that the spine faces the experimenter. Pull up the skin on the proximal part of the thigh and make a long posterolateral incision on the animal side to expose the biceps femoris muscle.
Locate the mid-thigh point. Holding blunt surgical scissors or course forceps vertically, penetrate the muscle at midpoint by approximately 10 mm and spread the branches in a rostrocaudal direction. Pulling the muscle away exposes the sciatic nerve and its branches (Figure 2, top left).
Note: It takes some effort to penetrate the muscle, and force should be applied to open it.
Using scissors and forceps, cut the muscles along the whole span of the sciatic nerve and beyond its trifurcation to the point where the branches are about to be cut (Figure 2, top right).
Use the hemostat or self-retaining retractor to keep the sciatic nerve and its branches open. The sciatic nerves in Sprague Dawley rats are relatively long and divide distally into branches (> 1 cm proximally). Using two retractors to expose the nerve with its branches might be helpful for careful dissection. We attempt to expose approximately 2 cm of nerve to the trifurcation into the tibial, common peroneal, and sural nerves. Exposing each branch will depend on the nerve of interest and the experimental aims.
Use curved forceps and spring scissors to gently separate the sciatic nerve from the surrounding fascia and large adipose tissue pieces. Insert the curved forceps underneath the proximal part of the nerve and move the forceps along the whole span of the nerve to ensure it is fully separated from the surrounding tissue.
Note: Older rats have more adipose tissue surrounding the nerves, especially at the point of trifurcation, making dissection more challenging.
Using spring scissors, cut the sciatic nerve branches to the desired length. Start from the lumbosacral region to maximize the length of the sciatic nerve and move distally to the anterior end of the nerve, cutting from side to side while lifting the top of the nerve to avoid nerve damage. Typically, the length of the cut sciatic nerve is approximately 20–30 mm.
Note: Careful nerve excision is critical in producing high-quality nerve preparation. We recommend cutting to the trifurcation point even if only the sciatic nerve is to be used, as it makes further manipulations easier.
Once the nerve has been cut to its desired length, grab it by the tip of the tibial nerve (the thickest branch) using coarse forceps to transfer the dissected nerve into the prepared Petri dish with Krebs bicarbonate solution (Figure 2).
Note: Never pinch or stretch the nerve, as it damages it.
Repeat the procedure for dissecting the nerve from the other side. After completing, gently transfer the second nerve to the Petri dish.
Note: Try to keep time for nerve dissection to the minimum possible and dissect both sciatic nerves within half an hour after euthanasia (10–15 min for each nerve).
Figure 2. Sciatic nerve dissection, showing various stages of nerve isolation from immediate exposure of the nerve (top left image) and its whole opening (top right) to both isolated nerves (bottom images). Notice connective and adipose tissues surrounding the nerves on the enlarged snapshot (bottom right).
Epineural sheath removal
Under visual control with a binocular or stereomicroscope, use a pair of spring scissors and fine forceps to carefully remove all surrounding tissue, such as fats and connective or fascia tissues, from the nerve on all sides (Figure 2, right bottom).
Note: Nerves must be very carefully handled during the procedure to avoid stretching them or producing accidental trauma to the tissue. If it is necessary to hold the nerve, do it by holding its tip.
Gently remove the epineurium, the sheath covering the nerve that significantly obstructs the drug permeability of nerve fibers [8], using two fine forceps. For this, tear the epineurium, starting from the trifurcation and further along the nerve, and gently pull the sheath along the nerve, taking care to minimize nerve stretching as much as possible. During this process, the sciatic nerve typically splits into its branches (Figure 3). Basically, the common peroneal branch could be separated along the whole span of the nerve. Splitting tibial and sural branches is more complicated, especially in the proximal part of the nerve.
Clean each nerve branch from which the recordings will be made, always maintaining the tissue in the Krebs solution (Figure 3 right). Once the epineurium has been removed, cut off the nerve ends that were held with the forceps during manipulation to ensure high-quality recordings from the nerve.
Figure 3. Nerve cleaning and epineurium removal. Left image, sciatic nerves after removal of connective and adipose tissue. Right image, the sciatic nerve split into separate branches after half of the nerve was stripped of the epineurium.
Transfer the cleaned nerve to a Petri dish with fresh and oxygenated medium.
Perform cleaning of the second nerve.
Measure the length of each nerve for further analysis (Figure 4).
Figure 4. Separate nerve branches after epineurium removal and cleaning. Notice the nerve length.
Fabrication of suction pipettes and nerve suction
A suction electrode is a valuable tool in electrophysiology experiments, and we use suction electrodes for both stimulating and recording the nerve.
Fabricate suction pipettes using a butane blow torch and glass capillaries to fire polish pipettes to a tip diameter close to the size of the nerve. Although suction electrodes are fabricated for each nerve individually, we recommend fabricating a set of pipettes with different tip diameters beforehand, as shown in Figure 5. Use the camera or the ocular to assess the tip diameter and choose the pipettes whose tips best fit the nerve end size.
Note: Fabrication of pipettes with various tip diameters allows for the accommodation of the nerve, depending on its diameter, and ensures that the nerve is held securely.
Figure 5. Glass pipettes fire-polished to various tip diameters for appropriate nerve suction
Prepare the electrophysiological rig, filling in the perfusion system with continuously oxygenated Krebs bicarbonate solution (95% O2 and 5% CO2) and circulating oxygenated medium through a recording chamber with a peristaltic pump. It is essential to circulate an oxygenated medium through the chamber to maintain the health of nerve tissue during prolonged recordings.
Note 1: We recommend using a Petri dish as a recording chamber. The flow rate of the medium will vary based on the design of the perfusion system, but it should be enough to maintain a consistent superfusion flow and provide adequate oxygen and nutrient levels to support the tissue. Excessive flow should be avoided as it may affect the recordings.
Note 2: The peristaltic pumps connected to the power supply typically produce electrical noise and should be ideally placed outside the Faraday cage.
Position two reference Ag/AgCl electrodes (one for the stimulator and one for the recording headstage) inside the chamber and connect suction pipettes to their wires. Let the pipettes fill with the medium.
Note: The medium should not reach the pipette holder, as it produces a substantial electrical noise that can be eliminated only by replacing the pipette holder. We recommend using a long-length suction pipette and a matching electrode wire.
Transfer one of the nerves to the recording chamber using closed forceps.
Note: Never grab the nerve with the forceps.
Focus on the nerve and assess the width of its distal and proximal ends using low magnification (e.g., objective 4×). Using closed forceps, push the distal end of the nerve to the very opening of the suction pipette connected to the wire and stimulator. Apply negative pressure to suck the tip of the nerve inside the pipette. Ensure the contact between the pipette tip and the nerve is tight. In particular, the nerve should not move further inside the pipette when the negative pressure is applied and should not slip out of the pipette once the negative pressure is released (Figure 6, bottom image).
Note: If the nerve does not fit inside the pipette, use a suction pipette with a broader or narrower tip diameter (see step D2).
Figure 6. Stimulating and recording suction electrodes with the nerve in for stable recordings
Repeat the previous step to proceed with the proximal end of the nerve for the recording pipette. Once both nerve ends are inside the suction pipettes, place the recording and stimulating electrodes in front of each other, avoiding stretching the nerve (Figure 6), which may affect the CAP recordings [9,10].
Note: Securing the nerve in the suction electrodes is critical for stable recordings, especially over a long time and under pharmacological applications.
Ensure that the stimulator is connected and ground electrodes are in place. Start testing the protocol by stimulating the nerve with a single pulse (1–2 mA, 1 ms duration) to initiate CAP generation by both myelinated (A-fibers) and non-myelinated C-fibers. Once the typical CAP is obtained (see section E), allow the nerve to settle for 15–20 min. This further improves the contact between the nerve and the pipettes and, hence, the stability of CAP recordings over time. Record at room temperature.
Nerve stimulation and recordings
Set the amplifier to the current clamp mode. As the recorded signals do not typically exceed 10 mV, we recommend using a 5 GOhms input resistor and digitizing the signals at 10–20 kHz. Set the Bessel filter signals at ≥ 2 kHz for the recordings from non-myelinated C-fibers. The Bessel filter should be increased to 5–10 kHz for recordings from the A-fibers, which are faster-conducting myelinated fibers.
Stimulate nerve fibers with square current pulses of positive polarity ranging from 0.1 to 2 mA. To activate the high-threshold C-fibers, pulses should be of 1 ms duration.
Note: This protocol also activates A-fibers, whose fast responses, however, overlap with the stimulation artifact. Activating C-fibers exclusively is possible by using 0.8–1.2 ms stimuli of negative polarity. In that case, A-fiber-driven CAPs do not appear due to an anodal block of conductance in A-fibers, while C-fiber-driven CAPs are delayed by several milliseconds.
Run multiple trials of CAPs generated with the above protocol (Figure 7).
Note: The CAPs are very reproducible; however, they may be prone to time- and use-dependent rundown of amplitudes and slowing down of conduction. Therefore, we recommend running 5–10 trials for each time point of interest with a gap of at least 10 s between trials.
Figure 7. Representative recordings of compound action potential (CAPs) generated by C-fibers (1 ms pulse duration). Red line shows the average trace of eight trials. Recordings are raw data collected using the Clampfit software.
For the recordings of the A-fiber-driven CAPs, we recommend using the protocols of 50 μs duration of stimuli (Figure 8).
Note: At high stimulus intensities, 50 μs pulses may also activate a substantial percentage of C-fibers (Figure 8, right bottom).
Figure 8. Representative recordings of mixed fiber stimulation (top traces) show A-type fiber response (lower left traces) and C-fiber stimulation (lower right traces). Red line shows the average of eight trials. Recordings are raw data collected using the Clampfit software.
Stimulus intensities required to elicit CAPs vary greatly between the nerves and their branches. It is, therefore, necessary to determine the minimum stimulus that produces CAP and the stimulus at which further increase in stimulating current does not produce any changes (i.e., saturating stimulus). For this, at the beginning of each experiment, start testing with stimuli of 1–2 mA (1 ms pulse duration) by incrementally increasing stimulus intensity to establish both the minimum and saturating currents for A-fiber-driven CAPs using 50 μs pulse protocol (Figure 9, left). The CAP magnitude increases nonlinearly with the stimulus (Figure 9, right).
Note: The typical amplitude of A-fiber-mediated CAPs driven by saturating stimulus ranges from 0.5 mV to several millivolts.
Figure 9. Representative recordings of A-type fiber response (left traces), showing the stimulus-intensity dependence (right graph)
Repeat the same for C-fiber-driven CAPs using 1 ms pulse protocol.
Note: The C-fiber-mediated CAPs driven by the saturating stimulus are smaller than for A-fibers, ranging between 0.05 and 0.5 mV.
After the minimum and the saturating stimuli are established, the following experimental protocols may test the A-fiber- and C-fiber-driven CAPs evoked by saturating currents before and after pharmacological treatment.
Note 1: The magnitude and signal profile of the CAP can vary between different nerves or even its branches or when recorded on the right or left nerve of the same rat.
Note 2: To expedite the process, stimulus intensity dependence can be excluded after gaining some experimental experience, and the optimal stimulus intensity can be found empirically by coarsely increased stimuli until it reaches the saturation level.
After completing the recordings, save the data for offline analysis.
At the end of the experiment, measure the distance between the tips of the suction electrodes to assess the conduction velocities of nerve fibers.
Note: C-fibers show substantial variability in their conduction velocities, and the CAP amplitude depends on the length of the nerve and the distance between two recording electrode tips [11]. We recommend using ~2 cm of nerve length for maximal quality recordings.
Data analysis
The shape and amplitude of CAPs depend on the nerve length and the quality of contacts (the resistance) between the suction electrodes and the nerve. These parameters vary significantly from preparation to preparation and, therefore, should be normalized for paired analysis. Also, CAPs may have complex shapes, including several peaks and variable profiles; thus, determining the amplitude might be challenging. We recommend using the normalized area under the curve as the primary signal readout for statistical analysis. Among other parameters to estimate, it may be the fiber conduction velocity, for either A- or C-fibers, calculated by dividing the distance between the tip of the suction electrodes by the latency between the onset of the stimulus and the start of the CAP. Data analysis may be performed using the Clampfit and/or Origin software.
Validation of protocol
To validate the protocol, we followed the steps above for electrophysiological recordings from the nerve, aiming to test the effect of lidocaine, a common local anesthetic used in clinics [12,13]. Also, a part of this protocol has been used and validated in the recently published research article (Figure 1 from Krotov et al. [14]).
Using two suction electrodes, we assessed both A- and C-fiber CAPs before and after applying lidocaine at a concentration of 2%. The pharmacological effect was prompt—we noticed a gradual decline in the magnitude of CAPs, which disappeared completely after 1–2 min of drug superfusion. The onset of the effect depends on the perfusion rate. Figure 10 displays an overlay of the two A-fiber conductance recordings to show the effect of lidocaine through paired recordings (from the same nerve before and after drug application). The statistical summary indicates the same therapeutic effect observed in several nerve preparations from different animals. This served as a proof-of-concept for the reliability of the method and its applicability to pharmacological testing.
Figure 10. Pharmacological treatment with lidocaine results in a complete block of A-fiber conductance. Left, representative recordings from the A-fibers before (control) and shortly after application of lidocaine at the concentration of 2% (1–2 min after superfusion). Right, quantification of the compound action potential (CAP) area under curve in different experimental conditions. n = 6 nerve samples independently tested.
Figure 11 demonstrates that the protocol allows for accurate measurements of the therapeutic effect of 2% lidocaine on the function of C-fibers. Similar to the A-fiber conductance, lidocaine completely blocked the CAP generated by nociceptive fibers within 1–2 min after drug superfusion. Previous research showed that lidocaine effectively blocks sensory and motor fibers by preventing the AP initiation and conduction along the nerve by blocking sodium ion channels, resulting in anesthetic effects [15,16].
Figure 11. Pharmacological treatment with lidocaine results in a complete block of C-fiber conductance. Representative recordings before (control) and shortly after the bath application of lidocaine (2%). Quantification of compound action potential (CAP) peak amplitude in different experimental conditions. N = 6 nerve samples pharmacologically tested.
Our findings provide functional evidence that the protocol we used is effective and reliable in examining nerve function in both physiological conditions and under pharmacological treatments. This model can be utilized in future studies to evaluate the effectiveness of pain-relieving drugs and other pharmacological treatments for various pathological conditions.
General notes and troubleshooting
The peripheral nerves have been considered important therapeutic targets for the treatment of pain, including chronic pain. However, accurately measuring nerve function is critical to assessing the efficacy of any therapeutic approach. Also, the readings on nerve function should be robust, reliable, and reproducible to examine the effects of pharmacological treatments. Here, we provide a detailed, step-by-step protocol for measuring nerve conductance in adult rats for sensory, motor, and nociceptive fibers. In summary:
After harvesting, sciatic nerves should always remain in a well-oxygenated fresh solution at room temperature. Tissues should not be kept on ice. Dissected nerves can be maintained for several hours.
While the protocol is relatively straightforward to implement, it requires practice. It generally takes approximately 2 h to complete nerve preparation. The most challenging and time-consuming steps of this protocol are removing epineurium and creating a tight contact between suction electrodes and the nerves. It is possible to omit the epineurium removal and record from the whole nerve rather than from individual branches, which might be a practical tool for studying the conduction of injured nerves. However, that would require higher intensities of electric stimuli necessary to activate nerve fibers and would impact wash-in and especially wash-off times of any pharmacological intervention.
We recommend using a Petri dish instead of a standard recording chamber coupled with a conventional perfusion system for the following reasons:
Standard recording chamber might be too small to accommodate long nerve preparations.
The use of a peristaltic pump for perfusion substantially increases the electrical noise of the recordings.
Vacuum-driven outflow requires large volumes of solutions that may not be suitable for pharmacological studies.
Electrophysiological recordings might be performed up to 12 h after dissection. However, a time-dependent rundown of the amplitudes of the responses should be considered. We recommend recording within 4 h after dissection and implementing experimental protocols of 0.5–1.5 h duration to avoid possible rundown.
Ensure temperature stability, as changes in 1–2 °C can affect the fiber conduction velocities, thereby changing the CAP shape.
Typically, in one rat, the right and left nerves are dissected, and the following measurements are recorded in control and testing conditions.
The protocol has been effective in using animals of different ages.
This protocol can be used for mouse nerve preparations. Due to their small size, we recommend using a stereomicroscope at higher magnifications to ensure better visualization of nerves and branches. Additionally, it is critical to handle the nerves with extra care throughout the procedure.
Depending on the research interests, the protocol is also adaptive to different genetic backgrounds and treatments.
Acknowledgments
This work was funded by the UCL-Welcome Trust Translational Partnership Pilot Award RM-TIN #178973 (O.K.).
Competing interests
Dr. Olga Kopach is an Associate Editor for Bio-protocol but did not participate in the editorial and peer review process of this article, except as an author. The authors declare no other conflict of interest.
Ethical considerations
Animals were used in accordance with the European Commission Directive (86/609/EEC) and the United Kingdom Home Office (Scientific Procedures) Act (1986).
References
Kopach, O. and Voitenko, N. (2021). Spinal AMPA receptors: Amenable players in central sensitization for chronic pain therapy? Channels 15(1): 284–297.
Rigaud, M., Gemes, G., Barabas, M. E., Chernoff, D. I., Abram, S. E., Stucky, C. L. and Hogan, Q. H. (2008). Species and strain differences in rodent sciatic nerve anatomy: Implications for studies of neuropathic pain. Pain 136(1): 188–201.
Kotamraju, B. P., Eggers, T. E., McCallum, G. A. and Durand, D. M. (2023). Selective chronic recording in small nerve fascicles of sciatic nerve with carbon nanotube yarns in rats. J. Neural Eng. 20(6): 066041.
Sun, S., Delgado, J., Behzadian, N., Yeomans, D. and Anderson, T. A. (2020). Ex Vivo Whole Nerve Electrophysiology Setup, Action Potential Recording, and Data Analyses in a Rodent Model. Curr. Protoc. Neurosci. 93(1): e99.
Zhao, D., Behzadian, N., Yeomans, D. and Anderson, T. A. (2021). In Vivo Whole‐Nerve Electrophysiology Setup, Action Potential Recording, and Data Analyses in a Rodent Model. Curr. Protocol. 1(11): e285.
Serra, J., Bostock, H. and Navarro, X. (2010). Microneurography in rats: A minimally invasive method to record single C-fiber action potentials from peripheral nerves in vivo. Neurosci. Lett. 470(3): 168–174.
Schulz, A., Walther, C., Morrison, H. and Bauer, R. (2014). In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves. J. Visualized Exp.: (86). DOI: 10.3791/51181-v.
Kagiava, A. and Theophilidis, G. (2013). Assessing the permeability of the rat sciatic nerve epineural sheath against compounds with local anesthetic activity: anex vivoelectrophysiological study. Toxicol. Mech. Methods 23(8): 634–640.
Heimburg, T. (2022). The effect of stretching on nerve excitability. Hum. Mov. Sci. 86: 103000.
Li, J. and Shi, R. (2007). Stretch-induced nerve conduction deficits in guinea pig ex vivo nerve. J. Biomech. 40(3): 569–578.
Lao, J., Li, Y., Zhao, X., Tian, D., Zhu, Y. and Wei, X. (2014). The optimal distance between two electrode tips during recording of compound nerve action potentials in the rat median nerve. Neural Regener. Res. 9(2): 171.
Cohen, S. P., Larkin, T. M., Weitzner, A. S., Dolomisiewicz, E., Wang, E. J., Hsu, A., Anderson-White, M., Smith, M. S. and Zhao, Z. (2023). Multicenter, Randomized, Placebo-controlled Crossover Trial Evaluating Topical Lidocaine for Mechanical Cervical Pain. Anesthesiology 140(3): 513–523.
Kalajian, T. A., Cannella, J. A., Vasudevan, A., Mizelle, J., Rendon, L. F., Nozari, A. and Ortega, R. (2023). An overview of local anesthetics in over‐the‐counter products. Pain Pract. 24(2): 364–373.
Krotov, V., Agashkov, K., Romanenko, S., Halaidych, O., Andrianov, Y., Safronov, B. V., Belan, P. and Voitenko, N. (2023). Elucidating afferent-driven presynaptic inhibition of primary afferent input to spinal laminae I and X. Front. Cell. Neurosci. 16: e1029799.
Gokin, A. P., Philip, B. and Strichartz, G. R. (2001). Preferential Block of Small Myelinated Sensory and Motor Fibers by Lidocaine. Anesthesiology 95(6): 1441–1454.
Huang, J. H., Thalhammer, J. G., Raymond, S. A. and Strichartz, G. R. (1997). Susceptibility to lidocaine of impulses in different somatosensory afferent fibers of rat sciatic nerve. J. Pharmacol. Exp. Ther. 282(2): 802–811.
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4,970 | https://bio-protocol.org/en/bpdetail?id=4970&type=0 | # Bio-Protocol Content
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Purification of Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography
PK Pachiyappan Kamarajan
JT John C. Timm
MG M. Paula Goetting-Minesky
EM Erin T. Malone
SG Sean Ganther
AR Allan Radaic
CT Christian Tafolla
JF J. Christopher Fenno
YK Yvonne L. Kapila
Published: Vol 14, Iss 7, Apr 5, 2024
DOI: 10.21769/BioProtoc.4970 Views: 380
Reviewed by: Kristin L. ShinglerEmmanuel Orta-ZavalzaAna Martinez
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Original Research Article:
The authors used this protocol in PLOS Pathogens Jul 2021
Abstract
Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research.
Key features
• This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins.
• We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola.
• The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell.
• We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).
Keywords: Treponema denticola Dentilisin Matrix metalloproteinases MMP-2 Gelatin zymography Periodontal ligament (PDL)
Background
Periodontitis is a chronic inflammatory disease resulting from bacterial dysbiosis and unfavorable host–bacterial interactions. This ultimately leads to the destruction of periodontal tissues. The development of dysbiotic oral biofilms is associated with the emergence of periodontal disease. This condition is often attributed to elevated levels of the "red complex" bacteria, which include Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia [2], while several other periodontopathogenic species have been identified in recent studies [3–5]. Previous studies using 16S rRNA and shotgun sequencing have established notable dissimilarities in the microbial communities of individuals with good oral health vs. those with periodontitis. Periodontitis is a complex condition that is not solely caused by one type of microbe. Instead, it is thought to be influenced by various factors, such as changes in inflammation and interactions between the host and microbes, particularly when keystone pathogens colonize [6]. Also, it is important to highlight that various factors play crucial roles in the development of periodontitis, as they can either support or hinder the equilibrium of oral microorganisms.
In this study, we focused on the contribution of the most well-known and well-characterized periodontal pathogenic oral spirochete, namely T. denticola, which is highly associated with periodontal disease. However, T. denticola is often below detectable levels in healthy gingival plaque [7,8]. T. denticola, the most readily cultivable oral spirochete, is the model organism for studying spirochete–host interactions in periodontitis. The severity of periodontitis is directly linked to an increase in the colonization levels of T. denticola, highlighting its significant contribution to the disease [9,10]. One of the factors contributing to the virulence of T. denticola is its protease complex called dentilisin. Dentilisin is an outer membrane–associated complex consisting of the acylated subtilisin-family PrtP protease and two other lipoproteins (PrcB and PrcA) that are unique to oral spirochetes (reviewed in [11]). Dentilisin is encoded as an operon in the chromosome consisting of prcB-prcA-prtP. Dentilisin proteolytic activity directly affects host cells and tissue: it disrupts intercellular junctions [12], contributes to tissue penetration [13], specifically cleaves host proteins critical to maintaining tissue homeostasis [14], and induces dysregulation of TLR/MyD88 and integrin/FAK signaling mechanisms [15–17]. The multifaceted action of T. denticola dentilisin not only facilitates T. denticola's adhesion to and harm of epithelial cells and fibroblasts but also aids in penetrating epithelial tissue; furthermore, it may be involved in T. denticola's strategies to evade complement-mediated bactericidal activity [12,18–20]. One of the unique features of the dentilisin complex is that it retains its stability and proteolytic activity when unheated samples are subjected to SDS-PAGE. We and others have exploited this feature by using electrophoretic methods to purify dentilisin and characterize its activity [14,21,22]. We have previously reported the use of preparative continuous polyacrylamide gel electrophoresis (PC-PAGE) to purify several T. denticola outer membrane–associated proteins [20,22–24]. Here, we describe a method for purifying large amounts of the active dentilisin complex using this method.
The present study focuses on exploring the link between dentilisin and MMP-2 activation in relation to periodontitis. Here, we describe a methodology for the purification of substantial amounts of the dentilisin protease complex from T. denticola and demonstrate its capacity to activate MMP-2, a key regulator of periodontal tissue homeostasis. We present the protocols used in this study in three sections: I. Detergent extraction of T. denticola outer membrane components; II. PC-PAGE for purification of T. denticola dentilisin; and III. Gelatin zymography to detect activation of MMP-2 by dentilisin.
Part I: Detergent extraction of T. denticola outer membrane components
Materials and reagents
Biological materials
Treponema denticola MHE strain [25]. This strain (derived from T. denticola ATCC 35405) is an isogenic mutant that lacks Msp, a prominent outer membrane protein. We used T. denticola MHE for the present study because it is difficult to separate dentilisin from Msp using the wild-type ATCC 35405 strain [20,24]. This strain is available from the authors upon request. While the mutation carried in T. denticola MHE was selected by resistance to erythromycin, antibiotic pressure is not required to maintain the mutation.
Reagents
Tryptone (Fisher Scientific, catalog number: 211705)
Brain heart infusion (Fisher Scientific, catalog number: 237500)
Yeast extract (Fisher Scientific, catalog number: 611805000)
Gelatin (Bio-Rad Laboratories, catalog number: 1706537)
(NH4)2SO4 (Thermo Scientific, catalog number: OXCM0635B)
MgSO4·7H2O (Thermo Scientific, catalog number: AAA144910I)
K2HPO4 (Fisher Scientific, catalog number: BP363)
KH2PO4 (Fisher Scientific, catalog number: BP362)
NaCl (Fisher Scientific, catalog number: S271)
KOH (Fisher Scientific, catalog number: P250)
KCl (Fisher Scientific, catalog number: BP366)
Na2HPO4 (Sigma Millipore, catalog number: 567547)
MgCl2 (Fisher Scientific, catalog number: BP214)
Thiamine pyrophosphate (TPP) (Sigma-Aldrich, catalog number: C8754)
Glucose (Sigma-Aldrich, catalog number: G5400)
L-cysteine HCl (Fisher Scientific, catalog number: BP376)
Sodium pyruvate (Fisher Scientific, catalog number: BP356)
Glacial acetic acid (Fisher Scientific, catalog number: A38S)
Propionic acid (Sigma-Aldrich, catalog number: P1386)
N-butyric acid (Sigma-Aldrich, catalog number: B10355-0)
N-valeric acid (Sigma-Aldrich, catalog number: V9759)
Isobutyric acid (Sigma-Aldrich, catalog number: I1754)
Isovaleric acid (Sigma-Aldrich, catalog number: 12954-2)
D,L-methylbutyric acid (Sigma-Aldrich, catalog number: 193070)
Rabbit serum*, heat inactivated (Pel Freeze Biologicals, catalog number: 31127)
Triton X-114 (Anapoe-X-114) (Anatrace, catalog number: APX114)
Acetone (Fisher Scientific, catalog number: A18)
Tris base (Fisher Scientific, catalog number: BP152)
HCl (12.1 N) (Fisher Scientific, catalog number: A144)
1 M Tris pH 6.8 (Fisher Scientific, catalog number: BP152)
DTT (Fisher Scientific, catalog number: BP172)
SDS (Fisher Scientific, catalog number: BP166)
Glycerol (Fisher Scientific, catalog number: BP229)
Bromophenol blue (BPB) (Fisher Scientific, catalog number: BP114)
Solutions
TYGVS growth medium and supplements for T. denticola (see Recipes)
1 M Tris, pH 8 (see Recipes)
Bromophenol blue (BPB) gel loading solution (see Recipes)
Phosphate buffered saline containing 5 mM MgCl2 (PBS) (see Recipes)
Recipes
TYGVS growth medium and supplements for T. denticola [26,27]
TYGVS base growth medium
Add to approximately 900 mL of distilled water:
10.0 g of tryptone
5.0 g of brain heart infusion
10.0 g of yeast extract
10.0 g of gelatin
0.5 g of (NH4)2SO4
0.1g of MgSO4·7H2O
1.13 of K2HPO4
0.9 g of KH2PO4
1.0 g of NaCl
pH to 7.2 with 4 N KOH in distilled H2O
Autoclave (121 °C, 20 min, 15 psi). Unsupplemented media can be stored for up to six months at 4 °C.
Add 100 mL of TYGVS supplements (described below) aseptically before use.
Complete media with supplements can be stored for up to one month at 4 °C.
TYGVS supplements
Combine the following together, adjust to pH 7.2 with 4 N KOH, and filter sterilize.
Amount per liter of final volume of medium:
0.0125 g of TPP
1.0 g of glucose
1.0 g of L-cysteine HCl
0.25 g of sodium pyruvate
0.27 mL of glacial acetic acid
0.1 mL of propionic acid
0.064 mL of n-butyric acid
0.016 mL of n-valeric acid
0.016 mL of isobutyric acid
0.016 mL of isovaleric acid
0.016 mL of D,L-methylbutyric acid
100 mL of rabbit serum*, heat inactivated
Mix well; pH to 7.2 with 4 N KOH.
Sterilize using a 0.2 μm vacuum filter unit.
*Note: Heat-inactivated horse or bovine serum can also be used, with little difference in growth.
1 M Tris, pH 8
Mix 12.11 g of Tris in 80 mL of H2O. Adjust to pH 8 by dropwise addition of 1M HCl.
Bromophenol blue (BPB) gel loading solution (10 mL)
Reagent Final concentration Quantity or Volume
1 M Tris pH 6.8 in distilled H2O 360 mM 3.75 mL
DTT 600 mM 0.93 g
SDS 12% 1.2 g
Glycerol 60% 6 mL
BPB 0.018% 18 mg
Phosphate buffered saline containing 5 mM MgCl2 (PBS)
NaCl 80 g/L
KCl 2 g/L
Na2HPO4 14.4 g/L
KH2PO4 2.4 g/L
MgCl2 1.0 g/L
Distilled H2O, 900 mL
pH to 7.4 with 4 N KOH; then, correct volume to 1 L with H2O
Laboratory supplies
Culture growth vessels. Size is limited by the ability to pass through anaerobic chamber port. We typically use 2 L Corning Pyrex bottles (Corning, catalog number: 13952L)
Spectrophotometry cuvettes: Polystyrene, 2 mL (Fisher Scientific, catalog number: 14955127)
Centrifuge tubes:
250 mL (nominal), Nalgene, polypropylene copolymer (Thermo Scientific, catalog number: 31410250). These tubes will fit a GSA-type rotor
50 mL (nominal), Oak Ridge style (Thermo Scientific, catalog number: 31190050) (polypropylene copolymer) and 31180050 (polycarbonate). These tubes will fit a SS-34-type rotor
15 mL, conical, polypropylene (Corning, catalog number: 430766 or equivalent). These tubes will fit the appropriate swinging bucket rotor insert in STR40R or equivalent centrifuge
50 mL, conical, polypropylene (Corning, catalog number: 430291) or equivalent. These tubes will fit the appropriate swinging bucket rotor insert in STR40R or equivalent centrifuge
Vacuum filter units (0.2 µm) (Thermo Scientific, catalog number: 5650020)
Equipment
Anaerobic chamber (Coy Lab Products or equivalent). We use the Coy Type B vinyl anaerobic chamber. Coy and other manufacturers also make rigid (metal- or polymer-walled) anaerobic chambers, either with gloves or gloveless, that are advertised as having equivalent performance to the “classic” vinyl chamber
Compressed gas tanks: Pre-purified nitrogen (NITPP; Cryogenic Gases, Detroit, MI); mixed gas (10% H2, 5% CO2, 85% N2) (5CO10H2N2; Cryogenic Gases, Detroit, MI). Specialty gas tank regulators (Grainger, Lake. Forest, IL)
Class II biological safety cabinet (Advance SG403, Baker, Sanford, ME). T. denticola is generally classified as an RG2 microbe requiring BSL2 containment and procedures
High-speed centrifuge with rotors for 15 mL, 50 mL, and 250 mL tubes
Sorvall RC2-B (or equivalent) with GSA rotor for 250 mL tubes; SS-34 rotor for 50 mL Oak Ridge tubes
Sorvall ST40R (or equivalent) equipped with swinging bucket rotor for 15 mL and 50 mL tubes
Vacuum trap system for biohazardous waste
SPE vacuum pump trap kit (Millipore Sigma, catalog number: 57120-U)
Whatman Vacu-Guard in-line filter (Cytiva, catalog number: 67225001)
Laboratory “house” vacuum system or small vacuum pump (Southern Labware, catalog number: 167300-11)
pH meter: Accumet AE 150 (or equivalent) (Fisher Scientific, catalog number: 13-636-AE153)
Spectrophotometer (Dayton, Model 1200, Unico)
Platform shaker (Labnet Orbit 1000, catalog number: S20301000B)
Procedure
Detergent extraction and phase partitioning of T. denticola for separation of outer membrane proteins. This procedure gently solubilizes the T. denticola outer membrane, releasing outer membrane and periplasmic proteins while leaving the protoplasmic cylinder intact. A solution of the nonionic detergent Triton-X114 is homogeneous at 0 °C but separates into an aqueous phase and a detergent phase above 20 °C [28–30]. This feature can be utilized to greatly enrich hydrophobic membrane-associated lipoproteins in the detergent phase while periplasmic proteins segregate in the aqueous phase. This methodology is used in spirochete research, both to isolate their abundant lipoproteins and to distinguish between soluble periplasmic proteins and outer membrane components [1,14,20,30–33]. While many investigators incorporate a protease inhibitor cocktail in this procedure, we do not include this step here because it would preclude our objective of obtaining a proteolytically active protein complex. The steps involved in this procedure for detergent extraction and preparation of samples for preparative electrophoresis are illustrated in Figure 1.
Grow T. denticola strain MHE [25] at 37 °C under anaerobic conditions (10% H2, 5% CO2, 85% N2) in complete TYGVS medium [26,27] to late log phase (3–4 days). The expected optical density at 600 nm is approximately 0.3–0.4 as measured in a standard spectrophotometer. The MHE strain is used for dentilisin purification because it lacks the major outer membrane protein Msp, which is difficult to separate from dentilisin using these methods. We typically grow up to 4 L of culture (up to 2 L per culture bottle) for this procedure, which will yield approximately 1.5 mL of the final step extract per liter of culture at a concentration of approximately 500 µg/mL [14].
Harvest culture by centrifugation (4,000× g, 10 min, 4 °C) using 250 mL Nalgene centrifuge tubes. Resuspend in half of the original culture volume of PBS (pH 7.4) containing 5 mM MgCl2, centrifuge as above, and repeat the wash/centrifugation step twice. At each step, suspend the pellet by pipetting gently in a small volume (10–20 mL) of the same PBS+MgCL2 buffer solution and then adding the remaining volume.
Resuspend the pellet as above in 1/40 of the original culture volume of PBS containing 5 mM MgCl2 and 1% Triton X-114.
Note: Triton X-114 is supplied as a 10% aqueous solution. Use small-volume containers of Triton X-114 because it easily becomes oxidized after exposure to air.
Rock the suspension gently overnight at 4 °C in Oak Ridge tubes on a platform shaker.
Centrifuge at 20,000× g for 10 min at 4 °C to separate and pellet unsolubilized material, which is primarily protoplasmic cylinders of spirochete cells [35].
Transfer the clear supernatant containing detergent-soluble material to new 15 mL conical tubes. Adding a drop of bromophenol blue solution (0.04%) to the mixture facilitates visualization of the detergent phase in the following separation step without affecting proteins of interest [32]. The extract may be processed immediately or stored at -80 °C until further processing, which is generally done within a few days.
Incubate the extract at 37 °C for 30 min. The solution will become cloudy.
Prewarm a rotor to 37 °C and then centrifuge the extract at 3,500× g for 10 min at 37 °C in a swinging bucket rotor. The extract should separate into an aqueous upper phase and a detergent lower phase of approximately 1/10 total volume. If good separation is not apparent, centrifugation speed and time can be increased to as much as 20,000× g for 1 h [1,30]. If this is necessary, perform centrifugation in polycarbonate Oak Ridge tubes, because they tolerate higher speeds and allow easier visualization of the detergent pellet than similar polypropylene tubes.
Carefully remove all traces of the aqueous phase from the lower phase.
Dilute the remaining detergent phase with PBS containing 5 mM MgCl2 (4 °C) to bring to the original volume before phase separation. This will return the Triton X-114 concentration to approximately 1%. Repeat phase separation steps 4–9 twice.
Transfer the final lower (detergent) phase to a fresh 50 mL polypropylene Oak Ridge tube. Note that polycarbonate tubes are incompatible with acetone.
Precipitate the detergent phase with acetone to remove Triton X-114 detergent from the sample prior to electrophoresis. Add 8 volumes of cold acetone (-20 °C) and incubate at -20 °C for at least 1 h but not more than 20 h. Centrifuge at 12,000× g at 4 °C for 30 min. Carefully pour off acetone from the soft pellet. Allow the pellet to partially dry and then dissolve in 1/2 volume of PBS containing 5 mM MgCl2. Detergent phase samples generally do not require further concentration before PC-PAGE.
The sample may be stored at -80 °C until purification on Prep Cell, as described in the following section. We generally try to proceed to the PC-PAGE step as soon as is practicable.
Part II: PC-PAGE for purification of T. denticola dentilisin complex
The dentilisin protease complex can be purified from the detergent phase extract prepared in the prior steps by preparative electrophoresis in a Model 491 Prep Cell. The complex, consisting of three lipoproteins (PrcB, PrcA, and PrtP) is remarkably stable under SDS-PAGE conditions when not heated prior to electrophoresis and retains proteolytic activity [13,21,24,36,37]. This PC-PAGE technique is appropriate for the purification of proteins whose apparent molecular weight differs by greater than 5% from their nearest neighbors in SDS-PAGE. In the case of dentilisin, the detergent extraction/phase-partitioning procedure removes the bulk of the proteins from the original crude preparation, and the approximately 95 kDa native protease complex can be recovered efficiently at a level of purity appropriate for many functional assays.
Pre-run preparation is done the day prior to starting the gel run. Including the pre-run, the whole process through sample collection takes approximately three days, not including characterization of fractions by SDS-PAGE and silver staining.
Materials and reagents
Biological materials
Detergent phase extract of T. denticola MHE prepared above
Reagents
30% Acrylamide/Bis solution, 37.5:1 (Bio-Rad Laboratories, catalog number: 161-0158)
Tris pH 8.8, 1.5 M in H2O
Tris-HCl pH 6.8, 1.25 M in H2O
Glycine (Fisher Scientific, catalog number: BP381)
Sodium dodecyl sulfate (SDS) (Invitrogen, catalog number: 15525017)
Tetramethylethylenediamine (TEMED) (Invitrogen, catalog number: 15524)
Ammonium persulfate (APS) (Thermo Scientific, catalog number: 32708)
Bromophenol blue 6× dye solution (Thermo Scientific, catalog number: J61337.AC)
CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) (Sigma-Aldrich, catalog number: C3023)
Bicinchoninic acid (BCA) protein assay kit (Thermo Scientific catalog number: 23225)
Horseradish peroxidase (HRP)-conjugated Goat Anti-Rabbit IgG (H+L) (Bio-Rad Laboratories, catalog number: 1706515)
SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, catalog number: 34580)
Dentilisin activity detection:
N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (SAAPFNA) (Sigma-Aldrich, catalog number: S7388)
N,N-dimethyl formamide (Thermo Scientific, catalog number: AC42364)
SAAPFNA stock: 50 mM in N,N-dimethyl formamide. Store at -20 °C
Reaction buffer: (1×) 50 mM Tris, 0.2 M NaCl, 5 mM DTT, pH 7.5. Make as 10× stock and store at -20 °C.
α-chymotrypsin, Type II as control. Dilute in reaction buffer before use: 1 mg/mL and further 1/10 dilutions (Sigma-Aldrich, catalog number: C4129)
Flat-bottom 96-well microplates (Fisher Scientific, catalog number: FB012931)
Solutions
6× Laemmli SDS sample buffer (see Recipes)
Upper electrophoresis chamber (cathode) buffer (see Recipes)
Lower electrophoresis chamber (anode) buffer (see Recipes)
Lower electrophoresis chamber (anode) buffer (see Recipes)
Elution buffer (see Recipes)
Post-electrophoresis buffer exchange (see Recipes)
APS 10% in H2O (freshly made)
Recipes
Standard SDS-PAGE preparative gel column
Resolving gel: 7.5% acrylamide for 28 mm diameter column with a gel height of 9.5 cm.
Scale appropriately for 37 mm column or for different acrylamide concentrations.
Note: Acrylamide monomer is a neurotoxin.
30% acrylamide/Bis 37.5:1 9.5 mL
1.5 M Tris pH 8.8 9.5 mL
H2O 18.4 mL
10% SDS 0.38 mL
TEMED 0.02 mL
10% APS 0.2 mL
Stacking gel: 2× sample volume (assuming 5 mL sample volume)
30% acrylamide/Bis 37.5:1 1.3 mL
1.25 M Tris-HCl pH 6.8 1.0 mL
H2O 7.54 mL
10% SDS 0.10 mL
TEMED 0.01 mL
10% APS 0.05 mL
6× Laemmli SDS sample buffer
375 mM Tris-HCl/Tris Base
9% SDS (w/v)
50% Glycerol (v/v)
9% 2-mercaptoethanol (v/v)
0.075% Bromophenol blue (w/v)
Upper electrophoresis chamber (cathode) buffer (500 mL)
25 mM Tris (pH 8.8)
192 mM glycine
0.1% SDS
Lower electrophoresis chamber (anode) buffer (2,000 mL)
25 mM Tris (pH 8.8)
192 mM glycine
Elution buffer (700 mL)
25 mM Tris (pH 8.8)
192 mM glycine
Notes:
We omit SDS from the anode and elution buffers so that there will be minimal residual detergent in the fractions collected. We have seen no effect of this modification on electrophoresis and fraction collection.
Degas all buffers and frits before use. Put the frits in a dish and cover with Tris/glycine buffer. Place them in the port of the anaerobic chamber with caps loosened, pull a vacuum, incubate overnight, then tighten caps, and hold at 4 °C until use.
Post-electrophoresis buffer exchange
PBS containing 0.1% CHAPS
Equipment
Chromatography refrigerator or cold room (Thermo Scientific, TSX2305CA)
Model 491 Prep Cell (Bio-Rad Laboratories, catalog number: 1702928)
Power supply (Bio-Rad Laboratories, model: PowerPac 3000)
Low-pressure chromatography system (Bio-Rad Laboratories, model: BioLogic LP)
ChemiDoc MP image system (Bio-Rad, model: Universal hood III, catalog number: 731BR01488)
Model 2128 fraction collector (flow rate: 1 mL/min; fractions: 5.0 mL; up to two racks of 128 tubes each) (Bio-Rad Laboratories, catalog number: 731-8123)
Variable speed buffer recirculation pump (Bio-Rad Laboratories, catalog number: 1702929)
Sample concentrators:
Millipore/Amicon stirred ultrafiltration cell. Several sizes are available: 50 mL (Millipore, catalog number: UFSC05001), 200 mL (Millipore, catalog number: UFSC20001), and 400 mL (Millipore, catalog number: UFSC40001). This is a stand-alone apparatus that requires a dedicated nitrogen gas tank to supply filtration pressure. Use with ultrafiltration disc membranes designed for use with stirred cells. Disc sizes are available for each size of stirred cell (Cole-Parmer, Inc., model: Millipore EW-29949)
Amicon Ultracel 30 centrifugal concentration unit (Millipore, catalog number: UFC9030). These are single-use filtration units appropriate for use in an ST40R centrifuge. They are available in a wide range of other volumes and molecular weight cutoffs. While relatively inexpensive, costs add up rapidly on large projects
Compressed nitrogen gas tank (NITPP; Cryogenic Gases, Detroit, MI)
Specialty gas regulator for nitrogen tank (Grainger, model: Harris KH1008). Stirred cell concentrators utilize gas pressure for sample concentration
Microplate reader (Molecular Devices, model: VERSAmax). A plate reader is used to assay dentilisin activity against the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (SAAPFNA) [21]
Procedure
Detailed instructions for assembly and operation of the Model 491 Prep Cell are provided in the Bio-Rad instruction manual. The steps involved in preparative electrophoresis of the sample prepared in Part I above are illustrated in Figure 1. The following are specific items for this application.
Assemble the 28 mm diameter glass gel tube assembly on its casting stand as described in the manual. For larger batch preps, it may be useful to use the 37 mm diameter glass gel tube, with gel component volumes scaled up accordingly.
Attach a continuous flow of water on ice through the cooling core after setting up on the casting stand.
Prepare 7.5% acrylamide resolving gel (total volume: 38 mL): 9.5 mL of 30% acrylamide/Bis 37.5:1; 9.5 mL of 1.5 M Tris pH 8.8; 16.55 mL of dH2O; 0.38 mL of 10% SDS; 0.038 mL of TEMED; 2.66 mL of 1% APS.
Pour resolving gel, one-third of the volume at a time, and then tap and shake to ensure there are no bubbles at the bottom or the side of the column each time. Top with 2 mL of H2O-saturated butanol.
After ~1 h polymerization, discard butanol, wash twice with distilled H2O, and cover with 25 mM Tris (pH 8.8), 192 mM glycine, and 0.1% SDS. This can be left for 48 h or longer at 4 °C or overnight at room temperature (RT) to ensure that polymerization is complete.
Pour an appropriate volume of stacking gel (= 2× sample volume). Note that it is not uncommon to load a 5 mL sample on the Prep Cell. For 10 mL of stacking gel: 7.66 mL of H2O, 1.0 mL of 1.25 M Tris-HCl pH 6.8; 0.1 mL of 10% SDS; 0.01 mL of TEMED; 0.07. mL of 1% APS. If the resolving gel is at RT, allow the stacking gel to polymerize for 30 min.
Disassemble the casting stand and assemble the gel column in the Model 491 Prep Cell according to the manufacturer's instructions, with particular attention to proper placement of support frit, elution frit, dialysis membrane, and O-rings.
In the chromatography refrigerator (or cold room), connect each of the following according to the instructions in the BioLogic LP chromatography manual:
Buffer cooling core inlet from the upper buffer chamber.
Buffer cooling core outlet into the buffer circulation pump.
Outlet from the pump to the spigot of the lower buffer chamber.
Elution outlet at the middle of the top of the cooling core to the inlet of BioLogic System pump.
Be sure you have a separate collection container for the elution buffer that comes off before initiation of fraction collecting.
Add 25 mM Tris (pH 8.8) containing 192 mM glycine to the lower buffer chamber above the level of the stacking gel.
Thaw the detergent phase extract on ice and add 1/5 volume standard 6× SDS-PAGE sample buffer containing reducing agent.
Add the sample with a Pasteur pipette via cut off plastic pipette guide as described in the Prep Cell Instruction Manual. It is useful to become familiar with this gel-loading procedure ahead of time. After finishing, remove the plastic pipette guide by sealing the top opening of the guide with your finger and gently pulling the guide out.
Fill the upper buffer chamber with 25 mM Tris (pH 8.8), 192 mM glycine, and 0.1% SDS. Fill the elution buffer chamber with 25 mM Tris (pH 8.8) and 192 mM glycine buffer.
Connect the electrodes of the Prep Cell to the Bio-Rad PowerPac. Set at 60 mA constant current, which should result in an initial voltage of approximately 550 V.
Connect the buffer circulation pump with 1.6 mm tubing and set the flow rate to 1 mL/min.
Do not turn on the fraction collector until needed (i.e., until after the dye front has passed through the gel). Set fraction collection to collect 5 mL fractions at a flow rate of 1 mL/min.
Screen every tenth fraction by silver-stained SDS-PAGE (10 µL per lane). Pool fractions containing the expected dentilisin complex migrating at 95–100 kDa.
Concentrate fractions of interest at least 10-fold at 4 °C in a stirred-cell ultrafiltration unit fitted with an ultrafiltration disc membrane filter. Subsequently, subject the sample to buffer exchange by washing three times with 10 volumes of PBS containing 0.1% CHAPS in the same stirred cell system. Alternatively, concentrate fractions and exchange buffer using Amicon Ultracel 30 centrifugal filter columns at 4 °C according to the manufacturer’s instructions.
Determine protein concentration using a bicinchoninic acid (BCA) protein assay following the manufacturer's instructions. If required, samples can be further concentrated using an Amicon Ultracel 30 centrifugal filter at 4 °C according to the manufacturer's instructions.
Determine dentilisin proteolytic activity using the chromogenic substrate SAAPFNA as described previously [21].
Load 50 µL of each protein sample and controls in sample buffer into the wells of a 96-well plate (negative control: protein sample buffer; positive control: protein sample buffer plus chymotrypsin).
Incubate at 37 °C for 5 min while making up enzyme substrate.
Dilute 10× reaction buffer 1:5 in distilled H2O.
Dilute SAAPFNA substrate stock 1:50 in the resulting 2× reaction buffer.
Add 50 µL of SAAPFNA substrate to each well. Use sample buffer plus substrate as a blank.
Incubate plate at 37 °C for 5–30 min, monitoring carefully for yellow color change in positive wells vs. blank wells.
Read absorbance value at 405 nm.
Store purified dentilisin at -80 °C in aliquots appropriately sized for intended uses. In our hands, properly stored dentilisin samples are stable for several years.
Each step of dentilisin purification including growth, harvesting, detergent extraction/phase separation, and PC-PAGE is shown in Figure 1. Detection of protein products of the T. denticola prcB-prcA-prtP operon in whole cells and purified dentilisin protease complex are shown in Figure 2.
Figure 1. Dentilisin purification workflow. Details of each step, including growth, harvesting, detergent extraction/phase separation, and PC-PAGE, are described in the text.
Figure 2. Detection of products of the Treponema denticola prcB-prcA-prtP operon in whole cells and purified dentilisin protease complex. A. Silver-stained SDS-PAGE of purified protease complex (0.4 µg per lane), unheated (lane 1) and boiled (lane 2). The locations of PrtP, PrcA1, PrcA2, and PrcB bands are indicated. Molecular weight standards in kDa (MW) are shown. B: Immunoblots of T. denticola whole-cell extracts and purified protease complex. All samples were boiled prior to electrophoresis. Samples were separated by standard SDS-PAGE and then transferred to nitrocellulose membranes. Lane 1: T. denticola whole-cell extract (equivalent to 10 µL of culture). Lane 2: purified dentilisin protease complex (0.4 µg per lane). Membranes were first probed with rabbit polyclonal antibodies specific for PrcB, PrcA1, PrcA2, PrtP-N-terminal domain, and mature PrtP [24,37,38] and then probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG. Protein bands of interest were visualized using the SuperSignal West Pico chemiluminescent substrate. The approximate size in kDa of each polypeptide is indicated (MW). Reprinted with permission from Godovikova et al. [24] (Figure 2) with minor modifications.
Part III: Gelatin zymography to detect activation of MMP-2 by T. denticola dentilisin
Native T. denticola dentilisin directly activates recombinant MMP-2
The oral spirochete Treponema denticola is highly associated with periodontal disease. The T. denticola outer membrane protein complex dentilisin contributes to the chronic activation of pro-MMP-2 in PDL cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells [14,39]. Despite these advances, there is no identified mechanism for dentilisin-induced MMP-2 activation. Gelatin zymography is an effective method for detecting gelatinolytic activity in various biological samples including culture media, cell extracts, tissue extracts, and biological fluids. This is accomplished by utilizing SDS-polyacrylamide gels that have been impregnated with gelatin. In this study, we explored the ability of T. denticola dentilisin to directly activate MMP-2. This is done by using both native purified T. denticola dentilisin and recombinant MMP2 in an in vitro setting, wherein MMP-2 activation is assayed by gelatin zymography and an in situ gelatin zymography assay.
A. Gelatin zymography
Materials and reagents
Reagents
Pierce BCA protein assay kit (Thermo Fisher, catalog number: 23225)
Pro-MMP-2 (R&D Systems, catalog number: 902-MP-010)
4× sample buffer (Bio-Rad, catalog number: 1610747)
10% Zymogram plus (gelatin) gel (Invitrogen, catalog number: ZY00100)
10× Gel electrophoresis running buffer (Novex, catalog number: LC2675)
Zymogram renaturing buffer (Novex, catalog number: LC2670)
Zymogram developing buffer (Novex, catalog number: LC2671)
Staining solution (Simplyblue safestain) (Invitrogen, catalog number: 465034)
Methanol (Fisher Scientific, catalog number: A452-4)
Acetic acid (Sigma-Aldrich, catalog number: A6283)
Solutions
Destaining solution (see Recipes)
Recipes
Destaining solution
10% methanol and 5% acetic acid in dH2O
Equipment
XCell SureLockTM electrophoresis cell (Thermo Fisher, model: EI0001)
PowerPacTM Basic (Bio-Rad, model: 041BR93879)
Eppendorf tubes (Eppendorf, catalog number: 0030 124.537)
ChemiDoc MP image system (Bio-Rad, model: Universal hood III, catalog number: 731BR01488)
Software and datasets
Image processing software (ImageJ version 2.14.0/1.54f, https://imagej.net/ij/download.html)
Procedure
Running and developing the gel
Conduct gelatin zymography as previously described [14].
Calculate protein concentrations for each sample using the Pierce BCA Protein Assay kit according to the manufacturer’s instructions.
Mix and incubate different concentrations of purified dentilisin and pro-MMP-2 for 30 min at RT and mix with 4× sample buffer. Then, load into each well and subject to SDS-PAGE on 10% gel containing gelatin.
After electrophoresis, remove SDS from the gels by washing them in renaturing buffer twice for 30 min. Then place gels in developing buffer for 30 min.
Replace the developing buffer and incubate gels at 37 for 16 h. Afterward, stain them with Simplyblue safestain for 30 min and subsequently destain using a solution of 5% acetic acid and 10% methanol until clear bands appear against a blue background.
Take zymogram pictures using ChemiDoc MP image system and measure the band intensity for active MMP-2 expression using ImageJ software (Figure 3).
For Figure 3, data was compared using one-way ANOVA Tukey’s multiple comparisons test. **p < 0.01; *** p < 0.001.
Figure 3. Dentilisin from Treponema denticola directly activates recombinant MMP-2. Top: Representative gelatin zymography image showing the activation of MMP-2. Bottom: The band intensity for active MMP-2 expression was measured using ImageJ software (n = 3). Data was compared using one-way ANOVA Tukey’s multiple comparisons test. **p < 0.01; ***= p < 0.001.
B. In situ gelatinase assay
Materials and reagents
Reagents
EnzChekTM gelatinase/collagenase assay kit (Invitrogen, catalog number: E12055)
Dye-quenched (DQ) gelatin fluorogenic substrate (Invitrogen, catalog number: E12054)
Pro-MMP-2 (R&D Systems, catalog number: 902-MP-010)
Pierce BCA protein assay kit (Thermo Fisher, catalog number: 23225)
Equipment
Fluorescent microplate reader (Molecular Devices, model: SPECTRA max, M2)
96-well plates (Thermo Fisher, catalog number: M33089)
Software and datasets
GraphPad Prism statistical software (GraphPad, Prism, version 10.1.1)
Procedure
Add 80 mL of 1× reaction buffer and 20 mL of the 1 mg/mL DQ gelatin fluorogenic substrate stock to each assay well.
Test different sample groups with control, purified pro-MMP-2, purified dentilisin, or both pro-MMP-2 and dentilisin with gelatin substrate and reaction buffer. Use Pierce’s BCA protein quantification kit according to the manufacturer’s instructions to determine the initial concentration of 1 mg/mL for each purified protein.
Achieve further dilution to a final concentration of 0.002 mg/mL for each protein with reaction buffer similarly to a previously described publication [40].
Add 100 mL of the final concentration of 1× reaction buffer (negative control), MMP-2 alone, or dentilisin alone to respective grouped wells.
Add 50 mL of the final concentration MMP-2 plus 50 mL of final concentration dentilisin to its respective well group.
Read samples in a fluorescent microplate reader at 495 nm absorption and 515 nm emission at 0, 30, 60, 90, and 120 min time points.
Data represents mean ± SD from two independent experiments (Figure 4).
Figure 4. Direct activation of pro-MMP-2 by dentilisin using in situ gelatin zymography. Fluorescent intensity of each group was plotted at each interval (0, 30, 60, 90, and 120 min). Sample groups were negative control, purified pro-MMP-2 alone, purified dentilisin, or both pro-MMP-2 and dentilisin. The plot represents an average of two experiments, each consisting of six separate samples in every group.
Data analysis
Data was compared using one-way ANOVA with Tukey’s multiple comparisons test (Figure 3, Statistical analysis, Front Cell Infect Microbiol. 2021 May 19; 11:671968).
Validation of protocol
The protocols for dentilisin purification and zymography described here have been previously reported and validated in:
Fenno et al. [20]. Infect. Immun. 66:1869–1877. doi: 10.1128/IAI.66.5.1869-1877.1998 (Figure 3).
Godovikova et al. [24]. Infect. Immun. 79(12):4868–75. doi: 10.1128/IAI.05701-11 (Figure 2).
Miao et al. [14]. Infect. Immun. 79(2):806–11. doi: 10.1128/IAI.01001-10 (Figure 2, 3B, 4A, 5A).
Miao et al. [39]. Arch. Oral Biol. 59(10):1056–64. doi: 10.1016/j.archoralbio.2014.06.003 (Figure 1A and B, 3C).
Malone et al. [17]. Front. Cell Infect. Microbiol. 11:671968. doi: 10.3389/fcimb.2021.671968 (Figure 4, 7A, 8A).
Ganther et al. [16]. PLoS Pathog. 17(7): doi: 10.1371/journal.ppat.1009311 (Figure 4b, S2).
Acknowledgments
These studies were supported by funding from the NIH as follows: R01 DE025225 to YLK and JCF; R01 DE018221 to JCF; Ruth L. Kirschstein Individual Predoctoral NRSA for MD/PhD, and other Dual Degree Fellowships (F30 DE027598) to EM; and Ruth L. Kirschstein NRSA Institutional Research Training Grant (T32DE007306) to SG. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript. The protocols described here were adapted from previously reported studies by the authors [14–16,20 22,24].
Competing interests
The authors declare no conflict of interest.
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Automated Layer Analysis (ALAn): An Image Analysis Tool for the Unbiased Characterization of Mammalian Epithelial Architecture in Culture
CC Christian Cammarota
DB Dan T. Bergstralh
TF Tara M. Finegan
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4971 Views: 2867
Reviewed by: Ralph Thomas BoettcherKaty RothenbergSusanne ReinhardtHsih-Yin Tan
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Original Research Article:
The authors used this protocol in Molecular Biology of the Cell Mar 2023
Abstract
Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo–two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity.
Key features
• This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion.
• ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy.
• ALAn code is written in Python3 using the Jupyter Notebook interactive format.
• Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland–derived and Caco-2 colon carcinoma cells.
• This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.
Graphical overview
Keywords: Epithelia Cell culture Image Analysis 3D Architecture MDCK cells Epithelial biology Quantitative microscopy analysis
Background
We developed a tool for the unbiased analysis of layer architecture (Automated Layer Analysis or ALAn) for two reasons. Firstly, cultured epithelial architecture can vary drastically between experiments, and we found this can be the case even across a single 1 mm2 culture well [1]. Secondly, epithelial tissues are commonly considered to be pseudo–two dimensional, meaning that architecture is primarily considered with respect to the tissue plane/surface. We found that this perspective is not always predictive of architecture with respect to tissue depth (the apical-basal axis) [1]. We are interested in the mechanical and biological parameters that contribute to epithelial layer architecture and we developed this tool to quantify architecture development over time and test the effect of plating density, culture time, and presence of cadherin-mediated adhesion on architecture. Due to the variability in the shape of layers and the complexity of attempting to perform 3D analysis manually, it was important to us to develop an unbiased and automated tool that requires as little user input as possible. As a consequence of developing ALAn, we identified a developmental series of layer architectures that progress through the process of epithelialization, whereby individual cells form a tissue layer [1,2]. Our quantifications of cell height, cell density, and circularity show distinct morphological regimes for each architecture.
Several tools exist for the analysis of epithelial topology in the plane of the tissue [3–6]. As far as we are aware, ALAn is the first to characterize and quantify the apical-basal topology of cultured layers. To do so, the tool makes use of two pieces of spatial information: 1) a projection of actin signal intensity and 2) the positions of cell nuclei in the apical-basal dimension. Using this information, ALAn outputs layer density and layer height and assigns each layer to one of four organized categories representing a developmental series (Immature, Intermediate A, Intermediate B, and Mature) or a fifth category of exclusion (Disorganized) [1,2]. ALAn also utilizes existing Python libraries to output in-plane quantifications of cell shape.
Confluent epithelial tissues in culture transition between distinct developmental architectures as they proliferate and therefore densify [1,2]. ALAn makes architectural classifications based on plots of the average spatial distributions of nuclei and actin across the apical-basal depth of a 3D-imaged region. At sparse densities, cultured epithelial cell layers have a squamous morphology and are characterized by a lack of defined lateral surfaces; these are classified as Immature. The spatial plots of Immature layers have the peak of nuclear distribution located at, or occasionally above, the peak actin intensity (Figure 1A). At intermediate densities, layers develop lateral cell–cell borders and rounded cell apices; these are classified as Intermediate. The Intermediate category is split into two. The first of these, called Intermediate A (IntA), are layers where cells exhibit an asymmetric actin intensity profile with a single peak closer to the apical surface than the nuclear profile (Figure 1B). If the plot of actin exhibits a shoulder (corresponding to distinct lateral and apical pools of actin), then layers are classified as Intermediate B (IntB) or Mature (Figure 1C–1D) [2]. ALAn uses the derivative of the actin intensity plot to detect this shoulder. If the derivative is two-peaked, the ratio of the right peak to the left peak is used. A ratio of less than 1 defines IntB layers, and a ratio of greater than 1 is classified as a Mature layer. The Disorganized architectural category of epithelial cell culture is characterized by a failure to achieve a regular monolayered architecture in the apical-basal axis and therefore is characterized by broad nuclear and actin profiles (Figure 1E). Disorganization occurs when the underlying cell culture substrate area is insufficient to accommodate the number of cells seeded onto the plate [1].
Figure 1. Epithelial architecture categories and the distinctive spatial distribution profiles of nuclei and actin positions in the apical-basal (z) axis that Automated Layer Analysis (ALAn) uses to categorize them
Materials and reagents
Reagents
Dulbecco’s phosphate buffered saline (dPBS) (Sigma-Aldrich, catalog number: SKU D1408
Phosphate buffered saline (PBS) (such as tablets from Thermo Fisher, Invitrogen, catalog number: 003002)
Paraformaldehyde, 37% (Fisher Scientific, catalog number: F79-500)
Tween 20 detergent (Sigma, catalog number: SKU 11332465001)
Vectashield antifade mounting medium with DAPI (Vector Laboratories, catalog number: SKU H-1200)
Fluorescein phalloidin (Thermo Fisher, Invitrogen, catalog number: F432)
Laboratory supplies
Collagen IV coated 8-well µ-slide, #1.5 polymer coverslip, sterilized (Ibidi, catalog number: 80822)
Equipment
Spinning disc confocal unit coupled to a Nikon Ti-E inverted microscope and Zyla 4.2 sCMOS camera; 405 nm and 488 nm laser lines; 40× NA 1.15 water immersion lens (Andor, Oxford Instruments model: Dragonfly 200)
Computer workstation with a minimum 8 GB RAM, AMD, or NVIDIA 2 GB Graphics card and monitor with 1,280 × 1,024 pixels.
Software and datasets
Fusion (Version 2.3, Andor). Optional; used for image acquisition
Imaris (Versions 9.7, 9.8, 9.9, or 10.0, Oxford Instruments). Optional, used for nuclei segmentation
FIJI, ImageJ2 distribution (Version 2.14.0) [7,8]
Microsoft Excel (Version independent)
Anaconda distribution (Version 3, 2023.07-2)
Code
Code can be obtained from GitHub: https://github.com/Bergstralh-Lab/ALAn (Access date, 03/13/2024)
Collation_Imaris2ALAn.ipynb
ALAn_v3.ipynb
Procedure
Cell culture and imaging (suggested approach)
Cell culture, fixation, and staining
Culture mammalian cells in Ibidi wells.
After the desired period of culture time, remove the culture media and wash cells once with 250 μL of dPBS.
Add 200 μL of a solution of 3.7% paraformaldehyde diluted in PBS with 2% Tween 20 to the well and incubate for 10 min at room temperature.
Wash the cells three times with 250 μL of PBS with 0.2% Tween 20, letting the wash solution sit in each well for 10 min between washing steps.
Dilute fluorescein phalloidin 1:500 in PBS with 0.2% Tween 20 and add 250 μL to each well. Incubate for 12–24 h at 4 °C.
Remove phalloidin solution and rinse with PBS with 0.2% Tween 20 three times.
Add approximately 500 μL (or at least enough to fully cover the cell layer) of Vectashield with DAPI to the well.
Confocal imaging
Cells can be imaged directly in the Ibidi wells through the #1.5 polymer coverslip bottom using an inverted confocal microscope. Image a region in XY with an area of at least 60 μm × 60 μm at Nyquist resolution at a minimum of 512 × 512 pixels.
Image actin stained by FITC-conjugated phalloidin using the 488 nm line and DNA stained by DAPI using the 405 nm line. Use standard imaging techniques to set acquisition parameters to ensure that you are taking advantage of the full dynamic imaging range and not saturating pixels.
Note: Any saturation will invalidate the use of ALAn.
Take confocal z-stacks at a spacing of no greater than 0.5 μm, preferably Nyquist sampling (select Auto step size in the Z-scan settings in Fusion), starting from beneath the bottom of the chamber slide where no actin (FITC) signal can be detected and ending above the layer where no more actin signal can be detected.
Preparation of data for input into ALAn
Segmentation of nuclei using Imaris
Save files using the following name format: “Identfier1_Identifier2_Identifier3_Identifier4.ims” where the identifiers are the experimental parameters that you would like to log (for example, “MDCKcells_GeneKnockOut1_PlatingDensity100k_48hrsGrowth.ims”).
Load the files to be analyzed into the Imaris arena using the Observe Path function.
Select the image to be analyzed and open in the Surpass view.
Go to the 3D View menu and select Cells .
Select the Add new Cells function to begin segmentation.
Use the default settings to segment nuclei and cell borders (although we will skip the cell borders segmentation) and deselect Segment only a Region of Interest.
Use the blue arrow to move to nuclei detection. Do not use the green arrow.
Set your source channel to the DNA-stained channel (DAPI-stained 405 channel using the above protocol).
Set the expected nucleus diameter based on the cell types. (For MDCK cells, we suggest a value of 7.3 μm.)
Ensure that the smooth feature is auto selected and set to a width of 1/10th of the nucleus diameter.
Select Split Nuclei by Seed Points.
Use the blue arrow to move to Filter Nuclei Seed Points.
Set the minimum Quality factor to 4.
Note: This value is set to be relatively low because, at this stage of the segmentation process, it is best to find as many nuclei as possible. It is easy to remove nuclei in ALAn, but impossible to add more.
Use the blue arrow to move to Nuclei Threshold.
Set the “Nuclei Threshold (absolute intensity)” to a value that accurately segments the nuclei in the interactive view (Figure 2A). (Check to ensure that this value does not include the peak on the histogram that corresponds to the background signal peak. If it does, then adjust the threshold up until it is past the peak.)
Figure 2. Screenshots showing key steps from the protocol. A. Screenshot from Imaris showing the nuclear segmentation step. Users must choose a nucleus threshold value such that nuclei are accurately segmented and segmented seeds are not touching where possible. B and C. Screenshots of Jupyter Notebook showing open Collation_Imaris2AlAn (B) and ALAn_v3 code (C).
Use the blue arrow to move to Filter Nuclei.
When the Thresholding ends, click the orange X. Do not do any further filtering at this point unless you disagree with the segmentation.
Save the segmented summary statistics by first selecting the Statistics option and then selecting the Export All Statistics to File option. It is helpful to save all Imaris segmentation files in one directory, separate from the original .ims image files. The Imaris output creates a folder of files that contain the positional and volumetric information of segmented nuclei.
Download Collation_Imaris2ALAn.ipynb from the Bergstralh lab GitHub. This data will convert the Imaris statistics into .csv data files that can be read into ALAn.
Open Jupyter Notebook from Anaconda navigator. This will open Jupyter Notebook in a new browser window of your default browser. A navigation menu of the files on your computer is shown.
Navigate to the location of the “Collation_Imaris2ALAn.ipynb” file on your computer and open this Notebook (Figure 2B).
Run the code in Jupyter Notebook cell 2, which imports the necessary Python packages.
Define the input directory path, where the Imaris Statistics files are located, on Jupyter Notebook cell 3, line 6.
Define the output directory name on Jupyter Notebook cell 3, line 11, and the directory location of this directory on Jupyter Notebook cell 3, line 14. This will be the location where .csv files containing the necessary nuclei positions will be saved.
Run through all code in the “Collation_Imaris2ALAn” file.
Required data if you segment nuclei using a method not using Imaris as described in Section 1
Segment nuclear positions and extract information about the volume and centroid of nuclei in the image file.
Generate a .csv file with four columns. Column 1: X coordinate of nucleus center; Column 2: Y coordinate of nucleus center; Column 3: Z coordinate of nucleus center; Column 4: Volume of Nucleus. Rows correspond to each segmented nucleus.
Add heading titles to row 1. A1 = Nucleus Position X; B1 = Nucleus Position Y; C1 = Nucleus Position Z; D1 = Nucleus Volume.
Scale XY confocal slices to 512 × 512 pixels.
This can be done in Imaris in the Edit menu by selecting Resample 3D and resizing the X and Y values as 512. Make sure to select Fixed Ratio X/Y, not Fixed Ratio X/Y/Z.
Determine the spatial bounds of the image.
Open the original image file using FIJI.
Select Show Info from the Image menu.
Identify the X, Y, and Z spatial bounds of the image file in the Show Info panel values ExtMax0 through ExtMin2. ExtMax0 =X max; ExtMax1 = Y max; ExtMax2 = Z max; ExtMin0 = X min; ExtMin1 = Y min; ExtMin2 = Z min.
Open the .csv file containing the segmented nuclei positions using Microsoft Excel.
Name column E “Positions.”
Paste values ExtMax0 through ExtMin2 into Excel notebook cells E2–E7.
Save confocal stacks as .tiff files in the same folder as the corresponding .csv files containing the nuclei segmentation information. This can be done by opening proprietary file images in FIJI and Selecting Save as > Tiff in the File menu.
Ensure that the corresponding .csv files containing the nuclear and layer positional information and the .tiff files containing the confocal stack images have the same name. ALAn will read both files and perform an analysis of these partner files.
Use ALAn to batch process and analyze data
Download “ALAn_v3.ipynb” from the Bergstralh lab GitHub. This is the main data analysis code.
Open Jupyter Notebook from Anaconda navigator. This will open Jupyter Notebook in a new browser window of your default browser. A navigation menu of the files on your computer is shown.
Navigate to the location of the “ALAn_v3.ipynb” file on your computer and open this Notebook (Figure 2C).
Run the code in Jupyter Notebook cell 2, which imports the necessary Python packages.
Change the code on Jupyter Notebook cell 3, line 7 to the path where your input partner .csv and .tiff files are located.
Run the code in Jupyter Notebook cell 3. The data will be imported into two lists. All of the information in the .csv files will be converted into a list of pandas DataFrames (list_of_dfs), and all .tiffs will be read in as a list of 4D numpy arrays (list_of_unshuffled_images). Any .tiff or .csv file that does not have a partner file with the same name will not be added to these lists. A third list (list_of_names) will be generated to keep track of which image name is stored in each of the elements.
Check that the file names, number of input images, and dimensions of the three lists are the same, as shown in the output text below the Jupyter Notebook cell.
Run the code in Jupyter Notebook cell 4. This will load all of the functions of ALAn. Note: A description of each function and a description of its inputs and outputs are in the README.md file on the Bergstralh-Lab/ALAn GitHub page.
Change the input parameters of the batch_process function on code line Jupyter Notebook cell 5, line 2.
First, define the channel that represents the image actin signal in the .tiff file (this can be determined by opening the .tiff in FIJI) (0 = first channel, 1 = second channel, etc.).
Second, change the name that you would like to save your output .csv file as.
Run the code in Jupyter Notebook cell 5. This will process all of the input images as a batch. A timer will output the time taken to run this function.
Open the output .csv file, which has the name as defined on Jupyter Notebook cell 5, line 4 as the last parameter of the batch_process function. The directory will be the same as your input files. Check that a valid output has been generated. The basic outputs of ALAn are:
Column A: Element number used by ALAn for each input.
Column B: Root name of input .tiff and .csv files.
Column C: Layer determination; ALAn makes a categorization of the layer into the following categories: Disorganized, Immature, Intermediate A, Intermediate B, and Mature. For a full explanation of these categories see Dawney et al. [1] and Cammarota et al. [2].
Column D: Number of cells identified in the basal tissue layer.
Column E: Number of cells identified above the basal tissue layer.
Column F: Total number of cells (sum of columns D and E).
Column G: Cell density of identified tissue layer per unit culture area.
Column H: Height of identified tissue layer.
Column I: Percentage of total cells above the layer.
Column J: Average area of cells in the plane of the tissue.
Column K: Average cell perimeter in the plane of the tissue.
Column L: Average cell circularity in the plane of the tissue.
Use of ALAn to make diagnostic/informative plots and visuals
For any dataset, the functions of ALAn can be used to generate diagnostic and data outputs and figures. Here, we provide a protocol for running the functions that provide the most informative figure outputs for the analysis of the data. An optional input of save_name = “string” allows you to save this plot as a .pdf. Figures should be generated in an embedded figure in the Jupyter Notebook (defined by code on Jupyter Notebook cell 2, line 16: “%matplotlib notebook”).
Determine the element number for the dataset of interest from the output .csv file of the batch_process function.
In a new Jupyter Notebook cell, define your dataset of interest by typing “I = n’ where n is the element number of your chosen dataset.
In any subsequent Jupyter Notebook cell, you can run the clear_debris function by typing “clear_debris(list_of_dfs[i], plot = True)” and run the Jupyter Notebook cell. This function provides data on which objects identified by the nuclear segmentation input are considered as real cells and used for analysis. A histogram showing the distribution of nuclear volumes in μm3 (X-axis) against the frequency of these volumes is output (Figure 3A). The objects discarded as debris are shown in blue, and the nuclei identified as valid are orange. A pandas DataFrame containing the nuclei identified as true datapoints is output (df_cleared) and printed below the figure.
Figure 3. Diagnostic and informative plots generated by Automated Layer Analysis (ALAn). A. The clear_debris function outputs a histogram showing the frequency of segmented objects with respect to their volume that was input into ALAn. The orange bars represent nuclei identified as real and the blue bars represent debris that is removed from the analysis. A dotted line indicates the threshold volume above which a nucleus is considered real. B. The nuclear_centroid_actin_overlay function outputs an image that displays the centroid position of the segmented nuclei overlaid onto the .tiff image of the actin signal projected on the XY plane of the tissue. C. The nuclei_distribution function outputs a plot showing both the projected signal of actin (red) and the nuclear distribution (blue), fit with a Gaussian, with respect to the depth of the tissue. The tissue layer top, bottom, and peak position of the actin signal are shown by dotted lines. D. The find_shoulders function outputs a plot of the actin signal (red) and the first derivative of this plot (blue) with respect to the tissue depth. The tissue layer top, bottom, and peak position of the actin signal are shown by dotted lines. E. The terrain_map function outputs a map of the position of the centroids of nuclei projected onto the XY plane of the tissue, displayed as dots. The size of the dots shows relative volumes of nuclei. The color of the dots represents the relative positions of the nuclei with respect to the depth of the tissue. The color scale goes from light orange (most basal) to purple (most apical). F. The sub_classify function outputs a map representing the XY field of view of the input 512 × 512 pixel .tiffs broken into 100 × 100 pixel sections, color-coded by the layer category as determined by the original version of ALAn. “lawngreen” = Immature, “forestgreen” = Intermediate A, “darkturquoise” = Intermediate B, “steelblue” = Mature, “orange” = Disorganized.
In any subsequent Jupyter Notebook cell, you can run the nuclear_centroid_actin_overlay function by typing “nuclear_centroid_actin_overlay(list_of_dfs[i], list_of_unshuffled_images[i]).” Use this function to test the input nuclear segmentation and clear_debris functions to ensure that the nuclei used by ALAn are reasonable. A figure is generated in the Notebook showing the nuclear centroid positions in the tissue plane are overlayed with the actin channel to view the position of nuclei within each cell (Figure 3B). Black dots indicate real nuclei and red dots indicate false nuclei as defined by clear_debris.
If you are not happy with the cutoff volume of real nuclei, you can change the value that is used to define what a real nucleus is in the clear_debris function. This is currently set to 1.5 standard deviations below the average nuclear volume (defined on Jupyter Notebook cell 4, line 97).
In any subsequent Jupyter Notebook cell, you can run the nuclei_distribution function by typing “nuclei_distribution(list_of_dfs[i], list_of_unshuffled_images[i], plot = True).” This function determines if the layer is organized into a monolayer or not and returns a classification as Organized or Disorganized. A plot showing the projected number of nuclei and actin intensity (double Y-axis) across the depth of the layer, Z position in µm (X-axis) is generated in the Notebook (Figure 3C). This is a useful figure to interrogate the organization of cells across the depth of the tissue.
In any subsequent Jupyter Notebook cell, you can run the find_shoulders function (Figure 1D) by typing “find_shoulders(list_of_dfs[i], list_of_unshuffled_images[i], plot = True).” This function determines whether the plot of actin intensity across the depth of the tissue has a shoulder. The output is a plot of actin intensity and the first derivative of the actin profile (double Y-axis) against the depth of tissue, Z position (µm) (X-axis). These plots are used to identify the correct subcategory of organized in terms of the developmental series of epithelialization. Immature layers are defined by two rules that do not pertain to this function; all other organized layers are identified by this function. Intermediate A layers have a one-peaked actin intensity derivative. Intermediate B and Mature layers have a two-peaked actin intensity derivative. By taking the ratio between the height of the apical peak to the lateral peak, we identify the difference between the two layer types. A ratio of less than one is classified as an Intermediate B layer, while a ratio of greater than one is classified as a Mature layer.
In any subsequent Jupyter Notebook cell, you can run the terrain_map function (Figure 3E) by typing “terrain_map(list_of_dfs[i], list_of_unshuffled_images[i]).” This function plots all of the nuclei in a confocal stack on the XY plane, positioned by their XY position, sized based on nuclear size, and colored based on the z-position of the nucleus (Figure 3E). This allows for the visualization of the position of extra layer cells or disorganized regions within the field of view.
In any subsequent Jupyter Notebook cell, you can run the sub_classify function (Figure 3F) by typing “sub_classify(list_of_dfs[i], list_of_unshuffled_images[i], plot = True).” This function breaks the full XY field of view into the smallest classifiable sections (100 px × 100 px) and outputs an ALAn classification of each of the subsections all_sections_analyzed list. This function outputs a figure showing the full field of view divided into a grid of 25 squares colored by layer type (Figure 3F).
In any subsequent Jupyter Notebook cell, you can run the xy_segmentation function by typing “xy_segmentation(list_of_dfs[i], list_of_unshuffled_images[i], plot = True).” This only works for Organized layer types. This function segments the basal actin of the underlying organized cell layer in the plane of the tissue to output shape parameters of cells in the plane of the tissue. This function uses region_props from the scikit-image package to extract shape parameters. The function will output four figures showing the stages of segmentation as compared to the projected actin signal. This output is useful to check that the actin signal has been correctly segmented for in-plane cell shape analysis. This function will output images for Disorganized layers if prompted, though results will be uninterpretable, so avoid running this function on a Disorganized layer.
Validation of protocol
This work was developed to investigate the effects of cell plating density and time and characterize the architectural development of MDCK monolayers in publications [1] and [2]. We developed an unbiased and thorough imaging strategy to image a comprehensive map of epithelial architecture, imaging the same 16 representative 300 × 300 μm regions from the 1 mm2 wells. We developed ALAn using a dataset of over 19,000 cells across 351 imaged regions from four repeats of three plating densities and two repeats of the 16 regions. We further validated our strategy and broad application of the tool to analyze epithelial architecture using Caco-2 and MCF-7 cells as shown in Dawney et al.[2], Figure S4.
General notes and troubleshooting
General notes
In principle, this tool can be applied to cultured layers imaged live. ALAn requires homogenous staining of actin across the layer. We have attempted to apply ALAn to MDCK layers imaged live using Hoechst or H2B- mCherry to segment nuclei and SiR-actin and LifeAct-GFP to visualize actin and had limited success. We found SiR-actin and LifeAct-GFP signals to be unpredictable in our hands; thus, cell borders were not always detectable in our samples, particularly at lower densities. The SiR-actin profile generally reflects the layer architecture profiles distinguished from phalloidin staining but the background signal above the layer changes the projected actin profile. We anticipate that if alternative dyes are used, particularly those used for live imaging, the rules of layer category assignment will need to be modified and optimized. Due to our difficulties in obtaining high-quality movie data, we have not extended the ALAn code such that it can automatically analyze 4D movies. However, high quality frames from 4D movies can be readily input into ALAn to analyze architecture changes across time.
An example microscopy dataset of wild-type MDCK cells is available for download: http://tinyurl.com/4kv7jfav.
Acknowledgments
We acknowledge the authors of Dawney et al. [1] and Cammarota et al. [2] whose work led to the development of this protocol. We thank the lab of Rene Marc Mege for requesting a detailed version of this protocol. We are grateful to past and present members of the Finegan-Bergstralh lab for their comments. This work was supported by a National Science Foundation CAREER award (PI: Bergstralh) and National Institutes of Health Grant R01GM125839 (PI: Bergstralh). We acknowledge the microscopy, computing, and software resources at the University of Missouri Advanced Light Microscopy Core facility, which were used to develop and optimize this protocol.
Competing interests
The authors declare no competing interests.
References
Dawney, N. S., Cammarota, C., Jia, Q., Shipley, A., Glichowski, J. A., Vasandani, M., Finegan, T. M. and Bergstralh, D. T. (2023). A novel tool for the unbiased characterization of epithelial monolayer development in culture. Mol. Biol. Cell 34(4): ee22–04–0121. https://doi.org/10.1091/mbc.e22-04-0121
Cammarota, C., Dawney, N. S., Bellomio, P. M., Jüng, M., Fletcher, A. G., Finegan, T. M. and Bergstralh, D. T. (2023). The Mechanical Influence of Densification on Initial Epithelial Architecture. bioRxiv. 2023.2005.2007.539758. https://doi.org/10.1101/2023.05.07.539758
Etournay, R., Merkel, M., Popović, M., Brandl, H., Dye, N. A., Aigouy, B., Salbreux, G., Eaton, S. and Jülicher, F. (2016). TissueMiner: A multiscale analysis toolkit to quantify how cellular processes create tissue dynamics. eLife 5: e14334. https://doi.org/10.7554/elife.14334
Farrell, D. L., Weitz, O., Magnasco, M. O. and Zallen, J. A. (2017). SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics. Development 144(9): 1725–1734. https://doi.org/10.1242/dev.146837
Guirao, B. and Bellaïche, Y. (2017). Biomechanics of cell rearrangements in Drosophila. Curr. Opin. Cell Biol. 48: 113–124. https://doi.org/10.1016/j.ceb.2017.06.004
Vicente-Munuera, P., Gómez-Gálvez, P., Tetley, R. J., Forja, C., Tagua, A., Letrán, M., Tozluoglu, M., Mao, Y. and Escudero, L. M. (2017). EpiGraph: an open-source platform to quantify epithelial organization. bioRxiv. e1101/217521. https://doi.org/10.1101/217521
Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T. and Eliceiri, K. W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinf. 18(1): e1186/s12859–017–1934–z. https://doi.org/10.1186/s12859-017-1934-z
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): 676–682. https://doi.org/10.1038/nmeth.2019
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4,972 | https://bio-protocol.org/en/bpdetail?id=4972&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells
AE Aaron Rodriguez Ehrenfried
SZ Stefan Zens
LS Laura K. Steffens
HK Hannes Kehm
AP Alina Paul
CL Claudia Lauenstein
MV Michael Volkmar
IP Isabel Poschke
ZM Zibo Meng
RO Rienk Offringa
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4972 Views: 3780
Reviewed by: Mary Luz UribeAndrea GramaticaDavide PallucciMarco Di Gioia
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Original Research Article:
The authors used this protocol in Science Translational Medicine Nov 2023
Abstract
The advent of single-cell RNA sequencing (scRNAseq) has enabled in-depth gene expression analysis of several thousand cells isolated from tissues. We recently reported the application of scRNAseq toward the dissection of the tumor-infiltrating T-cell repertoire in human pancreatic cancer samples. In this study, we demonstrated that combined whole transcriptome and T-cell receptor (TCR) sequencing provides an effective way to identify tumor-reactive TCR clonotypes on the basis of gene expression signatures. An important aspect in this respect was the experimental validation of TCR-mediated anti-tumor reactivity by means of an in vitro functional assay, which is the subject of the present protocol. This assay involves the transient transfection of mRNA gene constructs encoding TCRα/β pairs into a well-defined human T-cell line, followed by co-cultivation with the tumor cells of interest and detection of T-cell activation by flow cytometry. Due to the high transfectability and the low background reactivity of the mock-transfected T-cell line to a wide variety of tumor cells, this assay offers a highly robust and versatile platform for the functional screening of large numbers of TCR clonotypes as identified in scRNAseq data sets. Whereas the assay was initially developed to test TCRs of human origin, it was more recently also applied successfully for the screening of TCRs of murine origin.
Key features
• Efficient functional screening of—and discrimination between—TCRs isolated from tumor-reactive vs. bystander T-cell clones.
• Applicable to TCRs from CD8+ and CD4+ tumor-infiltrating T-cells originating from patient-derived tumor samples and syngeneic mouse tumor models.
• Rapid flow cytometric detection of T-cell activation by means of TNFα and CD107a expression after a 5 h T-cell/tumor cell co-cultivation.
Keywords: Tumor-infiltrating lymphocytes Anti-tumor reactivity T-cell receptor mRNA transfection T-cell activation
Graphical overview
Workflow for functional screening of T-cell receptors (TCRs) as transiently expressed in human T-cells by means of mRNA transfection followed by co-culture with autologous tumor cells and analysis of T-cell reactivity by means of flow cytometric detection of TNFα and CD107a (figure created with BioRender.com).
Background
Crucial to the further improvement of T-cell cancer therapy is detailed information on the anti-tumor reactivity and antigen-specificity of the tumor-infiltrating lymphocyte (TIL) repertoire in large numbers of human tumor samples from different cancer types. In recent years, single-cell RNA sequencing (scRNAseq) technology platforms have emerged to enable the identification of paired T-cell receptor (TCR)α/β sequences in the context of the complete transcriptome for thousands of tumor-infiltrating T-cells. Whereas initial studies primarily focused on discriminating between tumor-reactive and bystander TCR clonotypes on the basis of distinct transcriptomic gene signatures [1], more recent studies including our own encompassed experimental validation of the predicted tumor reactivity by means of molecular cloning of selected TCRα/β pairs and in vitro functional assays with TCR-transfected T-cells [2–7]. In the majority of cases, these screenings were based on the presumption that the immunodominant T-cell antigens in tumors are neoepitopes encoded by somatic mutations in the tumor genome, and therefore focused on TCR screening against libraries of potential neoepitopes. However, it is still an open question whether this class of epitopes truly represents the immunodominant antigens targeted by the natural anti-tumor T-cell response in human cancers.
In view of the latter, based on the low mutational burden of human pancreatic ductal adenocarcinoma (PDAC) [8,9] and the notion that only a small fraction of nonsynonymous mutations is expected to encode immunogenic T-cell epitopes [10–13], we chose to perform an unbiased functional screening of tumor-derived TCRs against autologous tumor cells instead of mutanome-based candidate neoepitopes [7,14]. Evidently, this required a highly robust assay with a favorable signal-to-background ratio. Whereas Jurkat T-cells are commonly used in this respect, the readout for TCR-mediated activation is limited to interleukin-2 production and reporter gene construct expression. Instead, our TCR screening assay is based on a human T-cell line, subsequently referred to as T222, that originated from an ex vivo–expanded culture of tumor-infiltrating T-cells isolated from a human PDAC sample (PDA222). In the context of prior studies, we noticed that the TCR repertoire of this T-cell culture had drastically changed during expansion, leading to the loss of all dominant TCR clonotypes as detected in the tumor sample [15]. Upon further in vitro cultivation according to a well-established rapid expansion protocol [16], we found that this T-cell line grew indefinitely and consistently. Moreover, T222 lacked background reactivity as measured on the basis of multiple commonly applied T-cell activation parameters when co-cultivated with various human tumor cells, including multiple PDAC, colorectal cancer, and melanoma cell lines and cultures, rendering it a potentially suitable tool for the functional screening of TCRs as identified in scRNAseq data sets of tumor-infiltrating T-cell isolates. With respect to TCR gene transduction, we chose transient mRNA transfection, based on the superior results obtained with this technology by colleagues in the field [17]. We selected TNFα and CD107a as markers for T-cell activation, not only because these reflect key aspects of the effector T-cell response (cytokine production and cytolytic activity), but also because both markers can be optimally measured in the context of a 5 h assay. By means of mRNA-transfected T222 cells, we successfully screened > 150 TCRs derived from nine human PDAC samples for reactivity against autologous tumor cells in 5 h co-cultivation assays [7]. In subsequent studies, our assay was also used successfully for the screening of TCRs derived from tumor-reactive CD4+ T-cells and of > 100 TCRs derived from TIL scRNAseq data sets of murine PDAC tumors.
Materials and reagents
Biological materials
T222 cell line as generated by in vitro expansion of tumor-infiltrating lymphocytes of human PDAC sample PDA222 [7]
Tumor cell lines derived from primary tumor samples of PDAC patients [7]
Murine tumor cell lines PDA30364 and PDA30364-OVA [18]
Reagents
T7 mScript standard mRNA production system (Cellscript, catalog number: C-MSC100625)
RNA 6000 Nano Kit for 2100 Bioanalyzer Systems (Agilent, catalog number: 5067-1511)
pcDNATM 3.1(+) (Invitrogen, catalog number: V79020)
Anti-human HLA-A, B, C antibody APC, W6/32 (BioLegend, catalog number: 311410)
Anti-human HLA-DR Antibody PE, LN3 (BioLegend, catalog number: 327008)
X-VIVO 15 (Lonza, catalog number: BE02-060Q)
Human serum type AB (Sigma-Aldrich, catalog number: H4522)
Gentamycin (Life Technologies, catalog number: 15750045)
Amphotericin B (Life Technologies, catalog number: 15290-026)
Human Interleukin-2 Proleukin® S (Clinigen Healthcare, catalog number: 17152.00.00)
Purified anti-human CD3 (Invitrogen, catalog number: 16-0037-85)
Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)
Human albumin 20% (CSL Behring, catalog number: PZN: 1468366)
OptiMEM serum-reduced media (Thermo Fisher, catalog number: 31985062)
Advanced DMEM/F12 (Gibco, catalog number: 12634010)
L-Glutamine, 200 mM (Gibco, catalog number: 25030-024)
Penicillin-Streptomycin, 10,000 IU/mL (Gibco, catalog number: 15140-122)
B-27 supplement, 50× (Gibco, catalog number: 17504044)
HEPES, 1 M, pH 7.0–7.6 (Sigma-Aldrich, catalog number: H0887)
Heparin sodium salt, grade I-A (Sigma-Aldrich, catalog number: H3149)
Glucose solution, 200 g/L (Gibco, catalog number: A24940-01)
RPMI 1640 (Gibco, catalog number: 52400-025)
Fetal bovine serum (FBS) (Biowest, catalog number: S181B-500)
DMEM, high glucose (Gibco, catalog number: 41965-039)
Sodium pyruvate, 100 mM (Gibco, catalog number: 11360039)
Recombinant human/murine interferon gamma (IFNγ) (Immunotools, catalog number: 11343536/12343536)
MEKi, GDC-0623, mitogen-activated protein kinase inhibitor (Active Biochem, catalog number: A-1181)
Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418)
EDTA, UltraPure 0.5 M, pH 8.0 (Invitrogen, catalog number: 15575020)
Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139)
Ionomycin calcium salt (Sigma-Aldrich, catalog number: I0634)
Anti-human CD107a Antibody PE/APC, H4A3 (BioLegend, catalog number: 328608/328620)
GolgiStop, Protein Transport Inhibitor (BD Biosciences, catalog number: 554724)
GolgiPlug, Protein Transport Inhibitor (BD Biosciences, catalog number: 555029)
LIVE/DEAD Fixable Aqua Dead Cell Stain kit (DCM) (Life Technologies, catalog number: L34957)
Anti-mouse CD16/32, 93 (BioLegend, catalog number: 101320)
Anti-mouse CD16.2, 9E9 (BioLegend, catalog number: 149502)
Anti-human CD3 antibody Brilliant Violet 711, OKT3 (BioLegend, catalog number: 317328)
Anti-human CD4 antibody APC/Fire 750, SK3 (BioLegend, catalog number: 344638)
Anti-human CD8a antibody Alexa Fluor 700, HIT8a (BioLegend, catalog number: 300920)
Anti-mouse TCR beta chain antibody Brilliant Violet 421, H57-597 (BioLegend, catalog number: 109230)
Anti-mouse CD8a antibody PerCP, 53-6.7 (BioLegend, catalog number: 100732)
Anti-mouse CD8b antibody PE, YTS156.7.7 (BioLegend, catalog number: 126608)
Anti-mouse CD4 antibody PE, GK1.5 (BioLegend, catalog number: 100408)
Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences, catalog number: 554714)
Perm/Wash buffer, included in Fixation/Permeabilization kit (BD Biosciences, catalog number: 51-2091KZ)
Anti-human TNF alpha antibody Alexa Fluor 488, MAb11 (BioLegend, catalog number: 502915)
Solutions
T-cell expansion medium (see Recipes)
Freezing medium (see Recipes)
T-cell medium (see Recipes)
Tumor cell medium I: human (see Recipes)
Tumor cell medium II: mouse (see Recipes)
FACS buffer (see Recipes)
Recipes
T-cell expansion medium
X-VIVO 15 supplemented with 2% human serum type AB, 100 IU/mL penicillin-streptomycin, 20 µg/mL gentamycin, 2.5 µg/mL amphotericin B, and 30 ng/mL purified anti-human CD3
Freezing medium
FBS supplemented with 10% DMSO
T-cell medium
X-VIVO 15 supplemented with 2% human albumin
Tumor cell medium I: human
DMEM/F12+ supplemented with 2 mM L-glutamine, 100 IU/mL penicillin-streptomycin, 2% B27 supplement (50×), 5 mM HEPES, 12 mg/mL heparin, and 6 g/L glucose
Tumor cell medium II: mouse
DMEM supplemented with 10% FBS, 100 IU/mL penicillin-streptomycin, and 1 mM sodium pyruvate
FACS buffer
PBS supplemented with 0.5% BSA and 5 mM EDTA
Laboratory supplies
T25 tissue culture flask (TPP, catalog number: TPP90026)
Cuvette, 0.4 cm gap Gene Pulser/MicroPulser Electroporation (Bio-Rad, catalog number: 1652088)
24-well plate, flat bottom, TC-treated (VWR, catalog number: 734-2325)
96-well U-bottom plate, round bottom, TC-treated (Avantor, catalog number: 734-2328)
96-well plate, flat bottom, polystyrene (TPP, catalog number: TPP92096)
96-well V-bottom plate, conical bottom, non treated, no lid (Costar, catalog number: 3897)
Pasteur pipette, glass 230 mm, no filter (WU Mainz, catalog number: 200763)
15 mL tube conical bottom (Greiner, catalog number: 188271-N)
50 mL tube conical bottom, polypropylene (Falcon, catalog number: 352070)
Equipment
2100 Bioanalyzer (Agilent)
Incubator Heracell 240i (Thermo Scientific, catalog number: 51032875)
Centrifuge 5810R refrigerated (Eppendorf, catalog number: EPP-022628187)
Electroporator, Gene Pulser Xcell Systems with ShockPod cuvette chamber (Bio-Rad, catalog number: 1652660)
LSRFortessaTM cell analyzer (BD Biosciences)
Software
FlowJo v10.9.0 (FlowJo LLC, Ashland, OR, USA)
Bio Render (https://www.biorender.com/)
Procedure
TCR cloning strategy and production of in vitro–transcribed mRNA for transfection
T-cell receptor V(D)J α- and β-chain nucleotide sequences from TCR clonotypes of interest as identified in scRNAseq data sets are synthesized as DNA oligomers. Seamless Golden Gate assembly is used for their insertion into the bicistronic TCR expression cassette pTRAC, as depicted in Figure 1. The template for synthesis of in vitro–transcribed mRNA is generated by linearization of the expression vector at the NotI site located downstream of the expression cassette. The T7 mScript Standard mRNA Production System is used for the generation of capped and poly(A)-tailed mRNA as outlined in the protocol provided by the manufacturer. mRNA quality is routinely checked for showing a single peak on the 2100 Bioanalyzer system using Agilent RNA 6000 Nano chips.
Figure 1. pcDNA3.1(+) vector with T7 promotor enabling in vitro transcription of mRNA encoding both a TCRβ and a TCRα chain. The variable VDJβ and VJα sequences are joined in-frame with the mouse TCRβ-constant (mTRBC) and TCRα-constant (mTRAC) domains, respectively, resulting in a bicistronic open reading frame in which the TCRβ and TCRα coding sequences are separated by a self-cleaving P2A site, as described in detail by Kropp et al. [19]. mTRBC and mTRAC sequences harbor the previously described T48C and S57C mutations, respectively, resulting in an additional interchain disulfide bond [20,21]. The latter mutations in combination with the use of murine constant regions reduce mispairing of the transfected T-cell receptor (TCR) with endogenously expressed TCR chains in human T-cells. Furthermore, this allows for flow cytometric detection of cell surface expression of the gene transduced TCR by means of a monoclonal antibody against the mouse TCRβ (mTCRβ) constant domain. Downstream of this open reading frame is a 3’UTR consisting of two tandem copies of the human β-globin gene that improve stability of the resulting transcript in cells [22]. The expression cassette is embedded in the pcDNA3.1(+) vector, allowing in vitro transcription through the T7 promoter sequence.
Testing of CD8+ TCRs in mRNA-transfected human T-cells for anti-tumor reactivity
The success of our assay for the functional screening of multiple TCRs stands or falls with the choice of T-cells and the preparation of the tumor cells, as discussed in detail in this paragraph. As mentioned in the Background section, we routinely use human T-cell line T222 for functional TCR screening experiments. This T-cell line was derived from a primary TIL culture of a human PDAC sample. Upon in vitro cultivation according to a well-established rapid expansion protocol [16], we found that this T-cell line grew indefinitely and consistently. T222 cells efficiently express the majority of TCRs upon mRNA transfection in approximately 90% of the cells (Figure 2), with the exception of unmatched TCRα/β pairs and some rare cases. We found T222 to lack background reactivity when co-cultivated with the vast majority of human tumor cells tested (see example in Figure 2), rendering it highly suitable for the functional screening of TCRs as identified in scRNAseq data sets of human TIL samples. In the context of the present protocol description, this raises two questions that are important to address:
How unique is the T222 line and could T-cell lines with similar properties be readily generated in other labs?
What would be the best strategy to generate similar T-cell lines?
Figure 2. Functional T-cell receptor (TCR) screening in a 5 h T-cell/tumor cell co-cultivation assay. Representative example of flow cytometry data showing the outcome of functional testing of four different CD8+ T-cell-derived TCRs, isolated from the same human pancreatic ductal adenocarcinoma (PDAC) tumor sample, upon transfection into human CD8+ T222 cells [7]. Left to right: mock-transfected T-cells, followed by T-cells transfected with a non-tumor-reactive (NTR) TCR and three different tumor-reactive (TR) TCRs. The tumor cells originated from the same patient-derived tumor sample as the four tested TCRs and were pretreated with IFNγ. The dot plots depict the expression of the mouse TCRβ-constant domain and intracellular TNFα in CD3+CD8+ T-cells, thereby demonstrating that T-cell reactivity is mediated by the transfected TCRs.
In the course of our work, we have evaluated the suitability for TCR screening of additional expanded TIL cultures as obtained from other human tumor samples, including settings with TILs and tumor cells from the same patients, with variable outcome. Some of these T-cell lines displayed similar qualities as T222, illustrating the feasibility of generating suitable TIL-derived T-cell lines in other laboratories. Other TIL-derived T-cell lines were found to maintain strong reactivity against the autologous tumor, which we found to be associated with persistence of tumor-reactive TCR clonotypes in spite of repeated expansion ([15] and unpublished data). Furthermore, some of the TIL-derived T-cell lines exhibited broader anti-tumor reactivity, presumably due to allo-reactivity. We postulate that the desirable properties of T222 and several other TIL-derived T-cell lines may be related to the greater clonality of these T-cell lines as compared to peripheral blood mononuclear cells (PBMC)-derived T-cell cultures [15], thereby reducing the likelihood that a fraction of the T-cells express allo/cross-reactive TCRs resulting in background reactivity in functional T-cell assays.
In view of the above, the best strategy to generate similar T-cell lines is to perform repeated rapid expansion [16] of approximately 10–20 available TIL cultures, freeze the resulting T-cell batches into aliquots, and evaluate the resulting T-cell lines for the above-mentioned desirable features.
The human PDAC tumor lines used in our studies [7] were all derived from patient-derived xenograft (PDX) models in NSG mice. More recently, we also successfully made use of tumor cell lines that were initiated as organoid cultures. To increase HLA class I–restricted antigen presentation on human tumor cell lines, the tumor cells are routinely pretreated with 333 IU/mL human IFNγ for 48 h. As shown in Figure 3, this significantly increases surface HLA expression and thereby the sensitivity of the assay, especially for the weakly reactive TCRs.
Figure 3. Importance of IFNγ-pretreatment of tumor cells for efficient detection of T-cell receptor (TCR)-mediated T-cell activation. (A) Impact of IFNγ-pretreatment on the cell surface HLA class I and II expression, as detected by pan-HLA class I and class II monoclonal antibodies (APC-conjugated W6/32 and PE-conjugated LN3, respectively) for a representative human pancreatic ductal adenocarcinoma (PDAC) tumor cell line. (B) Flow cytometry data showing the outcome of functional testing in a 5 h co-cultivation experiment of three different CD8+ T-cell-derived TCRs, isolated from the same human PDAC tumor sample, upon transfection into human CD8+ T222 cells. Left to right: mock-transfected T-cells, followed by T-cells transfected with a non-tumor-reactive (NTR) TCR and two tumor reactive (TR) TCRs. The dot plots depict the expression of CD107a and TNFα in CD3+mTCRβ+ T-cells. The data illustrate that detection of TCR reactivity is strongly promoted by IFNγ-pretreatment of the tumor cells, especially for weaker TCRs as exemplified by TCR1. The same applies, even to greater extent, for the detection of reactivity by TCRs derived from CD4+ T-cell clones (see Procedure C).
Maintenance and quality control of T-cell line
Cultivate cells according to the rapid expansion protocol as described by Dudley et al. [16]. See Figure S1 and appending text for details.
Validate each batch prior to use in TCR screening experiments for CD8+/CD4+ ratio and TCR transfection efficiency.
T-cell preculture (Day 0 of assay timeline as shown in Figure 4)
Figure 4. Assay timeline
Thaw T-cells 72 h prior to transfection. The preculture increases the viability after electroporation. See Note 1 and Figure S2 for estimation of required T-cell number, taking into account potential proliferation during the 72 h culture and cell loss due to electroporation.
Seed 3 × 106 T-cells (viable cells as counted with trypan blue staining, e.g., using Vi-CELL counter) per well in 1 mL of T-cell medium + 300 IU of IL-2 in a 24-well plate. The cells of all wells will be pooled before electroporation.
Culture T-cells for 72 h at 37 °C and 5 % CO2.
IFNγ pretreatment of tumor cells (Day 1)
Add 333 IU/mL of human IFNγ to tumor cell culture and incubate for 48 h. As shown in Figure 4, IFNγ pretreatment should be initiated 72 h before initiation of T-cell/tumor cell co-cultures. See Note 2 for IFNγ concentration.
mRNA-transfection of T-cells (Day 3)
T-cells are transfected in batches of 2.5 × 106–7 × 106 cells per TCR. The number of cells to be transfected depends on the number of conditions to be screened per TCR during the T-cell/tumor cell co-culture. For each condition, 2 × 105 transfected viable T-cells are needed on the day of co-culture. Take into account cell loss due to electroporation of the T-cell line concerned (Note 1). See Figure S2 for a calculation example. The following steps describe the transfection of 5 × 106 cells with 5 µg of TCR-encoding mRNA. This setting is based on extensive evaluation of different T-cell numbers and mRNA concentrations in pilot experiments. Adapt the amount of TCR-mRNA and transfection volume to the actual number of transfected cells. See Table 1 for orientation.
Table 1. Examples with respect to calculation of T-cell transfection volume
Parameters Example 1 Example 2 Example 3
T-cells per TCR transfection 2.5 × 106 5 × 106 7 × 106
TCR mRNA 2.5 µg 5 µg 7 µg
T-cell transfection volume 125 µL 250 µL 350 µL
Pre-chill cuvettes and OptiMEM at 4 °C on day of electroporation.
Pre-heat T-cell medium to 37 °C.
Keep all preparations/solutions containing RNA on ice to avoid degradation.
Prepare RNA-OptiMEM mix (4 °C):
Use 5 µg (per 5 × 106 cells) of in vitro–transcribed capped and tailed mRNA per TCR. Fill up with OptiMEM to 25 µL to reach 1/10th of the total transfection volume.
Collect precultured T-cells from 24-well plate in 50 mL tube and wash twice with ice-cold OptiMEM at 350× g for 5 min at 4 °C.
Set concentration to 20 × 106 cells/mL in OptiMEM. Keep cells on ice.
Pipette 250 µL of T-cell suspension (5 × 106 cells) into pre-chilled cuvette. Add 25 µL of ice-cold RNA-OptiMEM mix containing 5 µg of TCR-mRNA. Mix by pipetting thoroughly, avoiding air bubbles. Place lid on cuvette to keep sterile.
Carefully wipe outside of cuvette dry with tissue; wet cuvettes may cause malfunction of electroporation device.
Place cuvette in Bio-Rad ShockPodTM cuvette chamber and electroporate cells with one square-wave pulse of 500 V for 5 ms.
After electroporation, immediately add 1 mL of pre-heated (37 °C) T-cell medium to the T-cells and transfer these with a Pasteur pipette from the cuvette to one well of a 24-well plate.
Repeat for each TCR-RNA and collect in separate wells.
Transfer plate with electroporated T-cells to incubator after no more than four electroporations. Cells should be transferred at latest 5 min after electroporation into incubator to avoid excessive loss of cell viability (Note 3).
Culture transfected T-cells for 20–24 h before initiating T-cell/tumor cell co-culture.
Tumor cell seeding for T-cell/tumor cell co-culture (Day 3)
See Figure 5 for an example of a 96-well plate layout.
Seed IFNγ-pretreated, adherent tumor cells into relevant wells of 96-well flat-bottom plate 24 h before co-culture. Aim for tumor cell cultures to be 90%–100% confluent on the day of co-culture for optimal assay sensitivity (see Figure S4A). See Notes 4 and 5 for setting up assays with non-adherent or freshly trypsinized tumor/target cells.
Figure 5. Example of co-culture plate layout. Columns 1–12 are typically used to test T-cells transfected with different T-cell receptors (TCRs). In this context, rows A–H are used to test reactivity of TCR-transfected T-cells under different conditions, e.g., with media only (negative control), in the presence of PMA/ionomycin (positive control), in the presence of one or more different tumor cell lines, and/or in the presence of immunomodulatory substances such as anti-PD-L1 antibodies [23]. It is advisable to keep the row with the PMA/ionomycin condition separated from the rows in which reactivity to tumor cells is tested in order to avoid accidental spillover of PMA/ionomycin-containing media. In case all rows on the plate are used, use rows A and B for PMA/ionomycin and media controls, respectively, as shown in figure. In case six or less rows are used, use row A for media control, rows B–F for testing conditions, and row H for PMA/ionomycin control. Figure created with BioRender.com.
Initiation of T-cell/tumor cell co-culture (Day 4)
Setup of different assay conditions as shown in Figure 5:
Carefully remove supernatant from the wells in which tumor cells were seeded on Day 3 without affecting adherence of the tumor cells.
Wash with 200 µL of PBS, carefully remove supernatant, and add 50 µL of T-cell medium to the tumor cell–containing wells.
Prepare PMA/ionomycin master mix for positive control cultures, consisting of T-cell medium with 400 ng/mL of PMA and 2 µg/mL of ionomycin calcium salt (ionomycin).
Add 50 µL of PMA/ionomycin master mix to each positive control well as indicated in Figure 5. Final concentration during co-culture is 100 ng/mL PMA and 500 ng/mL ionomycin.
Add 50 µL of T-cell medium to each negative control well as indicated in Figure 5.
Addition of TCR-transfected T-cells:
Adjust concentration of transfected T-cells to 2 × 106 cells/mL in T-cell medium. In many cases, this can be done in the 24-well plates in which the T-cells were cultured after transfection.
According to the layout shown in Figure 5, add 100 µL of T-cell suspension containing 2 × 105 T-cells into each well.
Addition of anti-CD107a and IL-2:
Prepare CD107a/IL-2 master mix, consisting of T-cell medium with 1:20 anti-CD107a-PE antibody (see Note 6) and 1200 IU/mL IL-2.
Add 50 µL of CD107a/IL-2-mix to all wells, thereby reaching a final volume of 200 µL per well. Final dilution during co-culture is 1:80 anti-CD107a-PE antibody and 300 IU/mL IL-2.
Spin down T-cells by centrifugation of the plate at 50× g for 1 min at room temperature (21 °C).
Incubate for 1 h at 37 °C and 5% CO2.
Addition of GolgiStop and GolgiPlug after the first hour of co-culture:
Prepare master mix consisting of T-cell medium with 1:100 GolgiStop and 1:100 GolgiPlug-solution.
Carefully add 20 µL of GolgiStop and GolgiPlug mix per well. Do not mix by pipetting as this will whirl up the cells and interfere with T-cell/target-cell interaction.
Incubate for another 4 h at 37 °C and 5% CO2.
Proceed immediately with the flow cytometry staining protocol.
Flow cytometry staining (Day 4)
Perform all centrifugation steps at 700× g for 2 min at 4 °C. In order to avoid photobleaching of the fluorophores, use the minimum light exposure required for safe working and avoid direct light on the samples.
Dead cell marker (DCM) staining:
Prepare DCM mix, consisting of 1:40 DCM in PBS (Note 7).
When the assay is performed according to the default protocol in a flat-bottom plate, resuspend T-cells and carefully transfer into a V-bottom plate to minimize cell loss during the staining procedure. If detached tumor cells come along, this is no problem. (See Note 8 for co-culture assays performed in U-bottom plate.)
Centrifuge cells down and flick plate to discard supernatant.
Add 200 µL of PBS.
Centrifuge and flick plate to discard supernatant.
Wash again with 200 µL of PBS by centrifuging and flicking.
Resuspend pellets in 100 µL of DCM mix per well.
Incubate at 4 °C protected from light for 15 min.
Add 100 µL of FACS buffer.
Centrifuge and flick plate to discard supernatant.
Add 200 µL of FACS buffer.
Extracellular staining (ECS):
Prepare ECS antibody mix. See Table 2 for example.
Centrifuge and flick plate to discard supernatant.
Resuspend pellets in 100 µL of ECS antibody mix.
Incubate at 4 °C protected from light for 25 min.
Add 100 µL of FACS buffer.
Centrifuge and flick plate to discard supernatant.
Add 200 µL of FACS buffer.
Table 2. Extracellular (ECS) antibody mix. Antibodies were titrated to identify ideal staining concentrations for our cell lines and flow cytometer setup.
V per sample Target Fluorophore Clone
2.5 µL hCD3 BV711 OKT3
2.5 µL hCD8a AF700 HIT8a
0.5 µL hCD4 APC/Cy7 SK3
2.5 µL mTCRβ BV421 H57-597
92 µL FACS buffer
100 µL Total volume
Intracellular staining (ICS):
Centrifuge and flick plate to discard supernatant.
Resuspend pellets in 100 µL of Cytofix/Cytoperm.
Incubate at 4 °C protected from light for 15 min.
Proceed with step 3e for staining on the same day or pause overnight as follows:
Pause point:
i. Add 100 µL of FACS buffer.
ii. Centrifuge and flick to discard supernatant.
iii. Add 200 µL of FACS buffer.
iv. Centrifuge and flick to discard supernatant.
v. Resuspend pellets in 200 µL of FACS buffer.
vi. Store at 4 °C protected from light overnight.
vii. Prepare Perm/Wash working solution (WS): 1:10 dilution with Milli-Q H2O.
viii. Centrifuge and flick plate to discard supernatant.
ix. Resuspend pellets in 100 µL of Perm/Wash WS.
x. Incubate at 4 °C protected from light for 15 min.
xi. Proceed with step 3e.
Add 100 µL of Perm/Wash WS.
Centrifuge and flick plate to discard supernatant.
Wash again with 200 µL of Perm/Wash WS.
Prepare ICS antibody mix according to your scientific question. See Table 3 for example.
Centrifuge and flick plate to discard supernatant.
Resuspend pellets in 100 µL of ICS antibody mix.
Incubate at 4 °C protected from light for 25 min.
Add 100 µL of Perm/Wash WS.
Centrifuge and flick plate to discard supernatant.
Wash again with 200 µL of Perm/Wash WS.
Centrifuge and flick plate to discard supernatant.
Resuspend pellets in 100–200 µL of FACS buffer.
Acquire data by flow cytometric analysis of samples.
Table 3. Intracellular (ICS) antibody mix. Antibodies were titrated to identify ideal staining concentrations for our cell lines and flow cytometer setup.
V per sample Target Fluorophore Clone
4 µL hTNFα AF488 Mab11
96 µL FACS buffer
100 µL Total volume
Functional testing of TCRs derived from CD8+ versus CD4+ T-cell clones
As shown in Figure 3A, many human tumor cells including PDAC cells can, upon pretreatment with IFNγ, be induced to express HLA class II at the cell surface. This enables the functional screening for anti-tumor reactivity of TCRs originating from CD4+ TILs, which is of particular interest for CD4+ TILs expressing exhausted or Treg-related gene signatures [3,24–27]. The experiment shown in Figure 6 demonstrates that our functional TCR screening assay can effectively identify tumor-reactive TCRs derived from both CD8+ and CD4+ T-cell clones, provided that the T-cells used express the co-receptor matching that of the T-cell clones from which the TCRs were isolated (see Note 9). In view of the latter, we have—by means of FACS sorting and subsequent expansion—generated T222 lines predominantly (> 90%) consisting of either CD8+ or CD4+ T-cells and include determination of the CD8+/CD4+ ratio in our routine quality control for generation of cell batches.
Figure 6. Importance of CD8/CD4 co-receptor expression for efficient detection of T-cell receptor (TCR)-mediated T-cell activation. Representative example of a 5 h co-cultivation experiment in which tumor-reactive (TR) and non-tumor-reactive (NTR) TCRs, derived from human CD8+ and CD4+ tumor-infiltrating T-cells of a single pancreatic ductal adenocarcinoma (PDAC) tumor sample, were tested for reactivity against autologous, IFNγ-pretreated tumor cells. The TCRs were transfected into either human CD8+ (top panel) or CD4+ (bottom panel) T222 cells. The dot plots depict the expression of CD107a and TNFα in CD3+mTCRβ+ T-cells, thereby revealing that, as expected, CD107a is the predominant activation marker of CD8+ T-cells and TNFα is that of CD4+ T-cells. Furthermore, these data show that detection of TCR reactivity is strongly promoted by using human T- cells expressing the co-receptor that matches the T-cell of origin.
Functional testing of TCRs originating from syngeneic mouse tumor models
In a study parallel to the screening of TIL-derived TCRs from scRNAseq data sets of human PDAC samples, we performed analogous analyses on the basis of a transplantable mouse PDAC tumor model [18] using the functional screening assay with the human T222 cells as described above (Kehm et al. [28]). Our protocol, as described under Procedure B, works equally efficiently for TCRs derived from mouse CD8+ T-cells (Figure 7) and CD4+ T-cells (not shown), provided that the following three modifications are applied:
Efficient antigen recognition by TCRs derived from mouse CD8+ and CD4+ T-cells when expressed in human T-cells requires co-transfection of the murine CD8+ and CD4+ co-receptor, respectively. In view of this, co-transfect human T-cells with mRNA-encoding mouse TCR (5 µg per 5 × 106 cells) and mouse CD8A and CD8B (2.5 µg each per 5 × 106 cells) or CD4 (5 µg per 5 × 106 cells), respectively.
For enhancement of MHC-restricted antigen presentation, pretreat the mouse tumor cells with 3,000 IU/mL mouse IFNγ for 48 h, taking into account the considerations under Note 2. In order to achieve sufficient induction of MHC Class II on mouse tumor cells for screening of CD4+ TCRs, pretreat the tumor cells with IFNγ in combination with MEK inhibitor (100 nM MEKi GDC-0623) as described in Baumann et al. [18].
Adapt the flow cytometry staining panel to include anti-mouse CD8a and CD8b or CD4. See Table 4 for our staining panel. In case the mouse tumor cells, e.g., B-cell tumors, express Fc receptors, perform mouse Fc receptor block [18] before extracellular staining.
Table 4. Extracellular (ECS) antibody mix for CD8+ mouse T-cell receptors (TCRs). Antibodies were titrated to identify ideal staining concentrations for our cell lines and flow cytometer setup. Anti-human CD107a-APC was used during co-culture to avoid spectral overlap with mCD8b-PE. We used anti-mouse CD4-PE (1:100) instead of mCD8b to validate co-receptor expression when screening mouse CD4+ T-cell-derived TCRs.
Volume per sample Target Fluorophore Clone
2.5 µL hCD3 BV711 OKT3
1 µL mCD8a PerCP 53-6.7
1 µL mCD8b PE YTS156.7.7
2.5 µL mTCRβ BV421 H57-597
93 µL FACS buffer
100 µL Total volume
Figure 7. Functional testing of mouse-derived T-cell receptors (TCRs) requires co-transduction of mouse co-receptors. Representative example of an experiment involving the functional testing of TCRs isolated from a transplantable mouse pancreatic ductal adenocarcinoma (PDAC) model expressing the chicken ovalbumin (OVA) neoantigen [18], approximately 50% of which were found directed against OVA [28]. In view of the proven reliability of our TCR-screening setup for human tumor–derived TCRs, we applied the same protocols for testing the anti-tumor reactivity of > 100 mouse PDAC tumor–derived TCRs. Shown in this figure is the reactivity of human CD8+ T222 cells transfected with a TCR derived from a tumor-infiltrating CD8+ T-cell clone, as compared to the OVA/SIINFEKL-specific TCR derived from the OT-1 TCR-transgenic mouse strain against IFNγ-pretreated OVA-expressing mouse PDAC tumor cells and the OVA-negative parental cells. The dot plots depict the expression of TNFα and mouse-CD8a+ in CD3+mTCRβ+ T-cells, thereby showing that both TCRs only mediate T-cell reactivity against the OVA-expressing tumor cells. Furthermore, the data demonstrate that reactivity by the strong OT-1-derived TCR is readily detected in the absence of co-transfection of the mouse CD8+ co-receptor, whereas detection of the reactivity of weaker TCRs is markedly facilitated by mouse-CD8 co-transfection. Notably, this does not significantly increase T-cell reactivity against the OVA-negative tumor cells.
Data analysis
Samples were acquired with LSRFortessaTM and analyzed with FlowJo software. See Figure S3 for our gating strategy.
Validation of protocol
This protocol was used in the following research articles:
Meng et al. [7]. Transcriptome-based identification of tumor-reactive and bystander CD8+ T-cell receptor clonotypes in human pancreatic cancer. Sci. Transl. Med. 15(722): eadh9562.
Sun et al. [23]. ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation. Cell Chem. Biol. 30(7): 739–752.e8.
Offringa et al. [29] Antigen reactive T-cell receptors, International patent WO 2022/200457 A1.
Kehm, H., Baumann, D., Meng, Z., Zens, S., Riemer, A., Volkmar, M., Poschke, I, Offringa, R. Dissection of the tumor-reactive and bystander T-cell repertoires in a murine model for pancreatic cancer (manuscript in preparation) [28].
Rodriguez Ehrenfried, A., Steffens, L.K., Meng, Z., Poschke, I., Volkmar, M., Offringa, R. Identification and functional analysis of tumor-reactive CD4+ effector and regulatory T-cell subsets in human pancreatic cancer. (manuscript in preparation) [27].
General notes and troubleshooting
General notes
For our T222 line, we have observed up to 2-fold increase in cell numbers during the 72 h preculture phase and a 25%–50% cell loss due to electroporation. For further TIL-derived T-cell lines, we have found other gain/loss ratios. In order to end up with sufficient numbers of viable TCR-transfected T-cells for the intended experiment, it is important to have a good idea of this gain/loss ratio and, based on this, to decide how many T-cells in total need to be transfected. See Figure S2 for an example of T-cell number development throughout the assay protocol.
Preparations of IFNγ may differ considerably in biological activity. In view of this, the concentration as defined in IU/mL should only be taken as a rough indication, and pilot experiments to test the optimal concentration of IFNγ for increasing HLA/MHC surface expression on tumor cells are recommended. In order to maintain consistency, dispense IFNγ stock solutions into aliquots and avoid repeated freezing/thawing. Caution: excessive concentrations of IFNγ can affect tumor cell viability.
Work as efficient as possible. Cells are particularly sensitive to temperature after transfection. A short processing time is crucial for cell viability.
Whereas our default assay setup involves pre-seeding of the tumor cells to allow these to adhere, the assay can also be successfully performed by seeding non-adherent tumor/target cells or freshly trypsinized cells, followed immediately by addition of the T-cells. In this case, collect the tumor/target cells on the day of co-culture (Day 4; see Figure 4). Wash them twice with T-cell medium by centrifuging at 350× g for 5 min at room temperature (21 °C) and discarding the supernatant. Resuspend in T-cell medium at required concentration to seed 50 µL of cell suspension per well. Use a U-bottom 96-well plate instead of a flat-bottom plate. Add tumor cells to T-cell/tumor cell co-culture according to the assay layout shown in Figure 5. In order to achieve optimal assay sensitivity, use an effector to target cell ratio ranging from 1:1 to 1:8; see Figure S4 for further guidance in this respect.
In order to evaluate whether tumor-reactive TCRs would be directed against specific T-cell epitopes, e.g., neoepitopes as predicted by tumor mutanome analysis, our assay setup can readily be adapted to the use of different target cells such as peptide-pulsed or minigene-transfected antigen-presenting cells [7].
We found that addition of anti-CD107a antibody during culture resulted in stronger, cumulative signals while not affecting cell viability. Notably, this procedure is in line with the majority of published CD107a staining protocols.
Reconstitute dead cell marker (DCM) in 500 µL of DMSO and store in 10 µL aliquots to avoid freeze/thaw cycles.
In case the assay is performed with non-adherent or freshly trypsinized tumor/target cells, do not transfer the cells in V-bottom plate but perform staining in U-bottom plate. This will avoid unnecessary cell loss during transfer.
We found that the fraction of TCRs isolated from TIL clones with gene signature-based predicted tumor-reactivity displaying reactivity against autologous tumor cells is lower for CD4+ clonotypes than for CD8+ clonotypes. This discrepancy is most likely related to key differences between the HLA class I and II antigen processing pathways [11]. Consequently, most of the HLA class II-restricted epitopes are only presented in the tumor microenvironment by professional antigen-presenting cells such as dendritic cells and tumor-associated macrophages, whereas only a small fraction is also directly presented by tumor cells.
Acknowledgments
This protocol was adapted from our previous work as described in Meng et al. [7].
Funding sources: K. H. Bauer foundation (RO, IP, and MV). The German ministry of education and research (BMBF; IP, project TIL-REP, 01ZY1403A). The Helmholtz-Institute for Translational Oncology, Mainz (HI-TRON; ARE and MV). The Chinese Scholarship Council (ZM). The Graduate school of the German Cancer Research Center (LS and HK). The Cooperation Research Program of the German Cancer Research Center with the Ministry of Science, Technology and Space and the German-Israeli Helmholtz International Research School/Cancer-TRAX Program (SZ). The Helmholtz Foundation, Immunology and Inflammation Program (CL, program TCR Gene Therapy of Pancreatic Cancer). The German Comprehensive Cancer Center (CL, program NEO-ATT). The Sino-German Laboratory of Personalized Medicine for Pancreatic Cancer (ZM and RO). The Joachim Herz Foundation (LS).
Competing interests
The authors declare no competing interests.
Ethical considerations
The PDAC samples and tumor-infiltrating lymphocytes thereof were provided by University Hospital Heidelberg via the European Pancreas Center on the basis of informed written consent. The study was approved by the local ethics committees (Nr. S-708/2019) and conducted in accordance with the declaration of Helsinki.
All animal procedures were approved by the governmental authorities (Regional Administrative Authority Karlsruhe, Germany) and adhered to the institutional laboratory animal research guidelines.
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Supplementary information
The following supporting information can be downloaded here:
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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4,973 | https://bio-protocol.org/en/bpdetail?id=4973&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Imaging Single-Cell Ca2+ Dynamics of Brainstem Neurons and Glia in Freely Behaving Mice
AB Amol M. Bhandare
ND Nicholas Dale
RH Robert T. R. Huckstepp
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4973 Views: 896
Reviewed by: Bo LiangYueqing Peng Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in eLIFE Oct 2022
Abstract
In vivo brain imaging, using a combination of genetically encoded Ca2+ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum—a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond.
Key features
• We developed a protocol that allows imaging from brainstem neurons and glia in freely behaving rodents.
• Our refined method of GRIN lenses implantation and cell sorting approach gives the highest number of cells with the least postoperative complications.
• The revised deep brainstem imaging method paves way to understand complex behaviours such as cardiorespiratory regulation, sleep, swallowing, and digestion.
• Our protocol can be implemented to image cerebellar structures to understand their role in key functions such as balance, movement, motor learning, and more.
Graphical overview
Keywords: Intravital microscopy Brainstem imaging Rodents Ca2+ imaging In vivo imaging
Background
In vivo Ca2+ imaging of neurons and glia in freely behaving rodents offers an exciting opportunity to understand brain structures with cellular resolution of their function, something that cannot be gained with any other current methodologies. Over the last decade, since the invention of the first intravital microscope [1], a tremendous advance has been made in the field with wireless cameras [2], dual colour imaging, large-field imaging [3], geometric transformation adaptive optics [4], imaging combined with optogenetics [5], improved fluorescent Ca2+ indicators [6], use of cre-dependent GCaMP reporter mice [7,8], and intersectional genetics to target subpopulations of cells of interest [9]. However, one of the major challenges remaining in the field is to record reliably from hard-to-access and potentially mobile brain regions such as the brainstem and cerebellum.
We have recently published our studies targeting neurons from the ventral brainstem surface called the retrotrapezoid nucleus (RTN) and medullary raphe to investigate their role in central chemosensitivity [10] and investigate the effect of acute and chronic seizures on the RTN neurons [11]. Our refined surgical protocol provides a way to record brainstem neurons and glia with cell analysis and selection/rejection criteria to identify the cells of interest. Although there are surgical protocols for imaging neurons and glia from the forebrain [12] and rostral brainstem [13], these protocols cannot be directly applied for brainstem imaging due to: i) gradient refractive index (GRIN) lens implantation/insertion through cerebellar and brainstem regions and associated risk of damage to basic/vital life functions and ii) additional complexity of motion artefact due to movement of brainstem tissue outside the cranial cavity. Our protocol specifically deals with these issues by creating a guide track for lens insertion and implantation and rejecting cells displaying artefactual activity correlated with body movements. Despite the improved ways to image brainstem neurons and glia in freely behaving rodents, we might be limited to targeting the rostral brainstem region. Imaging from the most caudal brainstem nuclei would require implantation of lenses through, and baseplate attachment over, the occipital bone (at the posterior end of interparietal bone), which is angled and densely innervated with muscles. Whilst our improved methodology increases the duration of lens implantation surgeries, it allows for the best surgical outcomes with the least postsurgical complications.
Overall, our improved surgical and cell analysis and selection protocol paves the way to image and record the activity of brainstem structures, to investigate their role in previously studied complex behaviours such as breathing [14,15], circulation [15,16], sleeping [17], digestion [18], and swallowing [19]. It could be extended to study the role of glial cells in numerous neurological disorders [20,21] and cerebellar structures in balance, movement, motor learning [22], and many other.
Materials and reagents
Biological materials
AAV-9:pGP-AAV-syn-GCaMP6s-WPRE.4.641 (Vigene Biosciences, SKU BS1-NOSAAV9: USA)
AAV-5: pZac2.1 gfaABC1D-cyto-GCaMP6f (Addgene, 52925, USA)
C57BL/6 mice (Charles River: UK)
Reagents
Isoflurane (Piramal Critical Care, catalog number: 2800025)
Atropine (Macarthys Laboratories Ltd., catalog number: PL01883/616R)
Meloxicam (Boehringer Ingelheim, catalog number: Vm 04491/5025)
Buprenorphine (Reckitt Benckiser, catalog number: Vm 15052/4081)
Superbond Universal Kit (Sun Medical Co., catalog number: K058E)
PVA glue (Hobbycraft Trading Ltd., catalog number: 5672751000) or silicon adhesive (WPI, catalog number: Kwik-Cast)
Eye ointment (Dr Gerhard Mann, catalog number: MEVIS05)
Lubricating jelly (Thornton & Ross Ltd., catalog number: 24242902)
Vetscrub (NewGenn Ltd., catalog number: CD003)
70% ethanol (VWR Chemicals, catalog number: UN1170)
Sodium chloride (NaCl) (VWR International Ltd., catalog number: 27788.297)
Distilled water
Solutions
Sterile 0.9% saline solution (see Recipes)
Recipes
Sterile 0.9% saline solution
Reagent Final concentration Quantity or Volume
NaCl 0.9% 9 g
Distilled water n/a Up to 1,000 mL
Dissolve 9 g of NaCl in 600 mL of distilled water and make up the final volume to 1 L. Autoclave and sterilise the solution for 15 min at 15 psi and 121 °C.
Laboratory supplies
Nitrile gloves (Starlab International), sterilised by autoclaving
Cotton buds (Dutscher, catalog number: 1504), sterilised by autoclaving
Tissue paper, sterilised by autoclaving
Surgical coat, sterilised by autoclaving
Overhead cap (Pal International Ltd.)
Mask (3M, FFP2)
Scalpel blades (Swann Morton Ltd., catalog number: BS2982)
Capillary tubes (Drummond Scientific Company, catalog number: 2-000-001)
Double-edged blade (Scientific Laboratory Supplies Ltd., catalog number: BH10)
Superglue (RS Components Ltd., catalog number: 473-455)
Blunt needles, 24 gauge (Becton Dickinson, catalog number: MIC0067)
Eppendorf 0.2 mL PCR tube cap (Eppendorf, catalog number: 0030124332)
Silicone tubing with syringe for viral injection (Saint-Gobain Life Sciences, catalog number: E-3603)
Aldasorber (Aston Pharma, catalog number: AST501)
Aluminium foil
Cling film
Equipment
nVista epifluorescent imaging system/camera (Inscopix, model: nVista)
Baseplate holder (Inscopix)
Miniscope system with DAC box (Inscopix)
ProViewTM GRIN lens probe 0.6 mm diameter, ~7.3 mm length (Inscopix, catalog number: 1050-002208)
Baseplates (Inscopix, catalog number: 1050-002192)
Baseplate cover (Inscopix, catalog number: 1050-002193)
Surgical tools (Fine Science Tools)
Lens holder (Inscopix, catalog number: 1000-004238)
Stereotaxic apparatus with electrode holder (Kopf, model: 940)
Trimmer
Anaesthetic machine with induction chamber (VetTech Solutions Ltd., catalog number: AN003-K)
Capillary puller (Sutter Instrument Co., model: P-97)
Heating pad (Dreamland)
Thermocouple heating system (Physitemp Instruments Inc., catalog number: TCAT-2LV)
Drilling machine with bits (Foredom, catalog number: SKU K.1070-21)
Plethysmography chamber (custom-made 0.5 L Plexi glass with air inlet and outlet)
Gas analyser (Hitech Instruments, model: GIR250)
NeuroLog system (Digitimer, model: NL900D)
Microinjection system: custom-made by connecting a graduated glass pipette to silicon tubing, which was attached to a 1 mL syringe on the other end. Pressure was manually controlled in the tubing with the syringe to achieve desired injection speed
Surgical microscope
Software and datasets
Inscopix Data Processing Software (IDPS) (Version 1.6.0.3225)
Inscopix Data Acquisition Software (version 2.0)
Spike2 (Version 8.23)
ANY-maze video tracking software (ANY-maze: Ireland)
Procedure
Below, we describe the surgical protocol for viral transfection (this step is optional if using the cre-dependent GCaMP reporter mice), implantation of GRIN lens, and baseplate installation. This is followed by the procedure for Ca2+ imaging in freely moving mice and data analysis.
Presurgical calibration
Calculate and validate the injection coordinates before viral injection, using fluorescent bead or dye injections to confirm coordinates for both viral injection and implantation of the lens.
Calculate the viral titre by injecting different concentrations of virus and imaging the GCaMP expression.
Ideally, cell somata should express the GCaMP without much expression in nuclei (Figure 1). This is critical to make sure viral titre is not too low (as it will not give a sufficient signal) nor too high (as it will compromise neuronal activity and health) during core recording experiments.
Figure 1. Viral titre and GCaMP expression. Examples of high (A), moderate (B), and low (C) syn-GCaMP expressing cells. Scale bar, 20 μm.
Presurgical preparation for aseptic surgery
Autoclave all surgical tools, drapes, cotton buds, foil, and tissue to be used in the surgery.
Drape the equipment and area used for storing all surgical tools and sterile consumables with autoclaved surgical drapes.
Cover all the surfaces that will be handled/touched during surgery with sterile aluminium foil or cling film i.e., microscope head and bar, stereotaxic frame knobs, etc.
Viral transfection
Initial animal surgical preparation.
Check and report the weight of the animal before surgery. Ideally, adult male or female mice between 8 and 12 weeks old are used.
Place the animal in the anaesthesia induction chamber (connected to the scavenger) with 4% isoflurane balanced with oxygen (4 L/min).
Once the animal is anesthetised, transfer it over to the stereotaxic apparatus. Place the animal prone onto the stereotax, put teeth into bite bar, and connect to the isoflurane nosecone to maintain anaesthesia throughout the surgery [0.5%–2% isoflurane in pure oxygen (1 L/min)].
Give a presurgical subcutaneous injection of atropine (120 μg/kg) and meloxicam (2 mg/kg) prepared in sterile 0.9% saline solution. Atropine reduces bronchial secretion and keeps the trachea clear. Meloxicam provides long-term analgesic effects due to its longer half-life (20 h).
Lubricate the thermocouple probe in lubricating jelly, insert it into the animal’s rectum to regulate the body temperature. Body temperature is maintained at ~33 °C to reduce bleeding and improve recovery.
Cover the animal’s eyes with eye ointment to prevent drying and protect from the light.
Fix the animal’s head into the ear bars.
Trim hair over the head. Wet shave the surgical area with a double-sided razor blade, using a cotton swab to apply either 70% ethanol or vetscrub.
Sterilise the surgical area of skin over the head with three alternating scrubs of vetscrub and 70% ethanol: always start with vetscrub as it can cause contact dermatitis if not removed with the ethanol.
The surgeon should dress for aseptic surgery: hair net, facemask, sterile gloves, and autoclaved surgical gown. The surgeon may be aided by a non-sterile assistant throughout the surgery.
Cover the animal in a cling film or sterile drape.
Virus injection.
Make a sagittal incision down the skull of approximately 1–1.5 cm so that bregma and the injection site can be accessed.
Remove all the connective tissues from the top of the skull using a sterile cotton-tip applicator (Figure 2A).
Place an empty capillary into the holder and adjust the skull position so that bregma is level with 2 mm caudal to bregma. We checked the skull’s left-right levelling at 1 mm laterally; ± 40 μm variation is acceptable.
Drill a hole at the stereotaxically designated position from bregma. To target brainstem nuclei, change the stereotaxic arm anteroposterior angle up to approximately 10° to achieve the desired targeted injection. For example, the coordinates for the retrotrapezoid nucleus with a 9° stereotaxic arm anteroposterior angle are -1.0 mm lateral and -5.6 mm caudal from Bregma and -5.5 mm ventral from the surface of the cerebellum (Figure 2B).
Load the capillary with viral solution and place in the capillary holder. Volume of injection depends on the target region. Generally, smaller and tighter injections (50–200 nL) are better for the specificity.
Connect the capillary to plastic tubing connecting to a pressure delivery system, e.g., a 1 mL syringe or a picospritzer.
Place the tip at bregma for calculating coordinates. Move the capillary tip to the desired injection site (keeping in mind the angle of the capillary holder). Lower the capillary into the drill hole.
i. Zero the Z coordinates at the surface of the brain.
ii. Do not keep the capillary touching the brain surface for too long, as this may lead to blockage of the capillary with keratin in the cerebral spinal fluid.
Insert the capillary to the desired Z coordinates.
Gently apply pressure through the system. Make sure to observe the movement of viral solution meniscus in the capillary through the microscope. Adjust the pressure accordingly to inject viral solution at a speed of approximately 100 nL/min (Figure 2C).
Keep the capillary in place for 3–5 min after injection to prevent backflow up the capillary track and to allow time for virus to spread at the tip of capillary.
Gently remove the capillary.
Pull the skin together and seal the wound with tissue adhesive.
Remove the animal from the stereotaxic frame.
Inject buprenorphine intraperitoneally (100 μg/kg prepared in sterile 0.9% saline solution).
Place the animal back in the cage on a heating pad until recovery.
Observe the animal until full recovery and monitor daily for postsurgical recovery for up to two weeks.
Figure 2. Viral transfection. A) Skin cut and skull top preparation for virus injection by removing connective tissues. B) Calibrated injection coordinates. C) Injection of AAV9-Syn or gfaABC1D-GCaMP6s virus using calibrated coordinates. Abbreviations: 7 N, facial motor nucleus; Py, pyramidal tract; MVe, medial vestibular nucleus; sp5, spinal trigeminal nucleus; RTN, retrotrapezoid nucleus; RMg, raphe magnus; RPa, raphe pallidus; pFL, parafacial lateral region.
GRIN lens implantation
Prepare the animal as for steps B and C1.
After the incision is made, remove an additional 1 mm of tissue from the cut edge (Figure 3A). This creates space for superbond to build around the lens. Using a sterile cotton-tip applicator, remove all the periosteum and connective tissue from the skull’s surface (Figure 3A).
Place an empty capillary into the holder and adjust the skull position so that the bregma is level with 2 mm caudal to bregma and the skull is left-right level at 1 mm laterally; ± 40 μm variation is acceptable.
Drill a hole at the stereotaxically designated position from bregma, big enough for the GRIN lens (Figure 3Be) to pass through (crucial). For our surgery, we used 0.6 mm diameter lens, and a hole size of approximately 0.7 mm was big enough to insert the lens through (the hole should be ~0.1 mm bigger than the lens diameter). The hole size can be checked by aligning the GRIN lens tip against the hole. It is crucial that the hole is not too big as this will not provide skull support to the lens and can allow superbond solution to enter the wound, causing postsurgical complications.
Creating track for the GRIN lens (crucial).
Creating a lens track is a very important and essential step, as insertion of the lens without creating a track applies pressure on the cerebellar and brainstem nuclei and leads to severe postoperative complications.
The lens track is created by inserting an empty capillary (to a depth of ~3/4 of the lens track) from the cranial hole drilled at desired coordinates (Figure 3C).
Place the blunt needle (Figure 3Bf) of an approximately similar diameter to the GRIN lens into the stereotaxic arm holder.
Adjust the coordinates at bregma and move the needle tip over the hole. At this point, move a needle at the edge of the hole, touch its tip on the skull surface, and zero the Z coordinates. Then, move the needle to the desired lens implantation position and lower the needle until it touches the brain surface. Calculate the distance between skull surface and brain tissue.
Touch the needle tip to the brain surface and make Z coordinates zero.
Slowly (250 μm/min) insert the needle into the brain tissue until 250 μm, pull it back to zero, insert again until 250 μm, and place it in position for approximately 3 min. This initial insertion generates the pressure on cerebellum and gives time for the needle edges to cut through the brain tissue.
After 3 min, drive the needle 500 μm ventrally and then retract the needle 250 μm. This creates a 250 μm stepwise needle insertion, which does not create excessive pressure on the brain. Perform the 250-μm insertion-retraction steps continuously.
Stop the needle insertion approximately 300–500 μm above the target region of interest or 100 μm above where the tip of the GRIN lens will be. Keep the needle in place at last insertion for approximately 3 min. Keep in mind that the GRIN lens has a focal plane of approximately 300 ± 100 μm from the bottom surface of the lens [10].
Gently withdraw the needle (Figure 3D).
Figure 3. Creating track for the gradient refractive index (GRIN) lens. A) Skin cut and skull top preparation for creating lens track and implantation. B) Imaging kit includes (a) mini epifluorescent camera, (b) microscope objective lens cover, (c) lens holder during surgical implantation, (d) baseplate cover plate, (e) lens, (f) blunt needle, (g) baseplate, (h) baseplate holder for surgical implantation, and (i) DAC box. C) Blunt needle insertion path through the brainstem and D) a track created after removing a blunt needle.
Implantation of GRIN lens.
Place the GRIN lens (Figure 3Be) into the holder (Figure 3Bc) attached to the stereotaxic arm and place the camera (Figure 3Ba) into the holder (Figure 3Bc) by removing the camera cap (Figure 3Bb). Adjust the centre of the GRIN lens surface at the bregma.
Move the GRIN lens at the edge of the hole and lower it until it touches the skull surface. Then, move the lens to the desired XY coordinates, lower it down until it touches the brain surface [by a distance between the skull and the brain surface calculated above (2c)], and zero the Z-coordinates.
Slowly insert the GRIN lens using the same insertion-retraction 250 μm steps described in step D2f. Do this until 1 mm depth and wait for 3 min at this point.
From 1 mm onward, drive the lens 200 μm ventrally, then retract it by 100 μm and wait for 1 min before the next insertion.
When the bottom surface of the lens is 500 μm from implantation site (~800 μm from the viral injection site), reduce the insertion speed to 50 μm/step (driving by 100 μm, retracting by 50 μm, and waiting for 1 min) (Figure 4A). Check the GCaMP expression during every insertion step by turning on the camera through the Inscopix Data Acquisition Software and DAC box (Figure 3Bi) so the fluorescence can be seen. Stop the lens insertion when GCaMP expression is good and shows increased whole Ca2+ fluorescence (Figure 4B) compared to low signal (Figure 4C). This should be ~300 μm from the viral injection site. Keep in mind that cellular Ca2+ dynamics are suppressed under anaesthesia and will not be very bright during lens implantation. The purpose of this is to ensure the best possible lens implantation distance from the cells of interest. The lens implantation step may be combined with the viral injection, but performing these as separate surgical procedures ensures the best possible lens implantation and the best possible cellular imaging outcome.
Once the lens is in place, dry the skull surface with Kimwipes or a cotton swab. Mix the superbond polymer with monomer as per the kit’s instruction. Add the catalyst and mix well. Wait until the mixture thickens slightly to a gel-like consistency. Apply the mixture around and at the bottom surface of the lens. This is a very important step. Make sure to avoid application of the mixture to the lens holder (Figure 3Bc) and to keep it away from the animal’s eyes. This first application should cover the lens’s surface around the skull. Wait until the superbond is dry (this takes approximately 10 min; an alternative is to use UV fix superbond, which speeds up the process of solidifying polymer and monomer mixture).
When the superbond is well dried, loosen the lens (Figure 3Be) from the lens holder (Figure 3Bc) and remove it. To do this, loosen the side screw on the holder that securely holds lens into the holder. Make sure that there is no superbond on the joint between lens holder and lens; otherwise, this will move the lens.
Mix the second batch of superbond polymer and monomer. Wait until the mixture is of gel-like consistency, apply it along the skin edges, and build around the lens. Make sure that the superbond does not come above the lens’s top surface (Figure 4D). Wait until the superbond dries.
Cover the lens top surface with an Eppendorf cap cut to the shape and adhere it there with PVA glue (Figure 4D) (alternatively, silicon adhesive can be used to cover the lens top surface) that can be removed during baseplate implantation surgery. This will protect the lens until the baseplate installation.
After the second application of superbond has dried and cap is well sealed to the lens, take off the animal from stereotaxic frame, inject buprenorphine intraperitoneally, and place it back in the cage on a heating pad until recovery.
Watch the animal until full recovery and monitor daily for postsurgical recovery for up to two weeks.
Figure 4. Implantation of gradient refractive index (GRIN) lens. A) Slow insertion of GRIN lens through a track created using a blunt needle until a good GCaMP expression and cellular Ca2+ dynamics are visible. Examples of a good (B) and an inadequate (C) GCaMP expression. D) Securing GRIN lens in a place using a good-quality dental cement. Lens top is covered with an Eppendorf cap cut to the shape and adhered with PVA glue. Scale bar, 50 μm.
Baseplate implantation
The animal will be anesthetised for the baseplate implantation surgery and placed in the stereotaxic frame as per step C1a–g of the viral injection protocol. However, this is a non-invasive surgery and therefore does not require the same sterility protocol as above.
Implanting baseplate.
Peel off the PVA glue to remove the lens cap.
Clean the lens top surface with Kimwipes soaked in ethanol to remove any possible dirt from the surface of the GRIN lens.
Attach the baseplate (Figure 3Bg) to the holder (Figure 3Bh) and place it in position in the stereotaxic arm.
Stereotaxic skull levelling is not possible at this step; therefore, try to level the skull as much as possible.
Attach the camera to the baseplate and move it near the lens surface (Figure 5A). Turn on the recording software and watch the live imaging. Adjust the GRIN lens top surface in the middle of the field of recording and lower the camera slowly until the lens surface is visible (take precautions during this step to not compress the camera lens on to any other surfaces and leave a clear gap between camera and GRIN lens).
When the GRIN lens’ sharp edge is visible (Figure 5B) (at the lowest camera position), adjust the sharpness of all edges by moving the nose cone. This ensures that both the camera and lens surface are parallel to each other.
Move the baseplate position away from the lens at the best possible field of view (Figure 5C). This is generally ~300 μm from the sharp edge. This field of view can be checked by gently touching the animal’s back, which possibly activates the cells to check the Ca2+ signal (Figure 5C).
Viral expression time varies from virus to virus and, therefore, may need extra time for the optimal expression. In this case, the animal can be returned back to the cage (by attaching cap to the lens) and be revisited for the baseplate surgery in a week’s time.
When the baseplate is in position with an optimal GCaMP expression, mix the superbond polymer with monomer and add catalyst to it. When the mixture is of good consistency to form a layer between the side of the baseplate and superbond over the skull, apply it on the edges, making sure it does not run under the baseplate or touches the camera lens. This is a critical step. After the first application, wait until mixture is well dried. Loosen the screw on the baseplate that holds the camera. Retract the camera up and remove it from the stereotaxic holder.
Apply the second layer of superbond to cover and seal all the gaps around the baseplate. If the superbond colour is not dark, then a black nail polish layer can be applied to make it opaque and prevent any light penetration under the baseplate.
Once the baseplate is well fixed, attach the baseplate cover (Figure 3B-d). Take the animal off from the stereotaxic frame and place it back in the cage on a heating pad until recovery.
Watch the animal until full recovery and monitor it daily for at least one week before recordings start.
Figure 5. Implantation of baseplate. A) Baseplate is attached to the skull over the gradient refractive index (GRIN) lens so that the best possible cellular Ca2+ dynamics can be imaged. B) The GRIN lens’ sharp edge is visible when the camera is at its lowest position during baseplate implantation. C) Move the baseplate position away from the lens so that the cells can be seen with the best possible field of view. Scale bar, 50 μm.
Mice training
Train the mice with dummy camera in the environment where they will be recorded (Figure 6A). Training could last from 10 to 60 min and depends on the duration of the study protocol. One to three training sessions could be performed at least a day apart.
Ca2+ imaging in freely moving mice
Attach the imaging camera to the animal’s head by docking it in the baseplate and tightening the side screw on the baseplate.
Imaging:
Set the recording parameters as desired to ensure the least LED power to prevent bleaching. Set the optimal recording frame rate [glial Ca2+ transients are slower than neurons, hence can be recorded at slower frame rate (< 10 FPS)] and adjust the plane of focus to achieve the desired field of view for recording (Figure 6C, D). This has been discussed in detail in the previous protocols and can be found in the manufacturer’s manual [12].
Place the animal in the recording environment, e.g., in plethysmographic chamber to test hypercapnic breathing response by measuring changes in breathing tidal volume and frequency (Figure 6B).
Recording should be started depending on the study protocol. The study might require the animal to be settled in the recording environment before recording begins or it might require recording the activity as soon as the animal is placed in the environment. To measure the hypercapnic breathing response from chemosensitive neurons or glia, the animal will be allowed to settle in the plethysmographic chamber connected to a gas analyser (to analyse CO2 and O2) and pressure transducer to measure breathing volume. After the animal is settled, Ca2+ imaging is started [through TTL (Transistor-Transistor Logic) pulse] and baseline breathing recorded followed by hypercapnic challenge and recovery.
The duration of Ca2+ imaging depends on the protocol. However, it is advisable to keep the recording duration as short as possible to avoid fluorophore bleaching. In our study, we have successfully recorded up to 15 min/session from the same mouse multiple times (keeping a week gap between recordings). However, imaging frequency and duration depends on the type of GCaMP indicator and camera imaging settings.
Synchronised video recording:
Real-time animal video can be recorded and synchronised with imaging data using TTL pulse script. Detailed description to set up TTL pulse can be found on CED-Spike2 website. The TTL pulse can be delivered to the DAC box to trigger the Ca2+ imaging, and these can be synchronised during analysis. To start the TTL pulse, a command is given from the Spike software, and this will allow to see when the command was delivered. This command will start the Ca2+ recording and, later, Ca2+ imaging trace is aligned with the TTL pulse command on Spike recording (Spike recording could be EEG or any other feature such as plethysmographic trace).
Figure 6. Training and Ca2+ imaging in a freely moving animal. A) Training an animal with dummy camera in an environment where they will be recorded so they get habituated to it. B) Ca2+ imaging of brainstem neurons and glia along with behavioural responses, e.g., breathing reflexes to hypercapnia. Example of fluorescent image from brainstem retrotrapezoid nucleus (RTN) neuronal (C) and astrocytic (D) Ca2+ dynamics. Scale bar, 50 μm.
Data analysis
Data analysis and cell classification
There are multiple ways to process the raw data, e.g., semi-manual ROI analysis, PCA/ICA analysis, constrained non-negative matrix factorisation for microendoscopic data (CNMF-E), and CNMF-E integrated into an open-source package: calcium imaging analysis (CaImAn). All these methods have their merits and demerits, which are outside the scope of this protocol and have been reported previously [23]. We used Inscopix Data Processing Software (IDPS) that incorporates some of these options to analyse imaging data and identify the cells. After the preprocessing step, movements of the visual field that is evident during most recordings was corrected via the Inscopix motion correction software to allow ROI-based measurement of fluorescence to remain in register with the cells during the recording. This allows to correct excessive XY plane movement.
Irrespective of the method used to identify cells, the major challenge with brainstem imaging remains that of the movement artefact. Most of the Ca2+ imaging methods can correct for rigid movement artefacts caused due to the animal or light camera movement. However, brainstem movement is non rigid and cannot be corrected with the previously documented methods that are an inbuild part of the workflow of these analysis methods. Therefore, we designed a cell inclusion/exclusion criterion that allows us to identify and discount/reject the cells caused by artefacts (Figure 7).
After identification of cells using given criteria in specific methods, we aligned all the cells with the video data. We aligned Ca2+ imaging traces with the animal’s body movements in the video recordings using ANY-maze video tracking software and converted these movements into a colour-coded sonogram. Extracted cells were only accepted for categorization if the following criteria were met: (1) The features of the cell (e.g., soma, large processes) could be clearly seen; (2) they occurred in the absence of movement of the animal or were unaffected by animal movement; (3) fluorescence changed relative to the background; and (4) the focal plane had remained constant as shown by other nonfluorescent landmarks (e.g., blood vessels). For the second criterion, cells are not excluded for merely displaying fluorescent changes during body movements. However, calcium signalling should occur irrespective of body movement (except for the motor-related neuronal or glial activity), and when the cells were extracted and aligned with the body movement, the fluorescent changes (amplitude of Ca2+ signal) seen during body movement should not be significantly different from the changes when the animal is quiet.
This method allowed us to include the cells that met the above criteria (Figure 7Aa) and reject cells that are caused by movement artefact, due to the brainstem being in the extracranial space during head, neck, or body movement (Figure 7A-b). This is a very specific issue with brainstem imaging that does not cause a problem while recording from other brain regions, therefore not having been discussed before in other imaging studies.
Previous studies have used anchoring tungsten wires glued to the GRIN lens to stabilise the brainstem imaging field of view [17,24,25]. This additional measure could also possibly help to reduce movement artefact but still does not allow eliminating the changes in cell brightness caused by movement artefact.
Glial brainstem Ca2+ dynamics are very slow and transient (Figure 7B), which makes it easy to include or exclude these cells using above criteria.
Figure 7. Data analysis and cell classification using set criteria. A) Examples of neuronal GCaMP fluorescence traces that meet (i) and do not meet (ii) our cell inclusion/exclusion criteria to deal with brainstem motion artefact. B) Example of astrocytic GCaMP fluorescence trace. Expanded GCaMP6 signal (3 s) from grey rectangle, presented as a montage of pseudo-coloured fluorescence images, shows slow Ca2+ dynamics in brainstem glia (from no fluorescence at 0 s that peaks in the middle and decays at 3.0 s). Scale bar, 20 μm. Abbreviations: WBP, whole body plethysmography; Mov, movement.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s) [10,26]:
Bhandare et al. (2022). Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice. eLife (Figures 1–7) [10].
Bhandare et al. (2023). Neural correlate of reduced respiratory chemosensitivity during chronic epilepsy. Frontiers in Cellular Neuroscience (Figures 1, 2, 4, 5 and 7) [26].
General notes and troubleshooting
General notes
Despite this protocol being validated in mice, it could be applicable to rats.
In vivo imaging is technically challenging and, therefore, no single step is crucial; instead, all steps in this protocol are important and must be carefully attended. Optimisation of all steps contributes to successful imaging.
Before starting this protocol, it is very important to validate the brain coordinates for the target nuclei and also the optimal concentration of the virus.
Intravital microscopy imaging generates a large volume of data (in gigabytes, therefore requiring big storage space) and also requires high-performance computers (high CPU speed, large amounts of RAM) to process the data.
It is essential to consider the animal husbandry and housing after implanting GRIN lens and baseplate as the headmount might get stuck into the food hopper or co-housing could damage it.
The choice of GCaMP indicator is important; it is essential to consider the firing properties of the cells and the questions that need to be answered before the start of the study [27].
The re-use of baseplates is very much possible; however, the re-use of the GRIN lens depends on the quality of the lens after its recovery from the previous experiment. Both the baseplate and lens can be left in acetone overnight and gently cleaned the next day using a soft brush.
Troubleshooting
Problem 1: Excessive movement in the imaging that does not look biological.
Possible cause: Biological movement in the imaging sessions is caused by the neck movement where tissue underneath the lens surface shifts its position in a XY direction (shifting the cells position in XY direction up to 5 μm) and occasionally in the Y plane, where motion correction is difficult. The XY type of biological movement can be corrected with the motion correction option built in the data analysis software. Non-biological movement is caused due to the loose fitting of implants such as lens, baseplate, or camera. The loose fitting of the camera into the baseplate cavity can occur if the baseplate side screw is not tightened enough onto the camera. These types of non-biological movement cause the whole field of view being shifted by more than 20 μm in a XY direction or a synchronised up and down movement of the field of view (field of view goes in and out of focus).
Solution: Check the baseplate screw and fix it. If it is the baseplate implant, then this is a bit complicated; either reimplant baseplate or, if it is due to the low quality of the dental cement, then reconsider a good-quality alternative material.
Problem 2: Not enough Ca2+ signal.
Possible cause: Figure 1 shows an example of high, moderate, and low syn-GCaMP expressing cells. The low Ca2+ signal during GCaMP imaging in freely behaving mice could be due to low concentration or volume of the injected virus. Resendez et al. [12] have described optimal viral expression in Figure 4. This could also happen due to missed viral injection and lens implant as shown by Resendez et al. [12] in Figure 6, panel C, D.
Solution: First, try to change the plane of focus and adjust LED power and frame rate. If it still does not fix the problem, then sacrifice the animal and check the GCaMP expression. Sometimes, the problem could be due to the serotype of the virus and the cell types tropism [28]. Make sure to choose the right virus and serotype for your purpose.
Acknowledgments
This work was supported by an MRC Discovery Award (grant number: MC_PC_15070) and the Epilepsy Research UK (ERUK) Project Grant (grant number: P1903). A.B. is an Epilepsy Research Institute’s (ERI) Emerging Leader Fellow (grant number: F2203). R.H. is supported by the Biological Sciences Research Council (grant number: BB/X008290/1). This protocol is adapted from Bhandare et al. [10] and has also been used in Bhandare et al. [11].
Competing interests
The authors declare no competing interests.
Ethical considerations
Experiments were performed in accordance with the European Commission Directive 2010/63/EU (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) and the United Kingdom Home Office (Scientific Procedures) Act (1986) with project approval from the University of Warwick's AWERB.
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Protocol for the Implantation of Scaffolds in a Humanized Mouse Cutaneous Excisional Wound Healing Model
DG Dina Gadalla
MK Maeve M. Kennedy
DL David G. Lott
Published: Vol 14, Iss 18, Sep 20, 2024
DOI: 10.21769/BioProtoc.4974 Views: 301
Reviewed by: Vivien J. Coulson-ThomasSudhir VermaTarsis Gesteira Ferreira
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Original Research Article:
The authors used this protocol in Journal of Tissue Engineering and Regenerative Medicine Sep 2024
Abstract
Tissue-engineered constructs combine the mechanical properties of biomaterials with biological agents to serve as scaffolds that direct the wound-healing process and promote tissue regeneration. A limitation to studying wound healing in vivo is that mouse skin contracts to heal rather than exhibiting granulation tissue formation and epithelialization like human skin. Therefore, it became necessary to develop a mouse model to better recapitulate human wound healing. The first splinted excisional wound healing model in mice, described in 2004, utilized silicone splints to prevent skin contracture. This model has been used to test a variety of wound healing strategies; however, to our knowledge, this model has not been adapted to test the effect of implants on wound healing. In our established protocol, circular bilateral excisional wounds are made on the mouse’s dorsum. A circular implant made of porous polyethylene is sutured to the skin within the wound. A thin, donut-shaped silicone splint is secured to the skin surrounding the wound, and a thick, donut-shaped splint is placed on top to tent the wound dressing. Finally, the mouse’s abdomen is wrapped in a bandage and tape to protect the implants. Our protocol offers a significant enhancement to the existing model by enabling the testing of implants for wound healing, as well as using an additional splint that prevents direct contact between the wound dressing and the wound bed. This model can be used to study tissue-engineered implant designs in a relatively low-cost, simple, and high-throughput manner before advancing to larger animal studies.
Key features
• Builds upon methods developed by Galiano et al. [1] and extends their application to include scaffold testing.
• Utilizes a construct that protects wounds, thereby enabling unaffected wound healing.
• Can be adapted to test a wide variety of biomaterials for wound healing.
• Describes dressing details and exact methodologies that prevent animals from interfering with wound healing.
Keywords: Wound healing Splinted excisional model Murine Scaffold testing Dermal Epithelialization Granulation tissue
Background
Non-healing wounds, chronic ulcers, and significant anatomical defects pose important challenges to both healthcare providers and scientists. Current wound healing research encompasses a range of innovative strategies, including tissue-engineered constructs, laser therapy, cell therapy, and platelet therapy [2]. These investigations are driven by the recognition that interactions between various cell types within the wound microenvironment demand a shift from in vitro to more complex animal models.
Although rabbits and pigs have historically served as valuable subjects in wound healing research, rodents have emerged as the most commonly utilized animal models due to their relatively low cost and small size [3]. Rodent and human skin differ in several ways, but the most significant is the presence of the rodent subcutaneous panniculus carnosus, which promotes healing through contraction [4]. In contrast, human skin relies on granulation and proliferation for healing [4]. Therefore, extensive efforts have been devoted to replicating human physiology in rodent models.
The current murine excisional wound model, first introduced in 2004, addressed the issue of contraction by incorporating a splint [1]. In this model, circular excisional wounds are created on the mouse dorsum, and donut-shaped silicone splints are centered around the wounds and secured to the skin. The addition of the splint prolongs wound healing by preventing contraction while yielding neo-epithelization that more closely resembles native tissue. Researchers have employed this model in a variety of settings including ischemic wounds, chronic ulcers, and hypertrophic scarring [5–10]. In 2023, Fischer et al. highlighted data analysis and splint troubleshooting; however, this publication still does not describe how to perform implant placement in the wound [11].
Although the existing model has been used successfully, there is a noticeable absence of publications demonstrating its applicability in testing a tissue-engineered, 3D implant that promotes wound healing. In this paper, we present two modifications to the splinted excisional wound model that enable the testing of a rigid polymer implant. The first modification adds sutures that secure the implant within the wound while limiting its manipulation and ensuring complete coverage by the wound edge. The second modification introduces a second, thicker splint on top of the first splint to create a tenting effect for the wound dressing, thereby protecting the implant.
The major contribution of this protocol is its ability to assess physical implants or tissue engineering and regenerative medicine research. We have successfully used this model to evaluate a polyethylene implant, augmented with an electrospun polymer layer soaked in a protein mixture. Importantly, this approach can be adapted to test a diverse array of scaffolds and can potentially extend its application to organ repair beyond the skin.
Materials and reagents
Reagents
Sodium chloride injection 0.9%, USP (Hospira, NDC, catalog number: 0409 -4888-02)
TerrellTM isoflurane, USP (Piramal Critical Care, NDC, catalog number: 66794-019-25)
Buprenorphine extended release 1.3 mg/mL (Fidelis Animal Health, NDC, catalog number: 86084-100-30)
Laboratory supplies
FisherbrandTM sterile alcohol prep pads (Fisher Scientific, catalog number: 22-363-750)
Prolene 6–0 suture (Ethicon, catalog number: 8617G) (Figure 1iii)
Ethilon 8–0 suture (Ethicon, catalog number: 2808G) (Figure 1iv)
C57BL/6 mice (The Jackson Laboratory, catalog number: 000664)
FlexWrap E-Z Tear® bandage 4 in. vet wrap (Aspen Veterinary Resources, catalog number: 14100122)
TegadermTM film dressing (3M, catalog number: 1626W)
Depilatory cream (Veet gel cream hair remover, catalog number: 3116875)
Triple antibiotic ointment (Padagis, NDC, catalog number: 45802-143-01)
Sterile cotton-tipped applicators (Medline, catalog number: MDS202000H)
LubriFreshTM lubricant eye ointment (Major, NDC, catalog number: 0904-6488-38)
Coach athletic tape (Johnson & Johnson, catalog number: JJ5188)
DuraporeTM surgical tape (3M, catalog number: B00042420)
Cyanoacrylate brush-on glue (Krazy Glue, catalog number: KG92548R)
Model MEDPOR® implant (Stryker, catalog number: 83020)
Betadine swab sticks (Emerson Healthcare, catalog number: 6761815301)
Insulin syringe with needle 1 mL 27G (Becton Dickinson, catalog number: 329412)
Syringe with attached needle 1 mL 25G (Becton Dickinson, catalog number: 309626)
Instant sealing sterilization pouches (Fisher Scientific, catalog number: 01-812-54)
Non-sterile woven gauze sponges (Medline, NON25212H) (Figure 1ii)
Towel drapes (Dynarex, catalog number: 4410)
Disposable dissection board (Mopec, catalog number: 22-444-314) (Figure 1i)
Figure 1. Instruments utilized during surgery. (i) Sterile dissection board, (ii) autoclaved gauze sponges, (iii) 6–0 Prolene suture, (iv) 8–0 Ethilon suture, (v) 0.5 mm thin silicone splints, (vi) 1.6 mm thick silicone splints, (vii) 6 mm diameter model MEDPOR® implants, (viii) 5 mm disposable biopsy punches to create wounds, (ix) needle holder, (x) spring scissors, (xi) straight tip forceps, (xii) Adson forceps, (xiii) angled tip forceps, (xiv) Moria forceps, (xv) fine scissors, and (xvi) sterile surgical marker.
Equipment
Research stereomicroscope system (Olympus, model number: SZX16)
Dino-lite digital microscope (Dino-Lite Digital Microscope, catalog number: AF4915ZTL)
Tuttnauer digital autoclave (Tuttnauer, model number: 2340E-B/L)
Anesthesia system (Veterinary Anesthesia Systems, catalog number: VAS 2001R)
Vetiva® mini trimmer (Wahl, catalog number: 88420)
Premium king-size heating pad (Sunbeam, catalog number: 938-511-000R)
Disposable biopsy punches 5 mm (Acuderm Inc, model: P550) (Figure 1viii)
Disposable biopsy punches 6 mm (Acuderm Inc, model: P625)
Disposable biopsy punches 8 mm (Acuderm Inc, model: P825) (Figure 2Bii)
Figure 2. Splint preparation. A. Thin 0.5 mm silicone sheet with 15 mm cork borer to punch the outer circle and 7 mm cork borer to punch the inner circle. B. Thick 1.6 mm silicone sheet with 10 mm punch to punch the outer circle and 8 mm punch to punch the inner circle.
Disposable biopsy punches 10 mm (Acuderm Inc, P1050) (Figure 2Biii)
Cork borer set (Humboldt, catalog number: H-9665)
Spring scissors 6 mm cutting edge (Fine Science Tools, catalog number: 15021-15) (Figure 1x)
Adson forceps 1.5 mm tip width (Fine Science Tools, catalog number: 11027-12) (Figure 1xii)
Moria iris forceps 0.5 mm tip width (Fine Science Tools, catalog number: 11373-12) (Figure 1xiv)
S&T needle holder without lock (Fine Science Tools, catalog number: C-14W; 00088-11) (Figure 1ix)
S&T forceps 0.3 × 0.25 mm angled tip (Fine Science Tools, catalog number: 00109-11) (Figure 1xiii)
S&T forceps 0.3 × 0.25 mm straight tip (Fine Science Tools, catalog number: 00108-11) (Figure 1xi)
Fine scissors (Fine Science Tools, catalog number: 14029-10) (Figure 1xv)
Sterile surgical marker (Aspen Surgical Products, catalog number: 1002-00-PDG) (Figure 1xvi)
BambooTM pad (Wacom, catalog number: CTH300U/U0-AX)
Non-adhesive silicone sheet, 0.5 mm (Sigma-Aldrich, Grace Bio-Labs CultureWellTM, GBL664571-5EA) (Figure 2Ai)
Non-adhesive silicone sheet, 1.6 mm (Sigma-Aldrich, Grace Bio-Labs CultureWellTM, GBL664273-5EA) (Figure 2Bi)
Dino-lite adjustable precision mount (Dino-Lite Digital Microscope, catalog number: RK-06A)
Software
DinoCapture 2.0 (Dino-Lite Digital Microscope, dino-lite.com)
ImageJ (National Institutes of Health, imagej.nih.gov)
Excel (Microsoft, microsoft.com)
StatPlus LE (AnalystSoft, analystsoft.com)
Procedure
Materials preparation pre-surgery
Make donut-shaped splints.
Remove the sticky wrappers from each side of the thin 0.5 mm and thick 1.6 mm silicone sheets.
To make thin splints, press firmly the thin 0.5 mm silicone sheet on a soft surface using the 15 mm cork borer to punch out the outer circle followed by the inner circle using the 7 mm cork borer (center punch in the middle of the 15 mm circle) (Figure 2A and Figure 1v).
To make thick splints, press firmly the thick 1.6 mm silicone sheet on a soft surface to punch out the outer circle using the 10 mm punch followed by the inner circle using the 8 mm punch (center punch in the middle of the 10 mm circle) (Figure 2B and Figure 1vi).
Place two thin and two thick splints in each sterilization pouch (1 pouch/animal surgery).
Prepare Tegaderm squares.
Draw a 2 cm × 2 cm grid over Tegaderm sheets using a pen or marker.
Cut Tegaderm sheets along the grid tracings into 2 cm × 2 cm squares.
Prepare vet wraps.
Cut FlexWrap vet wraps into 2 cm wide and 8 cm long pieces (Figure 3K insert).
Figure 3. Excisional wounding. A. Mouse anesthetized. B. Mouse dorsum shaved, depilated, and wiped. C. Splints placed on each side of the mouse dorsum to mark the location of the two wound centers. D. Marked location of the wound center pinched with forceps and skin fold pulled parallel to the board. E. Marked lines on biopsy punch aligned with skinfold edges. F. Wounds created on either side of the mouse dorsum. G. Implant placed over each wound bed. H. Outer ring of implant sutured to surrounding skin and splint-glued to the perimeter. I. Outer ring of the splint sutured to surrounding skin. J. Tegaderm-thick splint construct applied centered to wound. K. Cut vet wrap wrapped around mouse abdomen. L. Vet wrap secured with athletic tape.
Punch out 6 mm diameter circular implants using the 6 mm punch (Figure 1vii).
Sterilize all non-sterile instruments and tools required for surgery by autoclaving at 135 °C for 25 min sterilization and 25 min drying time.
Place surgical tools required per animal in one sterilization pouch and into the autoclave machine.
Place four pieces of gauze per sterilization pouch (1 gauze pouch/animal) and into the autoclave machine.
Place the prepared sterilization pouch with splints into the autoclave machine.
Preparation of wound site
Weigh the mouse.
Briefly anesthetize the mouse with 3% isoflurane in 1 L/min oxygen flow in an induction chamber.
Note: Continuously monitor the mouse throughout and every time it is under anesthesia by observing its breathing pattern and using the toe pinch check. The mouse’s chest should be moving up and down in a slow and regular rhythm. Pinching the mouse’s foot pad assesses the pedal withdrawal reflex; if it causes a response, this means additional anesthesia must be supplied.
Administer a single dose of buprenorphine (3.25 mg/kg body weight) subcutaneously to control postoperative pain.
Turn off the anesthesia machine by setting it to 0% isoflurane at 0 L/min oxygen flow and wait at least 30 min for the buprenorphine to take effect.
Reinduce anesthesia in the induction chamber with 3% isoflurane at 1 L/min oxygen flow.
Remove the fully anesthetized mouse from the induction chamber and apply eye ointment to both eyes of the mouse.
Transfer the mouse from the induction chamber to the shaving area placing it in the prone position and reduce maintenance anesthesia to 1.5% isoflurane at 1 L/min oxygen flow delivered via face mask (Figure 3A).
Confirm anesthesia with a firm toe pinch (described in step B2).
Shave the dorsum of the mouse, making sure that the shaved area will fit the thin 15 mm splint on each side of the midline.
Note: Make sure to shave the sides of the abdomen so that there is no hair visible around the circumference of the abdomen that is visible while the mouse is in the prone position.
Apply depilatory cream to the shaved area using the cotton-tipped applicators for 30 s; then, wipe it off using a gauze pad.
Note: It is important not to keep the cream on for longer than 1 min as skin irritation will likely occur.
Apply sterile water to a gauze pad; then, wipe the mouse’s shaved skin to remove any remaining cream and fur (Figure 3B).
Cut a piece of Durapore tape and put it over the shaved area; then, gently peel it off (Figure 3B insert).
Note: The adherence capacity of the tape ensures the removal of any shaved hair that is stuck to the skin.
Wipe off the shaved area with an alcohol pad.
Prepare the surgical table with a surgical drape and all the required sterilized instruments and materials (Figure 1).
Transfer the mouse to the prepared surgical table over the cutting board and maintain anesthesia with 1.5% isoflurane at 1 L/min oxygen flow delivered via face mask.
Sterilize the skin for surgery. Use a betadine swab to coat the surgical field, moving in a concentric circle motion starting in the center and moving outward. Follow this with an alcohol prep pad using the same motion. Repeat this process two more times for a total of three rounds.
Apply another sterile drape over the mouse to expose only the surgical field (Figure 3C insert).
Excisional wounding
Plan the location of each of the two wound centers to be created bilaterally on each side of the mouse’s midline and mark with the surgical marker as a reference point (Figure 3C).
Note: You can temporarily place a thin splint on each side of the dorsum to help locate the center of the wounds to be created.
On the outer side of the metallic part of the 5 mm punches, use the surgical marker to make reference lines exactly across from each other as if marking a semi-circle (Figure 3E insert).
Note: When punching the skinfold, this will ensure that if both lines are visualized, then the wound will be a perfect circle.
Pinch the skin on the marked location of the wound center using the Adson forceps to create a skinfold. Use the forceps to place the skinfold parallel to the surgical board so that punching can take place on a flat surface (Figure 3D).
Note: The mouse might rotate on its side as the skin is pulled onto the flat board; adjust the mouse on the face mask as needed.
Carefully align the two marked lines on the biopsy punch with the skin fold edges and its center with the marked wound center location (Figure 3E).
Note: The center of the marked wound location and the two lines on the punch should all be in parallel and create a line along the diameter of the punch.
Excise the skin by pressing down with the punch and slightly twisting or rocking the punch as needed.
Note: This punches a semi-circle on the skinfold, a full 5 mm circle once the skin is unfolded back natively (Figure 3F).
Check that the created wound is 5 mm by gently placing the punch against the wound once the skin is back in place and comparing it to the wound size.
Ensure the removal of epidermis, dermis, hypodermis, and panniculus carnosus from each wound using the spring scissors as needed.
Note: Punched skin can be saved and used as control tissue in wound healing analysis.
Repeat steps C3–C7 for the wound on the other side using a new biopsy punch.
Note: This punching method is used in place of the common method where the skin at the mouse’s midline is pulled and a full circle punch is made at the skin folds to generate a full wound punch on each side of the midline, as this common method tends to create wounds that are too close to each other and not allow enough skin surface area for two splints.
Implant and thin splint placement
Gently place a 6 mm diameter implant over each wound bed (Figure 3G).
Note: Implant size is larger than the wound punch size, as wounds tend to stretch and become slightly larger than their original sizes. You can use control implant on one side in the same animal based on experimental objectives.
Suture the outer edge of the implant to the adjoining wound edge skin using six interrupted 8–0 sutures (Figure 3H).
Proceed to splint placement. Hold one edge of the thin splint with forceps and ask the surgical assistant to apply glue to it by spreading many small glue droplets all around on one side of the splint (Figure 3H insert).
Note: Avoid having bigger droplets to ensure that the glue does not seep through and disturb the wound/implant site.
Place the splint, glue side down, centered around the wound/implant perimeter (Figure 3H).
Gently press down on different areas of the splint using the sides of forceps.
Note: Allow the splint to adhere to the skin for at least 10 min and ensure adequate adherence by gently pulling the outer splint edges away from the skin prior to suturing it to the surrounding skin.
Whilst waiting for the splint to adhere to the skin, repeat steps D2–D5 for the wound on the other side of the midline.
Suture the outer edge of each splint to the surrounding skin using eight interrupted 6–0 sutures (Figure 3I).
Begin by suturing the splint that was adhered to the skin first and has had longer time to dry.
Take a picture of each wound using the Dino-lite microscope secured to its stand.
Place the microscope in the plane parallel to the plane of the implant.
Center implant in the middle of the image.
Note: The wound area measurement from these images taken on the day of surgery will be used as the wound baseline areas (at day 0).
Dressings
Carefully remove the backing from a Tegaderm square using forceps and place a thick splint in the center of the square on the sticky side (Figure 3J insert).
Flip this construct, apply it centered on top of each thin splint with the thick splint facing down, and press the Tegaderm down to stick on the surrounding skin (Figure 3J). The order of materials from bottom to top should be mouse skin, thin splint, thick splint, and Tegaderm.
Note: This is very important and unique to this protocol because this construct creates a tent like structure that protects the implant whilst providing a moist environment to prevent the wound from drying out.
Wrap a cut vet wrap (Figure 3K insert) around the mouse’s abdomen somewhat tightly (Figure 3K) and secure with athletic tape.
Make sure that the tape covers most of the wrap area to create a more rigid structure that prevents mice from pulling on flexible vet wrap (Figure 3L).
Note: Securing the tape in this specific manner is very important as it prevents mice from reaching, biting, and pulling on thin splints underneath the vet wrap, thus allowing thin splints to remain adhered to the skin during the study period.
Inject the mouse with 1 mL of warm sodium chloride injection subcutaneously to account for fluid loss during surgery.
Turn off the anesthesia machine, place the mouse in its cage on top of the heating pad, and monitor it until it is fully recovered.
Note: Mice are individually housed to minimize wound disruption.
Keep the mouse in a temperature- and light-controlled animal facility with free access to food and water for the duration of the study.
Save taken images from Dino Capture in Windows Bitmap (.bmp) format with a scale bar.
Note: The day of wounding is day 0 and images should be named and saved accordingly.
Observation and wound assessments
Monitor mice daily for general ambulatory ability, signs of distress, and behavioral changes.
Take wound images and change dressing every other day as described in the next steps.
Note: Imaging time points should be adjusted based on the experimental plan and research objectives.
Anesthetize the mouse with 3% isoflurane in 1 L/min oxygen flow in an induction chamber.
Transfer the mouse from the induction chamber to the table, placing it in the prone position, and reduce anesthesia to 1.5% isoflurane at 1 L/min oxygen flow delivered via face mask.
Carefully cut through vet wrap and tape at the mouse midline to remove them.
Carefully peel off each Tegaderm square from the skin surrounding the thin splints using forceps.
Note: Thick splint will also come off attached to Tegaderm.
Discard Tegaderm and place thick splints on an alcohol pad. Wipe to reuse them when reapplying dressing.
Take a photo of each wound using the Dino-lite microscope secured to its stand as described in step D8.
Reapply new dressing as described in steps E1–E3.
Turn off anesthesia.
Weigh the mouse.
Place the mouse in its cage over the heating pad and monitor it until it is fully recovered.
Save taken images from Dino Capture in Windows Bitmap (.bmp) format with a scale bar.
Note: Name and save images according to the day of study.
Explantation
Euthanize mice via carbon dioxide inhalation at the end of the desired study time point post-wounding.
Note: This can be done at different time points for different animals based on the experimental design.
Carefully remove the dressing as described in steps F5–F6.
Mark the area to be excised with a surgical marker, ensuring that an outer ring of native, unwounded tissue is also removed with the implant.
Remove the thin splint by carefully cutting through the sutures with scissors and peeling it off using forceps.
Carefully excise marked skin regions of the implant/wound using fine scissors whilst using forceps to peel the cut skin from the underlying tissue.
Process excised tissue according to desired analysis and subsequent studies (mechanical testing, histological and immunofluorescent staining, etc.).
Data analysis
Open wound image taken on day 0 (surgery day) on ImageJ.
Trace a line on the scale bar of the image (automatically generated from Dino Capture 2.0) using ImageJ straight-line drawing tool.
Set scale to that line according to scale bar.
Set scale is under the Analyze toolbar.
Enter the distance (of scale bar) corresponding to the pixels of the drawn line.
Trace the outer perimeter of the wound with the digital stylus of the pad using the ImageJ freehand drawing tool.
Record the wound area (the sketched region) with the ImageJ Measure tool under the Analyze toolbar.
Note: The wound area has a value of zero if the wound is completely covered with tissue.
Transfer the value to an Excel sheet.
Repeat steps 1–6 for wound images taken on all days post-wounding.
Calculate the wound area as a percentage on day x as follows:
W
o
u
n
d
a
r
e
a
a
t
d
a
y
×
(
%
o
f
o
r
i
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i
n
a
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i
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e
)
=
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e
a
D
a
y
x
w
o
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n
d
a
r
e
a
D
a
y
0
×
100
Create a wound healing curve using a standard X-Y plot [wound area (%) vs day post-wounding].
Analyze statistical differences on StatPlus LE using 3–5 animals for each time point being analyzed.
Transfer wound area (%) values from Excel to StatPlus LE.
Perform ANOVA test using Tukey’s HSD post-hoc test.
Consider p < 0.05 as statistically significant.
Note: Exclusion criteria were wounds in which the splint partially or completely detached prior to hair growth.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Gadalla et al. [12]. Polycaprolactone Fiber and Laminin and Collagen IV Protein Incorporation in Implants Enhances Wound Healing in a Novel Mouse Skin Splint Model. J Tissue Eng Regener Med.
Acknowledgments
We would like to acknowledge Galiano et al. (2004) for the wound splint methods first developed, which we built upon for the development of this protocol. This work was supported by the National Institute on Deafness and Other Communication Disorders of the National Institute of Health under award number RO1 DC019114-01.
Competing interests
The authors declare that there is no conflict of interest.
Ethical considerations
Animal studies were conducted in accordance with Mayo Clinic Institutional Animal Care and Use Committee (IACUC) under IACUC protocol number A00005810-21.
References
Galiano, R. D., Michaels, V, J., Dobryansky, M., Levine, J. P. and Gurtner, G. C. (2004). Quantitative and reproducible murine model of excisional wound healing. Wound Repair Regener. 12(4): 485–492. https://doi.org/10.1111/j.1067-1927.2004.12404.x
Mirhaj, M., Labbaf, S., Tavakoli, M. and Seifalian, A. M. (2022). Emerging treatment strategies in wound care. Int Wound J. 19(7): 1934–1954. https://doi.org/10.1111/iwj.13786
Sami, D. G., Heiba, H. H. and Abdellatif, A. (2019). Wound healing models: A systematic review of animal and non-animal models. Wound Med. 24(1): 8–17. https://doi.org/10.1016/j.wndm.2018.12.001
Zomer, H. D. and Trentin, A. G. (2018). Skin wound healing in humans and mice: Challenges in translational research. J Dermatol Sci. 90(1): 3–12. https://doi.org/10.1016/j.jdermsci.2017.12.009
Son, D. O. and Hinz, B. (2021). A Rodent Model of Hypertrophic Scarring: Splinting of Rat Wounds. Methods Mol Biol. 405–417. https://doi.org/10.1007/978-1-0716-1382-5_27
Chen, L., Tredget, E. E., Liu, C. and Wu, Y. (2009). Analysis of Allogenicity of Mesenchymal Stem Cells in Engraftment and Wound Healing in Mice. PLoS One. 4(9): e7119. https://doi.org/10.1371/journal.pone.0007119
Chen, L., Tredget, E. E., Wu, P. Y. G. and Wu, Y. (2008). Paracrine Factors of Mesenchymal Stem Cells Recruit Macrophages and Endothelial Lineage Cells and Enhance Wound Healing. PLoS One. 3(4): e1886. https://doi.org/10.1371/journal.pone.0001886
Park, D. J., Duggan, E., Ho, K., Dorschner, R. A., Dobke, M., Nolan, J. P. and Eliceiri, B. P. (2022). Serpin-loaded extracellular vesicles promote tissue repair in a mouse model of impaired wound healing. J. Nanobiotechnol. 20(1): e1186/s12951–022–01656–7. https://doi.org/10.1186/s12951-022-01656-7
Vuerich, R., Groppa, E., Vodret, S., Ring, N. A. R., Stocco, C., Bossi, F., Agostinis, C., Cauteruccio, M., Colliva, A., Ramadan, M., et al. (2023). Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels. npj Regener Med. 8(1): e1038/s41536–023–00283–6. https://doi.org/10.1038/s41536-023-00283-6
Wu, Y., Chen, L., Scott, P. G. and Tredget, E. E. (2007). Mesenchymal Stem Cells Enhance Wound Healing Through Differentiation and Angiogenesis. Stem Cells. 25(10): 2648–2659. https://doi.org/10.1634/stemcells.2007-0226
Fischer, K., Litmanovich, B., Sivaraj, D., Kussie, H., Hahn, W., Hostler, A., Chen, K. and Gurtner, G. (2023). Protocol for the Splinted, Human-like Excisional Wound Model in Mice. Bio Protoc. 13(3): e4606. https://doi.org/10.21769/bioprotoc.4606
Gadalla, D., Kennedy, M., Ganem, J., Suppah, M., Schmitt, A. and Lott, D. G. (2024). Polycaprolactone Fiber and Laminin and Collagen IV Protein Incorporation in Implants Enhances Wound Healing in a Novel Mouse Skin Splint Model. J Tissue Eng Regener Med. 2024(1): e1155/2024/2515383. https://doi.org/10.1155/2024/2515383
Article Information
Publication history
Received: Oct 1, 2023
Accepted: Mar 11, 2024
Available online: Sep 20, 2024
Published: Sep 20, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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4,975 | https://bio-protocol.org/en/bpdetail?id=4975&type=0 | # Bio-Protocol Content
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Preparing and Evaluating the Stability of Therapeutically Relevant Oligonucleotide Duplexes
SI Shreyas G. Iyer §
AK Andrea L. Kasinski
(§ Technical contact)
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4975 Views: 2996
Reviewed by: Chiara AmbrogioWendy Leanne HempstockCatherine Hurd
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Original Research Article:
The authors used this protocol in Oncogene Sep 2023
Abstract
The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively.
Key features
• Adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other unmodified and modified oligonucleotides.
• Does not necessitate any Biological Safety Level clearance and offers a rapid, cost-effective, and entirely in vitro procedure.
• Allows researchers to evaluate multiple modification patterns that, when coupled with targeting activity, allow for selecting the best modification pattern prior to in vivo analysis.
Keywords: Serum Stability RNA microRNA siRNA RNA Interference Oligonucleotide therapeutics Annealing Duplex Degradation kinetics Modified oligonucleotides Glycerol tolerant gel
Graphical overview
Background
The field of RNA therapeutics holds promise for applications in various diseases. However, the inherent instability of RNA poses a major obstacle, especially due to the harsh conditions encountered during circulation [1]. Therefore, a protocol for rapid in vitro evaluation of RNA stability in serum holds critical significance. This protocol presents a reliable and reproducible method for preparing oligo duplexes followed by the assessment of their stability in serum.
Fetal bovine serum (FBS) is used extensively in research settings as an additive to basal growth medium for cell and tissue culture applications. It serves as a rich source of proteins and growth factors that are vital for supporting cell growth in a cultured environment. FBS contains over 1,000 different components, including growth and attachment factors, lipids, hormones, essential nutrients, energy sources, electrolytes, carriers, and enzymes [2]. Notably, FBS also contains nucleases, which cause degradation of oligonucleotides over time [3,4]. Thus, FBS replicates a surrogate for the conditions faced by oligonucleotides during their circulation in the bloodstream, potentially even more rigorous than those encountered in circulation.
RNA instability, coupled with potential immunogenic effects associated with unmodified RNAs, are critical challenges in the development of RNA-based therapies. Unmodified oligos face rapid degradation by nucleases and elicit immunogenic effects, limiting their efficacy and necessitating high and frequent dosing in vivo [1]. Various chemical modifications, such as 2'-O-methyl and 2'-fluoro modifications to the ribose and phosphorothioate substitutions to the backbone, have been employed to enhance RNA stability [5–7]. Ribose modifications improve binding affinity and nuclease protection, while phosphorothioate bonds confer resistance to exonucleases [8]. The 2'-O-methyl modification also mitigates immune system stimulation triggered by delivered RNA. Although these modifications are advantageous, each RNA requires a unique pattern of modifications along both sense and antisense strands to limit degradation while maintaining target engagement. The protocol provided here allows one to evaluate "the stability" of multiple modification patterns and, when coupled with gene expression analysis (qRT-PCR or western), the researcher can easily identify the optimal modification pattern for a particular RNA.
Due to blood-born nucleases, these RNA modifications are essential, especially when the RNA is delivered using direct ligand conjugation [9–11]. Nonetheless, this protocol's relevance extends to studies involving therapeutic RNA encapsulation and studies using transfection of modified miRNA and siRNA, which are subject to intracellular degradation, effects that can be evaluated using this simple in vitro protocol.
While the concept of serum stability is not novel, the methodologies employed by researchers tend to exhibit variability [12,13]. This protocol aims to serve as a standardized reference for researchers. By utilizing this protocol, researchers can effectively compare the impact of various modifications on the stability of various oligos and evaluate the degradation kinetics of oligonucleotide therapeutics.
Materials and reagents
Biological materials
Fetal bovine serum Premium (Bio-Techne, catalog number: S11150)
Oligonucleotides (Integrated DNA technologies or other relevant source)
Complementary sense and antisense strands
Options to add modified bases including 2'-O-methyl and 2'-fluoro nucleotides, modifications to the backbone, including phosphorothioate backbone modification, replacing phosphodiester bonds in the oligo backbone at specific locations, or other modifications.
Reagents
Nuclease-free water (Invitrogen, catalog number: AM9932)
Tris base, molecular biology grade (Millipore Sigma, catalog number: 648310)
Concentrated hydrochloric acid (HCl) (Fisher Chemical, catalog number: SA49)
Sodium chloride (NaCl) (Millipore Sigma, catalog number: S9888)
EDTA (0.5 M), pH 8.0, RNase-free (Invitrogen, catalog number: AM9260G)
Taurine (Millipore Sigma, catalog number: T0625)
Acrylamide/Bis solution 29:1 (30%) (Bio-Rad, catalog number: 1610156)
Ammonium persulfate (APS) (Millipore Sigma, catalog number: A3678)
TEMED (tetramethylethylenediamine) (Bio-Rad, catalog number: 1610800)
2× RNA loading dye (Thermo Scientific, catalog number: R0641)
GelRed nucleic acid gel stain (Fisher Scientific, Biotium, catalog number: NC9594719)
Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: SH30256FS)
Solutions
NaCl (2.5 M) (see Recipes)
1 M Tris, pH 7.5-8.0 (see Recipes)
10× annealing buffer (see Recipes)
20× glycerol-tolerant gel buffer (see Recipes)
15% polyacrylamide glycerol-tolerant gel (see Recipes)
Recipes
NaCl (2.5 M)
Reagent Final concentration Quantity
NaCl (powder) 2.5 M 1.46 g
Nuclease-free H2O n/a 10 mL
Total 2.5 M 10 mL
1 M Tris, pH 7.5-8.0
Reagent Final concentration Quantity
Tris base 1M 121.1 g
Nuclease-free H2O n/a 800 mL
Concentrated HCl n/a 65 mL
Nuclease-free H2O n/a Add to bring final volume to 1 L
Total n/a 1 L
10× annealing buffer
Reagent Final concentration Quantity
Tris, pH 7.5–8.0 (1 M) (Recipe 2) 100 mM 400 µL
NaCl (2.5 M) (Recipe 1) 500 mM 800 µL
EDTA (0.5 M) 10 mM 80 µL
Nuclease-free H2O n/a 2,720 µL
Total n/a 4 mL
Store at -20 °C
20× glycerol-tolerant gel buffer
Reagent Final concentration Quantity
Tris base 1.78 M 216 g
Taurine 570 mM 72 g
EDTA (0.5 M) 1 mM 2 mL
Nuclease-free H2O n/a Bring to 1,000 mL
Total n/a 1,000 mL
Store at 4 °C
15% polyacrylamide glycerol-tolerant gel (see Note 4)
Reagent Final concentration Quantity
Acrylamide/Bis solution 29:1 (30%) 15% 7.5 mL
Glycerol-tolerant gel buffer (20×) (Recipe 4) 1× 750 µL
Ammonium persulfate (10%) 0.1% 150 µL
TEMED 1% 15 µL
Nuclease-free H2O n/a 6.6 mL
Total n/a 900 mL
Laboratory supplies
Standard pipette tips with a volume capacity of 2 µL, 20 µL, 200 µL, and 1 mL
Manual pipettes set of 2, 20, 200, and 1,000 µL (Mettler-Toledo, Rainin, catalog number: 17014393, 17014392, 17014391, and 17014382 or similar)
Premium microcentrifuge tubes 1.5 mL (Fisherbrand, catalog number: 05-408-129, or similar RNase-free microcentrifuge tubes)
Bel-Art® gel staining box with cover (Millipore Sigma, catalog number: BAF135511000-1EA)
Equipment
Standard dry block heaters (VWR, catalog number: 75838-318)
Criterion empty cassettes (Bio-Rad, catalog number: 3459901)
Criterion cell and PowerPac basic power supply (Bio-Rad, catalog number: 1656019)
VWR® compact UV transilluminator (VWR, catalog number: 76407-432)
Laboratory shaker (Reliable Scientific, model: 55)
High-resolution digital camera (iPhone, Android, or other cell phone cameras are acceptable)
Software and datasets
ImageJ is a Java-based (runs on Mac OS X, Linux, and Windows) freeware available for download at: http://rsb.info.nih.gov/ij/
Procedure
Oligo duplex preparation
Resuspend each single-stranded oligonucleotide (oligo) strand to a concentration of 200 µM or to the desired concentration using nuclease-free water (Note 1).
Combine 10 µL of each of the 200 µM RNA oligo solutions (sense and antisense) and 5 µL of 10× annealing buffer (Recipe 3) in a 1.5 mL microcentrifuge tube with 25 µL of nuclease-free water. The final volume is 50 µL (Note 2).
Incubate the solution for 5 min at 95 °C in a dry heat block and allow to cool down slowly to room temperature (Critical) (Note 3).
The solution can be stored frozen at -20 °C and freeze-thawed up to five times. The final concentration of oligo duplex is 40 µM and can be diluted to the desired concentration with nuclease-free water (Pause Point).
Analysis of oligo duplexes
Cast a 15% polyacrylamide glycerol-tolerant gel using either 12-well or 18-well combs (Recipe 5) (Note 4).
Place polymerized gels into the gel running apparatus and fill with 1× glycerol-tolerant gel buffer (dilution of 20× glycerol-tolerant gel buffer, Recipe 4).
Gently remove the combs from the gels and clean out the wells by forcefully pipetting the 1× glycerol-tolerant gel buffer into empty wells.
Mix 1 µL of the oligo sample of the desired concentration with 4 µL of water and 5 µL of 2× RNA loading dye (Notes 5 and 6).
Denature RNA/RNA loading dye at 70 °C for 5 min in a dry heat block and transfer samples back to ice quickly.
Load the samples on the gel and run at 100 V until the desired separation is achieved (Note 7).
Once the gel has finished running, carefully separate the glass cassette and remove the gel. Transfer the gel into a small dish for staining.
Stain the gel using GelRed nucleic acid gel stain by mixing 10 µL of GelRed with 10 mL of 1× glycerol-tolerant gel buffer. Incubate the gel in this mixture for 10 min at room temperature with gentle shaking (not to exceed 30% of maximum to avoid gel tear).
Visualize the oligo bands in the gel using a UV transilluminator to confirm successful annealing of the duplex and to assess the purity of samples.
Capture an image using a high-resolution camera (Figure 1). This can be used for quantification of band intensities with ImageJ or similar quantification software.
Figure 1. Polyacrylamide gel image for qualitative assessment of oligo duplex generation. Representative GelRed-stained polyacrylamide gel of a fully modified version of miR-34a (FM-miR-34a) containing 2'-O-methyl and 2'-fluoro bases and phosphorothioate backbone modifications, highlighting successful annealing of oligo duplex as indicated by mobility shifts on the gel. For exact sequence and modification pattern of oligos used for this experiment, refer to Abdelaal et al. [8]. Each lane was loaded with 40 pmol of oligo. A similar quality assessment of additional oligo duplexes can be found in Li et al. [14]; Orellana et al. [9].
Serum stability assay
Prepare 50 pmol of the oligo duplex in 50% FBS in a total volume of 10 µL (Note 8 and 9).
Prepare the correct number of tubes (at room temperature) to reflect the desired timepoints. Recommended timepoints include 0 min, 10 min, 30 min, 1 h, 6 h, 12 h, and 24 h.
Incubate the tubes at 37 °C until the timepoint is reached. At that point, mix 5 µL of the RNA/serum sample with 5 µL of RNA loading dye and store the respective tube at -20 °C (Note 10) (Pause Point).
After the last timepoint, analyze samples on a 15% polyacrylamide gel in glycerol-tolerant gel buffer followed by staining RNA using GelRed nucleic acid gel stain, as outlined in section B (Figure 2).
Figure 2. Serum stability gel. Representative GelRed-stained polyacrylamide gel of partially modified (PM) and fully modified (FM) miR-34a following exposure to 50% serum over a time period. For exact sequence and modification pattern of oligos, refer to Abdelaal et al. [8]. Each lane contains 50 pmol of oligo incubated with serum. Figure adapted from Abdelaal et al. [8]. A similar assessment of the stability of duplexes can be found in Li et al. [14]; Orellana et al. [9].
Notes
Throughout the protocol, the stock concentration of the single-stranded oligo is considered to be 200 µM.
The key requirement is a 1:1 equimolar ratio for the two strands, although the concentration of both strands can be higher or lower. The highest concentration tested for annealing is a final concentration of 50 µM for the oligo duplex.
This is accomplished by turning off the heat source but leaving the tube within the dry heat block, allowing it to cool down gradually to room temperature. Alternatively, a beaker of water can be used as a water bath to achieve the 95 °C temperature, and then the heat source is removed, allowing the beaker of water containing the tube of oligos to cool down gradually to room temperature.
This process is optimized for casting gels using Criterion empty cassettes. To avoid premature polymerization, 10% APS and TEMED should be the last two reagents added to the gel solution. After adding TEMED, mix and quickly transfer the appropriate volume of gel solution (~13 mL) to fill up the empty cassette. Insert comb into the gel and allow 20–30 min for the gel polymerization to occur. Do not discard the remainder (~2 mL) of gel solution, as it can be used to verify if the gel solution has completely polymerized. Based on the Criterion gels, gels are typically run at 100 V at room temperature with no concerns of overheating.
If samples were previously frozen, thaw them on ice.
Include necessary controls such as single-stranded oligos and, if desired, a DNA/RNA ladder.
Depending on the size of the oligo, different resolution times may be required to achieve the necessary separation. Run times are typically 90–120 min at room temperature for a microRNA (such as miR-34a), which is 19–25 nucleotides in size per strand.
This is achieved by diluting 1.25 µL of 40 µM duplex by adding 1.25 µL of nuclease-free water and 2.5 µL of 100% FBS. If setting up multiple tubes, a master mix can be created.
The oligo can be incubated with PBS instead of FBS for all timepoints or selected timepoints as negative controls. This ensures that any degradation is attributable to the serum rather than potential nuclease contamination in the sample or water.
For the 0-min timepoint, ensure that the RNA loading dye is added immediately after adding the serum and is frozen immediately.
Data analysis
Measure the band intensities using ImageJ or a similar software. The protocol for using ImageJ can be found in Davarinejad [15].
While one technical replicate is sufficient, at least three biological replicates are necessary due to variability associated with bovine serum.
Student’s t-test can be used to compare band intensities between two separate oligos. ANOVA can be used to compare band intensities across multiple timepoints employed in serum stability assay.
Validation of protocol
This protocol was used to generate and deliver functional microRNA-34a (miR-34a) duplexes, specifically and rapidly to tumor tissues. It was also used to evaluate the development of ligand-conjugated miR-34a [9,11]
This protocol was used to generate and assess the stability of a fully modified miR-34a, compared to the stability of partially modified miR-34a and unmodified miR-34a duplexes following incubation in 50% serum over time. While unmodified and PM-miR-34a were destabilized rapidly following exposure to serum, FM-miR-34a was completely resistant up to 24 h and remained intact for at least 72 h [8].
At least three independent biological replicates are recommended to generate accurate, reliable results.
General notes and troubleshooting
General notes
Originally designed for annealing two complementary synthetic RNA oligos, this protocol has been successfully validated with modified oligos, including 2'-O-methyl and 2'-fluoro ribose sugars, and other backbone modifications including phosphorothioate bonds. It is adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other modified oligos.
This protocol uses FBS for the serum stability assay. It can be modified to include any of the following bovine serum types as necessary: newborn calf serum, bovine calf serum, adult bovine serum, and donor bovine serum.
There is considerable variability between FBS batches, including differing levels of nucleases and other components that contribute to oligo degradation. When using a new batch of FBS, include appropriate controls to enable consistent comparisons with previous results.
For recipes detailed in the Recipes section, prepare the necessary stock solutions first.
The gel employed in this protocol is a polyacrylamide gel run using a glycerol-tolerant buffer. The gel composition has been slightly modified to incorporate glycerol-tolerant buffer within the gel. See Note 4 for additional tips regarding the gel casting process.
Troubleshooting
The final volume while loading might be less than 10 µL—this is likely due to evaporation during RNA denaturation process. This should not affect the outcome of the experiment.
The presence of unannealed single strands visualized in the gel can be corrected by precisely determining the concentration of single-strand products and adding an equal molar concentration during the annealing process.
It is useful to resolve both single-stranded oligos on the gel to gain a better understanding for any lower resolving products on the gel.
Acknowledgments
This work was funded in part by the National Institutes of Health (R01CA205420 and R01CA226259) to A.L.K., and a LCRP Idea Development Award from the Department of Defense (W81XWH-22-1-1085) to A.L.K. This protocol was adapted from Orellana et al. [11], Orellana et al. [9], and Abdelaal et al. [8].
Competing interests
A.L.K. is an inventor on U.S. patent PCT/US2017/061997 submitted by Purdue University that covers methods and uses of ligand-targeted delivery of miRNAs. A.L.K. is an inventor on U.S. provisional patent 63/454,177 submitted by Purdue University that covers methods and uses of modified miR-34a. S.G.I. has no competing interests. A.L.K. is a co-founder and CEO of LigamiR Therapeutics that works on ligand-mediated delivery systems for delivery of microRNAs.
References
Abdelaal, A. M. and Kasinski, A. L. (2021). Ligand-mediated delivery of RNAi-based therapeutics for the treatment of oncological diseases. NAR Cancer 3(3): e1093/narcan/zcab030.
Boone, C. W., Mantel, N., Caruso, T. D., Kazam, E. and Stevenson, R. E. (1971). Quality control studies on fetal bovine serum used in tissue culture. In Vitro 7(3): 174–189.
Eder, P. S., DeVINE, R. J., Dagle, J. M. and Walder, J. A. (1991). Substrate Specificity and Kinetics of Degradation of Antisense Oligonucleotides by a 3′ Exonuclease in Plasma. Antisense Res. Dev. 1(2): 141–151.
Yen, A., Cheng, Y., Sylvestre, M., Gustafson, H. H., Puri, S. and Pun, S. H. (2018). Serum Nuclease Susceptibility of mRNA Cargo in Condensed Polyplexes. Mol. Pharmaceutics 15(6): 2268–2276.
Biscans, A., Caiazzi, J., Davis, S., McHugh, N., Sousa, J. and Khvorova, A. (2020). The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy. Nucleic Acids Res. 48(14): 7665–7680.
Hassler, M. R., Turanov, A. A., Alterman, J. F., Haraszti, R. A., Coles, A. H., Osborn, M. F., Echeverria, D., Nikan, M., Salomon, W. E., Roux, L., et al. (2018). Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 46(5): 2185–2196.
Jackson, A. L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J. M., Lim, L., Karpilow, J., Nichols, K., et al. (2006). Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12(7): 1197–1205.
Abdelaal, A. M., Sohal, I. S., Iyer, S., Sudarshan, K., Kothandaraman, H., Lanman, N. A., Low, P. S. and Kasinski, A. L. (2023). A first-in-class fully modified version of miR-34a with outstanding stability, activity, and anti-tumor efficacy. Oncogene 42(40): 2985–2999.
Orellana, E. A., Abdelaal, A. M., Rangasamy, L., Tenneti, S., Myoung, S., Low, P. S. and Kasinski, A. L. (2019). Enhancing MicroRNA Activity through Increased Endosomal Release Mediated by Nigericin. Mol. Ther. Nucleic Acids 16: 505–518.
Rangasamy, L., Chelvam, V., Kanduluru, A. K., Srinivasarao, M., Bandara, N. A., You, F., Orellana, E. A., Kasinski, A. L. and Low, P. S. (2018). New Mechanism for Release of Endosomal Contents: Osmotic Lysis via Nigericin-Mediated K+/H+ Exchange. Bioconjugate Chem. 29(4): 1047–1059.
Orellana, E. A., Tenneti, S., Rangasamy, L., Lyle, L. T., Low, P. S. and Kasinski, A. L. (2017). FolamiRs: Ligand-targeted, vehicle-free delivery of microRNAs for the treatment of cancer. Sci. Transl. Med. 9(401): eaam9327.
Jafari, M., Xu, W., Pan, R., Sweeting, C. M., Karunaratne, D. N. and Chen, P. (2014). Serum Stability and Physicochemical Characterization of a Novel Amphipathic Peptide C6M1 for SiRNA Delivery. PLoS One 9(5): e97797.
Kajino, R., Sakamoto, S. and Ueno, Y. (2022). Synthesis, gene silencing activity, thermal stability, and serum stability of siRNA containing four (S)-5′-C-aminopropyl-2′-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine). RSC Adv. 12(18): 11454–11476.
Li, W., Wang, Y., Liu, X., Wu, S., Wang, M., Turowski, S. G., Spernyak, J. A., Tracz, A., Abdelaal, A. M., Sudarshan, K., et al. (2024). Developing Folate-Conjugated miR-34a Therapeutic for Prostate Cancer: Challenges and Promises. Int. J. Mol. Sci. 25(4): 2123.
Davarinejad, H. (2015). Quantifications of Western Blots with ImageJ [Protocol]. https://www.yorku.ca/yisheng/Internal/Protocols/ImageJ.pdf
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CRISPR/dCas9-Tet1-Mediated DNA Methylation Editing
JQ Junming Qian
SL Shawn X. Liu
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4976 Views: 1090
Reviewed by: Clara Morral MartinezPhilipp Wörsdörfer Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Translational Medicine Jan 2023
Abstract
DNA methylation is a key epigenetic mechanism underlying many biological processes, and its aberrant regulation has been tightly associated with various human diseases. Precise manipulation of DNA methylation holds the promise to advance our understanding of this critical mechanism and to develop novel therapeutic methods. Previously, we were only able to alter genome-wide DNA methylation by treating with small molecules (e.g., 5-Aza-2-deoxycytidine) or perturbing relevant genes (e.g., DNA methyltransferase) targetlessly, which makes it challenging to investigate the functional significance of this epigenetic mark at specific genomic loci. By fusing the catalytic domain of a key enzyme in the DNA demethylation process (Ten-eleven translocation dioxygenases 1, Tet1) with a reprogrammable sequence-specific DNA-targeting molecular protein, dCas9, we developed a DNA methylation editing tool (dCas9-Tet1) to demethylate specific genomic loci in a targeted manner. This dCas9-Tet1 system allows us to study the role of DNA methylation at almost any given loci with only the replacement of a single-guide RNA. Here, we describe a protocol that enables modular and scalable manipulation of DNA methylation at specific genomic loci in various cell cultures with high efficiency and specificity using the dCas9-Tet1 system.
Key features
• Precisely editing the DNA methylation of specific genomic loci in a targeted manner.
• Fine-tuning gene expression without changing DNA sequence.
• Applicable to many types of cell cultures and with the potential for ex vitro and in vivo applications.
Keywords: CRISPR Epigenome editing dCas9-Tet1 DNA methylation Gene regulation
Graphical overview
Overview of dCas9-Tet1-mediated DNA methylation editing
Background
DNA methylation is one of the most important epigenetic modifications, which refers to the covalent modification of the cytosine of DNA by a methyl group. In mammals, DNA methylation often occurs in the context of cytosine-phosphate-guanosine (CpG) residues [1]. It plays a critical role in various biological processes including gene regulation, genomic imprinting, and X chromosome inactivation, as well as the preservation of chromosome stability [2,3]. The proper regulation of DNA methylation is pivotal in maintaining normal cellular activities, and its dysregulation has been tightly associated with a myriad of human diseases [4].
Given its significance, the ability to manipulate DNA methylation with precision could revolutionize both basic research and therapeutic fields. Over the years, several epigenetic editing tools have been developed to manipulate DNA methylation at specific sites by tethering DNA methylation-associated effectors to sequence-specific DNA-binding domains, comprising mainly zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and the clustered regularly interspaced short palindromic repeats (CRISPR) system [5–7]. Our lab pioneered a CRISPR-based DNA-methylation editing system using the fusion of a catalytically dead Cas9 (dCas9) with Ten-eleven translocation 1 (Tet1) hydroxylase catalytic domain (dCas9-Tet1), allowing for the erasure of DNA methylation in the mammalian genome with high specificity [8]. It has been proved as a powerful technology in many aspects, including mechanistically dissecting the functional significance of DNA methylation as well as treating incurable diseases by rewriting the DNA methylation of disease-causing genes. For instance, we demonstrated that targeted demethylation of the MyoD distal enhancer by dCas9-Tet1 along with 5-Aza (5-Aza-2-deoxycytidine, a DNA methylation inhibitor) treatment facilitated myogenic reprogramming of fibroblasts [8]. With the application of dCas9-Tet1, we also studied the hypermethylation of the CGG repeat expansion mutation at the 5’ UTR of fragile X mental retardation 1 (FMR1) gene. We demonstrated that demethylation of the CGG repeats unlocked the epigenetic silencing of FMR1 and restored FMRP expression in Fragile X syndrome cells [9]. More recently, we reported that dCas9-Tet1-mediated DNA demethylation of the promoter of methyl CpG-binding protein 2 (MECP2) gene can reactivate the expression of MECP2 from the inactive X chromosome in Rett syndrome (RTT)-like human embryonic stem cells (hESCs) and in hESCs-derived neurons, which could be a potential therapeutic approach for Rett syndrome [10].
Compared to ZFPs- or TALEs-based approaches, our methods displayed higher efficacy, specificity, and resolution of DNA methylation editing [8]. Importantly, unlike ZFPs and TALEs, which require design and optimization of corresponding distinct proteins to target different DNA sequences, only a replacement of single-guide RNA (sgRNA) is needed for CRISPR/dCas9 system to target different genomic loci. The versatility to flexibly target almost any given genomic loci enable researchers to precisely manipulate DNA methylation with ease. Nevertheless, the size and structural complexity of dCas9-Tet1 system might limit its application in vivo.
Herein, we describe a step-by-step protocol for editing DNA methylation in cell cultures using the dCas9-Tet1 system. This protocol is divided into three subsections to outline 1) the identification of sgRNA target sequences and clone sequences into sgRNA scaffold construct, 2) the delivery of the constructs encoding dCas9-Tet1 and sgRNA into the cells of interest, and 3) the examination of editing results by pyrosequencing.
Materials and reagents
Biological materials
Human embryonic kidney (HEK) 293T cells (ATCC, catalog number: CRL-11268)
Human embryonic stem cells (hESCs) [National Institutes of Health (NIH) registration number: WIBR-2, #29]
Mouse embryonic fibroblasts (MEFs) (ATCC NIH3T3, catalog number: CRL-1658 or derived from other labs)
Stbl3 chemical competent cells (Invitrogen, catalog number: C737303)
pgRNA-modified (Addgene, catalog number: 84477)
Fuw-dCas9-Tet1-P2A-BFP (Addgene, catalog number: 108245)
Fuw-dCas9-dTet1-P2A-BFP (Addgene, catalog number: 108246)
PiggyBac transposase (Lab stock, available upon request)
dCas9-Tet1 on PiggyBac transposon vector (Lab stock, available upon request)
pCMV-dR8.74 (Addgene, catalog number: 22036)
pCMV-VSVG (Addgene, catalog number: 8454)
Reagents
DNA oligos (Eton Bioscience, customized order)
T4 ligase and its buffer (New England Biolabs, catalog number: M0202)
T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201)
AarI enzyme (Thermo Scientific, catalog number: ER1581)
Agarose (Fisher Scientific, catalog number: BP1356-500)
Zymoclean Gel DNA Recovery kit (Zymo Research, catalog number: D4007)
S.O.C medium (Invitrogen, catalog number: 15544034)
mTeSR1 medium (Stemcell, catalog number: 85850)
LB Broth, Miller (Fisher Scientific, catalog number: BP1426-500)
Agar (Fisher Scientific, catalog number: BP1423-500)
Carbenicillin disodium salt (Sigma Aldrich, catalog number: C1389)
E.Z.N.A. plasmid mini kit I (Omega Biotek Inc, catalog number: D6943)
E.Z.N.A. plasmid mini kit II (Omega Biotek Inc, catalog number: D6945)
ZymoPURE II plasmid purification kit, Maxiprep (Zymo Research, catalog number: D4203)
X-tremeGENE DNA transfection reagent (Sigma Aldrich, catalog number: 06365787001)
Opti-MEM I reduced serum medium (Gibco, catalog number: 11058021)
DMEM, high glucose (Gibco, catalog number: 11095092)
Fetal bovine serum (FBS) (Gibco, catalog number: 10082-147)
200 mM L-glutamine (Gibco, catalog number: 25030081)
MEM non-essential amino acids solution, 100× (Gibco, catalog number: 11140076)
Penicillin-Streptomycin (10,000 U/mL) (Gibco, catalog number: 15140122)
Trypsin-EDTA (0.25%), phenol red (Gibco, catalog number: 25200056)
DMEM/F12 medium (Invitrogen, catalog number: 11320-033)
Knockout serum replacement (Invitrogen, catalog number: 10828028)
β-mercaptoethanol (Life Tech, catalog number: 21985023)
Fibroblast growth factor 2 (FGF2) (Gibco, catalog number: PHG0263)
Calcium- and magnesium-free phosphate-buffered saline (PBS) (Gibco, catalog number: 10010023)
Bovine serum albumin (BSA) Fraction V solution (7.5%) (Gibco, catalog number: 15260037)
0.5 M EDTA, pH 8.0 (Invitrogen, catalog number: 15575038)
1 M HEPES (Hyclone, catalog number: SH30237.01)
DNeasy blood & tissue kit (Qiagen, catalog number: 69504)
QIAamp DNA micro kit (Qiagen, catalog number: 56304)
EZ DNA Methylation-Gold kit (Zymo Research, catalog number: D5006)
PyroMark PCR Master Mix kit (Qiagen, catalog number: 978703)
PyroMark Q48 Advanced CpG Reagents (4 × 48) (Qiagen, catalog number: 974002)
Rock inhibitor (Sigma-Aldrich, catalog number: S4317)
Doxycycline (Sigma-Aldrich, catalog number: D9891)
Solutions
HEK293T cell culture medium (see Recipes)
hESCs culture medium (see Recipes)
FACS buffer (see Recipes)
Recipes
HEK293T cell culture medium
Reagent Final concentration Volume
DMEM medium n/a 435 mL
FBS 10% 50 mL
200 mM L-glutamine 2 mM 5 mL
Non-essential amino acids 1% 5 mL
Penicillin-streptomycin 1% (100 U/mL) 5 mL
Total n/a 500 mL
hESCs culture medium
Reagent Final concentration Quantity or Volume
DMEM/F12 medium n/a 385 mL
FBS 15% 75 mL
Knockout serum replacement 5% 25 mL
200 mM L-glutamine 2 mM 5 mL
Non-essential amino acids 1% 5 mL
Penicillin-streptomycin 1% (100 U/mL) 5 mL
β-mercaptoethanol 0.1 mM 0.5 mL
FGF2 4 ng/mL 2 μg
Total n/a 500 mL
FACS buffer
Reagent Final concentration Volume
PBS n/a 142.62 mL
7.5% BSA 0.5% 5 mL
0.5 M EDTA 0.5 mM 150 μL
1 M HEPES 15 mM 2.25 mL
Total n/a 150 mL
Laboratory supplies
1.5 mL microcentrifuge tube (Axygen, catalog number: MCT-175-C)
PCR tube (Axygen, catalog number: PCR-0208-CP-C)
12-well plate (Corning, catalog number: 3513)
96-well plate (Corning, catalog number: 3596)
T-175 flask (Corning, catalog number: 431080)
0.22 μm filter (Corning, catalog number: 430513)
0.45 μm low protein binding filter (Thermo Scientific, catalog number: 121-0045)
Ultracentrifuge tube (Beckman Coulter, catalog number: 344058)
0.4 cm electroporation cuvette (Bio-Rad, catalog number: 1652081)
Equipment
Thermocycler (Applied Biosystem, ProFlex)
PyroMark Q48 Autoprep sequencer (Qiagen, catalog number: 9002471)
Bio-Rad gene pulser Xcell electroporation systems (Bio-Rad, catalog number: 1652660)
FACSAria cell sorter (BD, model: BD FACSAria™ II)
Fluorescence microscope (Nikon, model: Eclipse TS2R)
Centrifuge (Qiagen, model: 5810 with rotor A-2-DWP-AT)
Ultracentrifuge and rotor (Beckman, model: SW32Ti)
Software and datasets
PyroMark Assay Design (Version 2.0.2, 1/10/2024)
PyroMark Q48 Autoprep (Version 4.3.3, 1/10/2024)
Procedure
Identification of sgRNA target sequences and clone into sgRNA scaffold construct
A targeting region is often selected within the CpG island (CGI) of a given gene or a differentially methylated region (DMR) of interest between different cell types or conditions identified by publicly available databases or determined by experiments.
Design the protospacer sequence of sgRNA for targeting dCas9-Tet1 under the following guidelines:
The sequence should be immediately adjacent to a protospacer adjacent motif (PAM), which is 5'-NGG-3' for dSpCas9 used in our system. Do not include PAM sequence as part of target sequence.
The length is typically 20 nt, but can be as short as 17 nt.
Avoid overlapping with CpG sites, as it will protect them from being demethylated.
The average effective range of dCas9-Tet1 with a single sgRNA is 150–200 bp downstream PAM sequence in most cases. Multiple protospacer sequences may be designed to target as many CpG sites within the targeting region as possible.
We design all the possible sgRNAs based on NGG PAM availability within the target region and then run these sgRNAs via online design programs such as CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) and Off-Spotter (https://cm.jefferson.edu/Off-Spotter/) to pick up the one with high on-target score by CRISPRa mode and lower off-target prediction.
For each sgRNA, synthesize two oligos with overhangs to be compatible with sgRNA backbone construct. The overhang sequences are specific to the sgRNA scaffold construct used. Herein, we will use the one commonly used in our lab as an example (pgRNA modified).
sgRNA-sense: 5'-TTGG(G)-N1N2N3 N4N5N6N7N8N 9N10N11N12N13N 14N15N16N17N18N 19N20-3'
sgRNA-antisense:5'-AAAC-N*20N*19N*18 N*17N*16N*15N*14N* 13N*12N*11N*10N*9 N*8N*7N*6N*5N*4 N*3N*2N*1-(C)-3' (Nx and N*x are complemented)
Clone protospacer sequences into sgRNA scaffold construct.
Oligo phosphorylation and annealing. Set up the following reaction in a PCR tube on ice (Table 1):
Table 1. Reaction for oligo phosphorylation and annealing
Reagent Quantity
sgRNA-sense oligo (100 μM) 1 μL
sgRNA-antisense oligo (100 μM) 1 μL
10× T4 ligation buffer 1 μL
Nuclease-free water 6.5 μL
T4 PNK 0.5 μL
Total 10 μL
Incubate in a thermocycler using the following program (Table 2 ):
Table 2.Thermocycling conditions for the oligo phosphorylation and annealing reaction
Temperature (°C) Duration
37 30 min
95 5 min
Ramp down to 25 °C at 1 °C/min
4 ∞
Dilute the phosphorylated and annealed oligo duplex 1:50 in nuclease-free water. Annealed oligos can be stored at -20 °C and are stable through at least 2–3 freeze-thaw cycles.
Linearize the sgRNA scaffold vector by restriction endonuclease AarI digestion. Set up the following reaction in a PCR tube in a microcentrifuge tube on ice (Table 3):
Table 3. Reaction for the linearization of sgRNA scaffold vector by AarI digestion
Reagent Quantity
pgRNA-modified (Plasmid DNA) 1 μg
10× AarI buffer 2 μL
50× oligonucleotide (0.025 mM) 0.4 μL
Nuclease-free water Up to 30 μL
AarI 1 μL
Total 30 μL
Incubate at 37 °C for 1 h. Gel purify the digested plasmid using Zymoclean Gel DNA Recovery kit and determine the concentration.
Ligate the annealed oligo duplex with protospacer sequence to the digested sgRNA scaffold construct with a standard T4 ligase reaction. Set up the following reaction in a PCR tube or in a microcentrifuge tube on ice (Table 4):
Table 4. Reaction for ligating annealed phosphorylated oligos into sgRNA scaffold vector
Reagent Quantity
Diluted phosphorylated and annealed oligo duplex 1 μL
AarI-digested pgRNA-modified DNA 50 ng
10× T4 ligation buffer 1 μL
Nuclease-free water Up to 10 μL
T4 ligase 1 μL
Total 10 μL
Incubate at room temperature for 30 min or at 16 °C overnight followed by heat inactivation at 65 °C for 10 min. Chill the ligation reaction on ice.
Transform the ligation product to Stbl3 E. coli cells.
Thaw Stbl3 competent cells on ice.
Aseptically add 5 μL of ligation reaction to 20 μL of competent cells.
Incubate on ice for 30 min.
Heat shock at 42 °C for 45 s.
Immediately place the cells on ice for 2–3 min.
Add 250 μL of S.O.C medium. Shake at 37 °C for 1 h.
Spread 100 μL on a LB agar plate with carbenicillin. Invert the plate and incubate at 37 °C overnight.
Construct validation and amplification.
Pick 2–3 clones for each sgRNA construct and propagate them in 2 mL of LB medium with carbenicillin at 37 °C with shaking overnight.
Extract the plasmid DNA using Omega E.Z.N.A. Plasmid Mini kit I or II and following manufacturer’s instructions. Submit samples for Sanger sequencing. The following oligonucleotide can be used as the sequencing primer to read out the final protospacer sequence for each clone: 5'-GAAACTCACCCTAACTG-3'.
Delivery of the constructs encoding dCas9-Tet1 and sgRNA into the cells of interest
Considering the different ability to accept exogenous genes of various cell types, we will describe three most used gene delivery methods: 1) chemically transient transfection (HEK293T cells as an example); 2) electroporation (hESCs as an example); and 3) lentivirus transduction (hESCs and hESCs-derived neurons as examples). These three approaches could be generalized to any other tissue cultures, though optimizations of reaction conditions may be needed to achieve the best performance.
Chemically transient transfection for HEK293T cells
HEK293T cells are maintained in HEK293T cell culture medium (see Recipe 1) at 37 °C with 5% CO2. Chemically transient transfection experiment in HEK293T cells is performed in 12-well plates using X-tremeGENE 9 DNA transfection reagent.
Plate approximately from 1 × 105 to 2 × 105 cells/well in a sterile 12-well plate 24 h before transfection. Make sure cells are at optimal concentration (50%–80% confluency) at the time of transfection.
Prior to transfection, bring transfection reagent, reduced serum medium (Opti-MEM I reduced serum medium), and plasmid DNA to room temperature.
Add 700 ng of plasmids DNA encoding dCas9-Tet1 (Fuw-dCas9-Tet1-P2A-BFP) and 300 ng of plasmids DNA encoding target sgRNAs to a sterile microcentrifuge tube and vortex to mix thoroughly. Additionally, prepare dCas9-catalytically dead Tet1 (Fuw-dCas9-dTet1-P2A-BFP) + target sgRNA, and dCas9-Tet1 + scramble sgRNA mixture as experimental control groups. Blue fluorescence protein (BFP) gene is co-expressed with dCas9-Tet1 or dCas9-dTet1, and the puromycin resistance gene and the gene encoding a red fluorescence protein, mCherry, are co-expressed with sgRNAs.
For each experimental group, add 3 μL of transfection reagent (transfection reagent:DNA ratio = 1:3) to 100 μL of Opti-MEM I reduced serum medium. Incubate at room temperature for 5 min.
Add plasmids DNA mixture. Pipette to mix thoroughly. Incubate at room temperature for 15 min.
Add transfection complex to the cells in a dropwise manner. Gently shake or swirl the plate to ensure even distribution over the entire well.
Forty-eight hours post transfection, isolate cells that are successfully transfected.
Dissociating cells with trypsin/EDTA: remove and discard medium. Wash cells with PBS once. Add 300 μL of prewarmed trypsin/EDTA solution to each well and incubate at 37 °C for 5 min or until cells detach.
Add 600 μL of prewarmed growth medium to inactivate trypsin. Gently disperse the medium by pipetting and transfer the cell suspension to a tube.
Spin at 250× g for 5 min at room temperature. Remove and discard the supernatant.
Prepare single-cell suspension by resuspending cell pellet in FACS buffer.
Perform FACS to isolate live BFP- and mCherry-double positive cells.
Plate sorted cells into plate and culture at the same condition for 3–5 more days.
Five to seven days post transfection, harvest cells for further analysis.
Electroporation for hESCs
hESCs are maintained either with mTeSR1 medium on Matrigel-coated plates or on irradiated MEFs with standard hESCs medium (see Recipe 2). The electroporation is performed with Bio-Rad Gene Pulser Xcell electroporator. We generally use PiggyBac transposon system to create a stable cell line for the ease of experiment.
The day before electroporation, for each experimental group, feed one 6-well plate of hESCs at confluency with medium containing Rock inhibitor (10 μM).
Prepare single-cell suspension:
Remove medium and wash with PBS once.
Add 1 mL of Accutase to each well. Incubate at 37 °C for 5 min.
Harvest cells by pipetting gently to dissociate cells and collect with 1 mL of hESCs culture medium supplemented with Rock inhibitor.
Spin down the cells at 250× g for 5 min.
Resuspend the cell pellet with 450 μL of PBS pre-cooled on ice to make the final volume 500 μL.
In a sterile microcentrifuge tube, add 11 μg of plasmid DNA encoding PiggyBac transposase and 33 μg of plasmid DNA encoding dCas9-Tet1 and targeting sgRNA. Add PBS to 300 μL and vortex to mix thoroughly. Additionally, prepare dCas9-dTet1 + target sgRNA, and dCas9-Tet1 + scramble sgRNA mixture as experimental control groups. dCas9-Tet1/dTet1 is under control by a TRE3G promoter, whose expression can be induced by the addition of doxycycline (Dox) at the presence of reverse tetracycline-controlled transactivator (rtTA). The plasmid also contains constitutively expressed genes encoding rtTA and a selection maker (e.g., PuroR for puromycin selection).
Perform electroporation:
For each experimental group, pre-cool an electroporation cuvette on ice.
Add DNA mixture to single-cell suspension and gently pipette to mix thoroughly.
Transfer the total of 800 μL mixture into a pre-cooled 0.4 cm electroporation cuvette.
Place the cuvette on ice for 5 min.
Put the cuvette into the chamber in the required orientation. Electroporate with the following settings:
Exponential setting
Voltage = 250 V
Capacitance = 500 μF
Resistance = infinity
Cuvette = 4 mm
One pulse with expected time 12–14 μs
Place cuvette on ice for 5 min.
Plate the treated cells from the cuvette into one 6-well plate with medium containing Rock inhibitor.
Keep cells in medium with Rock inhibitor.
Three days post electroporation, switch to standard medium without Rock inhibitor.
The next day, add selection chemical (e.g., puromycin for cells receiving construct with PuroR genes).
Keep selection for extended days until visible colonies are formed. Change the medium every day during the process.
Add Dox to induce the expression of dCas9-Tet1/dTet1 for 5–7 days and harvest cells for further analysis.
Lentivirus transduction for hESCs and neurons
Lentivirus packaging:
HEK293T cells are maintained in HEK293T cell culture medium (See Recipe 1) at 37 °C with 5% CO2. Early passage is preferred.
The day before transfection, plate approximately 8 million cells per T-175 flask and plate two flasks per construct. Make sure cells are at optimal concentration (70%–80% confluency) at the time of transfection.
Prior to transfection, bring the transfection reagent, reduced serum medium, and plasmid DNA to room temperature.
Add 28 μg of pCMV-dR8.74 plasmid DNA, 4.7 μg of pCMV-VSVG plasmid DNA, and 37.3 μg of dCas9-Tet1/dTet1 or 19 μg of sgRNA plasmid DNA to a sterile microcentrifuge tube and vortex to mix thoroughly.
Note: All plasmids DNA should be prepared by endotoxin-free Maxi-prep kit (e.g., ZymoPURE II plasmid purification kit, Maxiprep).
For each construct, add 210 μL of transfection reagent to 5 mL of Opti-MEM I reduced serum medium. Incubate at room temperature for 5 min.
Add premixed plasmid DNA. Mix thoroughly. Incubate at room temperature for 15 min.
Add transfection complex to the cells in a dropwise manner (2.5 mL/flask). Gently shake or swirl the plate to ensure even distribution over the entire flask.
The next day, change to fresh medium. From this point forward, consider that everything that comes in contact with cells contains infectious lentivirus particles. Use BSL2 safety protocols with respect to PPE, decontamination of plasticware and media, etc.
Two days after medium change, harvest the medium and replace with fresh medium. Store the collected medium at 4 °C.
Two days after the first medium collection, harvest the medium. Store the collected medium at 4 °C.
Lentivirus concentration
Filter the collected medium with a 0.45 μm filter with a low protein binding membrane.
Spin down the lentivirus at 90,000× g for 1 h and 45 min at 4 °C.
Discard the supernatant. Resuspend the virus pellet in 200 μL of PBS per centrifuge tube.
Rotate the combined viruses in PBS at 4 °C overnight. Aliquot to the volume needed for single-use to avoid freeze-thaw cycles.
Lentivirus titration
Seed 1 × 104 HEK293T cells/well in a 96-well plate one day in advance.
Perform gradient dilution of the lentiviral particles to 1:10, 1:102, 1:103, 1:10 4, 1:105, and 1:106 in 100 μL of final volume in culture medium.
A total 100 μL of viral particle mixture should be added to each well with at least three replicates per virus.
Two days post infection, count the fluorescent positive cells using fluorescent microscopy and select the dilution factor with a proper fluorescent positive proportion (10%–30% positive cells/well). Count the triplicates and average the number of positive cells.
Estimate the functional lentivirus titer using the following formulation:
Virus titer (T) was calculated based on the infection efficiency for HEK293T cells, where T = (P*N)/(V), T = titer (TU/μL), P = % of infection positive cells according to the fluorescence marker, N = number of cells at the time of transduction, V = total volume of virus used.
Note: TU stands for transduction unit.
Lentivirus transduction:
Depending on the cell type, add virus to each well with a multiplicity of infection (MOI) of 10–100. For post-mitotic neurons, a high MOI of 50–100 is recommended to achieve high transduction efficiency and editing performance, while a MOI of 10 works well for dividing cells like HEK293T cells.
Incubate cells with viruses at 37 °C for 15 min.
Spin cells with viruses at 800× g for 1 h at room temperature (Qiagen 5810 with rotor A-2-DWP-AT).
Incubate for 24 h and then perform half media change.
Twenty-four hours later, do a full medium change.
Isolate infected cells by FACS or eliminate non-infected cells by chemical selection, depending on the selection marker on the used viral vector.
Harvest cells for further analysis.
Editing results examination by pyrosequencing
Extract genomic DNA of edited cells using Qiagen DNeasy blood & tissue kit or Qiagen QIAamp DNA micro kit following manufacturer’s instructions.
Perform bisulfite conversion of genomic DNA using EZ DNA Methylation-Gold kit following manufacturer’s instructions. Unmethylated cytosine (C) is converted to uracil during bisulfite conversion, which will be read as thymine (T) during PCR amplification. Methylated cytosine (mC) remains unchanged.
Pyrosequencing assay design.
The assay is designed using Qiagen PyroMark Assay Design software following software guidance.
Pick the high-scored primer set (forward and reverse PCR primer and sequencing primer) with no unspecific binding.
PCR to amplify the genomic region of interest from bisulfite-converted genomic DNA using PyroMark PCR kit. Set up the reaction on ice (Table 5):
Table 5. Reaction for pyro-PCR
Reagent Quantity
2× PyroMark PCR master mix 12.5 μL
10× CoralLoad Concentrate 2.5 μL
Forward primer (5 μM) 0.5 μL
Reverse primer (5 μM) 0.5
Nuclease-free water Up to 25
Bisulfite converted genomic DNA (10–20 ng)
Total 25 μL
Run the following program in a thermocycler (Table 6):
Table 6. Thermocycling conditions for the PCR reaction
Step Temp. (°C) Duration No. of cycles
Initial denaturation 98 1
Denaturation 98 10 s 45
Annealing 56 30 s
Extension 72
Final extension 72 1
Hold 4 ∞ -
Take a small aliquot of PCR product (10 μL) to run in an agarose gel. Make sure there is only one single and intense band with size matched as predicted.
Perform pyrosequencing to quantify the T:C ratio at each CpG site within assayed regions and convert into a percentage of DNA methylation.
Program the sequencing run using PyroMark Q48 Autoprep software following manufacturer’s instructions.
Add the reagents supplied by PyroMark Q48 Advanced CpG Reagents (4 × 48) kit (nucleotides, sequencing primer, buffer, substrate, and enzyme mix) into the appropriate wells of the cartridge according to the volume calculated by the sequencer software. Load PCR products into the corresponding wells of the pyrosequencing disc and place the disc into the sequencer.
Start the run.
Once the sequencing is done, analyze the results using PyroMark Q48 Autoprep software.
Data analysis
The analysis of DNA methylation by pyrosequencing has been reported previously [11,12]. We used the PyroMark Q48 Autoprep software by Qiagen. The result file generated by the sequencer was directly opened and analyzed by PyroMark Q48 Autoprep software with default settings. Figure 1 shows a typical program analyzing 11 CpGs in the promoter region of mouse Snrpn genes. The methylation percentage is displayed above bars highlighted in blue, which stands for every cytosine in the context of CpG within the assayed region. The background color of the percentage shows the quality assessment by the software: blue means passed QC, yellow for check, and red for failed. We normally only use the ones with high sequencing quality (blue and yellow). The bar with an orange background is the bisulfite treatment controls to assess successful bisulfite treatment; it is normally a cytosine site in non-CpG context and known to be unmethylated. As a result of successful bisulfite conversion reaction, this unmethylated cytosine will be fully converted to T after PCR and sequenced as T in pyrosequencing. Make sure the examined DNA is efficiently bisulfite converted.
Figure 1. Example program analyzing 11 cytosine-phosphate-guanosines (CpGs) in the promoter region of mouse Snrpn promoter. The y-axis shows the signal intensity in arbitrary units, while the x-axis represents the dispensation order. The dispensations corresponding to the cytosines of assayed CpGs are highlighted in blue. The methylation percentages at individual CpG site are displayed above the respective bars. The background color of percentage shows the quality assessment by the software: blue for passed, yellow for check, and red for failed.
To examine the editing outcome, compare the DNA methylation percentage of CpGs with high sequencing quality between samples with different treatment. If the targeted genomic region is successfully edited, the DNA methylation level of assayed CpGs from dCas9-Tet1 + target sgRNA-treated cells should be lower than the ones from untreated cells or dCas9-dTet1 + target sgRNA- or dCas9-dTet1 + scramble sgRNA-treated cells. For further examination of the direct outcome of DNA methylation change, RT-qPCR and western blotting may be needed to quantify the change in RNA and protein level of edited genes, respectively.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Qian et al. [10]. Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons. Science Translational Medicine (Figure 1, panel c).
General notes and troubleshooting
General notes
We design all the possible sgRNAs based on NGG pam availability within the target region and then run these sgRNAs via online design programs such as CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) and Off-Spotter (https://cm.jefferson.edu/Off-Spotter/) to pick up the ones with high on-target score by CRISPRa mode and lower off-target prediction. Two critical points to consider are: 1) the editing window for dCas9-Tet1 described here is within 150–200 bp downstream of PAM site and 2) the CpG sites within the sgRNA target sequence are often protected against methylation editing.
Compared to classical CRISPR-Cas9 knockout systems where sgRNAs are always designed within coding regions to disrupt gene expression, the sgRNAs for demethylation editing are recommended to be designed upstream of the transcription start site to avoid dCas9-mediated steric hinderance and transcription interference.
In order to achieve the highest editing efficiency, a small screening may be conducted to test multiple sgRNAs and their combinations to identify the most effective sgRNA(s).
The expression level of dCas9-Tet1 is critical to achieving high editing efficiency. The period of dCas9-Tet1 expression is critical to control off-target effects. Therefore, choosing the proper expression system (inducible or constitutive) and controlling the expression time for editors (dCas9-Tet1 and sgRNA) are key to specifically edit DNA methylation in a variety of targeted cells.
Demethylation in neurons is slower than in dividing cells. A high titer of lentiviral editors expressing dCas9-Tet1 and sgRNA are required to edit methylation in neurons. To avoid neuronal cell death upon lentivirus transduction, a high density of neuronal culture is preferred. Alternatively, a Dox-inducible dCas9-Tet1 expression cassette can be engineered in hESCs, and editing DNA methylation in neurons can be achieved after neuronal differentiation.
The dynamics of dCas9-Tet1-mediated demethylation largely depends on cell types. In general, the editing effect reaches its peak after 5–7 days of editing. However, it could be slower in non-dividing cells such as post-mitotic neurons, as both passive and active demethylation can operate in dividing cells, but only active demethylation occurs in cells that are not dividing.
The duration and reversibility are locus-specific and influenced by local chromatin environment as well as cell type. In general, edited DNA demethylation can be maintained in dividing cells but is not stable in post-mitotic cells such as neurons. For instance, the demethylation status of FMR1 on the active X chromosome can be maintained in vitro for weeks [9], whereas the demethylation status of MECP2 on the inactive X chromosome is not stable in post-mitotic neurons [10].
When doing lentivirus transduction, the MOIs will depend on the purpose of each experiment and the type of cell lines. We used higher MOIs (50–100) for post-mitotic neurons and lower MOIs (approximately 10) for dividing cells such as HEK293. Please note that this MOI is based on the virus titer (T) that is calculated based on the infection efficiency for HEK293T cells, where T = (P*N)/(V), T = titer (TU/µL), p = % of infection positive cells according to the fluorescence marker, N = number of cells at the time of transduction, V = total volume of virus used. Note that TU stands for transduction unit.
Troubleshooting
Problem 1: The efficiency of methylation editing is low as measured by pyrosequencing.
Possible cause: The titer of lentiviral dCas9-Tet1 is too low to induce methylation editing.
Solution: Increase the titer of lentiviral dCas9-Tet1 by performing ultracentrifugation of at least 38 mL of viral supernatant harvested from transfected HEK293T cells and then reconstituting in 100 μL of PBS for effective infection to deliver dCas9-Tet1 into targeted cells.
Acknowledgments
Funding was obtained from the NIH grant R00MH113813 (X.S.L.), Rett Syndrome Research Trust grant (X.S.L.), and Columbia University Startup grant UR011118 (J.Q. and X.S.L.). This protocol is previously described and validated in Qian et al. [10]. Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons. Science Translational Medicine. The graphical abstract was created with BioRender.com.
Competing interests
X.S.L. is cofounder of Epitor Therapeutics.
References
Jaenisch, R. and Bird, A. (2003). Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33: 245–254. https://doi.org/10.1038/ng1089.
Smith, Z. D. and Meissner, A. (2013). DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14(3): 204–220. https://doi.org/10.1038/nrg3354.
Mattei, A. L., Bailly, N. and Meissner, A. (2022). DNA methylation: a historical perspective. Trends Genet. 38(7):676–707. https://doi.org/10.1016/j.tig.2022.03.010.
Greenberg, M. V. C. and Bourc'his, D. (2019). The diverse roles of DNA methylation in mammalian development and disease. Nat. Rev. Mol. Cell Biol. 20(10): 590–607. https://doi.org/10.1038/s41580-019-0159-6.
Holtzman, L. and Gersbach, C. A. (2018). Editing the Epigenome: Reshaping the Genomic Landscape. Annu. Rev. Genomics Hum. Genet. 19: 43–71. https://doi.org/10.1146/annurev-genom-083117-021632.
Liu, X. S. and Jaenisch, R. (2019). Editing the Epigenome to Tackle Brain Disorders. Trends Neurosci. 42(12): 861–870. https://doi.org/10.1016/j.tins.2019.10.003.
Nakamura, M., Gao, Y., Dominguez, A. A. and Qi, L. S. (2021). CRISPR technologies for precise epigenome editing. Nat. Cell Biol. 23(1): 11–22. https://doi.org/10.1038/s41556-020-00620-7.
Liu, X. S., Wu, H., Ji, X., Stelzer, Y., Wu, X., Czauderna, S., Shu, J., Dadon, D., Young, R. A. and Jaenisch, R. (2016). Editing DNA Methylation in the Mammalian Genome. Cell 167(1): 233–247.e217. https://doi.org/10.1016/j.cell.2016.08.056.
Liu, X. S., Wu, H., Krzisch, M., Wu, X., Graef, J., Muffat, J., Hnisz, D., Li, C. H., Yuan, B., Xu, C., et al. (2018). Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene. Cell 172(5): 979–992.e976. https://doi.org/10.1016/j.cell.2018.01.012.
Qian, J., Guan, X., Xie, B., Xu, C., Niu, J., Tang, X., Li, C. H., Colecraft, H. M., Jaenisch, R. and Liu, X. S. (2023). Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons. Sci. Transl. Med. 15(679): eadd4666. https://doi.org/10.1126/scitranslmed.add4666.
Tost, J. and Gut, I. G. (2007). DNA methylation analysis by pyrosequencing. Nat. Protoc. 2(9): 2265–2275. https://doi.org/10.1038/nprot.2007.314.
Delaney, C., Garg, S. K. and Yung, R. (2015). Analysis of DNA Methylation by Pyrosequencing. Methods Mol. Biol. 1343: 249–264. https://doi.org/10.1007/978-1-4939-2963-4_19.
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Original Research Article:
The authors used this protocol in Science Advances May 2023
Abstract
The CTC1-STN1-TEN1 (CST) complex is a single-strand DNA-binding protein complex that plays an important role in genome maintenance in various model eukaryotes. Dysfunction of CST is the underlying cause of the rare genetic disorder known as Coats plus disease. In addition, down regulation of STN1 promotes colorectal cancer development in mice. While prior studies have utilized RNAi to knock down CST components in mammalian cells, this approach is associated with off-target effects. Attempts to employ CRISPR/Cas9-based knockout of CST components in somatic cell lines have been unsuccessful due to CST's indispensable role in DNA replication and cell proliferation. To address these challenges, we outline a novel approach utilizing a Cre-loxP-based conditional knockout in mouse embryonic fibroblasts (MEFs). This method offers an alternative means to investigate the function and characteristics of the CST complex in mammalian systems, potentially shedding new light on its roles in genome maintenance.
Key features
• Conditional depletion of mammalian STN1 using mouse embryonic fibroblast (MEFs).
• Analysis of oxidative damage sensitivity using STN1-depleted MEFs.
• This protocol requires Stn1flox/flox mice.
Keywords: STN1 CST Conditional knockout Mouse embryonic fibroblast (MEFs) Genome maintenance Oxidative damage 4-Hydroxytamoxifen (4-OHT) treatment
Graphical overview
Mouse embryonic fibroblast (MEF) isolation procedure. (Images created using BioRender)
Background
The CTC1-STN1-TEN1 (CST) complex is a single-stranded DNA-binding protein complex essential in several genome maintenance pathways [1–10]. Mutations in CST components, specifically CTC1 (Conserved Telomere Maintenance Component 1) and STN1 (homolog of yeast Stn1, also known as oligonucleotide/oligosaccharide binding fold containing 1 or OBFC1, and alpha accessory factor 44 or AAF44), have been implicated in the pathogenesis of Coats plus syndrome and Dyskeratosis congenita. Coats plus syndrome is a multisystem disorder with manifestations such as retinopathy, stunted growth, and early-onset aging signs [11–13], whereas Dyskeratosis congenita presents with bone marrow failure and notable skin changes [13,14].
Initially identified as co-factors for DNA polymerase α (POLα), CST's interaction with replication machinery, including the MCM helicase complex, signifies its broader regulatory role in DNA replication [15]. Beyond POLα regulation, CST plays a critical role in telomere maintenance by binding to telomeric ssDNA and facilitating C-strand fill-in, thereby preventing telomere over-extension by telomerase and maintaining telomere stability [1,3,5,16–19]. The importance of CST is further highlighted under replication stress conditions, where it protects nascent DNA at stalled replication forks from MRE11 (meiotic recombination 11 homolog A)-mediated degradation [20]. This protection is partly due to CST interaction with RAD51 (DNA repair protein RAD51 homolog 1). CST promotes RAD51 recruitment to replication forks to protect fork stability under stress [8,9,20,21]. Beyond DNA replication, CST is recruited to double-strand breaks by the Shieldin complex, favoring the non-homologous end joining pathway, especially in BRCA1-deficient cells. CST loss in such contexts leads to increased end resection and resistance to PARP inhibitors in BRCA1-deficient cells. Thus, it appears that CST participates in cellular response to PARP inhibitors, especially in BRCA1-deficient tumors [22–25].
Genome-wide association studies have identified that STN1 variants are linked to increased risk of multiple types of cancer [26–31]. Analyses from the TCGA Pan-Cancer project show that decreased CST expression is associated with adverse clinical outcomes [32,33]. In colorectal cancer, CTC1/STN1 expression is markedly reduced, correlating with higher tumor mutation burdens and poorer survival, suggesting their potential as prognostic biomarkers [34].
The crucial role of CST in the development of genetic diseases and cancer underscores the urgency of understanding its functions in genome maintenance pathways. Traditionally, most studies investigating the role of the CST complex in mammalian cells have utilized RNAi to reduce the expression of CST components. However, the effectiveness of RNAi methods may be limited due to unintended off-target effects. Here, we describe an alternative approach using a conditional knockout strategy in mouse embryonic fibroblasts (MEFs) to examine the function of Stn1. This method involves the utilization of tamoxifen-inducible Cre recombinase to delete the promoter region and the ATG start codon of the murine Stn1 gene. The tamoxifen-inducible nature of the Cre recombinase enables researchers to grow cells before administering tamoxifen and control the timing of Stn1 deletion. By employing this method, it becomes possible to specifically deplete the mammalian Stn1 protein, leading to the destabilization of the CST complex. Additionally, the study demonstrates that conditional knockout of Stn1 renders cells more susceptible to damage caused by hydrogen peroxide exposure, emphasizing the significance of the CST complex in countering DNA damage during replication stress and oxidative stress.
Materials and reagents
Biological materials
CreERT2 C57BL/6 mice harboring the conditional CreERT2 transgene (Jackson Laboratory, stock 004682)
Homozygous Stn1flox/flox (Stn1F/F) C57BL/6 mice (generated as described in Nguyen et al. [34])
CreERT2; Stn1F/F and control Stn1F/F C57BL/6 mice: generated by crossing with Stn1F/F with CreERT2 C57BL/6 mice to generate progeny heterozygous for Stn1Flox allele and hemizygous for the CreERT2 genotype. These mice were then bred to homozygous Stn1F/F mice to derive Stn1 conditional knockout CreERT2; Stn1F/F and control Stn1F/F genotypes as described previously [34].
Reagents
DMEM/F-12 (Gibco, catalog number: 11320033)
Fetal bovine serum (FBS) (Biowest, catalog number: S1620)
Antibiotic/antimycotic solution (HyClone, catalog number: SV30079.01)
Trypsin-EDTA 0.05% and 0.025% (Gibco, catalog number: 2530062)
DMSO (Sigma, catalog number: D-5879)
4-Hydroxytamoxifen (4-OHT) (VWR, catalog number: 89152-604)
KOD Hot Start DNA Polymerase kit (Millipore Sigma, catalog number: 710863, solutions provided in the kit)
Oligos:
oIMR5984: 5'-GCTAACCATGTTCATGCCTTC-3' (Cre transgene forward)
oIMR8744: 5'-CAAATGTTGCTTGTCTGGTG-3' (Cre transgene reverse)
oIMR8745: 5'-GTCAGTCGAGTGCATAGTTT-3' (Cre internal positive control forward)
oIMR9074: 5'-AGGCAAATTTTGGTGTACGG-3' (Cre internal positive control reverse)
Stn1loxPShift-F: 5'-TGTAATCCCAGCGCTCAGGAG-3'
Stn1loxPShift-R: 5'-GATCTGACAGAGATCTCCTGGCT-3'
Obfc1 5'wtF2: 5'-GACTCCTTAGCCCCAGATCTCCGTCAT-3'
Obfc1 5'mtR: 5'-CTTCGTATAGCATACATTATACGAAGTTATGGATCCACCGACT-3'
Obfc1 5'mtF: 5'-CCACATAGTCGGTGGATCCATAACTTCGT-3'
Obfc1 5'wtR: 5'-GGGCTAGGGTGTAGCTCAATGGTAGA-3'
Obfc1 3'wtF: 5'-CTCTGGGGAGACTGTCTCAGATGTTCA-3'
Obfc1 3'mtR: 5'-GAGGCTCAGGATCCATAACTTCGT-3'
Obfc1 3'mtF: 5'-GCCAGCCTGGTCTACGTGACAATAACT-3'
Obfc1 3'wtR: 5'-GGTCATATGGCCTCACCCTCTAAGA-3'
Glycerol (Fisher Scientific, catalog number: BP-229-1)
Agarose (Sigma-Aldrich, catalog number: A9539-500G)
DTT (Sigma-Aldrich, catalog number: D0632-5G)
Anti-STN1 antibody (Santa Cruz, catalog number: sc-376450), 1:500 dilution
Anti-β-actin (Sigma, catalog number: A2228) 1:60,000 dilution
Secondary horseradish peroxidase antibody: TrueBlot anti-mouse IgG HRP conjugated (Rockland, catalog number: 18-8817-30) 1:2,000 dilution
SuperSignal West Femto (Thermo Fisher, catalog number: 34095)
Hydrogen peroxide (30%, ACS grade) (Ward’s Science, catalog number: 470301)
Crystal Violet (Sigma, catalog number: C3886)
CHAPS (Fisher Scientific, catalog number: BP571-5)
Ethanol (Sigma-Aldrich, catalog number: 459844-4L)
Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500)
Bromophenol Blue (Sigma, catalog number: B-8026)
NaOH (Fisher Scientific, catalog number: S318-500)
NaCl (Fisher Scientific, catalog number: S271-1)
Tris-HCl (Sigma-Aldrich, catalog number: T3253-500G)
Na2HPO4 (VWR, catalog number: 0404-500G)
KH2PO4 (J.T. Baker, catalog number: 3246-01)
KCl (EMD, catalog number: PX1405-1)
Tween-20 (VWR, catalog number: M147-1L)
Non-fat dry milk (Nestle, catalog number: 12428935)
Solutions
2 mM 4-OHT stock solution (see Recipes)
DNA isolation lysis buffer (see Recipes)
DNA isolation neutralization buffer (see Recipes)
Phosphate-buffered saline (PBS), pH 7.4 (see Recipes)
CHAPS lysis buffer (see Recipes)
Loading buffer (see Recipes)
Crystal violet staining solution (see Recipes)
1% SDS in 70% Ethanol
PBS-Tween (PBST)
DMEM/F-12 culture medium (see Recipes)
Recipes
All solutions are prepared with either autoclaved Milli-Q water or DMSO.
2 mM 4-OHT stock solution
Reagent Final concentration Quantity or Volume
4-OHT 2 mM 7.75 mg
DMSO n/a 10 mL
Total n/a 10 mL
Store at -20 °C. Protect from light. For long-term storage, store the solution at -80 °C.
DNA isolation lysis buffer
Reagent Final concentration Quantity or Volume
NaOH 25 mM 20 mg
EDTA 0.2 mM 1.17 mg
H2O n/a 20 mL
Total n/a 20 mL
Store at room temperature (RT).
DNA isolation neutralization buffer (pH 5.5)
Reagent Final concentration Quantity or Volume
Tris-HCl 40 mM 69.9 mg
H2O n/a 20 mL
Total n/a 20 mL
Store at RT.
PBS
Reagent Final concentration Quantity or Volume
NaCl 137 mM 8 g
KCl 2.7 mM 0.2 g
Na2HPO4 10 mM 1.44 g
KH2PO4 1.8 mM 0.24 g
H2O n/a 1,000 mL
Total n/a 1,000 mL
Store at RT.
CHAPS lysis buffer
Reagent Final concentration Quantity or Volume
CHAPS 10% 1% 10 mL
Tris-HCl pH 7.4 30 mM 363.4 mg
NaCl 150 mM 876.6 mg
DTT 200 mM 3 g
H2O n/a 90 mL
Total n/a 100 mL
Store at -20 °C.
Loading buffer
Reagent Final concentration Quantity or Volume
SDS 20% 4% 20 mL
Tris-HCl pH 6.8 100 mM 1.21 g
Bromophenol Blue 0.2% 0.2 g
Glycerol 20% 20 mL
H2O n/a 60 mL
Total n/a 100 mL
Store at RT.
Crystal violet staining solution
Reagent Final concentration Quantity or Volume
Crystal Violet 0.5% 2.5 g
Ethanol n/a 500 mL
Total n/a 500 mL
Store at RT.
1% SDS in 70% Ethanol
Reagent Final concentration Quantity or Volume
SDS 20% 1% 50 mL
Ethanol 70% 700 mL
H2O n/a 250 mL
Total n/a 1,000 mL
Store at RT.
PBST
Reagent Final concentration Quantity or Volume
NaCl 137 mM 8 g
KCl 2.7 mM 0.2 g
Na2HPO4 10 mM 1.44 g
KH2PO4 1.8 mM 0.24 g
Tween-20 50% 0.05% 1 mL
H2O n/a 999 mL
Total n/a 1,000 mL
Store at RT.
DMEM/F-12 culture medium
Reagent Final concentration Quantity or Volume
DMEM/F-12 n/a 500 mL
FBS
100× antibiotic/antimycotic
13%
1×
75 mL
5 mL
Total n/a 580 mL
Laboratory supplies
96-well plates (Genesee Scientific, catalog number: 25-109)
6-well plates (VWR, catalog number: 10062-892)
10 cm tissue culture dishes (Nest Biotechnology, catalog number: 704201)
PVDF membrane (MilliporeSigma, catalog number: ISEQ00010)
Forceps (Fisher Scientific, catalog number: 08-880)
Dissecting scissors (Fisher Scientific, catalog number: 08-940)
Scalpel (Fisher Scientific, catalog number: 12-000-164 and 12-000-161)
Equipment
Inverted microscope (Fisherbrand, model: 03000013)
SDS-PAGE and western blot transfer apparatus (Bio-Rad, model: Mini-PROTEAN Tetra System)
Thermocycler (Bio-Rad, model: T100 Thermal Cycler)
iBright imaging system (Thermo Fisher, model: CL1000)
Plate reader (BioTek, model: Synergy H1)
Software and datasets
GraphPad Prism v9.3 (GraphPad, 11/15/2021)
ENSEMBL Mouse genome browser 110 (https://useast.ensembl.org/Mus_musculus/Info/Index)
Procedure
Design of CRISPR/Cas9 to insert loxP sites
The mouse Stn1 locus contains 10 exons, with the ATG start codon residing in exon 2 (Figure 1A). When loxP insertion sites were initially designed, the most convenient approach to delete murine Stn1 appeared to be deleting exons 5 and 6, which would create a frameshift and generate a truncated STN1 protein. However, a long non-coding RNA (lncRNA) with unknown function is present in this region. To avoid affecting this lncRNA, we targeted the upstream regions of the Stn1 gene by inserting two loxP sites upstream of the promoter region and downstream of the ATG start codon in exon 2 (Figure 1A). Sequence information and the exact loxP insertion sites are provided in Supplementary File 1. Cre expression resulted in the deletion of the promoter sequence and the translation start site, thereby inhibiting both transcription and translation of the gene (Figure 1A). Deleting the promoter region would also eliminate the possible complications caused by a truncated protein that could be synthesized from a downstream ATG codon. However, we have found that the inducible Stn1 deletion is incomplete, likely due to inefficient recombination caused by the long distance between two loxP sites, resulting in partial knockout or depletion of STN1.
Isolation of mouse embryonic fibroblasts
Set up the breeding pairs of CreERT2; Stn1F/F and Stn1F/F C57BL/6 mice, either male CreERT2; Stn1F/F paired with female Stn1F/F, or male Stn1F/F paired with female CreERT2; Stn1F/F. We have not observed breeding difference.
Euthanize 12–14 days (est.) pregnant mouse with CO2 followed by cervical dislocation.
Critical: Before transfer under the cell culture hood, spray mouse down thoroughly with 70% ethanol.
Open the abdominal cavity with sterile scissors. Be careful not to disturb uterine horns.
Note: Lifting the skin with a sterile forceps before incision and doing the same with the abdominal wall ensures no accidental damage.
Critical: It is important to keep any hair from being introduced into subsequent steps. Change instruments if necessary.
Extract embryos still within the uterine horns by gently lifting them with forceps and using scissors to cut them free close to the pelvis. Then, place them in a 10 cm dish with ~10 mL of PBS prewarmed to 37 °C.
Note: If they do not lift out freely, carefully check for locations to cut.
Critical: It is important not to tear or cut any part of the bowels to avoid bacterial contamination.
Cut open uterus and extract embryos still within amniotic sac; separate embryos by cutting the area between each and remove each embryo while keeping amniotic sac intact.
Open the amniotic sac with forceps and sterile scalpel, remove the embryo, and sever the head above the eye line. Place the severed head in a separate tube on ice for later DNA extraction.
Remove red organs with scalpel and place the remaining tissue in 2 mL of 0.05% trypsin in a 6-well dish.
Gently but thoroughly disrupt tissue with scissors until no piece larger than 1 × 1 × 1 mm remains.
Repeat steps B5–7 for each embryo and place dishes into incubator at 37 °C with 5% CO2 for 10 min.
Note: Start incubation within less than 5 min of processing the first embryo, depending on the number of embryos and time required for each. Incubation for separate batches may need to be staggered.
Critical: Swift processing is recommended to minimize cell death.
Gently pipette cell suspension up and down with a P1000 to further disassociate cells mechanically before they are transferred into 2 mL of DMEM/F-12 culture medium and resuspended again.
After 5 min, transfer the supernatant to a new tube, leaving the larger tissue fragments at the bottom of the tube.
Spin down the new tube at 500× g for 2 min and discard the supernatant; resuspend the cell pellet in 2 mL of fresh DMEM/F-12 culture medium, transfer into a 6-well dish, and place into the incubator overnight.
Note: Be careful not to mix or pool cells from different embryos.
The next day, replace the medium with fresh medium.
Monitor cells until each well reaches full confluency (1–2 days later) and transfer into a 10 cm dish for expanding and subsequent freezing down of cells.
Validation of genotype
Place a small piece of the tissue from cell isolation (see B6) in 50 μL of DNA isolation lysis buffer (see Recipe 2) and incubate at 98 °C for 1 h.
After incubation, add 50 μL of DNA isolation neutralization buffer (see Recipe 3), pipette the mixture up and down thoroughly to disrupt the rest of the tissue, and transfer into a fresh tube, leaving behind any significant tissue clumps.
Pause Point: DNA can be stored at -20 °C until needed.
Use 1 μL each of the final mixture to run a PCR reaction with Cre transgene primer pairs and Stn1flox primer pairs.
To determine if animals contain the CreERT2 transgene, the PCR reactions should contain:
1 μL lysate
2.5 μL dNTPs (2.5 mM stock)
2.5 μL 10× buffer for KOD Hot Start
1.5 μL oIMR5984 (10 μM)
1.5 μL oIMR8744 (10 μM)
1.5 μL oIMR8745 (10 µM)
1.5 μL oIMR9074 (10 µM)
0.5 μL KOD
1.5 μL MgSO4 (50 mM stock)
1.25 μL DMSO
9.75 μL H2O
25 μL Total
Run the PCR under the following thermocycler conditions:
95 °C for 5 min
95 °C for 30 s
55 °C for 30 s
72 °C for 1 min
Go to 2) for 31 cycles
72 °C for 5 min
10 °C infinite hold
To determine if the animal is homozygous or heterozygous of the Stn1 flox allele, the PCR reactions should contain:
1 μL lysate
2.5 μL dNTPs (2.5 mM stock)
2.5 μL 10× buffer for KOD Hot Start
1.5 μL STN1loxPShift-F (10 μM)
1.5 μL STN1loxPShift-R (10 μM)
0.5 μL KOD
1.5 μL MgSO4 (50 mM stock)
1.25 μL DMSO
13 μL H2O
Run the PCR under the following thermocycler conditions:
95 °C for 5 min
95 °C for 20 s
60 °C for 30 s
72 °C for 40 s
Go to 2) for 34 cycles
72 °C for 2 min
10 °C infinite hold
(Optional) To validate if the animal contains the loxP insertions in the correct Stn1 loci, four additional PCR reactions can be performed:
1 μL lysate
2.5 μL ldNTPs (2.5 mM stock)
2.5 μL 10× buffer for KOD Hot Start
1.5 μL Primer a, c, e, or g (10 μM) (see primer notes below)
1.5 μL Primer b, d, f, or h (10 μM) (see primer notes below)
0.5 μL KOD
1.5 μL MgSO4 (50 mM stock)
1.25 μL DMSO
13 μL H2O
Run the PCR under the following thermocycler conditions:
95 °C for 5 min
95 °C for 20 s
60 °C for 30 s
72 °C for 40 s
Go to 2) for 34 cycles
72 °C for 2 min
10 °C infinite hold
Notes:
Primer pair #1: produces a 547 bp product if 5' loxP is present.
Primer a: Obfc1 5'wtF2, anneals upstream of the 5' loxP site (Figure 1A)
Primer b: Obfc1 5'mtR, anneals at the 5' loxP site (Figure 1A )
Primer pair #2: produces a 226 bp product if 5' loxP is present.
Primer c: Obfc1 5'mtF, anneals at the 5' loxP site (Figure 1A )
Primer d: Obfc1 5'wtR, anneals downstream of the 5' loxP site (Figure 1A)
Primer pair #3: produces a 335 bp product if 5' loxP is present.
Primer e: Obfc1 3'wtF, anneals upstream of the 3' loxP site (Figure 1A)
Primer f: Obfc1 3'mtR, anneals at the 3' loxP site (Figure 1A )
Primer pair #4: produces a 550 bp product if 5' loxP is present.
Primer g: Obfc1 3'mtF, anneals at the 3' loxP site (Figure 1A )
Primer h: Obfc1 3'wtR, anneals downstream of the 3' loxP site (Figure 1A)
Figure 1. Genotyping of the Stn1F/F allele and the CreERT2 transgene. A. Targeting construct encoding the murine Stn1 gene, which contains 10 exons. Yellow arrowheads designate loxP sites. Arrows designate PCR primer sites. Open box: non-coding exon. The Cre protein is a site-specific DNA recombinase that recombines a pair of loxP sequences. The Cre enzyme binds to the loxP sites and brings the two sites together, catalyzing a recombination event between them, resulting in the excision of the DNA sequence between the two loxP sites. The excised DNA is usually degraded, and the ends of the remaining DNA are ligated together. B. Example of PCR products for detecting the presence of the Stn1F/F allele. C. Example of PCR products for detecting the CreERT2 transgene.
Add 5 μL of 6× loading buffer (see Recipe 6) to each PCR reaction and load 12 μL of the mixture onto a 2.5% agarose electrophoresis gel. Run at 120 V for 1 h.
Pause Point: PCR reactions can be stored at -20 °C for several days before electrophoresis.
Expected results and interpretation: The WT allele will produce a 184 bp PCR product, and the recombined flox allele will produce a 218 bp product. If the PCR reaction yields a single 218 bp band, it indicates that the animal is Stn1F/F; a single 184 bp PCR product means that the animal is Stn1 +/+; and a double band of 184 bp and 218 bp means the animal is Stn1F/+ (Figure 1B). The presence of the CreERT2 transgene generates a double band (Figure 1C).
Cultivation of MEFs and induced knockout of Stn1
Grow MEF cells in DMEM/F-12 culture medium at 37 °C with 5% CO2.
Passage cells when they reach near or full confluency at a 1:4 split ratio.
Note: When cell confluency is too low after passaging, cells take a long time to grow.
To induce Cre expression to deplete Stn1, add 4-OHT into media to a final concentration of 2 μM for 48–96 h.
Immortalization of MEFs
Primary cells are spontaneously immortalized by continuous passaging in DMEM/F-12 medium at 37 °C with 5% CO2 for several months (20+ passages). Surviving cells that continue to grow and do not senesce can be considered immortalized. Immortalized cells exhibit slightly different morphology (Figure 2).
To induce Cre expression to knockout Stn1, add 4-OHT into media at 2 μM for 48–96 h.
Figure 2. Cell morphology of primary and immortalized mouse embryonic fibroblasts (MEFs). A. Primary MEF cells tend to grow in aggregates. B. Immortalized MEFs grow more evenly distributed and spread out, cells are contracted and round, and spindles are narrower compared to primary MEFs. Images were taken with Zeiss inverted microscope Observer with a 10× phase contrast lens.
Analysis of protein levels after induction
Extract proteins from 20,000 cells by resuspending cells in 15 μL of CHAPS lysis buffer (see Recipe 5) and incubating on ice for 30 min. Then, spin down at 12,000× g for 15 min at 4 °C and mix the supernatant 1:1 with 2× loading buffer.
Load samples onto 10% SDS-PAGE gel and run at 160 V for 60 min in SDS gel running buffer.
Note: Exact run time may differ; refer to the location of the loading buffer band and run until it is near the bottom edge of the gel.
Transfer proteins from the gel onto a PVDF membrane in a Bio-Rad transfer chamber at ~100 V and 400 mA for 70 min.
Block membrane in PBST buffer + 5% non-fat milk for 1 h at RT.
Incubate the membrane in primary antibody (anti-STN1 and anti-β-actin) in PBST at 4 °C overnight.
Wash membrane five times in PBST for 10 min each.
Incubate the membrane in secondary HRP antibody in 5% non-fat milk PBST buffer at RT for 1 h.
Wash the membrane five times in PBST for 10 min each.
Apply SuperSignal West Femto mixture diluted 1:5 in ddH2O to the wet membrane.
Capture western blot signal with iBright CL1000 imaging system. Examples of western blot signals are shown in Figure 3A and 3B.
Figure 3. Induction of STN1 depletion leads to cell sensitivity to oxidative stress. A. Primary Stn1F/F and CreERT2, Stn1F/F mouse embryonic fibroblast (MEF) clones were treated with 4-OHT (2 μM) for 48 h. STN1 protein expression was analyzed by western blotting. B. Spontaneously immortalized Stn1F/F and Cre, Stn1F/F MEF clones were treated with 4-OHT (2 μM) for 48 h. STN1 protein expression was analyzed by western blotting. C. Immortalized MEF clones were seeded to 35 mm dishes and then treated with 4-OHT for 48 h. Cells were then trypsinized and seeded into 96-well plates with 5,000 cells per well. Once cells were attached, hydrogen peroxide was added at the indicated concentration for 24 h. Cell viability was determined by Crystal Violet colorimetric assay. Two independent experiments were performed. Representative results from one experiment are shown. Error bars: S.D.
Testing cell sensitivity to hydrogen peroxide
Grow MEFs in culture, trypsinized, and seed 5,000 cells into each well of a 96-well plate, following induction according to step D3. Maintain 4-OHT in the media. Seed cells in six wells for each sample and each treatment condition.
After cells have settled and have been allowed to attach to dishes for approximately 3 h, add hydrogen peroxide and incubate the plate at 37 °C and 5% CO2 overnight.
Remove media with a multichannel pipette, wash gently with 200 μL of PBS per well, and remove PBS.
Note: To avoid accidentally detaching cells, PBS should be added slowly along the side of the wells and in the same manner across the whole plate. Removal of the media and PBS should be done slowly with the plate slightly tilted and the pipette tip in one corner of the wells.
Add 100 μL of Crystal Violet solution (see Recipe 7) to each well and incubate for 15 min at room temperature.
Pour out the Crystal Violet solution and blot firmly against paper towels.
Add ddH2O with the multichannel pipette, pour out, and firmly blot against paper towels.
Immerse the plate in ddH2O for 15 min at RT, pour out, and blot against paper towels.
Air dry the plate until completely dry.
Pause Point: Dried plates can be stored in a dry and dark place at RT overnight if necessary.
Add 200 μL of 1% SDS in 70% ethanol (see Recipe 8) per well and carefully resuspend and mix each well with a multichannel pipette without introducing bubbles.
Measure absorbance at 570 nm with a plate reader. Results are shown in Figure 3C .
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Nguyen et al. [34]. Deficiency in mammalian STN1 promotes colon cancer development via inhibiting DNA repair. Science Advances (Figure 4, Figure 6, panels D and E, Figure 7, panel A).
General notes and troubleshooting
Troubleshooting
Problem 1: MEF cells grow slowly.
Possible cause: After a number of passages, primary MEFs senesce.
Solution: Use early passaged MEF cells. It is recommended to freeze down several aliquots of early-passaged primary MEFs for later use.
Problem 2: High background in anti-Stn1 western blot.
Possible cause: Use of regular HRP-conjugated secondary anti-mouse IgG antibody.
Solution: Use the TrueBlot HRP-conjugated anti-mouse secondary antibody.
Problem 3: Immortalized MEF cells lose Stn1 depletion.
Possible cause: We initially used SV40 large T-antigen to immortalize MEFs. However, we have found that MEFs immortalized with this method do not respond to 4-OHT treatment and appear to have lost STN1 depletion. Although the exact cause of this loss is unclear, we suspect that SV40 large T-antigen alters the repair of double-strand breaks, which could influence Cre-loxP recombination efficiency and precision. In addition, large T antigen expression leads to higher mutations and chromosome aberrations, which could impact the sites where Cre recombination acts.
Solution: Use spontaneously immortalized MEFs. Do not use SV40 large T-antigen to immortalize MEFs.
Problem 4: Inefficient STN1 depletion after Cre induction.
Possible cause: Tamoxifen treatment time is not long enough, or tamoxifen concentration is incorrect.
Solution: Cells need to be treated with 4-OHT for at least 48 h to achieve efficient Stn1 depletion. Check the 4-OHT concentration to make sure that it is correct.
Acknowledgments
We thank Duc Dinh Nguyen, Eugene Kim, Thanh Nguyen, and Olga Shiva for technical assistance. This protocol was described and validated in the original research paper: DOI: 10.1126/sciadv.add8023.
Competing interests
Authors declare no financial or non-financial competing interests.
Ethical considerations
Animals were housed and studied in specific pathogen-free animal facilities at Loyola University Chicago Health Sciences campuses. All studies were approved by Institutional Animal Care and Use Committee (IACUC).
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Supplementary information
The following supporting information can be downloaded here:
Sequence of murine Stn1 (aka OBFC1) locus including the upstream region. Location of the loxP insertion sites and inserted loxP sequence are highlighted. Long red arrows indicate the location of primers used in genotyping.
Article Information
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in Sporisorium reilianum
JW Janina Werner
WZ Weiliang Zuo
Gunther Doehlemann
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4978 Views: 681
Reviewed by: Shweta PanchalEugenio LlorensXiaofei Liang
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Abstract
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum.
Key features
• First CRISPR/Cas9 application in S. reilianum.
• Generation of S. reilianum mutants without genomic integration of resistance marker.
• Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.
Keywords: CRISPR/Cas9 Ribonucleoprotein Knockout Marker-free S. reilianum Smut fungi
Background
The smut fungi consist of more than 1,500 species, being highly economically important due to their infection of relevant crops such as barley, sorghum, wheat, and maize [1]. The majority of smut fungi infect their host systemically through the roots and replace the inflorescences with teliospores without causing symptoms during early infection stages [2,3]. One example of this systemic infection is Sporisorium reilianum f. sp. zeae, which is the causal agent of maize head smut. S. reilianum is closely related to the intensively investigated model organism Ustilago maydis. However, they differ in their mode of infection as well as in the site of symptom development [4,5]. In 2010, a genome sequence of S. reilianum f. sp. zeae was published, which, together with the U. maydis genome, provided the foundation for systematic identification and genetic manipulation of effector genes contributing to virulence [6,7]. Genome comparison of U. maydis and S. reilianum revealed conserved effector genes even though they differ drastically in their pathogenesis on the same host, Zea mays. To characterize effector genes and their contribution to virulence, knock-out mutants are generated and compared to the wild type. In the past, U. maydis knock-out mutants were generated using PCR-amplified donor templates with resistance markers for gene replacements [8]. Importantly, it was shown that not only the genomic locus but also the integration of resistance markers can negatively influence the expression of reintegrated genes [9]. Recently, the mutagenesis of U. maydis was drastically improved with a marker-free approach using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) [10,11] and further developed for a seamless gene conversion approach [12]. In contrast to U. maydis, the generation of knockout mutants in S. reilianum is still dependent on resistance markers, and multiple gene knockouts are hampered by the limited number (i.e., carboxine, hygromycin, nourseothricin, and phleomycin) of available resistance markers [8]. However, the plasmidbased CRISPR/Cas9 transformation as used in U. maydis has not been successful for S. reilianum.
CRISPR/Cas9, originating from the adaptive immune system of Streptococcus pyogenes, has been broadly adapted to many eukaryotic systems. It is a versatile tool for mutagenesis in various filamentous fungi [13]. The delivery strategies of CRISPR/Cas9 differ between fungal species: (i) stable genomic integration of cas9, (ii) transient delivery of Cas9 where the expression of Cas9 is dependent on selection pressure of a self-replicating plasmid or a telomere vector [10,14], or (iii) ribonucleoprotein complex (RNP)mediated transformation [15,14]. Here, we describe CRISPR/Cas9 applications in S. reilianum using an RNPmediated transformation approach. We demonstrate the generation of frameshifts as well as knock-out mutants mediated by RNPs, thereby generally improving the mutagenesis, and, for the first time, enable a marker-free editing in S. reilianum.
Materials and reagents
Biological materials
S. reilianum strains were stored at -80 °C in 30% glycerol. For transformation, S. reilianum wildtype strains SRZ1 and SRZ2 [7] were used.
Reagents
Single-guide RNA (sgRNA) synthesis
T4 DNA polymerase (New England Biolabs, catalog number: M0203S), storage: -20 °C
NEBufferTM r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg/mL BSA, pH 7.9), storage: -20 °C (New England Biolabs, catalog number: B7202S)
dNTPs (DNA) (Carl Roth, catalog number: K039.1), storage: -20 °C
NucleoSpin® Gel and PCR clean up (Machery and Nagel, catalog number: 740.609.250), storage: RT
HiScribe® T7 High Yield RNA Synthesis kit (New England Biolabs, catalog number: E2040S), storage: -20 °C
DNase I (Thermo Fisher, catalog number: EN0521), storage: -20 °C
DNase I buffer (Thermo Fisher, catalog number: EN0521), storage: -20 °C
RNA Clean & Concentrator 25 kit (Zymo Research, catalog numbers: R1017 and R1018), storage: RT
Purple loading dye (New England Biolabs, catalog number: B7024S); ingredients: 2.5% Ficoll®-400, 10 mM EDTA, 0.08% SDS, 0.02% Dye 1, 0.02% Dye 2, pH 8; storage: RT
Nuclease-free water, storage: RT
Formation of RNP and in vitro cleavage assay
EnGen® Spy Cas9 NLS + NEB buffer r3.1 (New England Biolabs, catalog number: M0667)
500 mM Ethylenediaminetetraacetic acid (EDTA) (Carl Roth, catalog number: 8043.2)
Proteinase K (Thermo Fisher, catalog number: EO0491)
100 bp ladder (New England Biolabs, catalog number: N3231S)
Universal agarose (Bio-Budget, catalog number: 10-35-1020)
1% Ethidium bromide solution (Carl Roth, catalog number: 2218.2)
Potato dextrose agar (PDA) plates (39 g/L) (BD, DifcoTM, catalog number: 213400)
Tris base (Sigma, catalog number: 102262896)
Acetic acid (VWR, catalog number: 20103.330)
EDTA 0.5 M pH 8.0 (Carl Roth, catalog number: 8043.2)
Protoplasting and transformation of S. reilianum
Novozym 234 [Novo Nordisc; Denmark, not available anymore; alternative: lysing enzyme from Trichoderma harzianum (Sigma, catalog number: SLBJ0553V)]
Sodium citrate (Carl Roth, catalog number. 3580.1)
Sorbitol (Sigma, catalog number: 102466217)
Citrate acid (Carl Roth, catalog number: X863.2)
Sorbitol (Sigma, catalog number: 102466217)
Tris-HCl (Carl Roth, catalog number: 9090.3)
CaCl2 (Sigma, catalog number: 1002825086)
Poly(ethylene glycol) PEG MW3350 (Sigma, P4338, catalog number: 102604683)
BactoTM-Yeast-Extract (Thermo Fisher, Gibco, catalog number: 212720)
BactoTMPeptone (BD, Difco, catalog number: 211820)
Sucrose (Carl Roth, catalog number: 4621.2)
Sorbitol (Sigma, catalog number: 102466217)
BactoTM-Agar (BD, catalog number: 214030)
Potato dextrose agar (PDA) plates (BD, DifcoTM, catalog number: 213400)
Carboxine (5 mg/mL) (Sigma, catalog number: 102085144)
Heparin sodium salt from porcine intestinal mucosa (15 mg/mL) (Sigma, catalog number: 1001937695)
Solutions
50× TAE buffer (see Recipes)
1× TAE buffer (see Recipes)
SCS buffer (see Recipes)
STC buffer (see Recipes)
STC/40% PEG (see Recipes)
Regeneration agar light (see Recipes)
Recipes
50× TAE buffer
Reagent Final concentration Quantity or Volume
Tris base 2 M (v/v) 242.0 g
Acetic acid 2 M (v/v) 57.1 mL
EDTA 0.5 M pH 8.0 10% (v/v) 100.0 mL
1× TAE buffer
Reagent Final concentration Quantity or Volume
50× TAE buffer 2% (v/v) 20.0 mL
Deionized water 98% (v/v) 980.0 mL
SCS buffer
Reagent Final concentration Quantity or Volume
Solution 1:
Sodium citrate, pH 5 0.6% (w/v) 5.9 ml
Sorbitol 18.2% (w/v) 182.0 g
Solution 2:
Citrate acid 0.4% (w/v) 4.2 g
Sorbitol 18.2% (w/v) 182.0 g
Solution 1 and 2 are mixed until pH 5.8 is reached (ratio ~5:1) and autoclaved.
STC buffer
Reagent Final concentration Quantity or Volume
Sorbitol 50% (v/v) 500.0 mL
Tris-HCl, 1 M pH 7.5 1% (v/v) 5.0 mL
CaCl2, 1 M, sterile-filtrated (100 mL total volume is enough) 10% (v/v) 50.0 mL
STC/40% PEG
Reagent Final concentration Quantity or Volume
STC buffer 60% (v/v) 600.0 mL
Poly(ethylene glycol) PEG, MW3350; sterilefiltrated, (50 mL total volume is enough) 40% (w/v) 400.0 g
Regeneration agar light
Reagent Final concentration Quantity or Volume
BactoTM yeast extract 1% (w/v) 10.0 g
BactoTM peptone 0.4% (w/v) 20.0 g
Sucrose 0.4% (w/v) 20.0 g
Sorbitol 18.2% (w/v) 182.2 g
BactoTM agar 1.5% (w/v) 15.0 g
Laboratory supplies
PCR machine (Bio-Rad, model: T100TM Thermal Cycler)
Microfuge for PCR tubes (VWR, model: Ministar)
Tabletop centrifuge (VWR, model: Microstar 17)
37 °C incubator (Memmert, model: UN110)
28 °C incubator/room
Optional: Polyacrylamide gel electrophoresis (SDS-PAGE) equipment (Bio-Rad, model: PowerPacTM Basic, Mini-Protean® Tetra System)
Agarose gel electrophoresis equipment
Nanodrop (Thermo Scientific, model: Nanodrop 2000c)
ChemiDocTM MP imaging system (or equivalent imaging system), with GFP filter (Bio-Rad, model: Universal Hood III)
Geldoc: visualization of DNA by UV radiation using a gel documentation unit (Peqlab/VWR, model: EBOX VX5)
Equipment
PCR tubes and 1.5 ml Eppendorf tubes
Sterile cut tips (1,000 µL and 20 µL)
Procedure
In vitro transcription of sgRNA
Design protospacer in CHOPCHOP sgRNA designer (https://chopchop.cbu.uib.no/) using S. reilianum as target organism. Choose the protospacer sequence starting with a G, which is needed for initiating the transcription by T7 RNA polymerase. If there is no desired protospacer starting with G, add an additional G upstream of the chosen protospacer sequence (21 nt).
Add T7 RNA polymerase promoter sequence and overlapping scaffold sequence upstream and downstream of the chosen protospacer sequence, respectively, and order the gene-specific oligonucleotide (Table 1). In addition, a reverse complementary constant oligonucleotide is needed, which harbors the scaffold and terminator sequence and a 20 nt overlap to the scaffold sequence of the gene-specific oligonucleotide (Table 1).
Table 1. Sequences of oligonucleotides for sgRNA synthesis
Oligo Sequence
Gene-specific CAAAATTCCATTCTACAAC-GNNNNNNNNNNNNNNNNNNN-GTTTTAGAGCTAGAAATAGCAAG
Constant AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
Note: The underlined sequence of the constant oligo depicts the overlapping part with the gene-specific oligo.
Mix both oligonucleotides in a 1:1 ratio as follows:
1 µL of protospacer oligo 100 µM stock
1 µL of constant oligo 100 µM stock
8 µL of H2O
10 µL total
Anneal the oligos using the following program in PCR machine:
95 °C for 5 min
95 °C to 85 °C at -2 °C/s
85 °C to 25 °C at -0.1 °C/s
4 °C pause
Add T4 DNA polymerase to fill in the overhangs:
2.5 µL of dNTPs (10 mM)
2 µL of NEBufferTM r2.1(10×)
5 µL of H2O
0.5 µL of T4 DNA polymerase
10 µL total
Incubate at 12 °C for 20 min in a PCR machine.
Purify the product with a PCR clean-up kit, measure the concentration with Nanodrop, and verify the PCR product on a 2%–3% TAE agarose gel.
Use 2 µg of the resulting DNA from above as template and the HiScribe T7 High Yield RNA Synthesis kit for the following reaction (NEB, protocol for small RNAs):
6 µL of NTPs (25 mM each in stock)
2 µL of 10× T7 buffer
1.5 µL of T7 RNA polymerase mix
X µL of Template (2 µg DNA template, step 6)
Y µL of nuclease-free H2O (add to 20 µL)
20 µL total
Flip the tube, vortex shortly, and incubate at 37 °C overnight.
The next day, add 14 µL of nuclease-free H2O, 4 µL of DNase I buffer (10×), and 2 µL of DNase I and incubate at 37 °C for 15 min.
Caution: Small RNA is easy degradable; work in a RNase-free space and use gloves and a lab coat for all following steps!
Purify the resulting sgRNA with the RNA Clean & Concentrator 25 kit and use the manufacturer's protocol (manual, page 5).
Optional: Check the quality of the RNA on 10% denaturing PAA gel using TBE buffer (89 mM Tris base, 89 mM boric acid, and 2 mM sodium EDTA) and 8 M urea and TBE as running buffer [14].
Measure the concentration by Nanodrop and proceed with in vitro cleavage assay (section B).
Pause point: You can freeze the sgRNA at -80 °C and continue the next day; long-term storage of sgRNA is also possible at -80 °C. See Troubleshooting 1.
In vitro cleavage assay
To test the in vitro efficiency of the designed sgRNA, mix 1.5 µL of Cas9 (NEB) and ~1.5 µg of the sgRNA (1:1 molar ratio) and incubate it for 10 min at RT (Figure 1B).
Figure 1. Graphical overview of the workflow of ribonucleoprotein complex (RNP)-mediated transformation in S. reilianum. (A) In vitro synthesis of sgRNA using T7 HiScribe kit. (B) RNP formation of in vitro–transcribed sgRNA with SpCas9. (C) Alternatively: To perform an in vitro cleavage assay, incubation at room temperature (RT) for 10 min and subsequent addition of a donor template (amplification of the gene of interest region) and incubation at 37 °C for 3 h is conducted. (D) Sampling of 10 µL of reaction mix after 1, 2, and 3 h (or alternatively overnight). (E) Visualization of in vitro cleavage on a 1.5% agarose gel using 100 bp ladder. (F) RNP incubation for 1 h at 37 °C prior to transformation into S. reilianum protoplasts. Figure was created with biorender.com.
Afterwards, add 333 ng of a DNA cleavage template (PCR product of the region of interest) (Figure 1C).
After 1, 2, and 3 h take 10 µL samples (Figure 1D) and stop the reaction by adding 1 µL of 500 mM EDTA, pH 8.
Subsequently, add 1 µL of proteinase K to the reaction and incubate the reaction mix for 30 min at 50 °C for degradation of Cas9.
Stop the reaction by the addition of 1× purple loading dye.
After the collection of all samples, check cleavage on an 1.5% agarose gel with 100 bp ladder (stained with ethidium bromide solution) visualized using a Gel-Doc (Figure 1E, see Troubleshooting 2).
Assembly of RNP for transformation into S. reilianum
Use 2 µg of the in vitro–transcribed sgRNA targeting the gene of interest and mix it with 6 µg of SpCas9.
Subsequently, add 1× NEBufferTM 3.1 and water in a minimum volume (Figure 1B).
After mixing and centrifugation, incubate the reaction for 1 h at 37 °C prior to transformation (Figure 1B).
Transformation of S. reilianum
Prepare S. reilianum protoplasts using Novozym 234 as described previously [8].
For RNP transformation (Figure 1E), thaw the protoplasts for 5 min on ice.
Add a self-replicating plasmid with antibiotic resistance cassette [e.g., pNEBUC—Carboxine (Cbx); Brachmann et al. [8], replicating in S. reilianum], the RNP (formed in section C), 1 µL of 15 mg/mL heparin, and, optionally, 1.5 µg of a donor template to the protoplasts (Figure 2).
Note: The self-replicating plasmid is lost when the selection for Cbx resistance is stopped. So far, we could not report an integration into the genome of S. reilianum.
Incubate the protoplasts for 10 min on ice.
Add 500 µL of STC/40% PEG and resuspend the protoplasts carefully with a tip-cut blue tip until the liquid looks homogenous without clumps (5–8 times pipetting up and down).
Incubate the protoplasts for another 15 min on ice.
Spread the protoplasts on a regeneration agar light plate with two layers [bottom layer: corresponding selective antibiotic (for pNEBUC—carboxin: 2.5 µg/mL), top layer: without antibiotic resistance].
The next day, flip the transformation plate upside down.
After four days, use a blue tip to single out transformants from regeneration agar to PDA + Carboxin (2.5 µg/mL) for 2–3 days.
Afterwards, transfer a single colony for two days to PDA plates to lose the resistance.
Subsequently, DNA is isolated [16] and used for further confirmation (see section E).
RNP-assisted homologous recombination to generate a knockout in S. reilianum
For the generation of an antibiotic-resistance-free knock-out mutant in S. reilianum, a CRISPR/Cas9-mediated homology-directed repair was exploited. To do this, a donor template is generated by cloning the 1 kb homology flanking regions of the target gene into a MOCLO vector TK#1_pAGM1311 by Gibson assembly (Figure 2).
For the transformation of S. reilianum protoplasts (see section D), add the donor template together with a self-replicating plasmid (pNEBUC), the RNP (with a sgRNA against the target region), and 1 µL of heparin.
Note: The transformation efficiency is high > 100 colonies; if your efficiency is lower, repeat protoplasting and transformation.
Transfer obtained transformants as described above (see section C).
Isolate DNA of the transformants and the wild type.
Conduct a PCR using the forward primer of the left flank and the reverse primer of the right flank (Figure 2C) and compare the band sizes to the wild type (Figure 2D).
Putative positive mutants from PCR are selected for further verification via Southern blot [17,18] using the deletion construct (left flank + right flank), previously used as a donor template, as probe for hybridization.
Figure 2. Transformation of S. reilianum using ribonucleoprotein complex (RNP) with and without a donor template. (A) Generation of frameshift mutants using RNP-mediated transformation. Generated frameshift mutants are screened by sequencing or as described in Figure 3 by the loss of fluorescence. (B) Generation of knock-out mutants using RNP-assisted homologous recombination with a donor template. (C) Donor template design for the generation of a knockout. 1 kb flanking regions of the coding sequence are amplified by PCR with overhangs for Gibson assembly into backbone TK#1_pAGM1311 (MOCLO backbone, Level -1). (D) Example of deletion mutant verification PCR using Left Flank (LF) forward primer and Right Flank (RF) reverse primer [expected sizes: WT, 1021 bp; mutant (+), 676 bp; efficiency: 62.5% (10/16)]. The efficiency can vary between different genomic loci. Figure 2A and B were created with biorender.com.
Validation of protocol
The efficiency of the RNP CRISPR/Cas9 can, for instance, be tested with GFP fluorescence as a readout (Figure 3). To test the efficiency in S. reilianum, a strain harboring a single integration of GFP controlled by pOTEF (constitutive promoter) was generated in the ip locus of SRZ2 strain and confirmed via Southern blot (Figure S1). For the transformation of S. reilianum protoplasts, a sgRNA against GFP together with the Cas9 in a RNP (see section C) and an auto-replicating plasmid (pNEBUC) for selection on regeneration agar was used. Transformants were singled out after four days of incubation at 28 °C on PDA + Cbx (2.5 mg/mL) and, after two days, were transferred to PDA plates and checked for their fluorescence using a Chemi-Doc.
Figure 3. GFP as target for ribonucleoprotein complex (RNP)-mediated transformation in S. reilianum. An example shows the efficiency of RNPmediated CRISPR/Cas9 transformation in S. reilianum. A S. reilianum SRZ2 strain expressing GFP under pOTEF promoter was generated. sgRNA+Cas9 targeting GFP coding sequence was transformed, and mutants with frameshift lose the GFP signal. Efficiency for GFP sgRNA: ~41% (34/83).
General notes and troubleshooting
Troubleshooting
No. Step Problem Suggestion/solution
1 sgRNA synthesis
Low concentration
(<500 ng/µL)
Do not proceed with transformation, repeat synthesis; high concentration in minimum volume is needed
2 In vitro cleavage assay No bands after cleavage
1) Test functionality of Cas9 enzyme (use a control)
2) Design of new sgRNAs
Acknowledgments
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. 771035), as well as funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy, EXC-2048/1- Project ID: 390686111. This protocol was adapted from previous work [14].
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Leisen, T., Bietz, F., Werner, J., Wegner, A., Schaffrath, U., Scheuring, D., Willmund, F., Mosbach, A., Scalliet, G., Hahn, M., et al. (2020). CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea. PLoS Pathog. 16(8): e1008326.
Foster, A. J., Martin-Urdiroz, M., Yan, X., Wright, H. S., Soanes, D. M. and Talbot, N. J. (2018). CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus. Sci. Rep. 8(1): 14355.
Hoffman, C. S. and Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformaion of Escherichia coli. Gene 57: 267–272.
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Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual (No. Ed. 2). Cold Spring Harbor Laboratory Press.
Supplementary information
The following supporting information can be downloaded here:
Figure S1. Southern blot of single integrated GFP into SRZ2 strain.
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4,979 | https://bio-protocol.org/en/bpdetail?id=4979&type=0 | # Bio-Protocol Content
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CD8α-CI-M6PR Particle Motility Assay to Study the Retrograde Motion of CI-M6PR Receptors in Cultured Living Cells
SR Shalini Rawat
Mahak Sharma
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4979 Views: 372
Reviewed by: Chiara AmbrogioDevashish DWIVEDI Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Oct 2022
Abstract
The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate.
Key features
• This assay is adapted from the methodology by Prof. Matthew Seaman for studying the retrograde trafficking of CI-M6PR by expressing CD8α-CI-M6PR chimera in HeLa cells.
• The experiments include live-cell imaging of surface-labeled CD8α-CI-M6PR molecules, followed by a chase in cells.
• Allows the monitoring of real-time motion of CD8α-CI-M6PR endosomes and facilitates calculation of kinetic parameters associated with endosome trajectories, e.g., speed and distance (run lengths).
Keywords: Trafficking Endosome motility CI-M6PR endosomes Trans-Golgi network Run lengths Retrograde motion
Background
At steady state, the cation-independent mannose 6-phosphate receptors (CI-M6PR) are primarily localized to the trans-Golgi network (TGN), with a perinuclear endosomal population and a minor fraction on the plasma membrane. Post cargo delivery to endolysosomal compartments, CI-M6PR receptors undergo trafficking from all the cellular locations to early/recycling endosomes; subsequently, they are retrieved back to TGN in a dynein-dependent manner. This endosome-to-TGN retrieval of CI-M6PR is crucial for maintaining lysosomal activity and is regulated by various proteins involved in sorting and packaging into carrier vesicles followed by their motility towards TGN. Dysregulation in CI-M6PR receptor trafficking is linked to neurodegenerative and lysosomal storage disorders such as Batten's disease and Parkinson's disease [1,2]. Initial studies on CI-M6PR trafficking either required radiolabeling newly synthesized CI-M6PR and tracking their journey from Golgi or predominantly relied on measuring the redistribution of CI-M6PR receptors from TGN to other cellular compartments [3]. However, these methodologies only provide information on the localization of the cargo upon its impaired trafficking without revealing the exact direction of trafficking defect. Recently, several studies have employed the CD8α-CI-M6PR chimera (developed by Matthew Seaman; [4]) to study retrograde trafficking of CI-M6PR, involving colocalization of CD8α-CI-M6PR with different endocytic markers at steady-state. This chimera, a fusion of the ectodomain of CD8 and the cytoplasmic tail of CI-M6PR, allows trafficking only in the retrograde direction towards the Golgi [5]. Here, we present a protocol that extends the use of CD8α-CI-M6PR chimera in a live-cell imaging setup, where surface receptors are labeled and then chased in cells as they move towards the Golgi (Figure 1). We also provide an approach to perform the quantitative analysis of CD8α-CI-M6PR motion as they move from early/recycling endosome to Golgi, including the calculation of kinetic parameters such as speed and distance associated with their motion.
Figure 1. Schematic illustrating the CD8α-CI-M6PR trafficking assay to track motility of CD8α-CI-M6PR endosomes internalized from the cell surface.HeLa cells expressing CD8α-CI-M6PR were incubated with primary (anti-CD8α) and secondary (Alexa Fluor 488-conjugated dye) antibodies on ice to specifically label surface receptors, followed by live-cell imaging in warm media at 37 °C after 10 min of chase.
Materials and reagents
Biological materials
70%–80% confluent HeLa cells (ATCC)
Note: We have used HeLa cells for this analysis because they are easy to transfect and considerably flat for 2D analysis of endosome motility; however, other cell lines can also be used.
Reagents
Plasmid CD8α-CI-M6PR-pIRES Neo2 (a kind gift from Prof. Matthew Seaman)
GibcoTM Dulbecco’s modified Eagle medium (DMEM), high glucose, with GlutaMAXTM, sodium pyruvate (Thermo Fisher Scientific, catalog number: 10569-010), storage: 2–8 °C
Dulbecco’s phosphate buffered saline (DPBS), without calcium and magnesium (Lonza Bioscience, catalog number: 17-512F), storage: 15–30 °C
Opti-MEM® reduced serum media (Thermo Fisher Scientific, GibcoTM, catalog number: 11058021), storage: 2–8 °C
Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco TM, catalog number: 10082147), storage: -20 °C
Antibiotic-antimycotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062), storage: -20 °C
GibcoTM 1 M HEPES (Thermo Fisher Scientific, catalog number: 15630-080), storage: 2–8 °C
GibcoTM 100× MEM non-essential amino acid solution (Thermo Fisher Scientific, catalog number: 11140050)
GibcoTM DMEM, high glucose, HEPES, no phenol red (Thermo Fisher Scientific, catalog number: 21063029), storage: 2–8 °C
X-tremeGENETM HD transfection reagent (Roche® Life Science Products, catalog number: 6366236001)
siRNA transfection reagent Dharmafect-1 (GE Healthcare, catalog number: T-2001-03)
Mouse anti-human CD8 monoclonal antibody (BD PharmingenTM, catalog number: 555631), storage: 4 °C
Alexa Fluor 488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, catalog number: A11029), storage: 4 °C
Citrate anhydrous (HiMedia, catalog number: GRM-1023)
Sodium citrate tribasic dihydrate (Tri sodium citrate dihydrate) (Sigma, catalog number: S4641)
Solutions
Citric acid buffer, pH 4.5 (see Recipes)
Recipes
Citric acid buffer, pH 4.5
Reagent Volume
Citric acid anhydrous 100 mL
Tri-sodium citrate dihydrate 100 mL
Total 200 mL
0.1 M Citric acid anhydrous
Reagent Quantity
Citric acid anhydrous 2.626 g
Total 125 mL
0.1 M Tri-Sodium Citrate dihydrate
Reagent Quantity
Tri-sodium citrate dihydrate 3.676 g
Total 125 mL
Adjust pH to 4.5 with sodium citrate.
Complete medium
Dulbecco’s modified Eagle medium (DMEM), high glucose, with GlutaMAXTM, sodium pyruvate supplemented with 10% FBS, 10 mM HEPES, 1× antibiotic-antimycotic, and 1× MEM non-essential amino acid solution
Laboratory supplies
35 mm μ-dish, high glass-bottom live-imaging dishes (ibidi, catalog number: 81158)
BD Falcon 15 mL centrifuge tubes (Corning, Falcon®, catalog number: 352096)
BD Falcon 35 mm cell culture dish (Corning, Falcon®, catalog number: 353001)
Equipment
Cell culture incubator (Eppendorf, New BrunswickTM, model: Galaxy ® 170 R)
Confocal microscope (ZEISS, model: LSM 710)
HulaMixerTM sample mixer (Thermo Fisher Scientific, catalog number: 15920D)
Software and datasets
Fiji software ( https://imagej.net/software/fiji/) 2.15.1
ZEN software, ZEN 2012 v. 8.0.1.273 (ZEISS)
Procedure
Experimental setup
Note: This protocol has been modified from CD8α-CI-M6PR trafficking assay [4], aiming to assess the motility of surface-labeled CD8α-CI-M6PR towards the Golgi in HeLa cells treated with siRNAs against a specific gene of interest.
Seed approximately 0.14 ×106 HeLa cells in a 35 mm glass-bottom live-imaging dish and incubate overnight in a cell culture incubator.
Note: For transfection, seed 0.25 × 106 HeLa cells and incubate overnight in a cell culture incubator.
On the following day, prepare siRNA transfection mix by combining 100 nM siRNA oligos and transfection reagent Dharmafect-1 (Dharmacon) in 200 μL of Opti-MEM and incubate the mixture at room temperature for 30 min. After incubation, add siRNA transfection mix dropwise over cells and incubate for 48 h.
After 48 h of siRNA treatment, transfect the cells with CD8α-CI-M6PR. To prepare the transfection mix, add 2 μg of CD8α-CI-M6PR-pIRES Neo2 plasmid DNA in 200 μL of Opti-MEM and X-tremeGENETM HD transfection reagent in a ratio of 1:1 (1 μg DNA: 1 μL transfection reagent). Incubate the transfection mix at room temperature for 30 min, then carefully add it dropwise over cells.
To fluorescently label the primary antibody, incubate the mouse anti-CD8 antibody along with Alexa Fluor 488-conjugated anti-mouse IgG in 1 mL of serum-free DMEM media at 4 °C for 30 min in a tube rotator.
Following 12–14 h of transfection, incubate the complex of anti-CD8α with Alexa Fluor 488-conjugated IgG with cells in a live-cell imaging dish on ice for 30 min to label the surface CD8-CI-M6PR receptors.
Note: As a control for the non-specific attachment of Alexa Fluor 488-conjugated antibodies to the plasma membrane, execute the antibody labeling and subsequent steps using untransfected cells.
After incubation, carefully aspirate the DMEM media containing anti-CD8α/Alexa Fluor 488-conjugated antibody complex.
To remove any unbound or non-specifically attached antibodies, wash the cells once with DPBS followed by two washes with ice-cold citric acid buffer, pH 4.5. Add approximately 1 mL of citric acid buffer to cells and incubate on ice for 5 min. Discard the buffer and repeat the washing step twice with citric acid buffer. Finally, add 1 mL of DPBS to remove any residual citric acid buffer from the cells.
Note: Prepare fresh citric acid buffer and use it ice-cold for washes.
After washing, add 2 mL of prewarmed phenol red–free DMEM media supplemented with 10% FBS on cells and incubate it for 10 min.
After incubation, transfer the dish to the humidified live-cell imaging chamber maintained at 37 °C with 5% CO2 and perform live-cell imaging of the transfected cells showing labeled CD8α-CI-M6PR receptors. Post 10 min chase, labeled CD8α-CI-M6PR receptors present on plasma membrane traffic to TGN due to the retrograde sorting signals present on the cytoplasmic tail of CI-M6PR.
Live-cell imaging and video acquisition
Use 512 × 512 frame size for higher acquisition speed and acquire a timelapse video of cells for approximately 200–300 frames minimum without any time interval.
Record the video of the labeled CD8α-CI-M6PR receptors for a duration not exceeding 15 min, following a 10 min chase, during which they are expected to reach TGN.
Image analysis and quantification using ImageJ/Fiji
Note: In the manuscript [6], we conducted particle tracking analysis using a semi-automated custom-written program and a MATLAB script [7]. However, in this protocol, we have presented an alternative protocol employing the TrackMate plugin for the analysis of CD8α-CI-M6PR particles.
Open the video file using Fiji. If there are multiple cells in a field, use the free-hand tool from the Fiji tool panel to select a single cell. Duplicate the selection by either using Ctrl+shift+D or go to Image menu and choose the Duplicate option. A pop-up window will appear; select the Duplicate stack option (Figure 2).
Figure 2. Duplicate video stack and select the cell for analysis
Remove the background fluorescence using the Edit menu. Select the Clear outside option (Figure 3). Choose yes to the process stack option allowing it to remove the background from all frames.
Figure 3. Clear outside from all frames of video
Subtract cytoplasmic background fluorescence from the cell to improve the endosome tracking process. Select Subtract background option in the Process menu in Fiji toolbar. Input the value of Rolling ball radius appropriately according to your background fluorescence in the video file (Figure 4).
Note: The value of Rolling ball radius should be higher than the largest object in the image.
Figure 4. Background subtraction in cell
Perform particle tracking on video file. Select Plugins > Tracking > TrackMate option. A dialog box will open with spatial and temporal information of the data. It provides information on pixel dimensions, number of frames, and also the dimensionality of the video file (2D or 3D lapse video). To proceed with tracking, press the Next button (Figure 5).
Figure 5. Particle tracking using TrackMate plugin
Next, a window will open with three options as object detectors. Choose the Difference of Gaussian (DoG) detector, designed to identify round particles like endosomes and being particularly effective for particle sizes below ~5 pixels. Proceed by clicking the Next button (Figure 6).
Figure 6. Selection of difference of Gaussian (DoG) detector
In the subsequent DoG detector window, input the values for a few parameters associated with particles to be analyzed, such as Estimated Blob diameter and Quality threshold. For Estimated Blob Diameter, enter the approximate diameter of the endosomes or measure it using the line tool in Fiji. Quality threshold allows you to limit your analysis to a specific number of endosomes, excluding spots/endosomes with quality below the threshold. Click the preview button to see the selected endosomes and assess how well the parameters fit the data. Adjust the Quality threshold to eliminate any false spot intensities from the analysis. Click on Preview to see the selected spots and accordingly adjust the threshold to choose the desired endosomes. You can also see the number of endosomes that were selected for the selected threshold (highlighted here in red outline) (Figure 7). Go to the next window once adjustments have been made.
Figure 7. Difference of Gaussian (DoG) detector parameters for particles
Next, choose the HyperStack displayer, which allows you to overlay the end results over the image (Figure 8).
Figure 8. Choose the HyperStack displayer
This window allows you to do auto or manual thresholding to select the particles. Choose Auto here. Click Next (Figure 9).
Figure 9.Thresholding on particles/spots
At this point, we can see the spots that are selected for analysis. We can run the video and observe the spots moving through all frames. In this window, you can set filters on the spots; however, for now, we will proceed with default settings and click Next (Figure 10).
Note: You can also filter spots based on parameters like intensity, quality, radius of the particles, frame, or positions (X, Y,) and do a comparative analysis of choices.
Figure 10. Set filters and choose particles or spots for analysis
Now, select the simple LAP (linear assignment problem) tracker, ideal for particles undergoing Brownian motion. This tracker creates tracks by linking particles across different frames in the video (Figure 11 ).
Figure 11. Selection of simple linear assignment problem (LAP) tracker
Next, a LAP tracker window will open, allowing you to input parameters for particle-to-particle linking. Enter 2 μm in the option for Linking max distance, which defines the maximum distance for two spots to be linked between two frames. Put the value of Gap-closing max distance as 2 μm, meaning that it will not link any two spots that are more than two frames apart. Set Gap-closing max frame gap to 1, which is the maximum frame difference allowed for a spot to be missed for linking (Figure 12).
Figure 12. Configuration for linking particles by simple LAP tracker
The next window allows the visualization of the tracks generated by LAP tracker. Click Next (Figure 13).
Figure 13. Tracks generated by LAP tracker
This window allows you to set filters on tracks. You can choose any option for viewing tracks, but here we have opted to set the color of the tracks based on maximal velocity. Click Next (Figure 14 ).
Figure 14. Color filter for visualization of tracks
The next window allows to further change the display options. Here, you can increase or decrease the size of the spot by changing its radius. Furthermore, you can also change the display of the tracks. Click on Next button. A new panel will open with options to plot different features associated with particle motility as a function of another. To continue, click on Next (Figure 15 ).
Figure 15. Display options for particles and tracks
Select Overlay option in the next window and click on Execute. A pop-up window will open, showing the total number of frames. Click on ok. You can save the video with all the captured tracks (Figure 16).
Figure 16. TrackMate features and analysis
To obtain measurements associated with particle/spot’s motion such as distance, displacement, maximum speed, and duration of a track, go back to the Display options window (shown in Figure 15) and click on Analysis. Three different windows will open, providing information on quantitative parameters for all the tracks associated with the particle motion (Figure 17).
Figure 17. Track analysis tables showing different kinetic parameters associated with particle motion
Data analysis
From the data extracted from the track statistics file or table, we can acquire values such as track duration, displacement, mean velocity, and maximum speed for all the tracks identified by the LAP tracker. In the video, we have detected roughly ~1,000 tracks by linking two spots over time. Each track encompasses values for displacement, duration, and velocity. Displacement signifies the distance between spots in consecutive frames, while duration indicates the time span. Velocity values denote the link velocity defined for each link between two spots. Subsequently, we can compute and plot the median, maximum, minimum, and average velocities from link velocities under different conditions or treatments. Analyze the tracks from approximately 7–10 cells and plot the values for each quantitative parameter against various treatments. Further, calculate the statistical significance of differences in mean values between two or more conditions by utilizing either an unpaired two-tailed Student’s t-test or analysis of variance (ANOVA), respectively. These tests are applicable when the data approximately follows a normal distribution and satisfies assumptions such as homogeneity of variances. For non-normal distributions, which lack symmetry, statistical comparisons are done between the medians of the two groups using Mann-Whitney U test.
Validation of protocol
This protocol has been used and published in the following research article:
Rawat, S. et al. (2022). RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes. J. Cell Biol. (Figure 10, panel D)
Note: This article has used a semi-automated custom-written program and a MATLAB script [7], but here we have employed an alternative approach using TrackMate plugin for the particle analysis.
Acknowledgments
The authors adapted and modified this protocol from a previously published study [4] and validated it in a recently described study [6]. The authors would like to thank Prof. Matthew Seaman (University of Cambridge, UK) for sharing the CD8α-CI-M6PR construct. S. Rawat acknowledges fellowship support from University Grants Commission. M. Sharma would like to acknowledge funding support from the Department of Biotechnology (DBT)/Wellcome Trust India Alliance Senior Fellowship (IA/S/19/1/504270), SERB-POWER Grant (SPG/2021/002790), and Janaki Ammal-National Women Bioscientist Award (BT/HRD/NWBA/39/01/2018-19). M. Sharma also acknowledges the infrastructure and financial support from IISER Mohali.
Competing interests
The authors declare no competing financial interests.
References
Calcagni, A., Staiano, L., Zampelli, N., Minopoli, N., Herz, N. J., Di Tullio, G., Huynh, T., Monfregola, J., Esposito, A., Cirillo, C. et al. (2023). Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation. Nat. Commun. 14(1): 3911. https://doi.org/10.1038/s41467-023-39643-7
Cui, Y., Yang, Z., Flores-Rodriguez, N., Follett, J., Ariotti, N., Wall, A. A., Parton, R. G. and Teasdale, R. D. (2021). Formation of retromer transport carriers is disrupted by the Parkinson disease-linked Vps35 D620N variant. Traffic 22(4): 123–136. https://doi.org/10.1111/tra.12779
Hirst, J., Futter, C. E. and Hopkins, C. R. (1998). The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment. Mol. Biol. Cell 9(4): 809–816. https://doi.org/10.1091/mbc.9.4.809
Seaman, M. N. (2004). Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J. Cell Biol. 165(1): 111–122. https://doi.org/10.1083/jcb.200312034
McKenzie, J. E., Raisley, B., Zhou, X., Naslavsky, N., Taguchi, T., Caplan, S. and Sheff, D. (2012). Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi. Traffic 13(8): 1140–1159. https://doi.org/10.1111/j.1600-0854.2012.01374.x
Rawat, S., Chatterjee, D., Marwaha, R., Charak, G., Kumar, G., Shaw, S., Khatter, D., Sharma, S., de Heus, C., Liv, N., et al. (2023). RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes. J. Cell Biol. 222(1). https://doi.org/10.1083/jcb.202108001
Mohan, N., Sorokina, E. M., Verdeny, I. V., Alvarez, A. S. and Lakadamyali, M. (2019). Detyrosinated microtubules spatially constrain lysosomes facilitating lysosome-autophagosome fusion. J. Cell Biol. 218(2): 632–643. https://doi.org/10.1083/jcb.201807124
Article Information
Copyright
© 2024 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 imaging > Live-cell imaging
Cell Biology > Cell-based analysis > Transport
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A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS)
SC Sek-Shir Cheong
TL Tiago C. Luis
MH Matthew Hind
CD Charlotte H. Dean
Published: Vol 14, Iss 8, Apr 20, 2024
DOI: 10.21769/BioProtoc.4980 Views: 461
Reviewed by: Alessandro DidonnaIvan Shapovalov Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Disease Models & Mechanisms Oct 2023
Abstract
Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets.
Key features
• Achieve permanent ex vivo gene modifications in complex tissue-based models within four days.
• Highly adaptable gene modification method that can be applied to induce gene deletion or activation.
• Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls.
• With optimisation, this method can be applied to precision-cut tissue slices of other organs.
Keywords: Precision-cut lung slices PCLS TAT-Cre recombinase Gene modification Floxed allele modification Ex vivo model TReATS Permanent gene manipulation Gene deletion Gene activation
Graphical overview
Workflow of TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS) and validation protocols of gene modification
Background
Precision-cut lung slices (PCLS) represent a 3D ex vivo platform that plays a crucial role in advancing respiratory research. The significance of PCLS lies in their ability to serve as an intermediate model bridging conventional in vitro models and in vivo studies. PCLS preserve the spatial and cellular complexities of the native lung microenvironment while mitigating challenges associated with in vivo experiments, such as ethical considerations, time constraints, and cost limitations [1]. As an ex vivo alternative, PCLS provide a controlled and physiologically relevant setting to investigate various aspects of lung biology, from responses to injury to the behaviour of specific cell types, all within the context of the native lung architecture [2–5]. This advances our understanding of the nuanced interplay between cellular and molecular components governing respiratory function.
Despite the versatility of PCLS, a notable gap has remained to enable effective and permanent gene manipulation in tissue slices, primarily due to the challenges associated with the inherent complexity of the tissue and the imperative to maintain tissue viability. Addressing this gap, our protocol describes a novel approach termed TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices). The TReATS method utilises TAT-Cre recombinase, a cell-permeant Cre protein, to effectively induce ex vivo genetic modifications in PCLS derived from floxed animals, resulting in permanent gene activation or deletion within a four-day timeframe [6]. This simple and effective technique circumvents the need for a Cre-containing mouse allele, eliminating the necessity for complex breeding strategies to generate transgenic mice of interest, thereby remarkedly reducing the time and costs involved and, most importantly, aligning with the 3Rs principles (Refinement, Reduction, and Replacement) by minimising the use of animals in research.
Application of the TReATS method in adult PCLS has important implications for genetic studies, as it overcomes issues related to embryonic or perinatal lethality that can occur upon early gene ablation or overexpression, thereby enabling the possibility to investigate gene function in adulthood [6]. While the TReATS method is useful for inducing ubiquitous pan-tissue gene manipulation across PCLS, its current limitation lies in its inability to induce cell type–specific gene modification. This can only be achieved by crossing with animals possessing specific Cre drivers.
The TReATS method is versatile and adaptable, as evidenced in the original manuscript, where the method is successfully applied to different loxP-modified alleles, inducing gene deletion or activation [6]. This adaptability holds great promise for accelerating the discovery of gene function, unravelling disease mechanisms, and exploring potential therapeutic interventions.
Materials and reagents
Biological materials
Rosa26-flox-stop-flox-EYFP mouse (10–12 weeks; male/female) (from Dr. Tiago Luis; strain: 006148; RRID: IMSR_JAX:006148). Abbreviation: R26R-EYFP
Wildtype C57BL/6J mouse (10–12 weeks; male/female; serves as a negative control) (Charles River, catalog number: 632)
Reagents
Dulbecco’s modified Eagle medium (DMEM-Glutamax) (Sigma-Aldrich, catalog number: 31966021)
Phenol red–free DMEM (Life Technologies, catalog number: 21063029)
Low-gelling temperature agarose (Sigma-Aldrich, catalog number: A9414)
Hank's balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H6648)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Life Technologies, catalog number: 15630080)
Penicillin-streptomycin 10,000 U/mL (Merck Life Science UK, catalog number: P0781)
Phosphate buffered saline (PBS) (Life Technologies, catalog number: 10010056)
Super glue liquid precision bottle 5 g (Loctite, catalog number: 2632836)
TAT-Cre recombinase (Merck, catalog number: SCR508)
ProlongTM live antifade reagent (Invitrogen, catalog number: P36975)
Cell proliferation kit (MTT assay) (Roche, catalog number: 11465007001)
Dimethyl sulfoxide (DMSO) (Merck Life Science UK, catalog number: D5879)
Absolute ethanol for molecular biology (Sigma-Aldrich, catalog number: 51976)
Methanol (Sigma-Aldrich, catalog number: 34860)
RNase Zap RNase decontamination solution (Life Technologies, catalog number: AM9780)
Ultrapure DNase/RNase-free distilled water (Life Technologies, catalog number: 10977035)
RNeasy mini kit (Qiagen, catalog number: 74104)
RNase-free DNase set (Qiagen, catalog number: 79254)
RNA screen tape (Agilent, catalog number: 5067-5576)
RNA screen tape sample buffer (Agilent, catalog number: 5067-5577)
High-capacity cDNA reverse transcription kit (Applied Biosystems, catalog number: 4368814)
TaqMan fast advanced master mix (Life Technologies, catalog number: 4444556)
(Optional) Mouse B2m gene expression assay (Life Technologies, catalog number: 4331182_Mm00437762_m1)
(Optional) YFP gene expression assay (Life Technologies, catalog number: 4331182_ Mr04097229_mr)
Solutions
HBSS/HEPES buffer (see Recipes)
Agarose solution 2% (w/v) (see Recipes)
Serum-free (SF)-DMEM culture medium (see Recipes)
Imaging medium (see Recipes)
MTT working solution (see Recipes)
70% (v/v) methanol (see Recipes)
70% (v/v) ethanol (see Recipes)
RLT/β-mercaptoethanol lysis buffer (see Recipes)
Recipes
HBSS/HEPES buffer (store at 4 °C for up to six months)
Reagent Final concentration Volume
HBSS n/a 495 mL
HEPES 10 mM 5 mL
Total n/a 500 mL
Agarose solution 2% (w/v) (freshly prepared and used)
Reagent Final concentration Quantity
HBSS/HEPES n/a 50 mL
Agarose 2% (w/v) 1 g
Total n/a 50 mL
SF-DMEM culture medium (store at 4 °C for up to six months)
Reagent Final concentration Volume
DMEM-Glutamax n/a 495 mL
Penicillin-streptomycin 100 U/mL 5 mL
Total n/a 500 mL
Imaging medium (freshly prepared and used)
Reagent Final concentration Volume
Phenol red–free DMEM n/a 49 mL
Penicillin-streptomycin 100 U/mL 500 µL
ProlongTM live antifade reagent 1% (v/v) 500 µL
Total n/a 50 mL
MTT working solution (freshly prepared and used)
Reagent Final concentration Volume
SF-DMEM n/a 2.7 mL
MTT solution (Cell proliferation kit) 10% (v/v) 300 µL
Total n/a 3 mL
70% methanol (store at room temperature)
Reagent Final concentration Volume
Methanol 70% (v/v) 14 mL
H2O n/a 6 mL
Total n/a 20 mL
70% ethanol (store at room temperature)
Reagent Final concentration Volume
Absolute ethanol 70% (v/v) 14 mL
H2O n/a 6 mL
Total n/a 20 mL
RLT/β-mercaptoethanol lysis buffer (freshly prepared and used)
Reagent Final concentration Amount
RLT buffer (RNeasy mini kit) n/a 1,980 µL
β-mercaptoethanol 1% (v/v) 20 µL
Total n/a 2 mL
Antibodies and fluorescent dyes
Alexa Fluor® 647-conjugated rat anti-lysosomal associated membrane protein 3 (LAMP3) (marker for mature differentiated alveolar type II (ATII) cells) (Dendritics, catalog number: DDX0192A647), dilution: 1:250
Podoplanin eFluor® 660 [alveolar type I (ATI) cell marker] (Life Technologies, catalog number: 50-5381-82), dilution: 1:500
Alexa Fluor® 647-conjugated platelet endothelial cell adhesion molecule (PECAM) (vascular endothelium marker) (BioLegend, catalog number: 102416), dilution: 1:200
Alexa Fluor® 647-conjugated CD11c (macrophage marker) (BioLegend, catalog number: 117312), dilution: 1:200
BioTrackerTM TiY vimentin live cell dye (fibroblast marker) (Sigma-Aldrich, catalog number: SCT059), dilution: 1:2,000
Armenian hamster IgG-PE isotype control (BioLegend, catalog number: 400907), dilution: 1:200
Rat IgG-Alexa Fluor 647 isotype control (BioLegend, catalog number: 400526), dilution: 1:200
4',6-diamidino-2-phenylindole (DAPI) 1 mg/mL (Life Technologies, catalog number: 62248), dilution: 1:500
Laboratory supplies
48-well clear TC-treated culture plate (Scientific Laboratory Supplies Ltd., catalog number: 3548)
96-well clear flat bottom TC-treated culture plate (Scientific Laboratory Supplies Ltd., catalog number: 3596)
24-well uncoated µ-plates (Ibidi, catalog number: 82421)
Hydrophilic PTFE 12 mm cell culture inserts with 0.4 µm pores (Millipore, catalog number: PICM01250)
Costar 5 mL stripette serological pipets (Scientific Laboratory Supplies Ltd., catalog number: 4487)
Costar 10 mL stripette serological pipets (Scientific Laboratory Supplies Ltd., catalog number: 4488)
Costar 25 mL stripette serological pipets (Scientific Laboratory Supplies Ltd., catalog number: 4489)
Syringe 5 mL luer (VWR, catalog number: 613-2042)
Swann-Morton surgical scalpels, No. 22 (Fisher Scientific Ltd., catalog number: 11758353)
Sterile bijou 7 mL (Greiner Bio-One Ltd, catalog number: 189171)
Weigh boats square 7 mL white (VWR, catalog number: 611-0093)
Qualfast Zinc-plated steel washer M6 (Cromwell, catalog number: QFT6455541N)
Petri dishes, 90 mm × 16 mm (Sarstedt, catalog number: 82.1472)
0.2 mL 8-strip non-flex PCR tubes (Starlab, catalog number: I1402-3700)
Homogenisation tubes, screw cap micro tube 2 mL (Sarstedt, catalog number: 72.609)
Homogenisation tube caps, standard screw cap (Starlab, catalog number: E1480-0100)
Homogenisation beads, fastPrep-24TM lysing matrix D 1.4 mm ceramic spheres (MP Biomedicals, catalog number: 1169131050-CF)
TapeStation loading tips (Agilent, catalog number: 5067-5153)
TapeStation sample tube strips (Agilent, catalog number: 4014128)
TapeStation sample tube caps (Agilent, catalog number: 4014125)
MicroAmpTM fast optical 96-well reaction plate (ABI Applied Biosystems, catalog number: 4346907)
MicroAmpTM optical adhesive film (ABI Applied Biosystems, catalog number: 4311971)
Ice and ice box
Laboratory tissue roll
Equipment
Automated compresstome (Precisionary Instruments, catalog number: VF-510-0Z)
Stainless steel blades for vibratome (Campden Instruments LTD, catalog number: 7550-1-SS)
Leica SP8 inverted confocal microscope (Leica)
SpectraMax® iD3 microplate reader (Molecular Devices, model: iD3, serial number: 37370-3811)
FastPrep-24TM classic bead tissue homogeniser (MP Biomedicals, catalog number: 116004500)
TapeStation 2200 (Agilent, catalog number: 18588)
VeritiProTM thermal cycler (Applied Biosystems, catalog number: A48141)
StepOnePlusTM real-time PCR system (Applied Biosystems, catalog number: 4376600)
NanoDrop spectrophotometer (Thermo Scientific, catalog number: ND-1000)
-20 °C freezer
Incubator (humidified, 37 °C, 5% CO2) (Sanyo, catalog number: MCO-17A)
Prism benchtop microfuge (Appleton Woods, catalog number: AA9760)
Centrifuge vortex for PCR plate CVP-2 (Grant Bio, catalog number: CVP-2)
IKA MS 3 basic shaker with MS 3.5 PCR plate attachment (IKA, catalog number: 0003617000)
Curved dissecting forceps, 10 cm, Serr/C (World Precision Instruments Ltd, catalog number: 15915)
Dissecting scissors, 10 cm, straight (World Precision Instruments Ltd, catalog number: 14393)
Surgical scissors, 14 cm, straight (World Precision Instruments Ltd, catalog number: 14192)
Spring scissors, 12 cm straight, 12 mm extra-fine blades (World Precision Instruments Ltd, catalog number: 14125)
Metallic spatula (Fisher Scientific Ltd., catalog number: 11523482)
Software and datasets
Leica Application Suite X software (Leica)
Fiji (ImageJ, version 2.9)
GraphPad Prism 10 (GraphPad)
2200 TapeStation System (Agilent, G2964AA)
StepOne Software (Applied Biosystems)
Procedure
Mouse dissection and lung inflation
Perform mouse dissection and lung inflation as previously described in Procedure A1 “Mouse dissection and lung harvesting” (refer to Video 1 and Figure 1) [7].
PCLS generation
Generate PCLS as previously described [7], with slight modifications (Video 1).
Video 1. Precision-cut lung slices (PCLS) generation using compresstome
Preparation
Cool the chilling block in the -20 °C freezer for 1 h before use.
Attach the stainless-steel blade to the blade holder using super glue. Use a paper clip to hold it in place and air dry at room temperature for 10 min before use.
Prepare 200 mL of HBSS/HEPES buffer (see Recipes) and a 48-well plate with ice-cold SF-DMEM (300 μL/well) and keep on ice until use.
Disinfect the surgical tools, spatula, specimen holder, and the buffer tray of the compresstome by wiping them with 70% ethanol (see Recipes). Let them air dry.
Prepare 50 mL of 2% (w/v) agarose solution (see Recipes) and an icebox with lukewarm water to keep the agarose warm and prevent it from solidifying throughout the slicing process.
Precision-cut lung slicing
In a 90 mm Petri dish, separate the fresh lung lobes using forceps and dissecting scissors.
Slice a tiny section off from the basal end of the tissue to form a flat surface using a scalpel blade. This ensures that the tissue can be securely glued to the specimen tube.
Squeeze a tiny drop of super glue onto the specimen tube base.
Note: Do not use too much super glue, as this will cause the specimen tube plunger to stick to the metal tube.
Place the lung lobe vertically onto the glue using forceps and allow the super glue to cure for 1 min.
Pull the specimen tube plunger downwards until the tissue is fully covered by the metal tube.
Use a 5 mL syringe and pipette enough lukewarm agarose solution to cover the tissue and ensure no bubbles formed around it.
Place the chilling block around the specimen tube for 1 min to solidify the agarose.
Once the agarose is solidified (turns from transparent to translucent), insert the specimen tube into the buffer tray fully until the stopper ring touches the adapter.
Adjust the micrometer until it touches the back of the specimen tube.
Slide the blade holder into the axial bar of the vibrating unit and secure it with the Allen key.
Add ice-cold HBSS/HEPES buffer into the buffer tray until the specimen tube is submerged in the buffer.
Set the compresstome to start slicing at a thickness of 300 μm, advance speed 4, and oscillation 4.
The desired thickness is 250 μm. Start slicing on continuous mode at 300 μm and reduce the thickness by 10 μm after each slice is produced (300 μm → 290 μm → 280 μm → 270 μm → 260 μm → 250 μm) until reaching 250 μm. Continue slicing at the thickness of 250 μm until the entire embedded tissue is completely sliced.
Note: The gradual reduction in thickness helps to produce slices with consistent and accurate thickness.
Collect the PCLS with a metal spatula and place them into the 48-well plate containing 300 μL of SF-DMEM (one PCLS per well) as the slicing progresses.
Note: As the number of live cells is a critical factor that determines the metabolic activity in MTT assays, it is recommended to use PCLS generated from the middle of the lung lobe to ensure size conformity.
Incubate the PCLS in the incubator (37 °C, 5% CO2) for 2 h before proceeding with the washing steps.
After incubation, wash the PCLS with 300 μL of prewarmed SF-DMEM per well. Incubate at 37 °C in 5% CO2 for 5 min during each wash and repeat the washing step three times.
Pause point: The PCLS can be incubated overnight at 37 °C in 5% CO2 before proceeding with the washing steps the next day. This does not significantly affect the efficacy of TAT-Cre treatment.
TAT-Cre treatment of PCLS
Experimental controls:
Negative control for Cre protein
Treat PCLS from the same R26R-EYFP mouse with only SF-DMEM. These PCLS serve as the negative control to exclude leaky transgene.
Control for artefacts due to the TAT-Cre treatment
Use PCLS from a wildtype mouse of the same background strain as the transgenic mouse as a control to exclude artefacts caused by the TAT-Cre treatment.
TAT-Cre recombinase treatment
After the washing steps, add 3 μM of TAT-Cre recombinase solution diluted in prewarmed SF-DMEM to the R26R-EYFP and wildtype mouse PCLS (250 μL/well) and incubate for 24 h at 37 °C in 5% CO2 (Figure 1A). Untreated R26R-EYFP PCLS with SF-DMEM are used as negative controls.
After 24 h of incubation, remove the TAT-Cre solution. Add fresh prewarmed SF-DMEM into the PCLS (250 μL/well) and incubate at 37 °C in 5% CO2 for a further 48 h.
See Troubleshooting.
Figure 1. TReATS method activates the expression of EYFP transgene in precision-cut lung slices (PCLS) generated from R26R-EYFP mouse. (A) Schematic showing a simplified structure of the loxP-modified allele in R26R-EYFP mouse and the structure of the targeted locus after Cre-mediated excision of the loxP-flanked stop sequence. TAT-Cre recombinase-mediated excision of the upstream loxP-flanked stop sequence allows the transcription and translation of EYFP transgene. (B) Tiled image shows ubiquitous expression of EYFP protein in TAT-Cre-treated R26R-EYFP PCLS. Image was generated using a confocal microscope.
Live staining of PCLS
To visualise in which alveolar cell types the TAT-Cre recombinase-mediated EYFP activation occurs (Figure 1B), at 72 h post-Cre treatment, immunolabel key alveolar cell type markers with fluorescent dye-conjugated antibodies, as listed in Materials and Reagents.
Incubate the PCLS with Alexa Fluor® 647-conjugated rat anti-LAMP3 (1:250), podoplanin eFluor® 660 (1:500), Alexa Fluor® 647-conjugated PECAM (1:200), Alexa Fluor® 647-conjugated CD11c (1:200), or BioTrackerTM TiY vimentin live cell dye (1:2,000) diluted in prewarmed SF-DMEM for 2 h at 37 °C in the dark. Use 250 μL of diluted antibody per PCLS per well in a 48-well plate.
To validate the absence of any non-specific binding of the conjugated fluorophores, stain PCLS with appropriate isotype IgG control antibodies diluted in prewarmed SF-DMEM for 2h at 37 °C in the dark: Armenian hamster IgG-PE (1:200) for TiY vimentin or rat IgG-Alexa Fluor 647 (1:200) for LAMP3, PDPN, PECAM, and CD11c. Use 250 μL of diluted antibody per PCLS per well in a 48-well plate.
After incubation, discard the culture medium with antibodies and add 250 μL of DAPI (1:500) diluted in prewarmed SF-DMEM for 15 min at 37 °C in the dark to counterstain the cell nuclei.
Remove DAPI, wash PCLS three times with 300 μL of prewarmed PBS, and immediately set up the PCLS for live imaging.
Note: It is recommended to image the immunostained PCLS on the same day to avoid fluorophore signal loss.
Live imaging of PCLS using confocal microscope
Pre-equilibrate the incubator chamber of an inverted confocal microscope with the following conditions: 37 °C, 5% CO2, and room air oxygen levels, approximately 21%, for 30 min.
Note: It is crucial to equilibrate the incubator chamber before imaging to maintain the viability of PCLS throughout the imaging process, particularly if the imaging is expected to take over an hour.
Meanwhile, add 100 μL of prewarmed imaging medium (see Recipes) into each well in an Ibidi uncoated 24-well µ-plate. Carefully transfer the immunostained PCLS from the 48-well plate onto the imaging medium using a metal spatula.
Using forceps, gently place a 12 mm Millicell® cell culture insert with 0.4 μm pores onto the PCLS so that the PCLS is positioned at the centre of the insert. Secure the insert in place by placing a flat metal washer on top of the insert (Figure 2A).
Add 200 μL and 400 μL of imaging media into the bottom chamber and upper chamber, respectively. Cover the 24-well plate with a lid.
Once the incubator chamber is equilibrated, place the Ibidi 24-well plate containing PCLS onto the motorised stage. Image using an HC PL APO 10×/0.40 air objective lens (Figure 2B, 2C) or HC PL APO 40×/1.30 oil objective lens (Figure 2D–2H). Use the following lasers: Diode 405 nm (for DAPI), argon 514 (for EYFP), Diode 561 (for vimentin), and Diode 633 (for LAMP3, PDPN, PECAM, and Cd11c) (Figure 2D–2H).
Note: Low magnification (10× objective lens) is recommended for an overview of EYFP activation across the entire PCLS; high magnification (40× objective lens) is recommended to visualise co-localisation of EYFP with different alveolar cell type markers.
Figure 2. Live imaging of precision-cut lung slices (PCLS) stained with specific cell type markers. (A) Schematic diagram shows the live imaging setup that utilises a transwell insert and a metal washer to secure the PCLS in place throughout the imaging process. (B-C) Representative images showing untreated R26R-EYFP PCLS (B) and TAT-Cre recombinase-treated R26R-EYFP PCLS (C). EYFP protein expression is shown in yellow and cell nuclei were labelled with DAPI (blue). Images were captured on a confocal microscope using an HC PL APO 10×/0.40 air objective lens. (D–H) Images showing co-localisation of EYFP with different alveolar cell type markers: LAMP3 (mature ATII cells) (D), PECAM for endothelial cells (E), PDPN for ATI cells (F), CD11c for macrophages (G), and vimentin for fibroblasts (H). Images were taken using 40× objective lens on a confocal microscope.
Image five random fields of alveolar regions per PCLS at approximately 40 μm from the bottom of the PCLS.
Note: A distance of 40 μm from the bottom of the PCLS is recommended as the PCLS are generally at the best focus at this distance. Imaging further from this distance is possible but can become challenging due to limited light penetrance through the depth of PCLS. To image the whole depth of PCLS, Z-stack is required, and this has been described in the original manuscript. It is also recommended to avoid imaging the PCLS less than 20 μm from the bottom of the PCLS, as the slicing process injures the surface of the PCLS, and an intact lung architecture may not be retained closer to the sliced surface. Thus, imaging at 40 μm from the bottom of the PCLS provides a convenient and representative snapshot of the PCLS.
Cell viability test (MTT assay)
At 72 h post-Cre treatment, perform MTT assays to examine whether the Cre treatment affects cell viability by assessing cell metabolic activities within PCLS.
Note: Use PCLS of equal size generated from the middle of the lung lobe for MTT assays.
Prepare 10% MTT solution (see Recipes). Use 250 μL of 10% MTT solution for each PCLS per well in a 48-well plate.
Positive control for dead cells: PCLS treated with 70% methanol serve as control for dead cells. Add 250 μL of 70% methanol (see Recipes) into the control PCLS and incubate at room temperature for 15 min. After 15 min, remove the methanol solution and wash the PCLS three times with 300 μL of room-temperature PBS. Proceed with step F4.
Remove the culture medium. Add 250 μL of 10% MTT solution into the PCLS and incubate at 37 °C and 5% CO2 in the dark for 45 min.
After incubation, discard the MTT solution. Add 250 μL of DMSO into each PCLS and incubate at 37 °C and 5% CO2 in the dark for a further 10 min.
After DMSO incubation, gently pipette up and down a few times and transfer 200 μL from each well into a new clear-bottom 96-well plate.
Read the absorbance at 570 nm and 690 nm and calculate cell viability (see Data analysis).
Note: Use four mice per genotype and three PCLS per treatment per experiment.
Validation of gene expression change
PCLS homogenisation and RNA extraction
Use RNase decontamination solution to clean the working surface.
Steps G1b–g are illustrated in Figure 3. In each homogenisation tube, add homogenisation beads to the level of the bottom cone line of the tube. Label each tube with the sample name.
Figure 3. Precision-cut lung slices (PCLS) homogenisation workflow
Add 600 μL of RLT/β-ME lysis buffer (see Recipes) into each homogenisation tube.
Discard the culture medium and gently wash the PCLS twice with 300 μL of room-temperature PBS.
Note: It is important to wash the PCLS with PBS. Incomplete removal of culture medium will inhibit lysis and may reduce RNA yield.
Use a metal spatula to transfer the PCLS into the homogenisation tube with RLT/β-ME lysis buffer. Pool three PCLS of the same treatment into each homogenisation tube.
Note: Pool three PCLS for an RNA sample and three RNA samples for each condition per experiment.
Load the tubes onto the rotor of the FastPrep-24 tissue homogeniser and secure them tightly with the rotor holder. Run the homogeniser for 45 s twice, with a 1-min resting interval.
After homogenisation, centrifuge the tubes at 13,000× g for 3 min at 4 °C. Collect the supernatant (approximately 600 μL) into a new microcentrifuge tube.
Add an equal volume (600 μL) of 70% ethanol (see Recipes) into the PCLS lysate and gently pipette up and down a few times.
Transfer 600 μL of the sample into the RNeasy spin columns. Centrifuge at 13,000× g at room temperature for 1 min. Discard the flowthrough. Repeat this by transferring the remaining 600 μL of lysate–ethanol mixture into the same RNA columns. Centrifuge again at 13,000× g at room temperature for 1 min and discard the flowthrough.
Add 350 μL of RW1 washing buffer (provided in the RNeasy mini kit) into each tube. Centrifuge again at 13,000× g at room temperature for 1 min and discard the flowthrough.
DNase digestion: prepare DNase I solution. For each RNA sample, add 10 μL of DNase I stock solution to 70 μL of RDD buffer (provided in the RNase-free DNase set). Mix by gently inverting the tube and centrifuge briefly.
Note: Do not vortex as DNase I is particularly sensitive to physical denaturation.
Add 80 μL of DNase I mix directly to the RNeasy spin column and incubate at room temperature for 15 min.
After incubation, add 350 μL of RW1 buffer to the column, centrifuge at 13,000× g at room temperature for 1 min, and discard the flowthrough.
Add 500 μL of RPE buffer to the column, centrifuge at 13,000× g at room temperature for 1 min, and discard the flowthrough.
Add 500 μL of RPE buffer to the column and centrifuge at 13,000× g at room temperature for 2 min.
Place the RNeasy spin column into a new collection tube and centrifuge at 13,000× g at room temperature for 1 min to remove residual solution.
Place the RNeasy spin column into a new collection tube. Add 30 μL of RNase-free water to the spin column and centrifuge at 13,000× g for 1 min. To increase the yield of RNA, transfer the eluent back to the spin column and repeat the centrifugation step.
Collect the total RNA sample into a new microcentrifuge tube. Store at -80 °C until use.
Assessment of RNA quality and concentration
Allow RNA ScreenTape sample buffer to equilibrate at room temperature for 30 min and vortex it before use.
Thaw total RNA samples on ice. Mix 5 μL of RNA sample buffer with 1 μL of RNA sample.
Spin down and then vortex using IKA vortexer and adaptor at 2,000 rpm for 1 min. Spin down to collect the solution at the bottom of the tube.
Denature the samples at 72 °C for 3 min using a thermal cycler, followed by 2 min cooling on ice.
Spin down to collect the sample at the bottom of the tube.
Load the samples and loading tips into the 2200 TapeStation instrument and start the run.
Note: Only RNA samples with RNA integrity number (RIN) > 8 are used for downstream experiments.
cDNA reverse transcription
Thaw the kit components on ice and prepare the RT master mix on ice according to Table 1:
Table 1. Reverse transcription reaction setup
Kit component Volume per reaction (μL)
10× RT buffer 2
25× dNTP mix (100 mM) 0.8
10× RT random primers 2
MultiScribeTM reverse transcriptase 1
RNA sample 200 ng
Top up with nuclease-free H2O to 20 μL (per reaction)
Pipette the mixture up and down a few times to mix.
Close the lids of the tubes and spin down to collect the solution to the bottom of the tubes.
Load the samples onto the thermal cycler and start the run using the following protocol: 25 °C for 10 min, 37 °C for 2 h, 85 °C for 5 min, followed by indefinite holding at 4 °C.
Store the cDNA samples at -20 °C freezer until use.
Quantitative real-time PCR (qRT-PCR)
Thaw cDNA samples on ice and measure the concentration of the cDNA samples using a Nanodrop. Use nuclease-free water as blank.
Dilute the cDNA samples to 50 ng/μL using nuclease-free water.
Thaw TaqMan® assays on ice.
Note: Use appropriate target assays accordingly. YFP (Assay ID: Mr04097229_mr) and B2m (Assay ID: Mm00437762_m1; reference gene) assays are used in this protocol (see Reagents).
Mix the Fast Advanced Master Mix by pipetting up and down a few times.
Calculate and prepare the qRT-PCR reaction mix in 8-tube PCR strips according to Table 2 (volume per reaction × number of reactions):
Table 2. qRT-PCR reaction setup
Kit component Volume per reaction (μL)
2× TaqMan® Fast Advanced Master Mix 5
20× TaqMan® Fast gene expression assay 0.5
cDNA template (50 ng/μL) 2
Nuclease-free water 2.5
Total volume per reaction 10
Note: Prepare three reactions (triplicates) per gene for each cDNA sample.
After all the components are added to the tubes, close the caps and vortex briefly to mix.
Briefly centrifuge the 8-tube PCR strips using a benchtop microfuge with 4 × 8 PCR strip rotor to spin down the contents.
Pipette 10 μL of the qRT-PCR reaction mix into each well of a 96-well PCR plate.
Seal the plate with an optical adhesive film. Centrifuge briefly using a microfuge with a 96-well plate rotor to eliminate the air bubbles.
Run the PCR on a StepOne Plus Real-Time PCR System using the following parameters: UNG incubation at 50 °C for 2 min, polymerase activation at 95 °C for 20 s, followed by 40 cycles of (denaturation at 95 °C for 1 s and annealing at 60 °C for 20 s).
Analyse the relative transcript levels using the 2−ΔΔCT method (see Data analysis).
Note: Use four mice per genotype, pool three PCLS per RNA sample, and three RNA samples per treatment per experiment.
Data analysis
For MTT assays, cell viability is calculated using the following formula:
in which,
ODAdj (Adjusted OD) = OD570nm – OD690nm
Use three PCLS per treatment (triplicates):
For qRT-PCR, gene expression change is calculated using the 2-ΔΔCT formula, in which
Pool three PCLS for each RNA sample and prepare three RNA samples per treatment per experiment (triplicates; nine PCLS required per treatment):
ΔΔCT = Mean ΔCTCre-treated PCLS – Mean ΔCTcontrol PCLS
2-ΔΔCT represents the fold change of the treated target gene relative to the control PCLS.
Repeat the experiments with a total of four mice (N = 4). Use non-parametric tests to compare the difference between Cre-treated and control PCLS.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Cheong et al. [6]. A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices. Disease Models & Mechanisms (Figure 1, panels A–E; Figure 2, panels A–E; Movie 1).
General notes and troubleshooting
If the efficacy of TAT-Cre recombination is insufficient, consider increasing the dosage of TAT-Cre and/or extending the incubation time of PCLS in the TAT-Cre solution. Refresh the SF-DMEM every other day if prolonged incubation of PCLS is required. Our experiments found that using up to 5 μM of TAT-Cre and incubating PCLS with the TAT-Cre solution for 72 h did not significantly impact the viability of PCLS.
As a reference, a minimum of 15 PCLS from a single animal are required per experimental condition. These include three PCLS for TAT-Cre treatment, three PCLS for MTT assay, and nine PCLS to generate three RNA samples for qRT-PCR analysis. For example, an experiment comparing untreated control PCLS vs. 3 μM TAT-Cre-treated PCLS will require 30 PCLS from a single mouse, and it is recommended to use four mice to confirm reproducibility.
Optional: If co-localisation staining is required, as described in this protocol, add three PCLS per condition per marker/antibody used. A single PCLS can be labelled with multiple markers/antibodies as long as the fluorophore spectra do not overlap.
A single RNA sample generated from pooling three PCLS generally has a yield of 1.5–3.0 μg (concentrations of 50–100 ng/μL in a total volume of 30 μL). The yield varies depending on the size of the PCLS used.
To validate the efficiency of the TReATS method, additional techniques beyond qRT-PCR and immunostaining, as described in this protocol, can be utilised. These include PCR to confirm DNA sequence changes as well as western blotting or flow cytometry to assess changes at the protein levels. Additionally, if the effect of gene modification is known, other readouts may also prove useful in validating the efficiency of the TReATS method. For instance, the lack of a functional Vangl2 gene is known to disrupt actin cytoskeleton remodelling, a phenotype that could be readily detected. In TAT-Cre recombinase-treated Vangl2flox/flox PCLS, highly disrupted filamentous actin (F-actin) was observed, indicating successful deletion of the Vangl2 gene in the PCLS. This was described in the original manuscript [6].
Acknowledgments
This project was initially reported in Cheong et al. [6] and funding was as follows: The Royal Brompton and Harefield Hospitals Charity (grant number: 123 P90719) and an award from Mr. and Mrs Youssef Mansour. T.C.L was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and The Royal Society (210424/Z/18/Z). This protocol is adapted from the method published in Cheong et al. [6]. Graphical overview and Figures 1A, 2A, and 3 were created with BioRender.
Competing interests
The authors declare no competing or financial interests.
Ethical considerations
All animal maintenance and procedures were conducted in compliance with the requirements of the Animal (Scientific Procedures) Act 1986. Animal work was approved by the South Kensington Animal Welfare and Ethical Review Body committee at Imperial College London.
References
Alsafadi, H. N., Uhl, F. E., Pineda, R. H., Bailey, K. E., Rojas, M., Wagner, D. E. and Königshoff, M. (2020). Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery. Am. J. Respir. Cell Mol. Biol. 62(6): 681–691.
Akram, K. M., Yates, L. L., Mongey, R., Rothery, S., Gaboriau, D. C. A., Sanderson, J., Hind, M., Griffiths, M. and Dean, C. H. (2019). Live imaging of alveologenesis in precision-cut lung slices reveals dynamic epithelial cell behaviour. Nat. Commun. 10(1): 1178.
Alsafadi, H. N., Staab-Weijnitz, C. A., Lehmann, M., Lindner, M., Peschel, B., Königshoff, M. and Wagner, D. E. (2017). An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices. Am. J. Physiol. - Lung Cell. Mol. Physiol. 312(6): L896–L902.
Kim, S. Y., Mongey, R., Wang, P., Rothery, S., Gaboriau, D. C., Hind, M., Griffiths, M. and Dean, C. H. (2021). The acid injury and repair (AIR) model: A novel ex-vivo tool to understand lung repair. Biomaterials 267: 120480.
Närhi, K., Nagaraj, A. S., Parri, E., Turkki, R., van Duijn, P. W., Hemmes, A., Lahtela, J., Uotinen, V., Mäyränpää, M. I., Salmenkivi, K., et al. (2018). Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras‐driven lung tumours. J. Pathol. 245(1): 101–113.
Cheong, S. S., Luis, T. C., Stewart, M., Hillier, R., Hind, M. and Dean, C. H. (2023). A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices. Dis. Model. Mech. 16(11): e050267.
Akram, K., Yates, L., Mongey, R., Rothery, S., Gaboriau, D., Sanderson, J., Hind, M., Griffiths, M. and Dean, C. (2019). Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices. Bio Protoc. 9(20): e3403.
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4,981 | https://bio-protocol.org/en/bpdetail?id=4981&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome
DG Dione Gentry-Torfer
EM Ester Murillo
CB Chloe L. Barrington
SN Shuai Nie
ML Michael G. Leeming
PS Pipob Suwanchaikasem
NW Nicholas A. Williamson
UR Ute Roessner
BB Berin A. Boughton
JK Joachim Kopka
FM Federico Martinez-Seidel
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4981 Views: 701
Reviewed by: Alba BlesaPiao YangOm Prakash Narayan
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Original Research Article:
The authors used this protocol in bioRxiv Nov 2022
Abstract
Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find “false” enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements.
Key features
• Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome.
• Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states.
• Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1].
• Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].
Keywords: Ribo-proteome SP3 beads Top-down proteomics Bottom-up proteomics Fractional protein synthesis rates
Graphical overview
Some of the illustrations in the graphical abstract were created and exported under a paid license with BioRender
Background
Throughout the tree of life (TOL), ribosomes play a pivotal role in synthesizing all proteins by decoding mRNA [3]. The most precise representations of the TOL to date have been crafted using aligned genomic sequences of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) [4–7]. This centrality underscores the significance of ribosomal components, many of which feature prominently in the universal gene set of life (UGSL) [8]. The UGSL comprises orthologous genes conserved across the phylogenetic TOL, altered only through speciation while retaining their fundamental functions [9–11]. The interaction of r-proteins and rRNAs throughout the TOL has necessitated a conserved, basic, structural riboproteome. In opisthokonts, the complex has significantly increased in size, both in terms of rRNA expansion segments and in number of associated r-proteins [3]. The r-proteins themselves have become more complex and, in many cases, acquired new functions. Among acquired functionality, ribosomes have gained the ability to selectively decide which transcripts to translate, a concept known as ribosome specialization [12,13]. For example, ribosomal populations enriched in RPL10 translate transcripts subsets [14], and ribosomes lacking RPS26 preferentially translate transcripts from stress-response pathways [15]. To what extent and what proportion of the r-proteome has acquired these types of adaptations remains unknown. Nevertheless, it is becoming increasingly clear that the composition of riboproteomes at a given time reflects structural and functional features of ribosomes [15–17] and grants them the ability to control translational output and shape protein expression.
The field of structural systems biology has progressed greatly in recent years due to the convergence of atomic structures, which provide an average scaffold of macromolecular complexes, and omics studies, which elucidate the relative or absolute abundances of their structural components in vivo [18]. Ribosomes have driven the convergence of these methods due to their ubiquitous and essential nature in the cellular environment. Ribosome structures can currently be highly resolved, reaching near-atomic resolution [19–21]. On the other hand, omics methods lag in providing tailored solutions to fully decipher the general status of riboproteomes. Although methods such as transcriptomics or translatomics can monitor gene expression in a significant percentage of the expressed genome, a gap remains between transcriptional regulation and the abundance of active protein pools [22]. Recent efforts include advanced mass spectrometry methods to elucidate different features of r-proteins [23], such as absolute quantification using synthetic peptide standards [14]. These examples make it clear that multiplexing methods will help to overcome the limitation of studying riboproteomes at the ribosome population level.
We focused on three major obstacles that currently prevent the thorough investigation of r-proteins. First, multiplexing requires the purification of riboproteomes in a pseudo-native state that conserves protein features to elucidate different aspects of their biology. Second, the analysis of r-proteins following protease digestion requires careful consideration of the amino acid composition, which includes many basic peptide stretches required for interacting with rRNA. Finally, many translational studies often fail to bridge the gap between translatome and proteome. Currently, the best method to monitor the translational output from ribosomes is Ribo-Seq [24], which has been optimized in plant systems [25] but fails to provide measurements of de novo synthesized proteins. Moreover, typical translational studies deal with organisms that transition between biological steady states. These transitions complicate the use of technologies such as in vivo stable-isotope labeling and mass spectrometry to monitor protein synthesis, as quantification of tracer incorporation assumes steady-state conditions.
Here, we set out to provide a standardized methodology to overcome these three limitations while addressing a previously unsolved biological question in plant translation [1]. To deal with the first limitation, we developed a strategy to purify the r-proteome as close as possible to its native state by using a chaotropic agent to dissociate r-proteins from rRNA. Our purification strategy enhances both top-down and bottom-up proteomics methods, as previously described by others [23], by capturing r-proteins with paramagnetic SP3 beads [26–28]. To bypass the second limitation, we carefully consider our choice of an optimal protease to digest ribosomal proteins across the TOL. We validate that our protease yields larger peptides and thus allows for a more comprehensive profiling of the r-proteome from bacteria to higher metazoans. To address the third limitation, we provide an R package called ProtSynthesis (https://github.com/MSeidelFed/ProtSynthesis), in which we developed physiological and data-based assumptions required to quantify tracer incorporation and fractional protein synthesis rates in organisms shifting between biological states, using plant cold acclimation as an example [1,29,30]. Our method uses organismal physiology in the treated vs. non-treated condition to make fractional synthesis rates comparable. First, we quantify the enrichment percentage in soluble amino acid pools and use them to constrain the maximum amount of nitrogen atoms that could have incorporated the label in specific peptide sequences, thus correcting for biases in 15N incorporation into soluble amino acid pools across conditions. Secondly, treatments often induce differences in growth, and we calculate relative growth rates to normalize fractional protein synthesis. Finally, because the most direct measurement of translation is protein content, we correct our relative growth rates to reflect the percentage of protein accumulated per unit of time. In summary, our calculations comprehensively incorporate organism physiology to enhance the comparison between fractional protein synthesis rates across different biological steady states, such as cold-acclimated plants vs. plants reared at optimal temperatures. Beyond cold acclimation, there are plant examples in which biological steady state shifts occur concomitantly to surmised translational reprogramming, which could profit from our methodology. For instance, 3',5'-cAMP supplementation induces spatially constrained rearrangements of the r-proteome in Arabidopsis [31]. Similarly, flg22 induces dissociation of the P-Stalk via the activation of Mitogen-activated protein kinase 6 pathway, influencing plant immunity to bacteria [16]. These examples set an ideal stage for inquiring about changes in the synthesis and assembly of the r-proteome and how it affects protein synthesis, i.e., what is the functional translational implication of remodeling the r-proteome. To summarize, we provide a comprehensive methodology to couple the most accurate methodology to study translational efficiencies (Ribo-Seq) to r-protein and proteome-wide fractional protein synthesis rates.
Summary
Our method contains five steps that are depicted in the graphical abstract. Step 1a entails labeling the biological specimens in a manner tailored to each organism. The required methodology, including materials and labeling considerations for plants, is discussed in detail elsewhere [2]. Step 1b entails a detailed phenotypic analysis of the organism of interest upon applying a specific treatment that produces a shift from the previous biological steady state. For phenotyping and growth analyses of plants, we use and recommend the classical definition of relative growth rates that has been discussed extensively in the literature over the last century [32–34] based on the first postulation of an efficiency index by Blackman, 1919 [35], which is equivalent to the relative growth rate. Step 2a accounts for differential labeling efficiency in soluble amino acid pools at the onset of the investigated physiological transition. The methodology for obtaining the primary metabolome and measuring metabolite abundances by gas chromatography–mass spectrometry has been described elsewhere [2]. The contents of the manuscript detail the procedure to purify and profile a complexOme or ribosomal proteome (Step 2b) and elaborate on the data analyses and further considerations needed to calculate protein fractional synthesis rates from protein components of isolated complexes (Step 3).
Materials and reagents
Biological materials
Apical root meristems of germinating seedlings from Barley (Hordeum vulgare cultivar Keel) obtained from The University of Melbourne from previous studies [36] (Note 1)
Escherichia coli 70S ribosomes (New England BioLabs, catalog number: P0763S); one tube of 1 mg is enough as internal standard for the protocol detailed below
Reagents
Guanidine hydrochloride (GuHCl) (Sigma-Aldrich, catalog number: G3272)
Trifluoracetic acid (TFA) (Thermo Fisher Scientific, catalog number: 85183)
PierceTM BCA Protein Assay kit (Thermo Fisher Scientific, catalog number: 23227)
Water, molecular biology grade, DNase and RNase free (Research Products International, catalog number: 248700)
Triethylammonium bicarbonate buffer (TEAB) (Sigma-Aldrich, catalog number: T7408)
Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (Macherey-Nagel, catalog number: 740395.107)
Iodoacetamide (IAA) (Sigma-Aldrich, catalog number: I5161)
Acetonitrile (Acn) (Sigma-Aldrich, catalog number: 271004)
Sera-MagTM SpeedBad carboxilated-modified magnetic particles [GE Healthcare, catalog number: 45152105050250 (hydrophilic); 65152105050250 (hydrophobic)]
Ethanol absolute, suitable for HPLC, ≥99.8% (Sigma-Aldrich, catalog number: 34852-M)
Endoproteinase Lys-C (New England Biolabs, catalog number: P8109S)
Trizma(R)-hydrochloride solution, pH 9.0, RNase free (Sigma-Aldrich, catalog number: 1185-53-1)
Nuclease-Free Water, for Molecular Biology (Sigma-Aldrich, 7732-18-5)
Potassium chloride (KCl) (2 M), RNase free (Sigma-Aldrich, catalog number: AM9640G)
EGTA (sigma-Aldrich, catalog number: 324626)
Magnesium chloride solution (MgCl2) (1 M), RNase free (Sigma-Aldrich, catalog number: M1028)
Chloramphenicol (Cm) (Sigma-Aldrich, catalog number: C0378)
Cycloheximide (CHX) (Research Products International, catalog number: 50488631)
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)
Protease inhibitor cocktail (Pi) (Sigma, catalog number: P9599)
Phenylmethylsulphonyl fluoride (PMSF) (Thermo Fisher Scientific, catalog number: 36978)
Solutions
Resuspension buffer (see Recipes)
6 M GuHCl (see Recipes)
50% (v/v) TFA (see Recipes)
500 mM TCEP (see Recipes)
55 mM IAA (see recipes)
Digestion buffer and endoproteinase Lys C working solution (see Recipes)
80% Acn, 0.1% TFA (v/v) (see Recipes)
Recipes
Resuspension buffer (10 mL)
*Prepare all necessary stocks before starting the protocol. The recommendation is to prepare a pre-resuspension buffer and store at 4 °C; all stocks, except for the small molecules, must be filtered through a 0.22 μm filter or autoclaved. Subsequently, just before usage in resuspension, add the translational stallers and reducing agents, i.e., Cm, CHX, DTT, PMSF, and Pi.
Reagent Final concentration Volume
TRIS HCl (1 M, pH 9.0) 200 mM 2 mL
KCl (2 M) 200 mM 1 mL
EGTA (500 mM, pH 8.0) 25 mM 500 μL
MgCl2 (1 M) 35 mM 350 μL
RNase-free water n/a 5.94 mL
CHX (180 mM) 0.18 mM 10 μL
DTT (1 M) 5 mM 50 μL
PMSF (200 mM) 1 mM 50 μL
Pi (100×) 1× 100 μL
Total n/a 10 mL
6 M GuHCl (10 mL)
*Prepare fresh every time.
Reagent Final concentration Quantity
GuHCl 6 M 5.73 g
RNase-free water n/a Up to 10 mL
Total n/a 10 mL
50% TFA (1 mL)
*Prepare fresh every time. Caution: prepare in fume hood.
Reagent Final concentration Volume
TFA 50% 500 μL
RNase-free water n/a 500 μL
Total n/a 1 mL
500 mM TCEP (750 μL)
*Prepare working solution of reducing agent TCEP according to manufacturer instructions. Dissolving the material in water might take several minutes; mix several times. After reconstitution, the solution should be stored at -20 °C and is stable for at least six months.
Reagent Final concentration Quantity
TCEP 500 mM 107 mg
RNase-free water n/a 750 μL
Total n/a 750 µL
55 mM IAA (3 mL)
* IAA is light sensitive. The solution should be freshly prepared, and the incubations should be performed in the dark.
Reagent Final concentration Quantity
IAA 55 mM 56 mg
TEAB (1 M) 25 mM 3 mL
Total n/a 3 mL
Digestion buffer and endoproteinase Lys-C
*The quantity of endoproteinase Lys-C needed depends on the number of samples and the protein amount in each sample. Consider that, even if the Lys-C protease stock is the same, the volume of 25 mM TEAB can be different if the amount of protein in the samples is different.
Reagent Final concentration Quantity or Volume
TEAB (1 M) 25 mM 10:1 (v/w) TEAB/protein
Endoproteinase Lys-C (20 µg) n/a 1:20 (w/w) Lys-C/protein
Total n/a n/a
80 % Acn, 0.1 % TFA (75 mL)
Reagent Final concentration Quantity or Volume
Acn 80% 60 mL
TFA (50 %) 0.1% 150 µL
RNase-free water n/a 14,850 µL
Total n/a 75 mL
Laboratory supplies
Ice
Ice bucket
Pipettes: 10 μL, 200 μL, 1,000 μL
Tips: 10 μL, 200 μL, 1,000 μL
Ultracentrifugation tubes, 1 mL, open-top thickwall polycarbonate (Beckman Coulter, catalog number: 343778)
Ultracentrifugation tubes (Beckman Coulter, catalog number: 326819)
2 mL safe-lock tubes (Eppendorf, catalog number: 0030120094)
1.5 mL safe-lock tubes (Eppendorf, catalog number: 0030120086)
96-well plates (Sarstedt, catalog number: 1581100)
Aluminum foil
pH indicator paper (Macherey-Nagel, catalog number: 90204)
Solid phase extraction (SPE) cartridges (Waters, catalog number: 186000383)
Equipment
-80 °C deep freezer (SANYO, model: MDF-U 72 V)
Balance XS105 (Mettler-Toledo, catalog number: 30132870)
SW55 Ti rotor (Beckman Coulter, Krefeld, model: 55 Ti)
Ultracentrifuge (Beckman Coulter, Krefeld, model: Optima XPN)
Optima MAX-XP ultracentrifuge (Beckman Coulter, catalog number: 393315)
MLA-130 fixed angle rotor (Beckman Coulter, model: Krefeld, model: MLA-130)
Vortex mixer (Bender & Hobein, model: G560-E)
Fume hood (Waldner, model: Airflow Controller AC2)
High-speed centrifuge (Eppendorf, model: 5417R)
Incubation room set to 37 °C (Note 2)
Thermomixer (Eppendorf, catalog number: 5382000015)
Microplate reader (BioTek Elisa ELx808, model: 25-315S)
Magnetic rack (Invitrogen, DynaMag-2, model: 12321D)
Speed-vac (LaboGene, model: SCANVAC CoolSafe100-9 PROSuperior XS)
Freeze-dryer (Christ, model: Alpha 2-4 LSCbasic)
Orbitrap mass spectrometer (Thermo Fisher Scientific, model: Eclipse) equipped with a nanoflow HPLC (Ultimate 3000 RSLC, Dionex)
Software and datasets
Absorbance plate reader software (BioTek, Gen5 2.09, 04/2022)
Microsoft Office software (Microsoft)
MaxQuant (above version 2.4, 04/2022)
R statistical language (above version 3.10, 06/2023)
R package ProtSynthesis (https://github.com/MSeidelFed/ProtSynthesis) (Access date, 05/2022)
Python (above version 3, 04/2021)
Python Script IsotopeEnrichment (https://github.com/mgleeming/isotopeEnrichment) (Access date, 05/2022)
Procedure
Pelleting of the polysome fractions
Plant monosome or polysome fractions can be purified in sucrose gradients [16,37,38].
Take polysome fractions previously isolated in the presence of known translational stallers such as chloramphenicol and cycloheximide. If stored at -80 °C, thaw on ice for 5 min (Note 3).
Transfer the polysome fractions to 1 mL, open-top thickwall polycarbonate ultracentrifugation tubes, and mix thoroughly (Note 4).
Add resuspension buffer up to 1 mL and resuspend thoroughly by pipetting up and down until the solution is clear (Note 5).
Tare all tubes with resuspended polysomes on a scale using resuspension buffer (standard deviation of 5 µg).
Centrifuge for 2 h at 110,000 RPM at 4 °C in an MLA-130 fixed angle rotor [g-force (avg) of 616,616× g, maximum 731,808× g, and K-Factor of 8] mounted on an Optima MAX-XP ultracentrifuge (Note 6).
Immediately after centrifugation, discard the supernatant by decanting while taking care not to disturb the pellet and acting quickly to avoid the possibility of resuspending the pellet in the buffer.
Place the tubes upside down on clean absorbent paper for 1 min to remove most of the supernatant, allowing the pellets to air-dry during this time interval. To fully dry excess liquid, briefly introduce absorbent paper inside the tubes without disturbing the pellet at the bottom. The polysome pellet has a flat jelly-like appearance at the bottom of the tube (Figure 1).
Figure 1. Ribosome pellets
Immediately transfer the tubes to ice and use or store the pellets at -80 °C until further use.
Resuspension for r-protein dissociation (Note 7)
Take the tubes containing polysome pellets from Step A8 and place them on ice.
Resuspend the ribosome pellets by adding 60 µL of 6 M GuHCl to the samples and slowly pipetting up and down until the pellet disappears.
Transfer the resuspension using a P1000 to 2 mL safe-lock tubes.
Acidify each sample by pipetting into the solution 1.2 µL 50% (v/v) TFA to reach a 1% (v/v) final volume.
Vortex briefly each sample.
Centrifuge at full speed (21,400× g) for 20 min at 4 °C to precipitate and remove the RNA.
Carefully transfer the supernatant using a P200 to a new 2 mL safe-lock tube without disrupting the pellet (Note 8), store the tubes on ice, and continue to the next section where protein contents are measured.
Determination and adjustment of r-protein concentration for beads and protease requirements
The following steps measure the total protein content of all samples using the classical BCA Protein Assay kit and an absorbance plate reader. Some of the steps have been modified within the ranges offered by the assay manual to fit the pipeline.
Take 6 µL from each sample to a 96 well-plate (e.g., Figure 2) and dilute at least 1× with RNase-free water to reach a concentration smaller or equal to 3 M GuHCl, which is below the compatibility threshold for the Pierce BCA Protein Assay.
Follow the Pierce BCA Protein Assay protocol to prepare the diluted BSA standards and working reagent and to determine the unknown protein concentration of each sample (Note 9).
First, pipette 10 µL of each standard in three replicates into a 96-well plate.
Add 200 µL of the working reagent to each well and mix the plate thoroughly on a plate shaker for 30 s.
Cover plate with aluminum foil and incubate at 37 °C for 30 min.
Cool plate at room temperature for 10 min. Measure the absorbance at or near 562 nm on a plate reader.
Subtract the average 562 nm absorbance measurement of the blank standard replicates (either protein-free buffer measurements or water, depending on the chosen procedure) from the 562 nm measurements of all other individual standard and unknown samples.
Prepare a standard curve by plotting the average blank-corrected 562 nm measurement for each BSA standard vs. its concentration in µg/mL. Use the standard curve to determine the unknown protein concentration of each sample.
Figure 2. 96-well plate layout. Columns in green refer to the three replicates of BSA standards and columns in gray represent up to 21 samples and their three technical replicates.
Determine and take the volume corresponding to 10 µg of protein for each sample.
Pipette the determined volumes into new 2 mL Eppendorf tubes and top up to 50 µL with 6 M GuHCl, 1% (v/v) TFA to standardize protein concentrations in all samples.
Increase the pH of the standardized protein solutions from acidic to approximately 8 by adding 10 µL of 1 M TEAB to each sample.
Monitor the pH of each sample by adding 1 µL to pH strips.
SP3 bead working solution
Remove the beads stocks A & B from 4 °C and let them equilibrate to room temperature for approximately 10 min. Stocks A & B correspond to the 45152105050250 (hydrophilic) and 65152105050250 (hydrophobic) parts that come together in the Sera-Mag SpeedBad carboxilated-modified magnetic particles kit. Mix both stocks by gently shaking to homogenize and allow the beads to come into suspension.
In a 2 mL Eppendorf tube, mix 100 µL of hydrophilic beads stock A with 100 µL of hydrophobic beads stock B and add 800 µL of RNase-free H2O.
Allow the beads to separate from the solution by placing the tubes in a magnetic rack for approximately 1 min and immediately discard the supernatant carefully without disturbing the beads.
To wash the magnetic beads, remove the tubes from the magnetic rack and add 1 mL of RNase-free H2O. Mix thoroughly by pipetting and place the tubes back in the magnetic rack. Let the beads separate and discard the supernatant after 1 min by removing it with a pipette without disturbing the beads. Repeat the washing step three times.
To obtain the working solution (20 µg/µL), add 500 µL of RNase-free H2O to the beads after removing the supernatant from the last wash. Working solution can be stored at 4 °C for a month.
Reduction, alkylation, and digestion
Considerations for the choice of a protease to digest a ribosomal proteome: RNA-protein binding domains are rich in amino acid residues such as histidine, arginine, and lysine, which are all basic amino acids. Basic amino acids populate ribosomal proteomes throughout the TOL because r-proteins must bind to rRNA. In fact, many of the r-protein interactions are directly mediated by rRNA. The nature of riboproteomes defines a clear expectation for tailored protease treatments to obtain adequate representation of all r-proteins. The protease trypsin is known to digest peptide sequences at the C-terminus of lysine and arginine. Therefore, cleavage of r-proteins could yield many small trypsinogenic peptides. In contrast, Lys-C digests protein sequences only at the C-terminus of lysine residues. According to in silico analyses, Lys-C cuts most r-proteins into significantly longer peptide pieces than trypsin (Figure 4). As a result, a better representation of the riboproteome can be achieved using Lys-C as compared with the tested proteases.
Figure 4. Volcano plot outlining the differences in mean peptide length produced with Lys-C compared with trypsin by in silico protease digestion applied to the ribosomal proteome of the model organisms across the tree of life. Related to Supplemental Table 1. In silico protease digestion was performed with the software Protein-Digestion-Simulator (https://github.com/PNNL-Comp-Mass-Spec/Protein-Digestion-Simulator). The resulting plot from analyzing the digestions contains, in the x-axis, the log2 of the ratio between mean peptide lengths from trypsin and Lys-C digestion. The y-axis contains the -log10 of the adjusted P value (Padj) from the statistical comparison of mean peptide lengths per each specific ribosomal protein. Note that in eukaryotic models mostly P-Stalk acidic proteins have x values > than 0; all the other ribosomal proteins have shorter mean peptide lengths when digested with trypsin as compared with Lys-C. The horizontal dotted line signals the significance boundary (P ≤ 0.05) transformed using a -Log10 function; thus, all the proteins above the line have significantly shorter mean peptide lengths when digested with Trypsin as compared with Lys-C.
Add 1.2 µL of 0.5M TCEP to each sample to reach a 10 mM concentration and shake with an incubator at 800 RPM and 37 °C for 45 min.
Work quickly: add 60 µL of freshly prepared IAA and shake in the dark or covered with aluminum foil with an incubator at 800 RPM and 37 °C for 45 min.
Add 160 µL of Acn to each sample to reach a 70% (v/v) final concentration.
Add 10:1 bead to protein ratio to each sample and mix thoroughly by pipetting to create a uniform solution.
Let the samples sit for 10 min at room temperature, mix by pipetting, and allow to sit for another 10 min at room temperature. Prepare digestion buffer while waiting.
Place tubes on the magnetic rack and allow them to separate for 30 s.
Critical: Work quickly and have all materials ready. All subsequent washing steps are performed quickly and without removing the safe-lock tubes from the magnetic rack.
Remove and discard supernatant with a pipette.
Add 1 mL of neat Acn to each tube, allow to sit for 10 s, and discard by drawing with a pipette.
Add 1 mL of 70% (v/v) ethanol to each tube, allow to sit for 10 s, and discard with a pipette. Remove tubes from the magnetic rack.
Add a 10:1 digestion buffer (µL) to protein (µg) ratio to each sample. The digestion buffer contains endoproteinase Lys-C at 1:20 protease to protein ratio, diluted in 25 mM TEAB.
Incubate at 37 °C by shaking at 200 RPM overnight (18–20 h).
Peptide wash and processing
Add 50% (v/v) TFA to reach 1% concentration to quench the digestion, e.g., for 130 µL add 2.6 µL of 50% (v/v) TFA per sample.
Mix thoroughly and spin for 2 s in a microcentrifuge.
Remove the magnetic beads by transferring the tubes to the magnetic rack, allow to separate for 60 s, and transfer the supernatant to new 1.5 mL safe-lock tubes.
Repeat this step by transferring the tubes with the supernatant back to the magnetic rack, allow to separate for 60 s, and transfer the supernatant to new 1.5 mL safe-lock tubes.
Centrifuge at maximum speed (21,400× g) for 10 min to get rid of any residual beads.
Without disrupting the pellet, transfer 90% of the supernatant volume to new 1.5 mL safe-lock tubes.
Corroborate that the pH is below 3 by placing 1 µL of each sample on a pH strip.
Mount SPE cartridges (one per sample) in a vertical manner in the arrangement of your choice.
Caution: Work in a fume hood.
Wash the SPE cartridges with 1 mL of 80% Acn, 0.1% TFA (v/v) to equilibrate, release, and discard the flowthrough by gravity.
Wash again twice with 1.2 mL of 0.1% (v/v) TFA, allow the flowthrough to be released by gravity, and discard it.
After washing, load the samples slowly to bind the peptides to the SPE cartridges.
Wash the bound peptides with 1.5 mL of 0.1% (v/v) TFA and discard the flowthrough by gravity.
Mount 1.5 mL safe-lock tubes below each SPE cartridge and collect the peptides by eluting with 800 µL of 80% Acn with 0.1% TFA (v/v).
Vacuum concentrate at RT to ~20% of the initial volume (at this preparation stage, Acn is almost completely removed).
Freeze the samples in a deep freezer (-80 °C) for 1 h.
Freeze-dry the samples for approximately 3 h or until tubes are completely dry.
Lyophilized peptides are ready to be resuspended in mass spectrometry loading buffer (2% ACN, 0.05% TFA) and measured in the LCMS platform of your choice.
Data analysis
The complete R and Python code used in our method is publicly available and fully documented via two GitHub repositories. Here, we include the documentation of the R package in Supplemental file 1 and its recommended usage on a compiled markdown in html format as Supplemental file 2, which contains detailed examples, input, and outputs for every function in our package. To visualize the figures embedded in the .md file, your computer must be logged into the internet. Additionally, we report the mathematical formulas, elaborate on their pragmatical implications and assumptions, and explain their results in the manuscript by Martinez-Seidel et al. [1].
Validation of protocol
This protocol or parts of it have been used and validated in the following research article(s):
Martinez-Seidel et al. [1]. Remodelled Ribosomes Synthesise a Specific Proteome in Proliferating Plant Tissue during Cold. bioRxiv (Figures 5 and 6).
To verify the methodological aims of our wet-lab methodology (Step 4), we used commercial preparations of Escherichia coli 70S ribosomes. We recommend the usage of such a control to confirm that the desired macromolecular complexes (in our case ribosomes) withstand with integrity all the purification procedures described herein.
Prepare 4 µL aliquots of E. coli ribosomes (Note 10).
Layer the ribosome aliquots on top of sucrose cushions (Martinez-Seidel et al. [1]) or sucrose gradients, as previously described in Siodmak et al. [16].
Centrifuge at high speeds as described in section A and follow the same procedure detailed in the sections of this manuscript.
We validated that our method enabled the recovery of native 70S E. coli ribosomes by assessing the completeness of the protein annotations generated by MaxQuant for three independent replicates (Figure 3).
Figure 3. Complete coverage of the Escherichia coli 70S ribosomal proteome from a commercially available preparation obtained by the ribosomal proteomics analysis pipeline. Related to Supplemental Table 2. The ribosomal proteomics pipeline was verified with three independent replicates from the same commercial preparation of Escherichia coli 70S ribosomes. The pipeline tested included ribosome extraction, subsequent purification through a sucrose cushion [following Procedure A using a 60% (w/v) sucrose solution as a cushion to filter the ribosomal particles from any other debris], resuspension of the pelleted complexes with a chaotropic agent to promote ribosomal protein dissociation, and rRNA removal before SP3 beads binding of the ribosomal proteins for protease digestion. The coverage of the 70S ribosomes was complete, with 21 proteins from the 30S small subunit and 33 from the 50S large subunit, plus a small set of expected ribosome-associated factors. The height of the triplicate bars in the plot (i.e., x-axis units) is the log10 value of the LFQ-ribosomal protein abundances as measured by LC-MS/MS from the control ribosomal complexes and quantified by MaxQuant. The y-axis contains the common name of the identified ribosomal proteins.
We can confidently report that in all replicates (Supplemental Table 2), we recovered and profiled all E. coli r-proteins at constant stoichiometries, thus validating that the proposed methodology recovers native and intact ribosomes and that the recovered riboproteome is not fundamentally altered by the purification procedures.
Mass spectrometry parameters: the gradient settings (at a flow rate 300 nL/min) were: solvent B 3%–23% in 59 min, 23%–40% in 10 min, 40%–80% in 5 min, which was maintained at 80% for 5 min before dropping to 3% in 0.1 min with subsequent equilibration at 3% solvent B for 9.9 min. An Eclipse Orbitrap mass spectrometer with nano electrospray ionization (ESI) source at positive mode was used for all LC-MS/MS experiments. The spray voltages, ion funnel RF, and capillary temperature level settings were 1.9 kV, 30%, and 275 °C, respectively. Mass spectra were acquired at 3 s per cycle per full MS scan spectra and as many data-dependent higher-energy C-trap dissociation (HCD)-MS/MS spectra as possible. The full scan MS spectra were set to capture ions between a m/z of 375 and 1500, with the maximum ion trapping time of 50 ms, and an auto gain control target value of 4e5; the resolution was 120,000 at a m/z of 200. The m/z isolation window was 1.6, with an auto gain control target value of 5e4, a normalized collision energy of 30%, with a maximum ion trapping time of 22 ms, and a resolution of 15,000 at a m/z of 200 was used to perform data-dependent HCD-MS/MS of precursor ions (with charge states ranging from 2 to 6).
The resulting dataset was deposited to the Proteome Xchange Consortium [39] via the PRIDE [40] partner repository under complete data submission, which means that all sequenced peptides and identified proteins can be actively browsed for. The dataset identifier is PXD032938 (DOI: 10.6019/PXD032938).
General notes and troubleshooting
General notes
We found that the simplest way to obtain apical root meristems from barley seedlings in a physiologically legitimate way was to germinate the plants in 15 cm dishes completely covered to avoid any light for 3–5 days (optimal germination time for barley). This ensured a dark environment for the root systems while providing the necessary nutrient solution to start germination. Imbibition is recommended in the dark 24 h before plating.
Any space or oven set to 37 °C works for the incubation steps.
We do not recommend standardizing the amount of polysomes that enter the pipeline, since these quantities are calculated based on rRNA absorbance, and as such are not a reflection of the r-proteome dynamic changes. Instead, we standardize the number of r-proteins that enter protease digestion. We do recommend recording and reporting the total amount of polysomes (i.e., derived from rRNA absorbances during fractionation of the ultracentrifuged sucrose gradients) as a reflection of active translation.
The volume may differ between samples. Total fractions volume should be between 400 and 800 µL. The maximum volume of ultracentrifugation tubes is 1 mL.
This will decrease the 60% (w/v) sucrose concentration, allowing all polysomes to pellet during ultracentrifugation. The recommendation is to prepare a pre-resuspension buffer without additions and store it at 4 °C. Before resuspension, add the translational stallers and reducing reagents.
Rotor and ultracentrifuge availability: The ultracentrifuge and rotor-specific runs that we describe here can be adjusted to whatever equipment you have access to by using our parameters and the Beckman-Coulter rotor conversion tool to find the equivalent run in your own equipment (https://www.beckman.com/centrifuges/rotors/calculator).
Always keep the samples on ice.
RNA pellets can be stored at -20 °C for later use.
Ideally, the standard curve should be prepared in the same solution as the test samples. Alternatively, always include controls without protein with the same solution as the test samples to check that there is no reactivity, thus validating a standard curve built in ultra-pure water.
Each aliquot contains approximately 2000 absorbance260 units, equating to approximately 102 µg of ribosomes and 23 µg of r-proteins.
Troubleshooting
Rotor and ultracentrifuge availability: The ultracentrifuge and rotor-specific runs that we describe here can be adjusted to whatever equipment you have access to by using our parameters and the Beckman-Coulter rotor conversion tool to find the equivalent run in your own equipment (https://www.beckman.com/centrifuges/rotors/calculator).
Incubation steps at 37 °C: Incubation steps were performed in a 37 °C room, but these can be done in any oven that can be set to the appropriate temperature.
Digestion times: Digestion times can vary from 12 to 24 h, but we recommend always keeping them reproducible irrespective of the range of your choice. Quenching the digestion with acid must be done as fast as possible to ensure reproducibility across samples.
Safe stopping points: We have successfully stored polysome pellets for up to six months at -80 °C. After resuspension in GuHCl, the safest stop point for undigested proteins is after reduction and alkylation when proteins will stay denatured until the following steps. We recommend not storing like this for more than a couple of weeks since TEAB buffer slowly loses its buffering capacity. After digestion, clean peptides can be stored preferably at -80 °C indefinitely, but we have stored peptides at room temperature for up to a month.
Beads solutions: Resuspended working solutions of magnetic beads are good for a month when stored at 4 °C.
Stable isotope titration and optimal labeling timing: Stable isotope labeling must be done long enough so that the metabolic targets (i.e., amino acids) reach a steady-state level of label incorporation, which typically lasts for a couple of days and allows the experimental period to be conducted within. This ensures that a fixed amount of your soluble amino acid pools is enriched when your experiment starts, and thus that the correction of peptide enrichments is legitimate. Failure to do this may result in irreproducible peptide enrichments.
SPE cartridges arrangement: We recommend gluing the SPE cartridges with regular tape onto an elevated surface in a way in which their bottom openings can fit into a receiving microcentrifuge tube. In this way, peptides will be eluted directly into the microcentrifuge tubes. SPE cartridges can dry without compromising the number of recovered peptides.
Acknowledgments
We thank the Mass Spectrometry and Proteomics Facility of The Bio21 Molecular Science and Biotechnology Institute at The University of Melbourne for the support of mass spectrometry analysis. F.M.-S. acknowledges the Max-Planck Society (Max Planck Institute of Molecular Plant Physiology) and the University of Melbourne for funding his research via the Melbourne-Potsdam PhD Programme (MelPoPP).
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary information
The following supporting information can be downloaded here:
Supplemental Table 1. Metainformation and analyses used to derive the differences in mean peptide length produced with Lys-C or trypsin as in silico proteases for digesting signature ribosomal proteomes across the tree of life. At = Arabidopsis thaliana, Sc = Saccharomyces cerevisiae, Hs = Homo sapiens, Ec = Escherichia coli. Tab A contains the FASTA sequences of the well curated ribosomal proteome. Tab B contains the results from an in silico digestion of the proteome in tab A using lys-C and obtaining, as a result, peptide monoisotopic masses between 200 and 6000 daltons. Tab C contains the same as tab B but using trypsin as protease (no P as digestion rule). Tab D contains the results from the comparison of both digestions in terms of Log2 fold changes and Padj values for peptide mean length per protein entry.
Supplemental Table 2. Protein content measurements in experimental samples using the BCA kit assay and subsequent ribosomal proteome profiling in E. coli control ribosomes. Tab A contains the measurements performed. Tabs B1 and B2 contain the full and reviewed E. coli ribosomal proteome from UniProt, respectively. Tab C contains the output proteinGroups file from the MaxQuant search using as FASTA target the SwissProt E. coli ribosomal proteome. Tab D contains the abundances of all ribosomal proteins and their graphical analysis.
Supplemental File 1. R documentation of the functions and methods outlined in the ProtSynthesis package version 0.1.0. This documentation is automatically saved onto your R library upon installation and can be accessed via the help call on the specific functions of the package from the R console. To install the package, please access the local repository in GitHub (https://github.com/MSeidelFed/ProtSynthesis).
Supplemental File 2. Development of a full usage example of the R package ProtSynthesis as to obtain the results on Martinez-Seidel et al., (2022). Supplementary data is also available to run independently the same example and can be accessed after installation of the R package via the ext_data method. The embedded images in the .html file require a network connection to be displayed, since their path is the local repository of the R package in GitHub (https://github.com/MSeidelFed/ProtSynthesis).
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Systems Biology
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4,982 | https://bio-protocol.org/en/bpdetail?id=4982&type=0 | # Bio-Protocol Content
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Apolipoprotein B Secretion Assay from Primary Hepatocytes
YW Yawei Wang
XL Xin Li
RH Runze Huang
XC Xiao-Wei Chen
XW Xiao Wang
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4982 Views: 466
Reviewed by: Ralph Thomas BoettcherKrishna Nakuluri Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Cell Biology Oct 2023
Abstract
Apolipoprotein B (APOB) is the primary structural protein of atherogenic lipoproteins, which drive atherogenesis and thereby lead to deadly cardiovascular diseases (CVDs). Plasma levels of APOB-containing lipoproteins are tightly modulated by LDL receptor–mediated endocytic trafficking and cargo receptor–initiated exocytic route; the latter is much less well understood. This protocol aims to present an uncomplicated yet effective method for detecting APOB/lipoprotein secretion. We perform primary mouse hepatocyte isolation and culture coupled with well-established techniques such as immunoblotting for highly sensitive, specific, and semi-quantitative analysis of the lipoprotein secretion process. Its inherent simplicity facilitates ease of operation, rendering it a valuable tool widely utilized to explore the intricate landscape of cellular lipid metabolism and unravel the mechanistic complexities underlying lipoprotein-related diseases.
Key features
• A pipeline for the isolation and subsequent culture of mouse primary hepatocytes.
• A procedure for tracking the secretion of APOB-containing lipoproteins.
• A rapid and sensitive assay for detecting the APOB level based on immunoblotting.
Keywords: Cardiovascular diseases Hyperlipidemia Lipoprotein secretion Primary hepatocytes Western blot
Background
Cardiovascular diseases (CVDs) continue to be the leading cause of death worldwide, accounting for over 20 million deaths each year. Hyperlipidemia, characterized by elevated plasma levels of cholesterol and triglycerides, is the most common risk factor for CVDs [1–3]. Due to their hydrophobic nature, lipids are loaded onto specialized carriers named lipoproteins before entering the circulation to meet the lipid demands of various body parts. Excess plasma lipids, especially those carried by apolipoprotein B (APOB)-containing lipoproteins, are the major cause of plaque buildup on the arteries, which leads to narrowed blood vessels, obstruction of blood flow, and ultimately CVDs. The APOB is the primary and unexchangeable structural protein of atherogenic lipoproteins, including low-density lipoproteins (LDL) and chylomicrons (CM), originating from hepatocytes and enterocytes, respectively [4–6]. Stoichiometrically, each APOB-containing lipoprotein contains one APOB molecule, which renders the plasma APOB levels a more reliable risk indicator for CVDs than plasma lipid levels per se.
Understanding the secretion process of lipoproteins is crucial for unraveling their roles in hyperlipidemia and CVDs. Abnormalities in lipoprotein secretion may result in elevated blood lipoprotein levels, further contributing to the progression of atherosclerosis. Therefore, developing an efficient and accurate method for detecting lipoprotein secretion is essential for a profound understanding of their biological functions and roles in disease development [7–11]. Here, we report a straightforward approach including the isolation and culture of primary mouse hepatocytes, followed by detecting the secreted APOB in the medium using established techniques like immunoblotting. Enzyme-linked immunosorbent assay (ELISA) can also be used for APOB secretion measurement, and the above methods are compatible. Our method provides a reliable, sensitive, specific, and semi-quantitative strategy for lipoprotein secretion detection, which can be feasibly applied to explore cellular lipid metabolism. This method may contribute to advancing the understanding of the regulatory mechanisms of lipoprotein export itinerary and holds promise for providing new insights into the prevention and treatment of CVDs.
Materials and reagents
25 mg/mL IV collagenase (Sigma-Aldrich, catalog number: C5138)
DMEM culture medium (Gibco, catalog number: 8123306)
Penicillin-Streptomycin solution (P/S), 100× (Caisson, catalog number: PSL01)
Phosphate buffered saline (PBS) (Meilunbio, catalog number: MA0015)
Fetal bovine serum (FBS) (Vistech, catalog number: SE100-011)
Acetone (TGREAG, catalog number: 105003)
APOB polyclonal antibody (Proteintech, catalog number: 20578-1-AP)
ALBUMIN monoclonal antibody (Proteintech, catalog number: 66051-1-Ig)
Glucose (HARVEYBIO, catalog number: SR1742)
NaHCO3 (TGREAG, catalog number: 114005)
NaCl (TGREAG, catalog number: 112008)
HEPES (Sigma, catalog number: V900477)
KCl (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10016328)
KH2PO4 (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10017628)
MgSO4·7H2O (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10013018)
Tris-HCl (Sigma, catalog number: V900483)
Nonidet P-40 (Sigma, catalog number: 56741)
Glycerol (Sigma, catalog number: G7893)
EGTA (Sigma, catalog number: 03777)
CaCl2 (TGREAG, catalog number: 104034)
Solutions
Krebs Ringer with glucose (KRG) buffer (see Recipes)
Solution C (see Recipes)
Buffer 1 (see Recipes)
Buffer 2 (see Recipes)
Lysis buffer (see Recipes)
Recipes
KRG buffer (1 L)
Note: KRG buffer (pH = 7.4) should be prepared immediately before use. Filter through a 0.22 μm membrane before use.
Reagent Final concentration Quantity or Volume
Glucose 20 mM 3.6 g
NaHCO3 25 mM 2.0 g
NaCl 120 mM 7.0 g
HEPES 1 M (pH 7.45) 5 mL
Solution C (Recipe 2) N/A 10 mL
Total N/A Set volume to 1 L with ddH2O
Solution C (1 L)
Reagent Final concentration Quantity or Volume
KCl 480 mM 35.79 g
KH2PO4 120 mM 16.33 g
MgSO4·7H2O 120 mM 29.58 g
Total N/A Set volume to 1 L with ddH2O
Buffer 1
Add 100 μL of 50 mM EGTA to 50 mL of KRG buffer.
Buffer 2
Add 100 μL of 1 M CaCl2 and 25 mg of IV collagenase to 50 mL of KRG buffer.
Lysis buffer
Note: Store at 4 °C and add protease inhibitor before use.
Reagent Final concentration
Tris-HCl (pH 7.4) 50 mM
Nonidet P-40 1%
Glycerol 10%
NaCl 150 mM
Laboratory supplies
Cell strainer (Falcon, catalog number: 352350)
Millex-GP Filter, 0.22 μm, PES 33 mm, non-sterile (Millex, catalog number: SLGP033NS)
Axygen MaxyClear Snaplock microtubes (Axygen, catalog number: MCT-150-C)
10 cm cell culture dish (JET, catalog number: TCD010100)
6-well cell culture dish (JET, catalog number: TCP010006)
50 mL centrifuge tube (JET, catalog number: CFT011500)
10 mL pipettes (JET, catalog number: GSP010010)
Perfusion set (BD Intima II, catalog number: 383405; TNTC-1066)
Eye scissors (Beijing Hongbai Technology Co., Ltd., catalog number: 2101157)
Forceps (Beijing Hongbai Technology Co., Ltd., catalog number: HC10704)
Nitrocellulose membrane (DiNing, catalog number: 66485)
Equipment
Centrifuge (Eppendorf, model: 5424R)
Centrifuge (Eppendorf, model: 5810)
GE Amersham Imager600 (GE Healthcare, catalog number: 29083463)
Western blot equipment (Bio-Rad, catalog number: 1658033)
Procedure
Isolation and culture of primary mouse hepatocytes
Sterilize eye scissors and forceps.
Preheat two 50 mL solutions of KRG buffer in a 37 °C water bath for 30 min.
Using preheated KRG buffer, prepare buffer 1 and buffer 2 (see Recipes).
Weigh and anesthetize the mice used for experiments by intraperitoneal injection of tribromoethanol according to the local animal handling protocols.
After complete anesthesia, fix the mouse on a clean foam board, spray with 75% alcohol, and wipe with sterile gauze.
Cut the abdominal wall, separate the subcutaneous tissue, and detach the abdominal skin on both sides with a needle. Open the abdominal cavity; the liver, portal vein, hepatic artery, and hepatic vein are exposed.
Prepare an infusion set. Insert the venous indwelling needle into the vena cava, open the infusion valve, cut the portal vein, and perfuse the liver at a rate of 2 mL/min using Buffer 1 to remove residual blood.
Add 25 mg of IV collagenase to buffer 2 close to the time of use.
After the blood is thoroughly washed out, switch to buffer 2 for liver digestion. The collagenase perfusion process lasts approximately 1–2 min.
When the liver becomes enlarged, white, and loose (such that pressing lightly leaves a small dent), place the liver into a 10 cm dish with the remaining buffer 2 (approximately 15 mL) and transfer to a biosafety cabinet for further processing.
After collagenase perfusion, the liver becomes loose and soft and can be easily cut up with a pair of 10 cm eye scissors. Cut the liver into small pieces in a culture dish. Pipette the digested liver tissue until dispersed with a 10 mL pipette. Filter the mixture through a 70 μm cell strainer into a 50 mL centrifuge tube to remove tissue debris.
Add pre-chilled DMEM to the tube for cell resuspension
Centrifuge at 50× g for 4 min at 4 °C, discard the supernatant.
Repeat steps A12–A13 three times until the supernatant is clear. Perform all washing steps with DMEM.
Discard the supernatant, add prewarmed complete medium (DMEM with 10% FBS and 1% P/S), and seed the cells in a culture dish according to the desired cell density for subsequent experiments. Recommended cell density: 4 × 105 cells per well of a 6-well dish.
It usually takes 3–4 h for cell adhesion and before the cells can be used for follow-up experiments.
Lipoprotein secretion detection in primary mouse hepatocytes
Change the medium to FBS-free DMEM preheated at 37 °C (other treatments can be performed simultaneously according to experimental requirements): 10 mL for a 10 cm dish and 2 mL per well of a 6-well dish.
Collect the medium after 4–8 h depending on the specific experimental conditions.
Centrifuge at 15,000× g for 15 min at 4 °C to remove cell debris.
Transfer the supernatant to a new centrifuge tube, add four times the sample volume of pre-chilled acetone, mix well, and precipitate at -20 °C overnight or at -80 °C for 2 h.
After centrifugation at 15,000× g for 10 min at 4 °C, resuspend the pellet using lysis buffer and perform western blotting.
Note: The conditions for western blot can be as follows: Run the gel on a 3%–8% tris-acetate gel at 100 V. When the 250 kDa marker reaches the middle, typically after 75 min, transfer to a nitrocellulose membrane overnight at 200 mA by using fresh transfer buffer. The primary antibodies used for overnight incubation were APOB polyclonal antibody and ALBUMIN monoclonal antibody.
The results of analyzing APOB and albumin in the culture medium by immunoblotting are shown in Figure 1.
Figure 1. Decreased apolipoprotein B (APOB) secretion after Mn2+ treatment. Primary hepatocytes from wild-type mice were treated with 125 μM MnCl2 for 8 h. APOB and albumin in the medium were analyzed by immunoblotting.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article: Wang et al. [12]. Manganese regulation of COPII condensation controls circulating lipid homeostasis. Nature Cell Biology (Figure 1, panel d; Figure 5, panel a).
Acknowledgments
The work is supported by the National Natural Science Foundation of China (NSFC: 32100947 to X.W., 32125021, 92254308, 91957119, 91954001, 31571213 to X.W.C) and the National Key R&D Program (2021YFA0804802). This protocol has been used in Nature Cell Biology [12].
Competing interests
All authors declare no conflict of interest.
Ethical considerations
All animal housing and use were approved by the Institutional Animal Care and Use Committees of Peking University, an AAALAC-accredited laboratory animal.
References
Roth, G. A., Abate, D., Abate, K. H., Abay, S. M., Abbafati, C., Abbasi, N., Abbastabar, H., Abd-Allah, F., Abdela, J., Abdelalim, A., et al. (2018). Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392(10159): 1736–1788.
Goldstein, J. L. and Brown, M. S. (2015). A Century of Cholesterol and Coronaries: From Plaques to Genes to Statins. Cell 161(1): 161–172.
Bentzon, J. F., Otsuka, F., Virmani, R. and Falk, E. (2014). Mechanisms of Plaque Formation and Rupture. Circ. Res. 114(12): 1852–1866.
Fisher, E. A. and Ginsberg, H. N. (2002). Complexity in the Secretory Pathway: The Assembly and Secretion of Apolipoprotein B-containing Lipoproteins. J. Biol. Chem. 277(20): 17377–17380.
Ference, B. A., Kastelein, J. J. P. and Catapano, A. L. (2020). Lipids and Lipoproteins in 2020. JAMA 324(6): 595.
Mehta, A. and Shapiro, M. D. (2021). Apolipoproteins in vascular biology and atherosclerotic disease. Nat. Rev. Cardiol. 19(3): 168–179.
Ginsberg, H. N., Le, N. A., Goldberg, I. J., Gibson, J. C., Rubinstein, A., Wang-Iverson, P., Norum, R. and Brown, W. V. (1986). Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo. J. Clin. Invest. 78(5): 1287–1295.
Björkegren, J. L. and Lusis, A. J. (2022). Atherosclerosis: Recent developments. Cell 185(10): 1630–1645.
Bhatia, H. S., Becker, R. C., Leibundgut, G., Patel, M., Lacaze, P., Tonkin, A., Narula, J. and Tsimikas, S. (2023). Lipoprotein(a), platelet function and cardiovascular disease. Nat. Rev. Cardiol. e1038/s41569-023-00947-2.
Tsimikas, S. and Witztum, J. L. (2023). Oxidized phospholipids in cardiovascular disease. Nat. Rev. Cardiol. 21(3): 170–191.
Willeit, P., Ridker, P. M., Nestel, P. J., Simes, J., Tonkin, A. M., Pedersen, T. R., Schwartz, G. G., Olsson, A. G., Colhoun, H. M., Kronenberg, F., et al. (2018). Baseline and on-statin treatment lipoprotein(a) levels for prediction of cardiovascular events: individual patient-data meta-analysis of statin outcome trials. Lancet 392(10155): 1311–1320.
Wang, X., Huang, R., Wang, Y., Zhou, W., Hu, Y., Yao, Y., Cheng, K., Li, X., Xu, B., Zhang, J., et al. (2023). Manganese regulation of COPII condensation controls circulating lipid homeostasis. Nat. Cell Biol. 25(11): 1650–1663.
Article Information
Copyright
© 2024 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-based analysis > Protein secretion
Medicine > Cardiovascular system
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4,983 | https://bio-protocol.org/en/bpdetail?id=4983&type=0 | # Bio-Protocol Content
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Peer-reviewed
Electrophoretic Mobility Assay to Separate Supercoiled, Catenated, and Knotted DNA Molecules
JC Jorge Cebrián
VM Victor Martínez
PH Pablo Hernández
DK Dora B. Krimer
MM María-Luisa Martínez-Robles
JS Jorge B. Schvartzman
MF María José Fernández-Nestosa
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4983 Views: 712
Reviewed by: Pilar Villacampa Alcubierre Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Nucleic Acids Research Feb 2015
Abstract
Two-dimensional (2D) agarose gel electrophoresis is the method of choice to analyze DNA topology. The possibility to use E. coli strains with different genetic backgrounds in combination with nicking enzymes and different concentrations of norfloxacin improves the resolution of 2D gels to study the electrophoretic behavior of three different families of DNA topoisomers: supercoiled DNA molecules, post-replicative catenanes, and knotted DNA molecules. Here, we describe the materials and procedures required to optimize their separation by 2D gels. Understanding the differences in their electrophoretic behavior can help explain some important physical characteristics of these different types of DNA topoisomers.
Key features
• Preparative method to enrich DNA samples of supercoiled, catenated, and knotted families of topoisomers, later analyzed by 2D gels (or other techniques, e.g., microscopy).
• 2D gels facilitate the separation of the topoisomers of any given circular DNA molecule.
• Separation of DNA molecules with the same molecular masses but different shapes can be optimized by modifying the conditions of 2D gels.
• Evaluating the roles of electric field and agarose concentration on the electrophoretic mobility of DNA topoisomers sheds light on their physical characteristics.
Keywords: DNA topology 2D gels Supercoiling Catenation Knots Topoisomerases
Graphical overview
Background
DNA molecules with different topology can be analyzed by the so-called single-molecule methods such as chromatin fiber autoradiography [1], dynamic molecular combing [2], transmission electron microscopy [3-5], atomic force microscopy [6], and magnetic tweezers [7,8]. DNA properties that are difficult to address experimentally can also be studied by computer simulations [9–13]. Two-dimensional (2D) agarose gel electrophoresis is the best experimental method currently available to allow the simultaneous identification of DNA molecules with different topology [e.g., supercoiled (Sc), catenated (Cats), and knotted (Kn) molecules]. This technique consists of two consecutive electrophoretic separations performed under different conditions and run at two orthogonal directions (4–8). The first dimension is resolved in a low-percentage (∼0.4%) agarose gel electrophoresis at a relatively low voltage (∼1 V/cm). The second dimension is run perpendicular to the first one so that an entire selected lane of the gel is used as replacement of the gel wells but in a higher-percentage (∼1%) agarose gel electrophoresis at higher electric field (∼5–6.6 V/cm). 2D gels were originally designed by Bell and Byers to separate branched and linear molecules [14], and it was early noticed that this method could also be successfully applied to study DNA topology. 2D gels were adapted to examine simultaneously thousands of molecules with different DNA topology, such as Sc forms, Kn forms, partially replicated forms (named pre-catenanes) with or without reversed forks, fully replicated catenanes (Cats), and replication intermediates (RIs) containing knotted bubbles [4,6,15–28]. 2D agarose gel electrophoresis has been extensively used to investigate the activity of topoisomerases in vitro and in vivo [29,30]. Additionally, 2D gels can also be used as a preparative method to enrich samples for specific DNA molecules that can be later examined by different techniques [4,6,18,19,31,32].
Plasmids are invaluable tools as a model to study DNA topology. The advantages of plasmids include their ease of isolation and the ability to quantitatively measure DNA supercoiling, knotting, and catenation in purified DNA samples [33]. Here, we present a protocol where 2D gels are used to analyze electrophoretic mobility of three families of topoisomers with the same molecular mass (8,766 bp): supercoiled dimers (ScDimers), monomeric-nicked catenanes (CatAs), and nicked-knotted dimers (KnDimers). We use monomeric (4,383 bp) or dimeric forms (8,766 bp) of pBR18, a derivative of pBR322 where the tetracycline resistance promoter was replaced with the poly-linker of pUC18 [34] to transform three different Escherichia coli strains. We describe the steps required to:
i. Obtain the three families of DNA samples for the analysis by 2D gels;
ii. Optimize their separation by 2D gels based on their extent of supercoiling, catenation, and knotting;
iii. Visualize them by non-radioactive detection.
Materials and reagents
Biological materials
Escherichia coli strains
DH5αF’: F’/gyrA96(Nalr) recA1 relA1 endA1 thi-1 hsdR17 (rk-mk+) glnV44 deoR Δ (lacZYA argF) U169[F80dΔ(lacZ)M15] (kindly gifted by Santiago Rodríguez de Córdoba) [35]
parE10: W3110 F- except [parE10 recA] (kindly gifted by Ian Grainge) [36]
LZ38 F-λ (P80 red114 xis-l cl857) zei-723::Tn10 parCK84::kanR (kindly gifted by Lynn Zechiedrich) [37,38]
Plasmid:
pBR18 (4,383 pb): a derivative of pBR322 where the tetracycline resistance promoter between EcoRI and HindIII was replaced with the poly-linker of pUC18 [32]
Reagents
Luria-Broth (LB) (Invitrogen, catalog number: 12795084)
Ethanol absolute, molecular biology grade (Sigma, catalog number: 64-17-5)
Agarose (Seakem LE, Lonza, catalog number: 50004)
Norfloxacin (Abcam, catalog number: 70458-96-7)
Ethidium bromide (Sigma, catalog number: 1239-45-8)
Digoxigenin-High Prime kit (Roche, catalog number: 11585614910)
Anti-Digoxigenin-AP conjugate antibody (Roche, catalog number: 11093274910)
CDP-Star (Perkin Elmer, catalog number: NEL601001KT)
Brij® 58 (Sigma-Aldrich, catalog number: P-5884)
Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750)
Tris-HCl (Sigma, catalog number: T2913)
Ethylenedinitrilotetraacetic acid, disodium salt (EDTA) (Sigma, catalog number: E7889)
Tris base (Sigma, catalog number: T1503)
Boric acid (Sigma, catalog number: PHR2664)
Hydrochloric acid (Sigma-Aldrich, catalog number: 258148)
Proteinase K, recombinant (Roche, catalog number: 3115879001)
Nb.BsmI 10,000 U/mL (New England Biolabs, catalog number: R0706S)
Calcium chloride (Flinn Scientific, catalog number: C0234)
Lambda DNA/HindIII Marker (Invitrogen, catalog number: SM0101)
BlueJuiceTM gel loading dye (10×) (Thermo Scientific, catalog number: 10816015)
Magnesium sulfate (MP Biomedicals, catalog number: 0219483394)
Sodium chloride (MP Biomedicals, catalog number: 0215257591)
Sucrose (Thermo Scientific Chemicals, catalog number: A15583.36)
Lysozyme (Thermo Scientific, catalog number: 10249843)
Polyethylene glycol (PEG) 6000 (Thermo Scientific, catalog number: J19972.A1)
Sodium hydroxide (Fisher Chemical, catalog number: 10675692)
Tri-sodium citrate (Fisher Chemical, catalog number: 10112880)
Disodium phosphate (MP Biomedicals, catalog number: 02191440.5)
Dextran sulfate (MP Biomedicals, catalog number: 11417880)
Sodium azide (Sigma, catalog number: S2002)
Sodium dodecyl sulfate (SDS) (Sigma, catalog number: 151-21-3)
Blotto (regular powdered milk at supermarket/pharmacy)
Sonicated and denatured salmon sperm DNA (Invitrogen, catalog number: 10605543)
Phenol:chloroform:isoamyl alcohol (25:24:1) equilibrated with 10 mM Tris–HCl, pH 8.0. (Sigma, catalog number: P3803)
Solutions
0.5 M EDTA, pH 8.0
For bacterial transformation (Section A):
1 M magnesium sulfate
0.1 M calcium chloride
For isolation of plasmid DNA (Section B):
Tris-EDTA (TE) (see Recipes)
Sodium chloride-Tris-EDTA (STE) (see Recipes)
25% sucrose (see Recipes)
Lysozyme–RNase A solution (see Recipes)
Lysis buffer (see Recipes)
25% PEG 6000 in TE (see Recipes)
Proteinase K buffer (20 mg/mL) (see Recipes)
For 2D agarose gel electrophoresis (Section E):
10× Tris Borate EDTA (TBE) (see Recipes)
For Southern blotting (Section F):
Depurination solution (see Recipes)
Transfer solution (see Recipes)
20× saline sodium citrate (SSC) (see Recipes)
For nonradioactive hybridization (Section G):
20× saline sodium phosphate EDTA (SSPE) (see Recipes)
20% dextran sulfate (see Recipes)
20% SDS (see Recipes)
Prehybridization/hybridization solution (see Recipes)
Recipes
For isolation of plasmid DNA (Section B):
Tris-EDTA (TE)
10 mM Tris-HCl
1 mM EDTA, pH 8.0
Ultrapure water
Note: Sterilize and store at room temperature. Stable for one year.
Sodium chloride-Tris-EDTA (STE)
0.1 M sodium chloride
10 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
Ultrapure water
Note: Sterilize and store at 4 °C. Stable for one year.
25% Sucrose
25% sucrose
0.25 M Tris-HCl, pH 8.0
Note: Sterilize and store at 4 °C. Stable for one month.
Lysozyme–RNase A solution
10 mg/mL lysozyme
0.2 mg/mL RNase A
0.25 M Tris-HCl, pH 8.0.
0.25 M EDTA, pH 8.0
Note: Prepare immediately prior to use; mix by inverting the tube and keep on ice.
Lysis buffer
50 mM Tris–HCl, pH 8.0
63 mM EDTA
1% Brij® 58
0.4% sodium deoxycholate
Ultrapure water
Note: Difficult to dissolve, use a magnetic stirrer. Stable for six months.
25% PEG 6000 in TE
25% PEG 6000
1.25 M sodium chloride
Note: Difficult to dissolve, use a magnetic stirrer. Store at 4 °C. Stable for two years.
Proteinase K buffer (20 mg/mL)
0.1 M sodium chloride
10 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
0.1% SDS
Ultrapure water
Note: Preheat at 37 °C and add 100 μg/mL of Proteinase K before use. Proteinase K buffer should be stored at -20 °C for up to two years to maintain its activity.
For 2D agarose gel electrophoresis (Section E):
10× Tris Borate EDTA (TBE)
1.00 M Tris base
1.00 M boric acid
0.02 M EDTA
Ultrapure water
pH 8.2–8.4
Note: Prepare a 1:10 dilution with ultrapure water to make 1× TBE. Store at room temperature. Stable for one year.
For Southern blotting (Section F):
Depurination solution
0.25 M hydrochloric acid
Ultrapure water
Note: Prepare immediately prior to use.
Transfer solution
0.4 M sodium hydroxide
Ultrapure water
Note: Prepare immediately prior to use.
20× saline sodium citrate (SSC)
3 M sodium chloride
0.3 M Tri-sodium citrate
Ultrapure water
Note: Store at room temperature. Stable for two years.
For nonradioactive hybridization (Section G):
20× saline sodium phosphate EDTA (SSPE)
3.6 M sodium chloride
0.2 M disodium phosphate
20 mM EDTA
Ultrapure water
Note: Store at room temperature. Stable for two years.
20% dextran sulfate
20 g of dextran sulfate
0.2 g of sodium azide
Ultrapure water
Note: Prepare aliquots and freeze (-20 °C). Stock solutions are stable for up to six months.
20% SDS
3 M sodium chloride
0.3 M sodium citrate
Ultrapure water
pH 7.0
Note: Store at room temperature. Stable for one year.
Prehybridization/hybridization solution
2× SSPE
1% SDS
0.5% Blotto
10% dextran sulfate
0.5 mg/mL sonicated and denatured salmon sperm DNA
Ultrapure water
Note: Prepare aliquots and freeze (-20 °C). Stock solutions are stable for up to 2 years.
Laboratory supplies
500 mL centrifuge bottles (Thermo Scientific, catalog number: 010-1493)
50 mL high-speed centrifuge tubes (Thermo Scientific, catalog number: 3118-0085)
50 mL Falcon tubes (Fisherbrand, catalog number: 11512303)
15 mL Falcon tubes (Fisherbrand, catalog number: 11765075)
1.5 mL microcentrifuge tubes (Thermo Scientific, PierceTM, catalog number: 10177443)
Whatman filter paper (GE Healthcare, catalog number: 3030917)
Zeta-Probe membrane (Bio-Rad, catalog number: 1620159)
Glass or plastic pipette
Film (Fuji, catalog number: 4741019236)
Cell spreader (Sigma-Aldrich, model: Cell Spreader silver stainless steel, bar L 33 mm, catalog number: HS86655)
Equipment
Superspeed floor centrifuge (Thermo Scientific, catalog number: 75006580)
Fixed-angle rotor for 500 mL bottles (Thermo Scientific, catalog number: 096-062375)
Swinging-bucket rotor for 50 mL round or conical tubes (Thermo Scientific, catalog number: 75003010)
Horizontal electrophoresis system with inter-electrode distances of at least 30 cm that can hold a 15 × 20 cm tray (Bio-Rad, catalog number: 1704403)
Basic power supply (Bio-Rad, catalog number: 11645050)
UV transilluminator (Biorad, model: Gel Doc XR+ System, catalog number: 1708195)
Microbiological shaking incubator (VWR, catalog number: 76628-472)
Thermoblock (Labnet, model: AccuBlock, catalog number: D1301)
Vacuum pump (Millipore, model: Millivac-Mini Vacuum Pump XF5423050)
Hybridization oven (Techne®, Hybrigene, catalog number: 445-0024)
X-ray film cassette or ChemiDocTM Imaging System (Bio-Rad, catalog number: 12003153)
Microcentrifuge (Eppendorf, model: 5418R, catalog number: EPP5401000013)
Software and datasets
ImageJ (https://imagej.nih.gov/ij/download.html) (Access date, 02/01/2024)
Procedure
We describe below the step-by-step procedure where 2D gels are used to analyze electrophoretic mobility of DNA molecules with the same mass (8,766 pb) but different topology: dimeric forms of a supercoiled molecule (ScDimers), nicked-catenated monomers (CatAs), and nicked-knotted dimers (KnDimers) (Figure 1). We use monomeric (4,383 bp) or dimeric forms (8,766 bp) of pBR18 to transform three different Escherichia coli strains (See Section A):
- E. coli LZ38 cells are recA+ and accumulate multimeric forms of the plasmid. In addition, they bear a mutation in the parC gene that turns Topo IV resistant to norfloxacin. Therefore, in these cells, norfloxacin inhibits only DNA gyrase, leading to the accumulation of poorly supercoiled molecules, and both monomeric (ScMonomers) and dimeric (ScDimers) forms become clearly distinguished in the same 2D gel.
- E. coli DH5∝F’ cells bear a mutation in the gyrA gene that turns DNA gyrase resistant to norfloxacin. Therefore, in these cells, norfloxacin inhibits only Topo IV, leading to the accumulation of catenated molecules (Cats).
- E. coli parE10 cells bear a thermosensitive mutation in the parE gene. Therefore, in these cells, norfloxacin inhibits only DNA gyrase, leading to the accumulation of relaxed molecules. In this way, supercoiled (Sc Dimers) and knotted molecules (KnDimers) become clearly distinguished in the same 2D gel.
Cell treatment with norfloxacin, an inhibitor of wild-type DNA gyrase and topoisomerase IV (Topo IV) [39], and DNA digestion with nicking enzyme Nb.BsmI are used to visualize Sc, CatAs, and Kn family of topoisomers in 2D agarose gels (Figure 1). The data generated using this protocol can be used to study the different reactions of Sc, CatAs, and Kn families of topoisomers to changes in agarose concentration and voltage during electrophoresis or it can be used as a preparative method to enrich samples for specific DNA molecules [6,18,31].
Figure 1. Cartoons of DNA molecules with different topology. Supercoiled monomer (ScMonomer), supercoiled dimer (ScDimer), nicked-catenated monomers (CatA), nicked-knotted monomer (KnMonomer), and nicked-knotted dimer (KnDimer) are represented. ScDimers, CatAs, and KnDimers were chosen in our study because all have the same mass and similar density of crossings, which allows us to study their electrophoretic mobility based only on their topology. Intramolecular crossings are marked with a black spot, while intermolecular ones are pointed with an asterisk. The catenated rings in CatA are drawn in blue and red and in green and red to distinguish each.
Bacterial transformation
Inoculate the selected bacterial strain in 30 mL of LB broth and grow culture in a shaking incubator at 250 rpm overnight.
Incubate DH5αF’ and LZ38 cells at 37 °C.
Incubate parE10 cells at 30 °C.
Make a 100-fold dilution in 30 mL of LB plus 10 mM magnesium sulfate.
Grow the culture up to an OD600 of 0.3–0.4 for approximately 3–5 h.
Incubate DH5αF’ and LZ38 cells at 37 °C.
Incubate parE10 cells at 30 °C.
Centrifuge 1 mL of the culture in a 1.5 mL Eppendorf tube at 9,300× g for 4 min at room temperature.
Carefully resuspend the pellet in 100 μL of 0.1 M cold calcium chloride.
Incubate the tube for 20 min on ice.
Add 10 ng of plasmid DNA, either monomeric (4,383bp) or dimeric forms (8,766 bp) of pBR18, in a 10 μL volume and incubate for another 10 min on ice:
To obtain ScDimers of pBR18, transform E. coli LZ38 cells with pBR18 monomers.
To obtain CatAs, transform E. coli DH5∝F’ cells with pBR18 monomers.
To obtain KnDimers, transform E. coli parE10 cells with pBR18 dimers.
Heat-shock the cells by placing the tube at 37 °C for 5 min or at 42 °C for 2 min.
Add 1 mL of LB and incubate at 37 °C for 1 h with moderate agitation.
Spread the cells on an agar plate containing 75 μg/mL ampicillin to select for cells with the plasmid.
Isolation of plasmid DNA (Note 1)
Inoculate the selected transformed bacterial strain (step A10) in 25 mL of LB broth and grow culture in a shaking incubator at 250 rpm overnight.
Dilute E. coli cells from the overnight culture 40-fold into 1 L of fresh LB medium and incubate in a shaking incubator at 250 rpm at 37 °C.
Grow the cells to exponential phase (OD600 0.4–0.6, approximately 3–5 h) and chill the culture quickly on ice. Transfer to 500 mL bottles and centrifuge at 3,500× g for 15 min at 4 °C in a floor centrifuge using a fixed-angle rotor.
Remove the media, resuspend the pellet in 20 mL of STE, transfer to a 50 mL Falcon tube, and centrifuge at 2,100× g for 15 min at 4 °C.
Harvest the cells by centrifugation and resuspend them in 50 mL high-speed centrifuge tubes with 5 mL of 25% sucrose (see Recipes). Keep the tubes on ice.
Add 1 mL of lysozyme–RNase A solution (see Recipes) and incubate on ice for 5 min.
Add 2 mL of 0.25 M EDTA, pH 8.0, mix by inverting the tube, and incubate on ice for 5 min.
Lyse the cells by adding 8 mL of lysis buffer (see Recipes) and inverting the tubes 3–4 times.
Incubate the samples for 1 h on ice.
Centrifuge at 30,000× g for 60 min at 4 °C to pellet the chromosomal DNA and other bacterial debris.
Transfer the supernatant with plasmid DNA (~10 mL) to a 50 mL high-speed centrifuge tube and precipitate it by adding 2/3 volumes of 25% PEG 6000 in TE (see Recipes).
Incubate overnight at 4 °C.
Pellet the precipitated DNA by centrifugation at 12,500× g for 15 min at 4 °C. Carefully remove the supernatant with a glass or plastic pipette.
Resuspend the pellet in 5 mL of a preheated proteinase K digestion buffer supplied with 25 μL of proteinase K (20 mg/mL) (see Recipes) and incubate for 30 min at 37 °C.
Under a fume hood, add v/v of phenol:chloroform:isoamyl alcohol (25:24:1) to your sample and incubate in tube rotator for 15 min at room temperature.
Centrifuge at 1,600× g for 5 min at room temperature. Carefully remove the upper aqueous phase and transfer the layer to a fresh tube. Be sure not to carry over any phenol during pipetting.
Repeat steps B15 and B16 at least once more.
Add one volume of chloroform:isoamyl alcohol (24:1) to your sample and incubate in a tube rotator for 15 min at room temperature.
Centrifuge at 1,600× g for 5 min at room temperature. Carefully remove the upper aqueous phase and transfer the layer to a 50 mL high-speed centrifuge tube.
Precipitate the DNA overnight at -20 °C with 2.5 volumes of cold absolute ethanol.
Centrifuge at 12,800× g for 60 min at 4 °C. Carefully remove the upper aqueous phase and wash the pellet with cold 70% ethanol.
Centrifuge at 12,800× g for 15 min at 4 °C. Aspirate the supernatant, air dry the pellet for 2 h, and resuspend the pellet in 150–200 μL of ultrapure water or 1× TE.
Inhibition of DNA Gyrase and Topo IV in vivo
Start growing cells as described above (Steps B1–B3) until they reach the exponential phase.
To accomplish inhibition of topoisomerases in vivo, add norfloxacin to a final concentration of 15 or 150 μM and incubate for 15–30 min with orbital shaking (see Note 2):
To partially relax supercoiled monomeric (ScMonomers) and dimeric (ScDimers) topoisomers, expose transformed E. coli LZ38 to 150 μM Norfloxacin.
To obtain catenanes monomers (Cats), expose transformed E. coli DH5∝F’ cells to 15 µM Norfloxacin.
To separate knotted dimers (KnDimers) and supercoiled molecules, expose transformed E. coli parE10 cells to 15 μM Norfloxacin.
To inhibit Topo IV in parE10 strain, start growing the culture at the permissive temperature (30 °C) until reaching the exponential phase and then incubate it at the restrictive temperature (43 °C) in shaking incubator at 250 rpm for 1 h.
Harvest the cells by centrifugation and proceed to plasmid DNA isolation as described before.
DNA treatments
To obtain nicked catenated monomers (CatAs) and nicked-knotted dimers (KnDimer): digest DNA from transformed E. coli DH5∝F’ and parE10 cells with Nb.BsmI (8 U/μg of DNA) for 1 h at 50 °C (see Note 3).
DNA (0.1 μg/μL) 10 μL
Buffer 10× 10 μL
RNase 1 mg/mL 10 μL
Ultrapure water 62 μL
Nb.BsmI (10,000 U/mL) 8 μL
Precipitate the DNA by adding 2.5 volumes (250 μL) of cold absolute ethanol.
Centrifuge at 16,800× g for 30 min at 4 °C. Carefully remove the aqueous phase and wash the pellet with cold 70% ethanol.
Centrifuge at 16,800× g for 15 min at 4 °C. Aspirate the supernatant, air dry the pellet for 15–30 min, and resuspend the pellet in 10 μL of ultrapure water or 1× TE.
2D agarose gel electrophoresis
We use a mix of plasmid DNAs isolated from the different bacterial strains to analyze the electrophoretic mobility of ScDimers and CatAs on one hand (Mix A) and ScDimers and KnDimers on the other hand (Mix B) (Figure 2).
Figure 2. Representative immunodetections of mixed DNA samples enriched for supercoiled dimers (ScDimers), nicked-catenated monomers (CatAs), and nicked-knotted dimers (KnDimers) analyzed by 2D agarose gel electrophoresis. A. Mixed DNA sample of ScDimers and CatAs. B. Mixed DNA sample of ScDimers and Kn Dimers. The first dimension was run at 1 V/cm (i.e., 30 V in our gel electrophoresis chamber) for 30 h in a 0.4% agarose gel; the second dimension was run at 5 V/cm (i.e., 150 V in our gel electrophoresis chamber) for 10 h in a 1% agarose gel. Interpretative diagrams are shown to the right. OC Dimers refers to the mobility of dimeric open circles used to align the different immunodetections.
Assemble a gel casting set with a 20-teeth (0.5 × 0.15 cm each) comb and pour a 0.4% agarose gel in 1× TBE. Let the gel solidify (15 × 20 cm).
Place the gel into a gel tank and pour 1× TBE buffer covering the gel. Carefully remove the comb.
Prepare the DNA samples in a final volume of 10 μL:
Mix A (ScDimers and CatAs):
1–3 μL of each DNA (200 ng of plasmid DNA).
1 μL of gel loading dye (10×) of 200 ng of plasmid DNA.
Fill with ultrapure water until a final volume of 10 μL (3–7 μL).
Mix B (ScDimers and KnDimers):
1–3 μL of each DNA (200 ng of plasmid DNA).
1 μL of gel loading dye (10×) of 200 ng of plasmid DNA.
Fill with ultrapure water until a final volume of 10 μL (3–7 μL).
Load the samples (Mix A or Mix B) in duplicates in two different lanes of the gel electrophoresis (e.g., lane 1 and lane 4). Lane 1 will be used only as a control to verify that the first dimension was carried out correctly (Note 4). Lane 4 (your target lane) will be used for the second dimension.
Run the first dimension at 1.0 V/cm (i.e., 30 V in our gel electrophoresis chamber) for 25–30 h at room temperature.
To prepare the second dimension, take the gel casting tray with the gel out of the tank and place it on a clean surface. Cut out the target lane from the first dimension (lane 4 in this protocol). Rotate the slice by 90º counterclockwise and place it on top of another gel casting (Figure 3).
Figure 3. Preparation of the second dimension. Cut out the target lane from the first dimension. Rotate the slice 90° and place it on top of another gel casting. Pour the dissolved agarose (0.8%–1.2%) around the excised agarose lane.
Melt 0.8%–1.2% agarose gel in 1× TBE, cool it down to 50–55 °C, and pour the dissolved agarose around the excised agarose lane from the first dimension. Let the gel polymerize at room temperature.
Pre-cool the 1× TBE buffer and the gel tray at 4 °C.
Place the gel into a gel tank and pour the cooled 1× TBE buffer covering the gel.
Run the second dimension at 5.0–6.6 V/cm (i.e., 150–200 V in our gel electrophoresis chamber) for 8–22 h in cold chamber at 4 °C.
After electrophoresis, transfer the DNA from the gels to an appropriate membrane by Southern blotting.
Southern blotting
Rinse the gel briefly with sterile distilled water.
To depurinate the DNA prior to transfer, submerge the gels for 10–15 min in depurination solution (see Recipes), with moderate shaking at room temperature. Transfer the gel to a clean recipient with sterile distilled water.
Set up the blot transfer as follows (see Figure 4): place three sheets of Whatman 3 MM paper that has been soaked with 0.4 N sodium hydroxide on top of a “bridge” that rests in a shallow reservoir of transfer solution. Place the gel on top of the three soaked sheets of Whatman 3 MM paper. Roll a clean pipette over the sandwich to remove all trapped air bubbles from between the gel and the paper.
Figure 4. Transferring DNA to a membrane. How to set up a Southern blotting of agarose gels by capillary transfer. Capillary transfer of the denatured fragments to a membrane uses the flow of the 0.4 N sodium hydroxide solution (transfer solution) to deposit the DNA fragments on the membrane. A wet sheet of blotting paper acts as a wick for the transfer solution (bridge), which is drawn up by a stack of dry paper through the gel/membrane sandwich. We use the alkaline transfer protocol described by López-Estraño et al. [40].
Cut a piece of positively charged Zeta-probe blotting membrane to the size of the gel. Pre-soak the membrane in sterile distilled water and place it on top of the gel. Use a pipette to eliminate air bubbles as above.
Add three sheets of Whatman 3 MM paper soaked in sterile distilled water, a stack of paper towels, a glass plate, and a 200–500 g weight (see Note 5).
Leave the blot transfer in transfer solution (see Recipes) overnight.
After the transfer, peel the membrane from the gel; rinse it twice in 2× SSC (see Recipes) for 5 min.
Nonradioactive hybridization
Label the DNA probes with digoxigenin using the Digoxigenin-High Prime kit following manufacturer’s recommendations.
In the meantime, prehybridize the membrane in a 20 mL prehybridization/hybridization solution for 4–6 h at 65 °C in hybridization bottles on a rotisserie inside a hybridization oven set at 65 °C.
Denature the labeled DNA probe by heating it at 95–100 °C for 5 min and chill it quickly in an ice bath.
Add the probe to the hybridization bottles to a final concentration of 20 ng/mL, place the bottles back into the oven, and hybridize overnight at 65 °C.
Wash the hybridized membranes sequentially with 2× SSC and 0.1% SDS for 5 min at room temperature twice, and then twice with 0.1× SSC and 0.1 % SDS for 15 min at 68 °C.
Perform the detection with an Anti-Digoxigenin-AP conjugate antibody and CDP-Star, according to the instructions provided by the manufacturer.
Data analysis
Scan the immunodetections and quantify the signals of interest by densitometry using Image J. The immunodetections are prepared according to the program manual, and the area and degree of saturation of each topoisomer are determined. The electrophoretic mobility values are obtained by measuring the distance of each topoisomer with respect to a signal common to all the immunodetections. We use the trivial knot, without supercoiling, called OC (Open Circle) as a reference. The electrophoretic mobility of individual spots of each family of topoisomers identified in immunodetections, with their corresponding increase in topological complexity (ΔC), expressed in mm/h, was compared for the different electrophoretic conditions (% agarose and voltage) used during the second dimension. (See Table 1 and Figure 5 for the electrophoretic mobility of topoisomers of Sc Dimers and CatAs under three different voltages applied during the second dimension.)
Table 1. Electrophoretic mobility of individual spots of ScDimers and CatAs identified in the immunodetections
Second dimension
ScDimers
1% agarose gel
Second dimension
CatAs
1% agarose gel
ΔC 150V 175V 200V ΔC 150V 175V 200V
1 5.5 10.55 14.19 1 2.65 4.91 5.50
2 5.5 10.59 14.25 2 2.35 4.32 4.50
3 5.5 10.64 14.38 3 2.5 4.55 4.56
4 5.5 10.64 14.44 4 2.8 5.14 4.81
5 5.6 10.64 14.50 5 3.25 5.82 5.38
6 5.65 10.68 14.56 6 3.8 6.64 6.19
7 5.8 10.68 14.69 7 4.45 7.45
8 5.9 10.73 14.75 8 5.05 8.32
9 6.15 10.77 14.81 9 5.75 9.09
10 6.35 10.91 14.88
Figure 5. Comparison of electrophoretic mobility of supercoiled dimers (ScDimers) and monomeric-nicked catenanes (CatAs) under three different voltages during the second dimension of 2D gels. Electrophoretic mobility as a function of topological complexity (ΔC) is expressed in mm/h for the ScDimers and CatAs under three different voltages during the second dimension. A. 1% agarose gel run at 5 V/cm (i.e., 150 V in our electrophoretic chambers) for 10 h. B. 1% agarose gel run at 5.8 V/cm (i.e., 175 V) for 10 h. C. 1% agarose gel run at 6.6 V/cm (i.e., 200 V) for 10 h.
Validation of protocol
This protocol has been used and validated in the following research article:
Cebrián et al. (2015). Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules. Nucleic Acids Res. Doi: 10.1093/nar/gku1255 (Figures 1, 2 and 5; Table 1).
General notes and troubleshooting
General notes
Commercial miniprep kits produce random DNA breaks in the plasmids molecules during their isolation and are not suitable for obtaining enriched samples of supercoiled, catenated, and knotted forms of the plasmids [34].
Treatment with suboptimal concentrations of the drug turns plasmids poorly supercoiled and, in this way, monomeric as well as dimeric topoisomers become clearly distinguished. In this way, ScDimers and KnDimers can be visualized in the same 2D gel. A similar result could be obtained using chloroquine 2D gels. However, chloroquine is not useful to resolve and identify nicked DNA (KnDimers and CatAs) as in these molecules the DNA is not under a torsional constraint (supercoiling relaxation), hence chloroquine does not have any topological effect.
DNA digestion with nicking enzymes releases torsional constrain (supercoiling relaxation) of the plasmids. In this protocol, DNA digestion with nicking enzyme Nb.BsmI is performed to remove DNA supercoiling present in our molecules; therefore, we convert all type of catenanes into CatAs and knotted DNA molecules are revealed.
A duplicate sample is loaded in the first lane of the gel only to check that first dimension of electrophoresis has been carried out correctly. Once the first dimension has finished, cut out the lane, place it carefully in ethidium bromide, and verify the correct migration of DNA samples with a UV transilluminator before proceeding to the second dimension of electrophoresis. This procedure is performed only after the first dimension. Once migration is verified, discard the gel and cut out your target lane (Figure 3). The ethidium bromide step must be performed with an extra sample (in a different lane) because ethidium bromide is a planar molecule that intercalates between the two strands of the DNA double helix altering DNA topology. Your target lane should not be exposed to ethidium bromide at any time during the electrophoresis to prevent DNA topology modification.
The capillary method uses paper towels to draw the transfer buffer through the gel to the membrane, placed on top of your gel. Make sure there is no shortcut between the wick and the paper towels; otherwise, if your paper towel layer touches the wick, the capillary action will bypass your gel and the DNA will not migrate from the gel onto the membrane. Parafilm or other wrap is often placed around the gel to frame its edges and prevent shortcuts.
Acknowledgments
The authors acknowledge the critics and continuous support of Andrzej Stasiak and all the current and former members of their groups. This work was sustained by grant BFU2011- 22489 to J.B.S. from the Spanish Ministerio de Economía y Competitividad. MJFN and VM were partially supported by an award granted by the National Council of Science and Technology (CONACYT), as Active Researchers of the National Incentive Program for Researchers (PRONII). We dedicate this work to the memory of our friend, mentor, and colleague, Jorge B. Schvartzman.
Competing interests
Authors declare no competing interests.
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Cebrian, J., Monturus, E., Martinez-Robles, M. L., Hernandez, P., Krimer, D. B. and Schvartzman, J. B. (2014). Topoisomerase 2 is dispensable for the replication and segregation of small yeast artificial chromosomes (YACs). PLoS One 9(8): e104995. https://doi.org/10.1371/journal.pone.0104995.
Sogo, J. M., Lopes, M. and Foiani, M. (2002). Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297(5581): 599–602. https://doi.org/10.1126/science.1074023.
Santamaria, D., Hernandez, P., Martinez-Robles, M. L., Krimer, D. B. and Schvartzman, J. B. (2000). Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins. J. Mol. Biol. 300(1): 75–82. https://doi.org/10.1006/jmbi.2000.3843.
Cebrian, J., Kadomatsu-Hermosa, M. J., Castan, A., Martinez, V., Parra, C., Fernandez-Nestosa, M. J., Schaerer, C., Martinez-Robles, M. L., Hernandez, P., Krimer, D. B., et al. (2015). Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules. Nucleic Acids Res. 43(4): e24. https://doi.org/10.1093/nar/gku1255.
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4,984 | https://bio-protocol.org/en/bpdetail?id=4984&type=0 | # Bio-Protocol Content
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Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells
UK Urs Kindler
HZ Holm Zaehres
LM Lampros Mavrommatis
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4984 Views: 1156
Reviewed by: Samantha HallerWilliam C. W. Chen Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in eLIFE Sep 2023
Abstract
Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors’ identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions.
Key features
• Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis.
• Analysis of early embryonic and fetal myogenesis.
• Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture.
• In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.
Keywords: Skeletal muscle Organoids Myogenesis iPS cells Satellite cells PAX7 Disease modeling Muscle dystrophy
Graphical overview
Skeletal muscle organoid induction and timeline of differentiation media applications and growth factor compositions
Background
Human stem cell–based in vitro models are becoming more frequently used specifically for human disease modeling, increasingly substituting artificial animal models. Using patient-derived cells for reprogramming opens the opportunity to generate cell lines from each genetic disease to study the specific phenotype and their underlying molecular mechanisms [1]. Even the U.S. Food and Drug Administration Modernization Act 2.0 in 2022, is paving the way for primarily using cell cultures for testing novel drugs.
For several years, various protocols have been proven effective in the directed differentiation of pluripotent stem cells (PSCs) into skeletal muscles and used to study the developmental stages of muscle differentiation [2–7], reviewed in Kim and Perlingeiro, Chien et al. and Yan et al. [8–10].
Importantly, the PSCs need to be guided toward paraxial mesoderm, which can be achieved by WNT pathway activation and BMP pathway inhibition along with FGF [2,3,11,12]. The early paraxial mesoderm is specified by WNT activation, while BMP inhibition prevents its shift toward the lateral-plate mesoderm. The transition from the posterior to the anterior somitic mesoderm can be confirmed by PAX3 expression [3]. As stated by the protocol of Chal and colleagues, the treatment by trophic factors such as bFGF, IGF, and HGF allows the PAX3 population to induce the myogenic program [4].
Recently, 3D differentiation has come into focus to mimic the environmental heterogeneity and the interactions among the different cell types and within tissues. In general, the maintenance and regeneration of tissue is aided by creating stem cell niches, which promote stem cell regeneration through their microenvironments. In case of skeletal muscles, various cell types are necessary to establish the stem cell niche–like fibroadipogenic progenitors, endothelial cells, pericytes, and macrophages, as well as transient neural crest structures that influence the skeletal muscle stem cell fate through various extracellular signals [13,14]. Besides establishing the stem cell niche, organoids can resemble early embryonic development and fetal cell maturation. These processes are essential for the initial pathological signs and early phenotypes in disorders such as Duchenne muscular dystrophy (DMD) [15,16].
The well-documented progression of diseases narrows the focus of the investigations to specific tissue and cell types. The prolongation of cultivation in vitro represents a challenge that overcomes embryonic gene expression and allows the observation of myogenesis until the onset of the disease in fetal or adult age. However, this appears mandatory to model genetic disorders like muscular dystrophies that have a relatively late onset. To overcome the lack of continued observation along myogenesis and disease onset, long-term culture approaches are essential to examine the mechanisms leading to muscle loss during myogenesis. We have established a 3D skeletal muscle organoid (SMO) system, which delivers a stable population of skeletal muscle progenitors to investigate human myogenesis during early embryonic and fetal development [17,18]. Induction of paraxial mesoderm is mediated by BMP inhibition, Wnt activation, and bFGF application as in other 2D protocols [4]. The application of retinoic acid, Shh, and WNT1A distinguishes this protocol from other 2D protocols and specifically leads to anterior somitic mesoderm and neural crest formation. The early Matrigel embedding is the decisive difference to all 2D protocols. In comparison to 2D protocols, our 3D protocol patterns Pax7-positive skeletal muscle progenitor cells with dormant, activated, and committed signatures of late fetal stages partially overlapping with adult satellite cell developmental scoring, which can be maintained for up to 14 weeks of culturing. Our 3D differentiation protocol does not go beyond 2D protocols to provide maturated physiologically responsive skeletal muscle cells, which we have demonstrated with the electrophysiological recording of organoid-derived cells of different origins [18].
However, structural distinctions like the posterior paraxial mesoderm on day 5, specified neural crest dermomyotome on day 17, myogenic progenitor migration on day 23, and neural crest lineage arrest on day 35 cannot be similarly mimicked with PSC-differentiation in 2D protocols. Recently, three other groups have described protocols for skeletal muscle development within 3D organoid differentiation systems [19–21]. In comparison to these protocols, we have focused on the myogenic progenitor cell identity in comparison to satellite cells by scRNAseq in greater detail (e.g., dormant, activated, and committed signatures) [18]. We see the strength of our system as being able to retain Pax7-positive myogenic progenitors/satellite-like cells constantly even during long-term cultivation to study their alterations in muscular dystrophies when generated from patient-derived induced pluripotent stem cells (iPSCs).
Materials and reagents
Biological materials
Human CD34 cord blood iPSCs [22]
Human LGMD2A and LGMD2A-isogenic iPSCs [23]
Human DMD iPSCs [24,25]
Human CD34 cord blood iPSC [26] (GibcoTM Episomal iPSC Line, catalog number: A18945)
HsdCpb: NMRI-Foxn1nu mice (NMRI-Foxn1nu/Foxn1nu, Janvier, St Berthevin Cedex, France)
Reagents
DMEM/F12 (GibcoTM, catalog number: 21331020)
ITS-G (GibcoTM, catalog number: 41400045)
ITS-X (GibcoTM, catalog number: 51500056)
L-Glutamine (GibcoTM, catalog number: 25030081)
Nonessential amino acids (NEAA) (GibcoTM, catalog number: 11140050)
Retinoic acid (Sigma-Aldrich, catalog number: R2625)
LDN-193189 (Sigma-Aldrich, catalog number: 1062368-24-4)
CHIR-99021 (Biogems, catalog number: 2520691)
Recombinant human FGF-basic (bFGF) (Peprotech, catalog number: 100-18B)
Recombinant human HGF (Peprotech, catalog number: 100-39H)
Recombinant human Sonic Hedgehog (SHH) (Peprotech, catalog number: 100-45)
Recombinant human Wnt-1 (WNT1A) (Peprotech, catalog number: 120-17)
Penicillin/streptomycin (P/S) (GibcoTM, catalog number: 15070063)
Poly-(methacrylacid-2-hydroxyethylester) (Sigma-Aldrich, catalog number: P3932)
Polyvinyl alcohol (PVA) (Sigma-Aldrich, catalog number: 363065)
TeSRTM-E8TM (StemCell Technologies, catalog number: 05990)
StemFlexTM medium (Thermo Fischer Scientific, catalog number: A3349401)
TrypLETM Select (GibcoTM, catalog number: 12563011)
Y-27632 (StemCell Technologies, catalog number: 72304)
Papain from papaya latex (Sigma-Aldrich, catalog number: P4762)
Corning® Matrigel® growth factor reduced (Corning, catalog number: 354230)
Anti-Brachyury (R&D Systems, 1:250, catalog number: AF2085)
Anti-TBX6, 1:200 (Abcam, catalog number: ab38883)
Anti-PAX3, 1:250 (DHSB, catalog number: N/A)
Anti-PAX7, 1:250 (DHSB, catalog number: N/A)
Anti-SOX10, 1:125 (R&D Systems, catalog number: AF2864)
Anti-TFAP2A, 1:100 (DHSB, catalog number: 3B5)
Anti-FastMyHC, clone MY-32, 1:300 (Sigma-Aldrich, catalog number: M4276)
Anti-SOX2, clone Btjce, 1:100 (Thermo Fisher Scientific, catalog number: 14-9811-82)
Anti-Dystrophin, 1:20 (Leica, catalog number: NCLDYS1)
Anti-Lamin A + Lamin C antibody, 1:150 (Abcam, catalog number: ab108595)
PE anti-human CD82 antibody (BioLegend, catalog number: 342103)
Goat anti-Mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568, 1:1,000 (Invitrogen, catalog number: A-11063)
Goat anti-Rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 488, 1:1,000 (Invitrogen, catalog number: A32731)
HISTOPRIME normal goat serum, sterile (NGS) (BIOZOL, catalog number: LIN-ENG1000-100)
Rhodamine RedTM-X (RRX) AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 1 specific, 1:100 (Jackson ImmunoResearch Laboratories, catalog number: 115-295-003)
Alexa Fluor 488, Goat Anti-Rat IgG (H + L) cross-adsorbed secondary antibody, 1:500 (Thermo Fisher Scientific, catalog number: A-11006)
Alexa Fluor 488, Donkey Anti-Mouse IgG (H + L) cross-adsorbed secondary antibody, 1:500 (Thermo Fisher Scientific, catalog number: A32766)
Alexa Fluor 488, Donkey Anti-Goat IgG (H + L) cross-adsorbed secondary antibody, 1:500 (Thermo Fisher Scientific, catalog number: A32814)
Alexa Fluor 568, Donkey Anti-Rabbit IgG (H + L) cross-adsorbed secondary antibody, 1:500 (Thermo Fisher Scientific, catalog number: A10042).
Cardiotoxin (CTX) (Sigma-Aldrich, catalog number: 217503-1MG)
Sodium citrate, dihydrate (Fisher Scientific, catalog number: 15538154)
Ethylenediamine tetraacetic acid disodium salt dihydrate (CARL ROTH, catalog number: 8043.2)
Citric acid monohydrate (Sigma-Aldrich, catalog number: C7129)
Single-cell 3’ Library & Gel Bead kit v3 (10× Genomics, catalog number: PN-1000075)
Single-cell B Chip kit (10× Genomics, catalog number: PN-1000073)
i7 Multiplex kit (10× Genomics, catalog number: PN-120262)
Bovine albumin (BSA) fraction V (7.5% solution) (GibcoTM, catalog number: 15260037)
Glycine (Sigma-Aldrich, catalog number: G8898)
L-cysteine (Sigma-Aldrich, catalog number: 168149)
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127)
Sucrose (Sigma-Aldrich, catalog number: 84100)
Tween-20 (Sigma-Aldrich, catalog number: 11332465001)
Tissue-Tek® O.C.T. compound (Sakura Finetek, catalog number: SA62550-01)
Triton X-100 (Sigma-Aldrich, catalog number: X100-100ML)
DAPI ready-made solution (Sigma-Aldrich, catalog number: MBD0015-5ML)
PBS (10×), pH 7,4 (GibcoTM, catalog number: 70011044)
Solutions
Coating solution (100 mL) (see Recipes)
Differentiation media 1 (Di-CL) (100 mL) (see Recipes)
Differentiation media 2 (Di-CLF) (100 mL) (see Recipes)
Differentiation media 3 (Di-CLFR) (100 mL) (see Recipes)
Differentiation media 4 (Di-LSW) (100 mL) (see Recipes)
Differentiation media 5 (Di-HF) (100 mL) (see Recipes)
Differentiation media 6 (Di-H) (100 mL) (see Recipes)
FACS buffer (25 mL) (see Recipes)
Blocking buffer (20 mL) (see Recipes)
Papain solution (30 mL) (see Recipes)
Sodium citrate solution (0.1 M) (see Recipes)
Citric acid solution (0.1 M) (see Recipes)
EFTA stock solution 0.5 M pH 8.0
Recipes
Coating solution (100 mL)
Reagent Final concentration Quantity or Volume
EtOH 95% 95 mL
ddH2O n/a 5 mL
Poly(methacrylacid-2-hydroxyethylester) 120 mg/mL 1.2 g
Total n/a 100 mL
The coating solution is not sterilized after mixing, as the high ethanol content makes contaminations unlikely. After application to the culture plate, it is sterilized by UV treatment. Alternatively, the coating solution can be sterilized through a 0.25 μm filter.
Differentiation media 1 (Di-CL) (100 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 96 mL
L-Glutamine 2 mM 1 mL
ITS-G n/a 1 mL
NEAA n/a 1 mL
P/S 500 U/mL 1 mL
CHIR-99021 3 μM 100 μL
LDN-193189 0.5 μM 100 μL
Total n/a 100 mL
Differentiation media 2 (Di-CLF) (100 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 96 mL
L-Glutamine 2 mM 1 mL
ITS-G n/a 1 mL
NEAA n/a 1 mL
P/S 500 U/mL 1 mL
CHIR-99021 3 μM 100 μL
LDN-193189 0.5 μM 100 μL
bFGF 10 ng/mL 50 μL
Total (optional) n/a 100 mL
Differentiation media 3 (Di-CLFR) (100 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 96 mL
L-Glutamine 2 mM 1 mL
ITS-G n/a 1 mL
NEAA n/a 1 mL
P/S 500 U/mL 1 mL
CHIR-99021 3 μM 100 μL
LDN-193189 0.5 μM 100 μL
bFGF 5 ng/mL 25 μL
Retinoic acid 10 nM 10 μL
Total n/a 100 mL
Differentiation media 4 (Di-LSW) (100 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 96 mL
L-Glutamine 2 mM 1 mL
ITS-G n/a 1 mL
NEAA n/a 1 mL
P/S 500 U/mL 1 mL
LDN-193189 0.5 μM 100 μL
SHH 34 ng/mL 100 μL
WNT1A 20 ng/mL 200 μL
Total n/a 100 mL
Differentiation media 5 (Di-HF) (100 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 96 mL
L-Glutamine 2 mM 1 mL
ITS-G n/a 1 mL
NEAA n/a 1 mL
P/S 500 U/mL 1 mL
HGF 10 ng/mL 100 μL
bFGF 10 ng/mL 50 μL
Total n/a 100 mL
Differentiation media 6 (Dix-H) (100 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 96 mL
L-Glutamine 2 mM 1 mL
ITS-X n/a 1 mL
NEAA n/a 1 mL
P/S 500 U/mL 1 mL
HGF 10 ng/mL 100 μL
Total n/a 100 mL
FACS-Buffer (25 mL)
Reagent Final concentration Quantity or Volume
PBS 1× n/a 18.24 mL
EDTA 2 mM 0.1 mL
BSA 2% 6.66 mL
Total n/a 25 mL
Blocking buffer (20 mL)
Reagent Final concentration Quantity or Volume
PBS 1× n/a 18.24 mL
BSA 2% 5.33 mL
Goat serum (NGS) 10% 2 mL
Total n/a 20 mL
Papain solution (30 mL)
Reagent Final concentration Quantity or Volume
DMEM/F12 n/a 30 mL
Papain 0.3 M 36 mg
EDTA 2% 8 mg
L-Cysteine 10% 8 mg
Total n/a 30 mL
Sodium citrate solution (0.1 M)
Reagent Final concentration Quantity or Volume
Sodium citrate 0.1 M 29.41 g
ddH2O n/a 1,000 mL
Total n/a 1,000 mL
Citric acid solution (0.1 M)
Reagent Final concentration Quantity or Volume
Citric acid 0.1 M 21.1 g
ddH2O n/a 500 mL
Total n/a 500 mL
Laboratory supplies
Cell culture plate, 24 well, surface: standard, flat base (SARSTEDT, catalog number: 83.3922)
Cell strainers 40 μm (SARSTEDT, catalog number: 83.3945.040)
Parafilm (Bemis, catalog number: 11772644)
Serological pipette, with tip, plugged, 5 mL, sterile (SARSTEDT, catalog number: 86.1253.025)
Serological pipette, with tip, plugged, 10 mL, sterile (SARSTEDT, catalog number: 86.1254.025)
Tissue culture dish, 35 × 10 mm, surface: standard (SARSTEDT, catalog number: 83.3900)
Tissue culture dish, 100 × 20 mm, surface: standard (SARSTEDT, catalog number: 83.3902)
Equipment
Cell culture microscope (Olympus, model: CKX41)
Leica modular stereo microscope (Leica, model: MZ10F)
CryoStar NX50 (Thermo Scientific, catalog number: 957210)
FACS sorter (Beckman Coulter, MoFlo Astrios EQ Cellsorter, catalog number: B52102)
Neubauer chamber (Marienfeld, catalog number: 0640131)
ZEISS slide scanner (Carl Zeiss Microscope, model: Axio Scan.Z1)
ZEISS confocal laser scanning microscope (Carl Zeiss Microscope, model: LSM 800)
Eppendorf centrifuge 5804R (Eppendorf, catalog number: EP022628146)
Software and datasets
Summit (Beckman Coulter, version: 6.3.1)
Kaluza Analysis (Beckman Coulter, version: 2.1)
Zen Lite Blue (Carl Zeiss Microscope, version: 4.0.3, June 2021)
R v4.2 (https://www.r-project.org/, April 2022)
Procedures
Human pluripotent stem cell maintenance
Human PSCs are cultured as colonies on 35-mm dishes, coated with 4% Matrigel with TeSRTM-E8TM or StemFlexTM medium. Cells are passaged at 70% confluency.
Day -3: Split cells to 1:3/1:4 ratio of a 70% confluent hiPSC culture.
Discard media and rinse the dish with PBS to remove non-adherent cells and incubate them with TrypLE for chemical dissociation for 3–5 min at 37 °C.
Stop dissociation using basal media DMEM/F12.
Spin the cells at 400 rpm for 5 min, discard the DMEM/F12 resuspended, and plate the cells in TeSRTM-E8TM supplemented with 10 µM Y-27632 onto new Matrigel-coated 35 mm dishes. (Dissolve 0.5 mL of Matrigel in 24.5 mL of DMEM/F12. Add 2 mL of the solution to a 35 mm dish and polymerize the Matrigel at 37 °C for 2 h or overnight at room temperature (RT), followed by UV sterilization before use.)
Culture the cells for two days and exchange TeSRTM-E8TM daily. Use 2 mL of media for a 35 mm dish.
Generation of embryoid bodies (EBs) and differentiation of organoids
Day -4: Prepare low-attachment coated plates by adding 150 μL of coating solution into each 24-well plate well. Let the ethanol evaporate overnight and store plates at RT until use.
Day -3/-2: Passage hiPSCs culture at 70%–80% confluency using the enzymatic dissociation approach to 1:3/1:4 ratio. The following day, refresh media without Rock inhibitor and, if:
Culture is at 30%–40% confluence, proceed with generating EBs the following day.
Culture is above 50% confluence, proceed with generating EBs the same day after refreshing the media without Rock inhibitor and culture the cells for at least 4–5 h before dissociating again for performing hanging drop method to generate EBs.
Day -1: Generation of Ebs via hanging drop method:
Dissolve 40 mg of PVA in 10 mL in TeSRTM-E8TM/StemFlexTM, followed by sterilization via filtration (0.25 μM) and supplemented with 10 μM Y-27632 and 1% P/S.
Discard media, rinse the dish with PBS to remove non-adherent cells, and incubate them with TrypLE for chemical dissociation for 3–5 min at 37 °C.
Stop dissociation using basal media DMEM/F12.
Spin the cells at 400 rpm for 5 min, discard the DMEM/F12, and resuspend in TeSRTM-E8TM/StemFlexTM supplemented with 10 μM Y-27632 and 1% P/S and PVA.
Count cells and adjust cell density to 200,000 cells/mL (=4,000 cells/20 μL).
Use the lid of a 10 cm cell culture dish and place 20 μL drops onto it. Fill the dish with PBS for humidified condition and place the lid again on the dish.
For EB formation, incubate the 10 cm cell culture dish overnight in the incubator at 37 °C and 5% CO2.
Day 0:
EBs should have a size of 200–250 μm.
Note: If a cloud of dead single cells surrounds the EB, and the EB is below 200 μm in size, while repeating step B3, increase the concentration of Rock inhibitor to 1.5–3× to enhance cell survival.
Thaw Matrigel on ice in the fridge; it should stay cold (<4 °C) to avoid premature polymerization throughout the process.
Wash EBs into a dish using DMEM/F12.
Note: The following step takes place under a stereoscope outside the hood after thorough disinfection of the used area. The application of 1.5× P/S in the medium prevents possible contaminations.
Place individual EBs onto a Parafilm® embedding surface using a 200 μL pipette. Remove excessive media but never leave EB without media (should remain approximately 5 μL of DMEM/F12). Well-formed EBs should not take any harm from it.
Place a 30 μL Matrigel drop onto each EB, resuspend to ensure a homogeneous mix of EBs within the Matrigel droplet, and place the EB in the center of the drop using a 200 μL pipette.
Note: To ensure homogeneous Matrigel polymerization: Resuspend 2–3× up and down and only place the EB within the Matrigel. Matrigel polymerizes faster due to the light source of the stereoscope.
Incubate the embedded EBs in the incubator at 37 °C for 20–25 min.
Note: The following step takes place under a cell culture hood.
Wash EBs into a 10 cm cell culture dish using DMEM/F12.
Note: Embedded EB droplets appear reddish due to the Matrigel. Use fresh DMEM/F12 for better visualization when collecting them with a cut-off 1,000 μL pipette tip. The pipette tips are not coated before handling cells, EBs, or SMOs throughout the protocol.
Prepare Di-CL media and place 1 mL in each low-attachment coated-plate well and warm it in the incubator.
Use one cut-off 1,000 μL pipette tip to transfer embedded EBs into a low-attachment coated-plate well and incubate them at 37 °C and 5% CO2.
Exchange media partly every day.
Note: Developing organoids are quite small. Control after media change if organoids are still present. To avoid destroying organoids during media change, hold the culture plate at 45 degrees and carefully remove media by pointing at the top of the well and aspirating slowly. Organoids should never be dried out during media change.
Important: Because myogenesis and migration take place within the Matrigel droplet, damaging the Matrigel during media change should be avoided at all times.
Note: This step requires extra caution to not lose or destroy the developing organoids, which can be challenging for beginners in all organoid protocols. The poor visibility of the embedded EB is a considerable criterion. A dark base under the cell culture plate helps to make it easier to recognize. Tilting the plate by just under 45° to the experimenter causes the organoids to shift to the lower edge and thus reduces the probability of damaging them.
Day 3: Change media from Di-CL to Di-CLF media by removing 75% and adding the same volume of media.
Day 5: Change media from Di-CLF to Di-CLFR media by removing 75% and adding the same volume of media.
Day 7: Change media from Di-CLFR to Di-LSW media by removing 75% and adding the same volume of media. Change media every second day.
Day 11: Change media from Di-LSW to Di-HF media by removing 75% and adding the same volume of media. Change media every second day.
Day 15: Change media from Di-HF to DiX-H media by removing 75% and adding the same volume of media. Change media every second day.
From Day 30 on: Change media every third day.
Steps 5–10 are depicted in the protocol outline of Figures 1A and 3A.
Figure 1. Representative images of different stages of skeletal muscle organoid development. A. Brightfield images of myogenic development stages, with corresponding cytokines/growth factor applications. B. Representative immunocytochemistry pictures of mesodermal, neural, paraxial mesodermal, and neural crest origin during early stages. C. Representative immunocytochemistry pictures depict neural lineage arrest, myogenic progenitor migration, and skeletal muscle organoid formation at more mature stages following organoid culture progression. Dashed lines indicate the initial EB embedding site and growth before migration takes place. Scale bars: 500 μm in (C), 200 μm in A (Day 18–Day 60) and B (Day 17), 100 μm in A (Day -1–Day 17) and B (Day 5, Day 7, Day 11) (modified from Mavrommatis et al. [18]).
Immunostaining of organoids at different stages
Fix organoids from different stages in 4% PFA (in PBS) overnight at 4 °C under shaking conditions.
Following PFA fixation, dehydrate organoids with 30% sucrose solution in PBS overnight and embed them in Tissue-Tek® O.C.T. compound.
Acquire cryosections of 20–30 μm thickness on a cryotome.
For the immunostaining process, rehydrate cryosections with PBS, permeabilize them first with 0.1% Tween-20 (10 min) in PBS, and then rinse 3× with 1× PBS.
Permeabilize further the cryosections with 0.1% Triton X-100 in PBS (10 min) and rinse 3× with PBS.
Block cryosections with the blocking buffer for 1 h at RT.
Note: For primary antibodies of goat origin, use blocking solution that contains only 2% BSA.
Incubate with primary antibodies overnight at 4 °C and secondary antibodies for 2 h at RT.
Mount cryosections with mounted media containing DAPI.
Acquire images on a confocal microscope and analyze them using Zen Lite Blue. Cryosections can be stable for months in the fridge; however, this cannot be generalized for all antibodies. Representative images are shown in Figures 1B and 1C.
Profiling skeletal muscle organoids from mature stages with single-cell RNA sequencing
Prepare single cells by incubating for 1 h with papain solution. Alternatively, an incubation with TrypLE Select for 15 min at 37 °C followed by mechanical dissociation by pipetting up and down using a 1,000 μL pipette upon resuspension also generates single cells suitable for scRNAseq pipeline.
Note: For TrypLE gentle dissociation, cut the 1 mL pipette tip before resuspending.
Upon dissociation, estimate cell number and viability. Resuspend cells in a solution containing 0.5% BSA in PBS to reach a concentration of 390 cells per μL.
Prepare cDNA libraries using the Chromium Single Cell 3' Reagent kit (v3) (Single Cell 3' Library & Gel Bead Kit v3, Single Cell B Chip Kit, and i7 Multiplex Kit) or an equivalent method according to the manufacturer’s instructions.
Sequence cDNA libraries on an Illumina HiSeq 3000 or equivalent method with 150 bp paired-end reads.
Exemplary downsteam bioinformatic analysis in R is illustrated in Figure 2.
Figure 2. Representative single-cell RNA-seq profiling of late fetal myogenic progenitors at mature stages during skeletal muscle organoid development. A. t-SNE visualization of color-coded clustering (n = 4323 cells) at 12 weeks post differentiation highlights the predominant presence of skeletal muscle lineage, represented by clusters corresponding to myogenic progenitors (n = 1625 cells, 37% of total population) in non-dividing (n = 1317 cells) and mitotic (n = 308 cells) state, myoblasts (n = 731 cells), myocytes (n = 1147 cells), and myotubes (n = 442). Additionally, mesenchymal and neural lineages are represented by two smaller clusters of fibroadipogenic (n = 165 cells) and neural (n = 213 cells) progenitors, respectively. B. t-SNE plot visualization of color-coded clustering indicates myogenic progenitor subcluster with distinct molecular signatures: “dormant” PAX7high/CHODLhigh/FBN1high, “activated” CD44high/CD98+/MYOD1+, and “mitotic” KI-67+/CDK1+/TOP2A. C. Circle plot illustrates the aggregated cell–cell communication network for all clusters at week 12 of human skeletal muscle organoids development. Circle sizes are proportional to the number of cells in each cell group and edge width represents the communication probability. D. Pseudo-time ordering for myogenic progenitors and myoblast-corresponding clusters highlight distinct developmental trajectories promoting myogenic commitment and self-renewal. E. Ridge plots of developmental score distribution of myogenic progenitors across in vivo or in vitro stages, based on the difference between upregulated satellite cell and embryonic markers from human reference atlases for week (Wk)-5–18 embryonic and fetal stages, years (Yr)-7–42 adult satellite cells and skeletal muscle organoids (modified from Mavrommatis et al. [18]).
In vivo potential of SMO-derived skeletal muscle progenitors
FACS isolation of CD82-positive skeletal muscle progenitor cells.
Dissociate the SMOs with TrypLE Select for 45 min at 37 °C including a mechanical dissociation step after 25 and 45 min by pipetting up and down using a 1000 μL pipette. The bulges of SMOs on day 84 are enriched with progenitors (Figure 3B, day 84).
Pipetting up and down 3–4 times every 15–20 min helps to dissociate the SMOs into a single-cell suspension.
Stop the reaction with FACS buffer by adding 3–4 times the volume of TrypLE Select.
Discard cell clumps by filtration through a 40 μm cell strainer followed by a centrifugation step.
Label the cells with PE anti-human CD82 antibody and APC anti-human CD56 (NCAM) antibody in FACS buffer (5 μL of each antibody in 100 μL of FACS buffer for 1 × 106 cells) and incubate the cell suspension on ice for 1 h, followed by a centrifugation step at 400 rpm for 5 min at RT.
Resuspend cells in FACS buffer supplemented with DAPI.
Use unstained SMO cells as baseline controls to exclude autofluorescence and sort the stained cells using a FACS sorter (Figure 3C).
FACS data are captured using summit software and analyzed using Kaluza Analysis software.
Transplantation of CD82-positive cells.
Cause an injury in the tibialis anterior (TA) muscle of 2–3-months-old male HsdCpb:NMRI-Foxn1nu mice by injecting 10 μL of CTX (40 ng/mL) 24 h before transplantation.
Under anesthesia, inject 1 × 105 cells into the TA muscle on one side of the mice.
Note: The injection was applied methodically as pictured in Feige & Rudnicki [27], Figure 4.
After six weeks, sacrifice the mice and isolate the TA.
Fix the TA in 4% PFA (in PBS) followed by embedding into O.C.T. compound media.
Slice the embedded TA in cross sections of 10–20 μm thickness using a CryoStar NX50.
Rehydrate slices with PBS, heat in citrate buffer (final concentration 19 nM, mix 82 mL of sodium citrate solution and 18 mL of citric acid solution plus 900 mL of ddH2O) until the buffer boils, and cook continuously for 15 min at 95 °C for antigen heat retrieval.
Treat the slices with an AffiniPure Fab Fragment Goat Anti-Mouse IgG for 60 min at RT to prevent unspecific bindings.
Permeabilize the slice with 1% (vol/vol) Triton X-100 and 125 mM glycine in PBS for 20 min at RT.
After blocking with blocking buffer for 60 min at RT, incubate the slides with the primary antibody overnight at 4 °C.
After washing three times for 10 min with PBS, incubate the slides with the secondary antibody for 2 h at RT.
Use Zeiss Scan.Z1 to scan the slides and process them using Zen Lite Blue (Figure 3D).
Figure 3. In vivo potential of skeletal muscle organoids (SMO)-derived skeletal muscle progenitors. A. Illustration of the differentiation protocol including the timeline of culture media addition and factor incubation (C: CHIR99021, L: LDN193189, F: bFGF, H: HGF, R: retinoic acid, S: Sonic hedgehog, W: WNT1A). B. Brightfield microscopy images of iPSC-derived SMOs on the selected days of a 84-day culture period (scale bar: 100 μm). C. FACS of organoid-derived CD82-positive skeletal muscle progenitors and D. their evaluation 6 weeks after transplantation into the CTX-injured tibialis anterior of an immunodeficient mouse [staining with huLamin A/C (green), dystrophin (red), DAPI (blue); scale bar 50 μm].
Validation of protocol
The reproducibility of the protocol has been described within various paragraphs of the corresponding article Mavrommatis et al. (2023). The organoid approach was evaluated with six hiPSC lines with independent genetic backgrounds, with more than five independent derivations per line, for the control line (CB CD34+) with more than 20 derivations, always obtaining similar results. The organoids show very reproducible sizes during their development (Figure 1B of Mavrommatis et al. [18]). To further evaluate the reproducibility of organoid development, diffusion map analysis on qPCR-based expression analysis of 32 genes was applied at early stages, as well as integrative analysis on scRNAseq datasets of mature stages of organoid development from four independent iPSC lines. The data indicate highly conserved cluster representation of myogenic progenitors at all states, together with skeletal muscle myofibers, fibroadipogenic progenitors, and neural progenitors-related clusters (Figure 4, Supplemental Figure 6, Mavrommatis et al. [18]).
Data analysis
RNA sequencing datasets produced by Mavrommatis et al. 2020, 2023 [17,18] are deposited in the Gene Expression Omnibus (GEO) under accession code GSE147514. To review GEO accession GSE147514: Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147514.
Acknowledgments
We thank all co-authors from our corresponding eLife (2023) manuscript for their contributions and support during the project development. The establishment of the protocol was supported by research grants from FoRUM F873-16, Medical Faculty, Ruhr University Bochum, from Deutsche Gesellschaft für Muskelkranke e.V. (DGM Foundation), Freiburg and Deutsche Duchenne Stiftung, Duchenne Deutschland e.V.
Competing interests
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Ethical considerations
All animal experiments were approved by the local authorities (81-02.04.2020.A476) and performed following the guidelines for Ethical Conduct in the Care and Use of Animals.
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Updated Pseudo-seq Protocol for Transcriptome-Wide Detection of Pseudouridines
YP Yi Pan
HA Hironori Adachi
XH Xueyang He
JC Jonathan L. Chen
YY Yi-Tao Yu
PB Paul L. Boutz
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4985 Views: 335
Reviewed by: Anna Sloutskin Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Cell Feb 2023
Abstract
Pseudouridine (Ψ), the most prevalent modified base in cellular RNAs, has been mapped to numerous sites not only in rRNAs, tRNAs, and snRNAs but also mRNAs. Although there have been multiple techniques to identify Ψs, due to the recent development of sequencing technologies some reagents are not compatible with the current sequencer. Here, we show the updated Pseudo-seq, a technique enabling the genome-wide identification of pseudouridylation sites with single-nucleotide precision. We provide a comprehensive description of Pseudo-seq, covering protocols for RNA isolation from human cells, library preparation, and detailed data analysis procedures. The methodology presented is easily adaptable to any cell or tissue type with high-quality mRNA isolation. It can be used for discovering novel pseudouridylation sites, thus constituting a crucial initial step toward understanding the regulation and function of this modification.
Key features
• Identification of Ψ sites on mRNAs.
• Updated Pseudo-seq provides precise positional and quantitative information of Ψ.
• Uses a more efficient library preparation with the latest, currently available materials.
Keywords: Pseudo-seq Transcriptome-wide pseudouridine mapping Next-generation sequencing Illumina NextSeq mRNA modification
Background
Many genetic diseases are caused by various mutations in specific disease genes. A significant proportion (~15%) of these mutations are nonsense mutations that create a premature termination codon (PTC) [1,2]. Consequently, the nonsense-mediated mRNA decay (NMD) surveillance pathway degrades a large fraction of PTC-containing mRNA [3]. Translation of the remaining undegraded PTC-containing mRNA terminates at the PTC, leading to no production of full-length protein and hence disease. Thus, suppressing NMD and translation termination at PTCs has become an attractive strategy for combating these diseases.
To address diseases caused by nonsense mutations in particular genes, substantial efforts have focused on altering PTC-containing mRNA associated with the condition. This alteration, occurring at the RNA and not DNA level, aims to convert the PTC back into a sense codon [4]. Inspired by this concept and considering the distinct chemical properties of pseudouridine (Ψ) compared to uridine, we have introduced a pioneering approach termed RNA-guided RNA pseudouridylation (U-to-Ψ conversion) [5,6]. This strategy targets the uridine within a PTC (UAA, UAG, or UGA), effectively inhibiting nonsense-mediated decay (NMD) while facilitating PTC read-through, leading to the production of a full-length functional protein in the cell. Our observations in yeast cells demonstrate a substantial increase in nonsense read-through upon converting the invariant U of a PTC into a Ψ [5,7].
The targeting of nonsense codons in yeast involves the expression of a designer box H/ACA guide RNA (gRNA), which possesses the capability to site-specifically direct the conversion of U to Ψ within the nonsense codon [8]. Box H/ACA gRNAs, abundant in archaea and eukaryotes, naturally direct pseudouridylation of rRNAs, snRNAs, and mRNAs in eukaryotes at specific sites [9–12]. Existing in the cell as a ribonucleoprotein complex (box H/ACA RNP), each box H/ACA gRNA directs site-specific pseudouridylation via distinctive base-pairing between the gRNA guide sequence and the substrate RNA [13].
Based on these observations, we have recently developed a novel approach, namely targeted PTC pseudouridylation, to suppress nonsense mutations in human cells [14]. By co-transfecting human cells with a designer box H/ACA gRNA gene targeting the PTC, we showed that targeted pseudouridylation suppressed both NMD and translation termination at PTCs. Targeted pseudouridylation appears to be the first RNA-directed gene-specific therapeutic approach that suppresses NMD and concurrently promotes PTC read-through.
To rule out the off-target effects of the gRNA transfection, we designed and performed Pseudo-seq to detect transcriptome-wide pseudouridylation. Recently, a number of Ψs have been predicted and experimentally detected by next-generation sequencing techniques [11,12,15,16]. In these techniques, RNA is first treated with carbodiimide N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC), which forms covalent adducts with the bases in guanidine, uridine, and Ψ [17]. Subsequently, alkaline hydrolysis removes the adducts from guanidine and uridine, but the CMC adduct at the N3 position of Ψ is resistant. The remaining CMC adduct on Ψ bases is an effective barrier to reverse transcriptase, which terminates one nucleotide before the modified Ψ. By mapping these strong reverse-transcription stop sites globally, the positions of Ψs can be determined. These methods are powerful and precise, but the original protocol has become obsolete due to a couple of factors. First, the adapters utilized in the initial Pseudo-seq are no longer compatible with current sequencers. Consequently, we substituted these adapters with a new set commonly employed in eCLIP [18]. Secondly, also based on eCLIP technical advances, adaptor ligation demonstrates higher efficiency than circularization [18]. Hence, we replaced the DNA circularization step with adapter ligation. With this revised Pseudo-seq technique, transcriptome-wide Ψs can be detected more efficiently in less-abundant mRNA.
Materials and reagents
Cells, reagents, and enzymes
HEK293T (ATCC, catalog number: CRL-11268)
DMEM (Gibco, catalog number: 11965)
FBS (Gibco, catalog number: 26140-079)
Trypsin (Gibco, catalog number: 25300054)
Opti-MEM (Gibco, catalog number: 31985070)
PEI MAX 40000 (Polysciences, catalog number: 49553-93-7)
TRIzol reagent (Invitrogen, catalog number: 15596018)
Glycogen (Thermo Scientific, catalog number: R0561)
Oligo d(T)25 magnetic beads (New England Biolabs, catalog number: S1419S)
T4 PNK (Thermo Scientific, catalog number: EK0031)
FastAP thermosensitive alkaline phosphatase (Thermo Scientific, catalog number: EF0651)
RQ1 RNase-free DNase (Promega, catalog number: M6101)
CMC [1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide Metho-p-toluenesulfonate] (TCI, catalog number: C0793)
T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs, catalog number: M0437M)
SYBR Select Master Mix (Applied Biosystems, catalog number: 4472908)
ExoSAP-IT (Applied Biosystems, catalog number: 78200.200.UL)
AMPure XP Bead-Based Reagent (Beckman Coulter, catalog number: A63881)
SYBR gold nucleic acid gel stain (Invitrogen, catalog number: S11494)
Dynabeads MyOne Silane (Invitrogen, catalog number: 37002D)
RLT buffer (QIAGEN, catalog number: 79216)
2× Q5 PCR master mix (New England Biolabs, catalog number: M0492S)
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (New England Biolabs, catalog number: E7335L)
3' RNA linker (RiL19): /5phos/rArGrArUrCrGrGrArArGrArGrCrGrUrCrGrUrG/3SpC3/ (Integrated DNA Technology, custom RNA oligo)
RT primer (AR17): dAdCdAdCdGdAdCdGdCdTdCdTdTdCdCdGdA (Integrated DNA Technology, custom DNA oligo)
3' DNA linker (rand3Tr3): /5phos/NNNNNNNNNNdAdGdAdTdCdGdGdAdAdGdAdGdCdAdCdAdCdGdTdCdTdG/3SpC3/ (Integrated DNA Technology, custom DNA oligo; see Note 1)
Chloroform (Thermo Scientific, catalog number: AC158210010)
Isopropyl alcohol (Thermo Scientific, catalog number: AC167880010)
RQ1 RNase-free DNase (Promega, catalog number: M6101)
Phenol:chloroform:isoamyl alcohol 25:24:1 (Thermo Scientific, catalog number: AAJ62336AN)
Sodium acetate (NaOAc) (Thermo Scientific, catalog number: AA1155430)
Sodium chloride (NaCl) (Thermo Scientific, catalog number: BP358-10)
Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA) (Thermo Scientific, catalog number: S311-500)
Sodium dodecyl sulfate (SDS) (Thermo Scientific, catalog number: BP166-500)
Tris base (Thermo Scientific, catalog number: BP152-1)
Lithium chloride (Thermo Scientific, catalog number: L121-500)
Lithium dodecyl sulfate (Thermo Scientific, catalog number: AC413300250)
Potassium acetate (Thermo Scientific, catalog number: P171-500)
Magnesium acetate (Thermo Scientific, catalog number: AC212550010)
Acetic acid (Thermo Scientific, catalog number: A38-212)
MES (Thermo Scientific, catalog number: BP300-100)
Bicine (Thermo Scientific, catalog number: AC327711000)
Sodium hydroxide (Thermo Scientific, catalog number: AC327715000)
Sodium carbonate (Thermo Scientific, catalog number: S263-500)
Bromophenol blue (Thermo Scientific, catalog number: AAA1846909)
Xylene cyanol (Thermo Scientific, catalog number: C422690050)
Formamide (Thermo Scientific, catalog number: BP228-100)
Boric acid (Thermo Scientific, catalog number: A73-1)
Urea (Thermo Scientific, catalog number: U15-3)
Acrylamide:Bis-Acrylamide 19:1 (40% solution/electrophoresis) (Thermo Scientific, catalog number: BP1406-1)
Micro Bio-Spin chromatography columns (Bio-Rad, catalog number: 7326204)
Dimethyl sulfoxide (DMSO) (Thermo Scientific, catalog number: BP231-1)
ATP solution (100 mM) (Thermo Scientific, catalog number: FERR0441)
Polyethylene glycol 8000 (PEG 8000) (Thermo Scientific, catalog number: BP233-1)
dNTP Mix (10 mM each) (Thermo Scientific, catalog number: FERR0192)
SuperScript III reverse transcriptase (Thermo Scientific, catalog number: 18080093)
DTT (dithiothreitol) (Thermo Scientific, catalog number: FERR0861)
Solutions
G50 buffer (see Recipes)
dT-lysis/binding buffer (see Recipes)
dT-wash buffer 1 (see Recipes)
dT-wash buffer 2 (see Recipes)
Low-salt buffer (see Recipes)
dT-elution buffer (see Recipes)
5× RNA shatter buffer (see Recipes)
2× Stop/PNK buffer (see Recipes)
BEU buffer (see Recipes)
Sodium carbonate buffer (see Recipes)
2× RNA loading dye (see Recipes)
4× TBE (see Recipes)
Recipes
G50 buffer (100 mL)
Store at 15–25 °C.
Reagent Final concentration Quantity or Volume
Tris base (1 M, pH 7.5) 20 mM 2 mL
Sodium acetate 300 mM 2.46 g
EDTA (0.5 M, pH 8.0) 2 mM 0.4 mL
SDS 0.2% 0.2 g
Total n/a 100 mL
dT-Lysis/Binding Buffer (100 mL)
Store at -20 °C for up to one month.
Reagent Final concentration Quantity or Volume
Tris base (1 M, pH 7.5) 100 mM 10 mL
Lithium chloride (1 M) 500 mM 50 mL
Lithium dodecyl sulfate 0.5% 0.5 g
EDTA (0.5 M, pH 8.0) 1 mM 0.2 mL
DTT 5 mM 0.077 g
Total n/a 100 mL
dT-wash buffer 1 (100 mL)
Store at -20 °C for up to one month.
Reagent Final concentration Quantity or Volume
Tris base (1 M, pH 7.5) 20 mM 2 mL
Lithium chloride (1 M) 500 mM 50 mL
Lithium dodecyl sulfate 0.1% 0.1 g
EDTA (0.5 M, pH 8.0) 1 mM 0.2 mL
DTT 5 mM 0.077 g
Total n/a 100 mL
dT-wash buffer 2 (100 mL)
Store at 4 °C.
Reagent Final concentration Quantity or Volume
Tris base (1 M, pH 7.5) 20 mM 2 mL
Lithium chloride (1 M) 500 mM 50 mL
EDTA (0.5 M, pH 8.0) 1 mM 0.2 mL
Total n/a 100 mL
Low-salt buffer (100 mL)
Store at 4 °C.
Reagent Final concentration Quantity or Volume
Tris base (1 M, pH 7.5) 20 mM 2 mL
Lithium chloride (1 M) 200 mM 20 mL
EDTA (0.5 M, pH 8.0) 1 mM 0.2 mL
Total n/a 100 mL
dT-elution buffer (100 mL)
Store at 15–25 °C.
Reagent Final concentration Quantity or Volume
Tris base (1 M, pH 7.5) 20 mM 2 mL
EDTA (0.5 M, pH 8.0) 1 mM 0.2 mL
Total n/a 100 mL
5× RNA shatter buffer (100 mL)
Store at 15–25 °C.
*Note: pH is adjusted by adding acetic acid to pH 8.2.
Reagent Final concentration Quantity or Volume
Tris base 200 mM 2.42 g
Potassium acetate 500 mM 4.91 g
Magnesium acetate 150 mM 2.14 g
Acetic acid n/a see note*
Total n/a 100 mL
2× Stop/PNK buffer (100 mL)
Store at -20 °C for up to 1 month.
Reagent Final concentration Quantity or Volume
MES (1 M, pH 6.0) 200 mM 20 mL
DTT 10 mM 0.154 g
EDTA (0.5 M, pH 8.0) 40 mM 8 mL
Acetic acid 0.15% 150 μL
Total n/a 100 mL
BEU buffer (100 mL)
Store at -20 °C for up to one year; check pH after long storage.
*Note: High pH is very important. Adjust the pH to 8.6–9.0 with NaOH. If the pH gets lower while under storage, remake it fresh.
Reagent Final concentration Quantity or Volume
Bicine 50 mM 0.815 g
EDTA (0.5 M, pH 8.0) 4 mM 0.8 mL
Urea 7 M 42 g
Sodium hydroxide n/a Up to pH ~9
Total n/a 100 mL
Sodium carbonate buffer (100 mL)
Store at 15–25 °C.
*Note: Filter sterilize (do not autoclave).
Reagent Final concentration Quantity or Volume
Sodium carbonate (1 M, pH 10.4) 50 mM 5 mL
EDTA (0.5 M, pH 8.0) 2 mM 0.4 mL
Total n/a 100 mL
2× RNA loading dye (100 mL)
Store at 4 °C.
*Note: Make stock solutions of EDTA, SDS, and the dyes. Mix into 95% formamide at the time of use. Discard any leftover.
Reagent Final concentration Quantity or Volume
EDTA (0.5 M, pH 8.0)
SDS
Bromophenol blue
Xylene cyanol
Formamide
5 mM
0.025%
0.01%
0.005%
95%
10 mL
0.025 g
0.01 g
0.005 g
see note*
Total n/a 100 mL
4× TBE (1 L)
Store at 15–25 °C.
*Note: TBE is used at a final 0.5× concentration. Dilute 8 times before use. Usually, pH adjustment is not necessary.
Reagent Final concentration Quantity or Volume
Tris base 0.52 M (pH 7.5) 43.2 g
Boric acid 180 mM 22 g
EDTA (0.5 M, pH 8.0) 8 mM 16 mL
Total n/a 100 mL
Laboratory supplies
6-well plates (Thermo Fisher Scientific, catalog number: 353046)
1.5 mL microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-129)
PCR tubes (Axygen, catalog number: PCR-0208-CP-C)
384-well real-time PCR plates (VWR, catalog number: 89218-294)
Equipment
CO2 incubator
Refrigerated microcentrifuge (VWR, model: 76019-208)
Benchtop mini microcentrifuge (Corning, model: 6770)
Dyna Mag-2 magnetic stand (Invitrogen, model: 12321D)
Owl gel electrophoresis apparatus (Thermo Fisher Scientific, catalog number: P9DS)
Amersham Typhoon RGB (Cytiva, model: GEH29187193EA)
Thermal cycler (Bio-Rad, model: T-100)
Thermo Mixer C (Eppendorf, model: 2231001005)
QuantStudio 5 Real-Time PCR System (Applied Biosystems, model: 384-well)
NextSeq 550 sequencing System (Illumina, model: SY-415-1002)
Software and datasets
Cutadapt version 4.1 (free, available at: https://cutadapt.readthedocs.io/en/stable/)
UMI-tools version 1.1.2 (free, available at: https://umi-tools.readthedocs.io/en/latest/QUICK_START.html)
STAR aligner version 2.5.3 (free, available at: https://github.com/alexdobin/STAR)
Bedtools version 2.26.0 (free, available at: https://bedtools.readthedocs.io/en/latest/)
R version 3.4.2 or later (free, available at: https://www.r-project.org/)
Procedure
We describe below the step-by-step procedure for performing the updated Pseudo-seq using HEK293T cells with modifications/improvements implemented at various steps. Although this protocol follows the workflow of the original Pseudo-seq method [19], the data generated using this protocol is different and we will show the data analysis method in detail.
Store all materials at 4 °C except for SDS-containing buffers and perform the procedure on ice. Room temperature is defined as 22 °C throughout this protocol. All washing steps throughout the protocol are performed with a volume of 1 mL unless stated differently.
Cell culture, transfection, and RNA collection
HEK293 cells were cultivated in DMEM medium with 10% FBS. Cells grown for under 20 passages are desired, but older cells can be used as long as they are of normal morphology.
Cell passage:
Wash the cells with 37 °C PBS once.
Add trypsin to the dishes and incubate at 37 °C for 3 min.
Add the FBS-containing medium to the dishes to inactivate trypsin.
Collect the cells by centrifuging at 300× g for 5 min and then count and split the cells for different purposes.
Seed the cells at 20% confluency.
Maintain cells at 37 °C with 5% CO2 and passage every 2–3 days at 80%–100% confluence.
The day before transfection, seed a certain number of cells in 6-well plates and incubate for 24 h to 80%–90% confluency.
Transfect the plasmids into HEK293T by PEI MAX 40000:
Mix 150 μL of Opti-MEM and 10 μL of PEI and incubate for 5 min.
Add 2 μg of gRNA plasmid and incubate for another 15 min.
Add the transfection mixture directly to the cells.
Collect the cells at 24–48 h after transfection.
Collect total RNA from one well of the 6-well plate with TRIzol reagent.
Add 1 mL of TRIzol and collect the cell lysate with a cell scraper.
Let the sample stand for 5 min at room temperature.
Add 200 μL of chloroform.
Vortex and incubate at room temperature for 3 min.
Centrifuge the samples at 16,000× g for 5 min at 4 °C.
Transfer the aqueous phase to a fresh tube.
Add 500 μL of isopropyl alcohol.
Incubate at room temperature for 10 min.
Centrifuge at 16,000× g for 15 min at 4 °C.
Discard supernatant and wash the pellet with 1 mL of 70% ethanol.
Centrifuge at 16,000× g for 5 min at 4 °C.
Dry the RNA pellet and resuspend it with 40 μL of ddH2O. Add 5 μL of 10× DNase I buffer and 5 μL of DNase I.
Incubate at 37 °C for 1 h.
Add 350 μL of G50 buffer (see Recipes) and mix with the DNase-treated RNA sample with phenol/chloroform/isoamyl alcohol (25:24:1).
Shake it vigorously and centrifuge at 16,000× g for 3 min at 4 °C.
Transfer the supernatant to a new tube, and add 2 μL of 20 mg/mL glycogen and 1 mL of 100% ethanol.
Shake it vigorously and centrifuge at 16,000× g for 20 min at 4 °C.
Dump the supernatant without disturbing the RNA pellet and wash with 1 mL of 70% ethanol.
Centrifuge at 16,000× g for 5 min at 4 °C.
Remove all supernatant and air-dry pellet for 5 min (do not dry longer).
Resuspend the RNA pellet with 500 μL of the dT-lysis/binding buffer (see Recipes).
Incubate at room temperature for 5 min with gentle agitation.
Place the 1.5 mL tube containing the Oligo d(T)25 magnetic beads and dT-lysis/binding buffer into the magnetic rack and pull the magnetic beads to the side of the tube.
Remove dT-lysis/binding buffer and add the RNA sample to the equilibrated magnetic beads.
Place the RNA–beads mixture on the agitator and incubate at room temperature for 10 min.
Place the tube into the magnetic rack, pull the beads to the side of the tube, and remove and discard supernatant.
Add 500 μL of dT-wash buffer 1 (see Recipes) to the beads and mix with agitation for 1 min.
Place the tube into the magnetic rack, pull the beads to the side of the tube, and remove and discard supernatant.
Wash the beads with dT-wash buffer 1 one more time, dT-wash buffer 2 (see Recipes) once, and low-salt buffer (see Recipes) once in the same way.
Place the tube into the magnetic rack, pull the beads to the side of the tube, and remove and discard supernatant.
Add 200 μL of dT-elution buffer (see Recipes) and vortex gently to suspend beads.
Incubate at 50 °C for 2 min with occasional agitation to elute poly(A)+ RNA.
Place the tube in the magnetic rack and pull the magnetic beads to the side of the tube. Transfer eluent to a new tube.
Repeat the elution step one more time in the same way.
Note: At this point, the protocol can be paused overnight, and the sample can be stored at -20 °C.
To remove the contaminated beads, perform phenol extraction followed by ethanol precipitation in the same way as in steps A21–A27, except for adding 1/10 volume of 3 M NaOAc (40 μL per tube here) instead of adding G50 buffer (Note 2).
Fragmentation/dephosphorylation
Heat the thermal cycler up to 95 °C with a hot lid.
Dilute the RNA into 1× shatter buffer on ice:
5 µL of 5× RNA shatter buffer (see Recipes)
20 µL of RNA in ddH2O
Place the RNA sample into the thermal cycler. Heat for 120 s.
After heating, immediately and quickly slam the tube on ice and add 25 µL of ice-cold stop/PNK buffer (see Recipes) to stop (Note 3).
Add 2.5 µL of T4 PNK.
Incubate at 37 °C with lid at 40 °C for 30 min in the thermal cycler to resolve and remove 2'-3' cyclic phosphates.
After 30 min, add the premixed mixture below:
5 µL of 10× FastAP buffer
5 µL of FastAP enzyme
37.5 µL of ddH2O
Incubate at 37 °C with lid at 40 °C for 15 min in the thermal cycler.
RNA cleanup.
Prepare beads:
Place 20 μL of MyONE silane beads into a 1.5 mL Eppendorf tube for each sample. Put the tube on the magnet and allow to separate; then, remove supernatant.
Wash once with 900 μL of RLT buffer.
Resuspend the beads in 300 μL of RLT buffer.
Bind RNA:
Add 300 μL of RLT buffer (3 volumes) with resuspended beads to each sample and mix thoroughly.
Add 10 μL of 5M NaCl (1/10th volume).
Add 615 μL of 95% or 100% ethanol (1.5 final volume).
Mix by rotating at room temperature for 15 min.
Wash beads:
Place tube on the magnet and allow beads to separate; remove supernatant carefully.
Resuspend beads in 1 mL of 75% ethanol and transfer to a new tube.
Place tube on the magnet and allow beads to separate; remove supernatant carefully.
Wash twice more with 75% ethanol, allowing beads to sit for 30 s each time.
Place tube on the magnet and allow beads to separate; remove supernatant carefully, getting all residual liquid possible.
Allow beads to air-dry for 5 min.
Elute RNA:
Resuspend beads in 30 μL of ddH2O and incubate for 5 min at room temperature.
Place tube on the magnet and allow beads to separate.
Transfer 18 μL of the supernatant to a new tube (for +CMC) and 12 μL to a second tube (for -CMC).
Note: The difference in volume between CMC+/- is to account for the reduction in precipitation efficiency of the CMC-modified RNA.
CMC treatment
Preheat the thermal cycler up to 80 °C with the hot lid.
Add 2.9 µL of 40 mM EDTA to both samples, heat for 3 min at 80 °C to denature, and then place them on ice.
Add 100 µL of CMC in BEU buffer (see Recipes) to the +CMC tube (Note 4) and 100 µL of BEU buffer alone to the -CMC tube. Incubate at 40 °C for 45 min at 1,000 rpm on the Thermomixer.
Perform ethanol precipitation. Add 2 µL of glycogen, 50 µL of 3M NaOAc, and 1 mL of ethanol. Precipitate overnight at -20 °C or for more than 30 min at -80 °C, and then centrifuge at maximum speed (16,000× g) for 30 min at 4 °C.
Wash twice with 500 μL of ice-cold 70% ethanol followed by spinning down at maximum speed for 10 min at 4 °C.
Dry for 2 min at room temperature.
Resuspend the pellets (+CMC and -CMC) in 30 µL of sodium carbonate buffer (see Recipes). Incubate at 50 °C for 2 h at 1,000 rpm on the Thermomixer.
Add 2 µL of glycogen, 3.33 µL of 3M NaOAc, and 88 µL of ethanol. Precipitate overnight at -20 °C or for more than 30 min at -80 °C, and then centrifuge at maximum speed for 30 min at 4 °C.
Wash twice with 500 μL of ice-cold 70% ethanol followed by spinning down at maximum speed for 10 min at 4 °C.
Dry for 2 min at room temperature with the cap open.
Resuspend pellet in 12.5 µL of water.
Note: At this point, the protocol can be paused overnight, and the sample can be stored at -20 °C.
PAGE separation and isolation
Wash plates, spacers, and combs of the Owl system with 5% SDS solution to eliminate RNase activity; then, dry and perform a 95% ethanol wash. Pour 8% Acrylamide gel containing 7 M Urea and 0.5× TBE. Pre-run the gel with aluminum heat sink to heat to 55 °C, gradually increasing the power. Gel should be pre-run for approximately 1 h including reaching temperature (Note 5).
Prepare fresh 2× RNA loading buffer, 95% formamide. Dilute DNA ladder: 0.125 µL of 10 bp ladder/lane (1/8 µL) into 12.5 µL total.
Add 12.5 µL of 2× RNA loading dye (see Recipes) to RNA samples and ladder (Note 6).
Heat samples to 95 °C for 1 min and then slam on ice. Before loading, use a syringe to blow out wells to remove excess urea.
Run xylene cyanol to near the bottom of gel, disassemble, and stain with SYBR Gold stain (1:10,000 dilution in 0.5× TBE) for 5 min.
Cut out gel fragments of appropriate sizes, 10–20 nucleotide bands (80–100, 100–120, 120–140). Collect gel slices in 0.6 mL microfuge tubes (Figure 1).
Figure 1. Gel image of the first RNA purification step. RNAs were treated with CMC and fractionated by Mg2+ ions. Excise the RNA lane in the three different areas shown as red. The gel was stained with SYBR Gold.
Using a 20-gauge needle, make a hole in the bottom of the tube and place it in a standard 1.5 mL tube.
Spin at maximum speed for 1 min. Check to make sure all gel fragments have gone through. You can reposition the remaining fragments and re-spin if necessary.
Add 360 µL of ddH2O and 40 µL of 3 M NaOAc.
Freeze at -80 °C (or on dry ice) and then quickly thaw.
Rotate overnight at 55 °C.
Spin down gel fragments and place supernatant into a Bio-Rad Micro-bio spin chromatography column. Spin at 1000× g for 2 min.
Add 1 µL of glycogen and 1 mL of ethanol and precipitate overnight at -20 °C or for more than 30 min at -80 °C. Then, centrifuge at maximum speed for 30 min at 4 °C.
Wash twice with 500 μL of ice-cold 70% ethanol followed by spinning down at maximum speed for 10 min at 4 °C.
Dry for 2 min at room temperature with the top open and resuspend in 10.5 µL of ddH2O. Preheat PCR machine up to 80 °C with hot lid.
3' RNA linker ligation
In a PCR tube, mix the reagents below:
10 μL of RNA (from above)
3 μL of 100% DMSO
1 μL of 3' RNA linker (RiL19, 40 μL)
Incubate at 65 °C for 2 min and place on ice for at least 1 min.
Prepare ligation master mix, 26 μL per sample:
4.0 μL of 10× NEB ligase buffer (commercially attached)
0.4 μL of 0.1 M ATP
0.6 μL of 100% DMSO
16.0 μL of 50% PEG 8000
2.6 μL of RNA ligase
2.4 μL of ddH2O
Thoroughly mix with pipette; then, add 26 μL of ligation master mix to each sample and mix well. Incubate for 75 min, flicking tube to mix approximately every 15 min.
RNA cleanup.
Prepare beads:
Place 20 μL of MyONE silane beads into a 1.5 mL Eppendorf tube for each sample. Put tube on magnet and allow to separate and then remove supernatant.
Wash once with 900 μL of RLT buffer.
Resuspend the beads in 120 μL of RLT buffer.
Bind RNA:
Add 120 μL of RLT buffer (3 volumes) with resuspended beads to each sample and mix thoroughly.
Add 120 μL of 95% or 100% ethanol.
Mix well and incubate at room temperature for 15 min, mixing every 5 min with a pipette.
Wash beads:
Place tube on the magnet and allow beads to separate; remove supernatant carefully.
Resuspend beads in 1 mL of 75% ethanol and transfer to a new tube.
Place tube on the magnet and allow beads to separate; remove supernatant carefully.
Wash twice more with 75% ethanol, allowing beads to sit for 30 s each time.
Place tube on the magnet and allow beads to separate; remove supernatant carefully, getting all residual liquid possible.
Allow beads to air-dry for 5 min.
Elute RNA:
Resuspend beads in 9 μL of ddH2O and incubate for 5 min at room temperature.
Place tube on the magnet and allow beads to separate.
Transfer the supernatant to a new tube.
Reverse transcription
In a PCR tube, mix the reagents below:
8 μL of the ligated RNA sample
1 μL of gel-purified RT primer (25 μM stock) (Note 7)
1 μL of 10 mM dNTPs (each)
3 μL of ddH2O
Incubate at 65 °C for 2 min, then immediately transfer to ice.
Prepare an extension master mix (per sample) as follows:
4 μL of 5× First-strand buffer
1 μL 0.1 M DTT
1 μL of ddH2O
1 μL of Superscript III
Add 7 μL of the extension mix to the annealing reactions and incubate at 42 °C for 1 h.
Cleanup cDNA.
ExoSAP-IT treatment:
Add 3.5 μL of ExoSAP-IT to each sample. Vortex to mix well, then spin tube to collect liquid at the bottom.
Incubate for 15 min at 37 °C in a thermal cycler.
Add 1 μL of 0.5 M EDTA and pipette to mix.
RNA removal:
Add 3 μL of 1 M NaOH and pipette to mix.
Incubate for 12 min at 70 °C in a thermal cycler.
Add 3 μL of 1 M HCl (to neutralize the NaOH) and pipette to mix thoroughly.
Silane cleanup cDNA.
Prepare beads:
Place 10 μL of MyONE Silane beads into a 1.5 mL Eppendorf tube for each sample. Put the tube on the magnet and allow to separate, then remove the supernatant.
Wash once with 500 μL of RLT buffer.
Resuspend the beads in 93 μL of RLT buffer.
Bind RNA:
Add the beads to each sample and mix thoroughly.
Add 111.6 μL of 95% or 100% ethanol.
Mix well and incubate at room temperature for 5 min.
Wash beads:
Place the tube on the magnet and allow the beads to separate; remove the supernatant carefully.
Resuspend beads in 1 mL of 75% ethanol and transfer to a new tube.
Place the tube on the magnet and allow the beads to separate; remove the supernatant carefully.
Wash twice more with 75% ethanol, allowing the beads to sit for 30 s each time.
Place the tube on the magnet and allow the beads to separate; remove the supernatant carefully, getting all residual liquid possible.
Allow beads to air-dry for 5 min.
Elute RNA:
Resuspend beads in 12.5 μL of ddH2O and incubate for 5 min at room temperature.
Place the tube on the magnet and allow the beads to separate.
Transfer the supernatant to a new tube.
Note: At this point, the protocol can be paused overnight, and the sample can be stored at -20 °C.
Second PAGE separation and isolation
Repeat the steps in D; the example of the second gel is shown below (Figure 2).
Figure 2. Gel image of the second DNA purification step. cDNAs were obtained from the reverse-transcription step, SYBR Gold staining. Useful parts (i.e., partial length cDNA) are those from the primers to the full-length, shown as red.
3' DNA linker ligation and library preparation
To the 5 μL of cDNA obtained from the ethanol-precipitated samples above, add:
0.8 μL of rand3Tr3 adapter (80 μM stock)
1 μL of 100% DMSO
Heat at 75 °C for 2 min, then place immediately on ice for a minimum of 1 min.
Prepare 12.8 μL of ligation master mix per sample, on ice:
2 μL of 10× NEB RNA ligase buffer (with DTT)
0.2 μL of 0.1M ATP
9.0 μL of 50% PEG 8000
0.5 μL of T4 RNA ligase (high concentration)
1.1 μL of ddH2O
Mix thoroughly and briefly spin down. Add 12.8 μL of master mix to each sample slowly with stirring.
Add an additional 1 μL of RNA ligase to each sample and mix by flicking the tube.
Incubate in a Thermomixer at 1,200 rpm for 30 s at room temperature. Then, transfer to bench top, flicking to mix every hour for a few hours.
Incubate overnight at room temperature.
RNA cleanup.
Prepare beads:
Place 5 μL of MyONE silane beads into a 1.5 mL Eppendorf tube for each sample. Put tube on the magnet and allow to separate, then remove supernatant.
Wash once with 500 μL of RLT buffer.
Resuspend the beads in 60 μL of RLT buffer per sample.
Bind RNA:
Add 60 μL of RLT buffer with resuspended beads to each sample and mix thoroughly.
Add 60 μL of 95% or 100% ethanol.
Mix well and incubate at room temperature for 5 min; gently flick to mix periodically.
Wash beads:
Place the tube on the magnet and allow the beads to separate; remove the supernatant carefully.
Resuspend beads in 1 mL of 75% ethanol and transfer to a new tube.
Place the tube on the magnet and allow the beads to separate; remove the supernatant carefully.
Wash twice more with 75% ethanol, allowing beads to sit for 30 s each time.
Place the tube on the magnet and allow the beads to separate; remove the supernatant carefully, getting all residual liquid possible.
Allow beads to air-dry for 5 min.
Elute RNA:
Resuspend beads in 27 μL of 10 mM Tris-HCl pH 7.5 and incubate for 5 min at room temperature.
Place the tube on the magnet and allow beads to separate; then, transfer 25 μL of sample to a new tube
Note: At this point, the protocol can be paused overnight, and the sample can be stored at -20 °C.
qPCR to quantify cDNA (in order to determine how many PCR cycles to use).
Prepare 9 μL of qPCR master mix, mixing per sample:
5 μL of SYBR Select Master Mix
3.6 μL of ddH2O
0.4 μL of qPCR primer mix (10 μM each D5 and D7 primers mixed together)
Add 1 μL of 1:10 diluted (in ddH2O) cDNA to each well of a 384-well qPCR plate. Mix master mix, add 9 μL to each well, and pipette to mix on ice.
Run qPCR according to standard procedure.
As a starting point for the final PCR, use 3 cycles less than the Ct of the 1:10 diluted sample (Note 8) (sample results are shown in Figure 3).
Figure 3. Sample qPCR results for cDNA quantification. A. Amplification plot. Threshold is automatically defined by software for Ct calculation and lined in bold. B. Ct plot. Three technical replicates for each sample.
PCR amplify cDNA.
Prepare PCR master mix on ice:
25 μL of 2× Q5 PCR master mix
5 μL of ddH2O
2.5 μL of 20 μM primer D50x (x = multiplexing barcode)
2.5 μL of 20 μM primer D70x
Add 35 μL of master mix to 8-well PCR tube strips for each sample. Then, add 12.5 μL of cDNA + 2.5 μL of ddH2O and mix thoroughly.
Program for PCR:
98 °C for 30 s
98 °C for 15 s → 68 °C for 30 s → 72 °C for 40 s (6 cycles)
98 °C for 15 s → 72 °C for 60 s (qPCR Ct, 3 cycles)
72 °C for 1 min
SPRI cleanup library:
Make sure AMPure XP beads are thoroughly resuspended prior to use.
Add 90 μL of AMPure XP bead suspension to each 50 μL of PCR reaction. Mix well and incubate for 10 min at room temperature. Periodically flick tube to mix.
Place tube on the magnet and allow beads to separate; remove supernatant carefully.
Resuspend beads in 1 mL of 75% ethanol and allow beads to sit for 30 s.
Place tube on the magnet and allow beads to separate; remove supernatant carefully.
Wash a second time with 75% ethanol, allowing beads to sit for 30 s.
Place tube on the magnet and allow beads to separate; remove supernatant carefully, getting all residual liquid possible.
Allow beads to air-dry for 5 min on magnet.
Remove tube from magnet and resuspend the beads in 12 μL of ddH2O. Incubate for 5 min at room temperature.
Place tube on the magnet and allow beads to separate; transfer 10 μL of the supernatant to a new tube.
Quantitate library on Bioanalyzer and submit for sequencing.
Sequencing should be performed on an Illumina NextSeq system (a NextSeq 550 was used in this protocol development; single-end 150-nucleotide read length is sufficient).
Data analysis
Filter and map reads, catalog 3' ends: Once the raw sequencing data have been acquired, the adaptor sequences must first be removed. Several free, publicly available software tools can be used (e.g., Cutadapt [20]). The 3' adaptor contains a unique molecular identifier (UMI) that allows duplicate reads arising from PCR overamplification to be removed. We used UMI-tools [21] for this purpose. Next, the reads are mapped back to the genome using any of the free available alignment tools; we used STAR aligner [22]. The final preprocessing step is to extract the coordinates of the 3' end of each read and determine the density of 3' ends at each nucleotide position within the genome. This can be accomplished using the genomeCoverageBed function of Bedtools [23]. A separate bedgraph should be produced for each strand in each sample.
Identify putative strong-stop peaks, assign to exons, measure background in surrounding region: In order to eliminate 3' ends generated by random reverse-transcriptase termination, we filter the genome-wide bedgraphs to require a minimum of 10 reads to identify putative RT-stop sites (peaks). After filtering, the peaks are then assigned to exons of genes using the intersectBed function of Bedtools [23]. A reference transcriptome of choice can be used for gene/exon assignment, for example GENCODE [24]. In the next step, the background signal for reverse transcriptase termination sites must be determined, in order to identify peaks that are of statistically significant enrichment. With the peak coordinate in the center position, a 100-nucleotide window is generated surrounding the peak within its assigned exon. If the window reaches the ends of the exon on either side, it is ended so as not to include intronic sequence, which is depleted of reads, in the window. Thus, some windows may be shorter than 100 nucleotides. The 3' end read depth at every nucleotide of each window was determined using the coverage function of Bedtools [23] with the –d option and using the previously created bedgraphs as input. The read depth at the peak position and the distribution of per-nucleotide 3' end read depth within the window for that peak were used in hypothesis testing as described below.
Identification of high-confidence Ψs: To test each putative reverse-transcription strong-stop site for significance above the background of random terminations, the read depths at each nucleotide position within the window surrounding each peak position are fitted with a Poisson distribution. The Poisson distribution variable mu is derived using a maximum-likelihood estimation. Then, the read count at the peak position is tested against the null hypothesis that it was sampled from the same Poisson distribution found in the surrounding window, in order to derive a p-value. A false-discovery rate (FDR) is then estimated using the Benjamini-Hochberg procedure; we set the threshold for a positive at 0.05. Finally, peaks that are significant in the CMC+ sample but not in the CMC- sample and that have a genomic “T” base at the position directly 3' of the putative RT-stop allow us to assign the adjacent “T” as a high-confidence pseudouridylation site.
Validation of protocol
This revised protocol was used in a recent publication to detect transcriptome-wide Ψs to confirm that the exogenous gRNA has the specificity and does not show the off-target effects [14].
The revised Pseudo-seq identified a total of 1,370 Ψs in polyadenylated transcripts, which were significant in two independent replicates, and another 3,979 that met the statistical threshold in a single replicate. Note that the numbers of sites identifiable in any specific experiment may depend on many factors, such as the species and cell-type in which the experiment is conducted and the sequencing read depth. Figure 4 shows that there were no significant differences in the magnitude of the Ψ stop signals between the two samples of mRNA isolated from cells before and after (or with and without) transfection of a β-thalassemia PTC-specific gRNA, suggesting that our approach has no significant off-target problem. Table 1 shows that even mRNAs that have a similar sequence to the target of the gRNA did not cause a significant difference between the “minus gRNA” and “plus gRNA” samples.
Figure 4. Reverse transcription (RT)-stop peak height over background is highly similar in control and gRNA-transfected cells [14]. Transcriptome-wide Ψ mapping was carried out. HEK293T cells were transfected with the plasmid containing the β-thalassemia PTC-specific gRNA gene or left untransfected (control). mRNA was recovered and the Pseudo-seq libraries were constructed (see Materials and Methods). Ψs were identified and compared between the two samples (control and gRNA-transfected cells). Each point represents a putative Ψ with a minimum of 10 3' ends stopping at the adjacent upstream nucleotide (peak). Putative Ψs meeting statistical significance [false-discovery rate (FDR) ≤ 0.05] are colored orange. The log2 of the peak height normalized to the 3' reads within a surrounding 100-nucleotide window is plotted for both the control cells (x-axis) and the gRNA-transfected cells (y-axis). The normalized peak heights show strong correlation (R2 = 0.88) between control and gRNA-transfected cells, indicating little difference in pseudouridylation sites between the two samples. Similar numbers of off-diagonal peaks are present in both samples, and none of the sites with six or more contiguous matches to the gRNA-target sequence are among them.
Table 1. Targeted pseudouridylation has no significant off-target effects [14]. From the set of peak positions genome-wide, those with a minimal match to the gRNA target sequence (ACCΨNGA) were extracted. This yielded 22 peaks. The gene and gene position (CDS, 5'UTR, or 3'UTR) are shown in columns 1 and 2. The extended surrounding nucleotides were then identified and, as shown in column 3, matches to the gRNA target sequence are shown in bold capital letters, mismatches in lowercase, and the putative Ψ genomic DNA is shown as a bold red T. In column 4 the number of contiguous matching nucleotides (without counting unpaired ΨN) is shown. Columns 5, 6, 7, and 8 provide the p-values of the enrichment of peak read counts over background for the control and gRNA-treated samples, respectively. Statistical significance was considered for peaks for which the CMC+, but not the CMC- sample, reached the significance cutoff. Peaks that are significant in the gRNA-treated CMC+, but not CMC- or CMC+ control sample, are shaded pink.
General notes and troubleshooting
General notes
The 3' DNA linker (rand103Tr3) is with 10 nt unique molecular identifier (UMI), denoted as NNNNNNNNNN.
For ethanol precipitations, make sure you do not use ammonium acetate, as the ammonium ions can carry over and are potent inhibitors of T4 PNK. Use sodium acetate that has been pH-adjusted to 5.5–6. Unadjusted 3 M NaOAc is basic and will result in alkaline hydrolysis of your RNA!
Allow shattered RNA solution to cool off before adding enzyme.
N-cyclohexyl-N0-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) should be prepared freshly at 0.5 M (212 mg/mL) in BEU buffer. CMC is sometimes described as 1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide Metho-p-toluenesulfonate.
Do not turn up the voltage too quickly while prewarming the denaturing polyacrylamide gel, as the plates may crack.
To make the density of the sample higher (to load the sample on the well easily), adding 1.2–1.5 times of 2× loading dye would help.
According to the original Pseudo-seq protocol [19]: “It is very important that the RT primer be gel-purified to ensure that it is a uniform length, allowing robust separation of truncated from full-length cDNAs. Gel-purification should be performed in house, as gel-purified primers obtained commercially can be heterogeneous.”
It may be necessary to empirically determine whether the Ct values obtained from a specific real-time qPCR protocol yield values that give the correct amplification range. Use the Bioanalyzer quantitation to zero in on the correct cycle number for subsequent experiments.
Acknowledgments
This work was supported by Grants GM138387, GM141544, and CA241111 from the NIH, Grant YU20G0 from the Cystic Fibrosis Foundation, and Grant GFF 521008 from the Gilbert Family Foundation. This protocol was used in Adachi et al. [14].
Competing interests
The authors declare no competing interests.
Ethical considerations
We have carried out all of the experiments under University of Rochester Biological Ethics Guideline.
References
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4,986 | https://bio-protocol.org/en/bpdetail?id=4986&type=0 | # Bio-Protocol Content
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Peer-reviewed
Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry
HL Hui Jing Lim
HM Hamish E. G. McWilliam
Published: Vol 14, Iss 9, May 5, 2024
DOI: 10.21769/BioProtoc.4986 Views: 651
Reviewed by: Keisuke TabataYu Hui KangAndrea Gramatica
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Sep 2022
Abstract
Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell–cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry–based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level.
Key features
• These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry.
• They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available.
• The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states.
• The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells.
• These assays have been extensively used on suspension cells but have not been tested on adherent cells.
Keywords: Plasma membrane Protein internalisation Protein recycling Flow cytometry Protein trafficking Oligonucleotide-antibody probe Cleavable biotin-antibody probe Major histocompatibility complex (MHC) molecules
Background
Cell surface proteins mediate many important physiological functions. How long they dwell at the cell surface is critical to their activity, and this is defined by a dynamic process of internalisation and often recycling from within the endosomal compartment back to the plasma membrane. For example, diverse cells display major histocompatibility complex (MHC) proteins, which allow patrolling T lymphocytes to monitor for pathogens or cancer [3]. MHC proteins are regulated by distinct mechanisms, and this can vary depending on the cell type or activation state [4–6]. Such protein trafficking is still being understood for diverse cell types; therefore, accurate methods are required.
Two major strategies to accurately quantify surface protein internalisation and recycling are microscopy-based or biochemical assays, but these have limitations. Microscopy allows the visualisation of subcellular localisation of the labelled surface molecule. However, it can be challenging to track molecules of low abundance or to differentiate internalised from surface molecules, especially at low resolution [7]. Further, microscopy can only accommodate a limited number of cells, and biochemical methods generally observe the sum of a single-cell population, making the analysis of specific cells in heterogenous samples impractical.
Here, we describe methods to quantitate the internalisation and recycling of membrane proteins in different cell types using functional probes conjugated to specific antibodies and detected by flow cytometry [4]. When combined with specific markers to differentiate cell types, protein dynamics can be analysed in a complex cell mixture at the single-cell level. Similar flow cytometry–based assays involve acid stripping the cell surface, but these have been shown to be harsh on cells, destroying some epitopes that prevent cell phenotyping, and lead to undesired cellular activation [8–11]. Here, we detail assays that allow gentle, rapid, and quantitative measurement of cell surface protein trafficking.
For internalisation, we detail the method developed by Liu et al. [1,2]. A fluorophore-labelled oligonucleotide is conjugated to a surface protein-specific antibody, termed the fluorescent internalisation probe (FIP) (Figure 1A). Cells are labelled and then allowed to internalise their labelled surface cargo. The fluorescence of the FIP-labelled proteins remaining on the cell surface is quenched with a complementary oligonucleotide-conjugated quencher, leaving only the internalised molecules fluorescent. Hence the cellular fluorescence remaining after quenching reflects the internalised molecules at each time point (Figure 1A).
Figure 1. Assay schematics. The internalisation assay (A) employs the fluorescent internalisation probe to label the surface protein of interest (i); then, after a cohort of these internalise (ii), a quencher distinguishes those that remain on the surface from those internalised (iii). The recycling assay (B) uses a recycling probe to label the surface protein (i); then, these are allowed to internalise (ii). The label on the probe bound to those remaining on the surface is cleaved (iii) and then recycling is allowed to occur (iv). Finally, those molecules that recycled are detected with fluorescently labelled streptavidin (SA) (v).
For recycling, a biochemical assay is adapted for flow cytometry [12,13]. Surface proteins are labelled with an antibody conjugated to a cleavable biotin group (Figure 1B). Proteins are allowed to internalise; then, the biotin tag is cleaved from non-internalised proteins with a membrane-impermeable reducing reagent. Proteins are allowed to recycle, and then any biotin probe–labelled proteins that re-emerge from inside the cell are detected with fluorophore-conjugated streptavidin. This assay then calculates the proportion of original surface molecules that recycle after a defined period of internalisation [4,14].
With these methods, we have measured the internalisation and recycling of the MHC class I-related protein 1 (MR1) and transferrin receptor in a range of cell types including peripheral blood mononucleated cells (PBMC) [4,14]. These methods can be used to study any membrane protein of interest in suspension cells but may also be adapted to adherent cells.
Materials and reagents
Biological materials
Human peripheral mononucleated cells (PBMCs) are isolated from buffy coats using Ficoll Paque PLUS (Cytiva, catalog number: 17144002) according to the manufacturer’s instructions. Buffy coats from healthy human donors were obtained from the Australia Red Cross Blood Service with written and informed consent and with ethics approval from the University of Melbourne Human Research and Ethics Committee (#1035100).
Reagents
RPMI 1640 medium (Gibco, catalog number: 11875093)
Fetal bovine serum (FBS) (Gibco, catalog number: 5256701). Heat-inactivate at 56 °C for 30 min with interval mixing and store at -20 °C
Ethylenediaminetetraacetic acid-balanced salt solution (EDTA-BSS): 147 mM NaCl, 3.6 mM KCl, 0.01 mM KH2PO4, 0.02 mM K2HPO4, 14.5 mM HEPES, 6 mM EDTA (prepared in-house by dissolving each chemical in distilled water)
Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher, catalog number: 10010023)
10 mM sodium bicarbonate, pH 8.0, diluted from 1 M (Thermo Fisher Scientific, catalog number: J62495.AP)
Purified antibody specific for the cell surface protein to be assessed for internalisation or recycling (at a concentration of at least 2 mg/mL)
Cell type–specific surface markers for cell phenotyping, e.g.:
PerCP conjugated anti-CD3 (BD, catalog number: 345766)
BUV805 conjugated anti-CD14 (BD Horizon, catalog number: 612902)
Fixable viability dye eFluor780 (eBioscience, catalog number: 65-0865-14)
Click-iTTM sDIBO Alkynes (Invitrogen, catalog number: C20025), dissolved in anhydrous DMSO at 1 mg/mL
Cy5-oligo-azide (5' Cy5-TCA GTT CAG GAC CCT CGG CT-N3 3') (Integrated DNA Technologies), dissolved at 150 μM in nuclease-free water and stored at -20 °C
Quencher (5' AGC CGA GGG TCC TGA ACT GA-BHQ2 3') (Integrated DNA Technologies), dissolved at 600 μM in nuclease-free water and stored at -20 °C
EZ-LinkTM Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific, catalog number: 21331)
Streptavidin-phycoerythrin (PE) (BioLegend, catalog number: 405203)
1 M Tris buffer, pH 8.0 (prepared in-house by dissolving Tris in distilled water and then adjusting pH with 1 M hydrochloric acid)
Sodium chloride (NaCl) (Spectrum Chemical, catalog number: SO160)
0.5 M EDTA, pH 8.0 (prepared in-house by stirring the required amount of EDTA powder in distilled water and then adjusting pH with solid sodium hydroxide pellets)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906)
Sodium 2-mercaptoethanesulfonate (MESNa) (Sigma-Aldrich, catalog number: M1511)
Laboratory supplies
Zeba spin desalting column (7K, MWCO 0.5 mL) (Thermo Fisher Scientific, catalog number: 89882)
Amicon Ultra centrifugal filters (30 kDa, MWCO 0.5 mL) (Millipore, catalog number: UFC5030)
Low-protein binding 1.5 mL centrifuge tubes (Thermo Scientific, catalog number: 90410)
Round bottom 96-well plates (Corning, catalog number: 3799)
Solutions
Cell culture media (see Recipes)
Staining buffer (see Recipes)
Recycling assay buffer 1 (see Recipes)
Recycling assay buffer 2 (see Recipes)
MESNa incubation buffer (see Recipes)
Cell surface marker staining solution (see Recipes)
Recipes
Cell culture media (500 mL)
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
RPMI 1640 n/a 450 mL
FBS 10% (v/v) 50 mL
Total n/a 500 mL
Staining buffer (510 mL)
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
EDTA-BSS n/a 500 mL
FBS 2% (v/v) 10 mL
Total n/a 510 mL
Recycling assay buffer 1 (500 mL)
Note: Dissolve the NaCl completely in milli-Q water before adding to the solution. Store at room temperature.
Reagent Final concentration Quantity or Volume
1 M Tris buffer, pH 8.0 50 mM 25 mL
NaCl 100 mM 2.9 g
0.5 M EDTA, pH 8.0 1 mM 1 mL
Milli-Q water Top up to 500 mL
Total n/a 500 mL
Recycling assay buffer 2 (50 mL)
Note: Prepare on the day of the assay. Scale down or up as required.
Reagent Final concentration Quantity or Volume
Recycling assay buffer 1 n/a 50 mL
BSA 0.2 % (w/v) 0.1 g
Total n/a 50 mL
MESNa incubation buffer (2 mL)
Note: Prepare on the day of the assay. Scale down or up as required.
Reagent Final concentration Quantity or Volume
Recycling assay buffer 2 n/a 2 mL
MESN 100 mM 0.033 g
Total n/a 2 mL
Cell surface marker staining solution
Note: Prepare on the day of the assay. Scale down or up as required. Replace with antibodies to differentiate your cells of interest.
Reagent Dilution Volume
PerCP conjugated anti-CD3 1:50 20 μL
BUV805 conjugated anti-CD14 1:200 5 μL
Fixable viability dye 1:1,000 1 μL
Staining buffer n/a 1,000 μL
Equipment
Air-cooled benchtop centrifuge (Beckman Coulter, model: Microfuge 22R Centrifuge)
Cell culture incubator at 37 °C and 5%–10% CO2 (dependent on the recommended CO2 level for your cells or media)
Flow cytometer (BD, model: LSR Fortessa FACS Calibur)
Software and datasets
FlowJo (version 10.4, 09/15/2017); can be purchased and downloaded online from https://www.flowjo.com/solutions/flowjo/downloads
Graph Pad Prism (version 10.1.0, 10/19/2023); can be purchased and downloaded online from https://www.graphpad.com
Procedure
Probe preparation
FIP
Overview: FIP is prepared by conjugating the antibody with Cy5-oligo using click chemistry of Click-iTTM sDIBO Alkyne and the azide group.
Dilute 200 μg of antibody to 2 mg/mL in 10 mM sodium bicarbonate (pH 8.0).
Add 7 μL of Click-iTTM sDIBO Alkyne and incubate for 2 h at 4 °C.
Remove the bottom closure of 7K Zeba spin desalting column and place it in a 1.5 mL tube.
Centrifuge the column at 1500× g for 1 min and discard the storage solution.
Add 300 μL of PBS to the column and centrifuge at 1500× g for 1 min. Discard flowthrough. Repeat twice.
Place the column in a 1.5 mL low-protein binding tube and add the protein-DIBO solution.
Centrifuge at 1500× g for 2 min and collect the eluate (containing the DIBO-labelled antibody eluate).
Add 20 μL of Cy5-oligo-azide to the eluate and incubate for at least 2 h (or ideally overnight) at 4 °C. This allows the reaction of conjugating the Cy5-oligo-azide to the DIBO-antibody forming the FIP.
Equilibrate the Amicon Ultra centrifugal filter by adding 500 μL of PBS and centrifuging at 13,000× g for 1 min. Discard flowthrough and any remaining PBS in the filter.
Top up the FIP solution (from step A8) to 500 μL in total with PBS.
Add to the Amicon Ultra centrifugal filter and centrifuge at 13,000× g for 5 min to remove excess unconjugated reagents. Discard flowthrough.
Repeat step A11 by rinsing the filter with 500 µL of PBS until the flowthrough is clear (approximately three washes).
Recover the purified FIP by placing the filter upside down in a new 2 mL tube and centrifuge at 1,000× g for 2 min.
Store the FIP in the dark at 4 °C for short-term use (<2 weeks) or aliquot and store at -20 °C for long-term storage.
FIP should be titrated to find the optimal staining dilution prior to beginning the assay as for normal flow cytometry antibodies (See Troubleshooting 2).
Recycling probe
Overview: Recycling probe is prepared by biotinylating antibody with EZ-LinkTM Sulfo-NHS-SS-Biotin that reacts with the primary amines on the antibody.
Dilute 200 μg of antibody to 2 mg/mL in PBS.
Prepare a 10 mM solution of Sulfo-NHS-SS-Biotin according to the manufacturer’s instructions.
Add 2.7 μL of Sulfo-NHS-SS-Biotin to the antibody (step B1).
Incubate the reaction on ice for 2 h to allow the conjugation of the Sulfo-NHS-SS-Biotin to the antibody.
Remove the bottom closure of 7K Zeba spin desalting column and place it in a 1.5 mL tube.
Centrifuge the column at 1,500× g for 1 min and discard the storage solution.
Add 300 μL of PBS, centrifuge at 1,500× g for 1 min, and discard flowthrough. Repeat twice.
Place column in a 1.5 mL low-protein binding tube and add the protein/Sulfo-NHS-SS-Biotin solution. Centrifuge at 1,500× g for 2 min and collect the antibody-SS-Biotin (the recycling probe) in the eluate.
Store the recycling probe in the dark at 4 °C for short-term use (<2 weeks) or aliquot and store at -20 °C for long-term storage.
Recycling probe should be titrated to find the optimal staining dilution prior to beginning the assay as for normal flow cytometry antibodies (See Troubleshooting 2).
Internalisation assay
Surface protein labelling
Prechill the centrifuge, plates, tubes, and solutions at 4 °C.
Add cells in 2 million cells per millilitre of suspension in cell culture media to a 1.5 mL centrifuge tube. Centrifuge at 620× g for 2 min. Wash cells once with staining buffer after removing supernatant.
Stain the cells with FIP diluted in staining buffer on ice for 20 min.
Wash the cells twice with ice-cold staining buffer.
Resuspend the cells in prechilled cell culture media at 2 million cells per millilitre.
Internalisation at 37 °C
Transfer 100 µL of the cell suspension (200,000 cells) into the appropriate wells of 96-round-bottom plates, on ice. Use two plates and perform each sample/time point in triplicate:
Plate 1 (control plate, always on ice) requires two control samples:
Tint = 0 –Q: Cells are not allowed to internalise and are not treated with quencher (Q).
Tint = 0 +Q: Background control—cells are not allowed to internalise and are treated with Q.
Plate 2 (internalisation plate, always in incubator at 37 °C).
Tint = X +Q: Internalisation sample where cells are allowed to internalise for X time. Repeat for each time point.
Keep Plate 1 on ice. Incubate Plate 2 at 37 °C for the desired duration for internalisation to occur.
Transfer Tint = X +Q samples from Plate 2 to Plate 1 (on ice) at the end of each time point, terminating internalisation for that sample.
Quenching the un-internalised labelled protein and cell phenotyping surface marker staining
At this point, only Plate 1 remains. Wash the cells by adding 150 μL of ice-cold staining buffer to each well. Centrifuge the plate at 300× g for 5 min. Decant the supernatant. Repeat the wash.
Resuspend the cells in cell surface marker staining solution with or without Q (dilution 1/1,000) as indicated and incubate on ice for 15 min.
Wash twice with ice-cold staining buffer.
Measuring the percentage of internalised protein of interest
Run the cells on a flow cytometer.
Measure the geometric mean fluorescent intensity (gMFI) of the Cy5 (FIP) for each cell population using FlowJo (refer to Figure 2 for typical results). Calculate internalisation (see Data Analysis 1).
Figure 2. Results from the internalisation or recycling assays for major histocompatibility complex (MHC) class I related protein 1 (MR1) using human peripheral blood mononucleated cells (PBMCs). (A) T cells and monocytes can be differentiated based on markers CD3 and CD14. The percentage of each population is shown in the plot. After identifying the cell population, (B) the fluorescence internalisation probe Cy5 signal (FIP-Cy5, using the antibody clone 8F2.F9 specific for MR1) for the internalisation assay or (C) the recycling probe–labelled Streptavidin-PE (using antibody clone 8F2.F9) for the recycling can be measured by flow cytometry. With the corresponding controls [Tint = 0 with and without quencher (Q) or MESNa cleavage (M)], the percentage of internalised or recycled molecules can be calculated. TINT indicates internalisation time. TREC indicates recycling time. Fluorescence minus one (FMO) indicates where the cells were stained with cell phenotyping antibodies and secondary staining but not FIP-Cy5 (B) or recycling probe (C). FMO served as the background control for the spread of signal in the multicolour panel and to know the quenching or cleaving effect in internalisation and recycling assays, respectively. Shown here for each assay, the typical results of one experiment (representing > 10) are shown by the geometric mean fluorescence (gMFI) and the calculated proportion of molecules that have been internalised (B) or recycled (C).
Recycling assay
Surface protein labelling
Prechill the centrifuge, plates, tubes, and solutions at 4 °C.
Add cells in 2 million cells per millilitre of suspension in cell culture media to a 1.5 mL centrifuge tube. Centrifuge at 620× g for 2 min. Wash cells once with staining buffer after removing the supernatant.
Stain the cells with staining buffer with the recycling probe on ice for 20 min.
Wash the cells twice with ice-cold staining buffer.
Resuspend the cells in prechilled cell culture media at 2 million cells per millilitre.
Internalisation at 37 °C
Transfer 100 µL of the cell suspension (200,000 cells) into the appropriate wells of 96-round-bottom plates, on ice. Use two plates and perform each sample/time point in triplicate:
Plate 1 (control plate, always on ice) requires one control sample:
–INT –M: Cells are not allowed to internalise (INT) and are not treated with MESNa (M).
Plate 2 (internalisation/recycling plate, always in incubator at 37 °C except for Step C).
+INT +M Trec = 0: Background control—Cells are allowed to internalise, treated with M, but not allowed to recycle.
+INT +M Trec = X: Cells are allowed to internalise, treated with M, and then allowed to recycle for time X. Repeat for each time point.
Keep Plate 1 on ice. Incubate Plate 2 at 37 °C for the desired duration for internalisation to occur (typically 20–120 min). The optimal time for internalisation is the time required for a significant amount to be internalised—typically ~50%–100%; for MR1, this is 1 h (Figure 2B). This should be empirically tested for each molecule and cell type.
Cleaving biotin from the un-internalised labelled protein
Note: This step is conducted on all +M samples in plate 1 and 2 on ice.
Wash the +M samples once by pelleting down the cells by centrifuging at 300× g for 5 min and adding 200 µL of ice-cold recycling assay buffer 1 to the cell pellet in each well. Centrifuge again and decant the supernatant.
Resuspend the +M samples in 40 µL of ice-cold MESNA incubation buffer.
Incubate for 10 min on ice.
Wash the +M samples once with ice-cold recycling assay buffer 2.
Repeat steps C2–C4 once.
Wash once with culture media. Resuspend cells in culture media.
Transfer +INT +M Trec = 0 samples from Plate 2 to Plate 1 (on ice) at the end of the internalisation and keep on ice.
Recycling at 37 °C
Note: At this point, only Trec = X samples remain in Plate 2.
Incubate samples remaining in Plate 2 at 37 °C for time X, to allow recycling of the protein to occur.
At the end of each incubation, transfer the samples to Plate 1 on ice.
Staining of the recycled labelled protein and cell phenotyping surface marker staining
Wash all the samples twice with ice-cold staining buffer.
Resuspend the cells in cell phenotyping staining solution including Streptavidin-PE (1:200) and incubate on ice for 15 min.
Wash twice with ice-cold staining buffer.
Measuring the percentage of internalised protein of interest
Run the cells on flow cytometer.
Measure the gMFI of PE of each cell population using FlowJo (refer to Figure 2 for typical results). Calculate recycling (see Data Analysis 2).
Data analysis
Calculate the percentage of protein internalisation and recycling based on the ratio of the signal measured at the desired time point comparing to the total signal collected at time point 0 (when no internalisation/recycling occurs). Remove background signal in the calculation by subtracting the background control from these values.
Calculate the percentage of internalised protein at each internalisation time point (TINT = X) as a fraction of the surface level before internalisation, subtracting the background signal (TINT = 0+Q); where Q = quencher:
Calculate the percentage of recycled protein at each recycling time point (Trec = X) as a fraction of the surface level before internalisation (–INT –M), subtracting the background signal (+INT +M Trec = 0); where M = MESNa; INT = internalisation step:
Plot a time-course line graph using Graph Pad Prism if membrane protein internalisation or recycling are examined at multiple time points.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Lim et al. [15] “A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells”, (Journal of Cell Biology), Figure 1A-C, 2D-E, 2H, 3F-G, 7B-C. Performed on human PBMC, human B lymphoblast cells line C1R cells and human monocyte cell line THP-1 cells.
McWilliam et al. [16] “The intracellular pathway for the presentation of vitamin B–related antigens by the antigen-presenting molecule MR1” (Nature Immunology), Figure 5A. Performed on human C1R cell line.
Howson et al. [17] “Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1” (Science Immunology), Figure 5C. Performed on human C1R cell line.
General notes and troubleshooting
General notes
These methods can be adapted to study different surface proteins in suspension cells from different hosts.
These methods are not suitable for adherent cell lines. Adherent cells may attach to the plate during the incubation at 37 °C. Trypsin treatment for the cell lifting may alter the cell behaviour and cell membrane biology, leading to inaccurate measurement in the assay.
The choices of fluorophores mentioned are those used by our studies; however, other fluorophores can also be used.
The recycling assay here measures the percentage of the originally labelled surface molecules that are recycled back to the cell surface after a period of internalisation. For the interpretation of the results, it is important to keep in mind that this rate is dependent on both the rate of recycling and the length of time used for the internalisation step. Hence, the length of time used for the internalisation step should be reported for this assay.
It has not been determined if fixation affects the performance of these assays. It is possible that fixing the cells after the assay is compatible with accurate measurements of internalisation and recycling, but this should be verified first by comparing the results of fixed vs. unfixed cells.
The protocol here uses the gMFI of the probes to measure the internalisation or recycling rate for the whole population of interest. It is possible, however, to use other measures or calculations distinct from gMFI to determine if there are heterogenous rates of internalisation within each population.
Troubleshooting
Problem 1: Too few cells acquired during flow cytometry.
Possible cause: Cells are lost during the washing steps, or the assay started with a low number of cells.
Solution: Increase the number of cells used in the assay.
Problem 2: Signal from positive controls (–Q or –M) is low.
Possible cause: The probes (FIP or recycling) are too diluted during the staining, or the expression of the protein being studied has a low abundance.
Solution: Optimise the concentration of the probe for the staining, until a stronger signal is gained. Use the best dilution of probes that give best signal (–Q or –M) to background (+Q or +M) ratio.
Problem 3: Inconsistent results.
Possible cause: Plates used for storing samples or controls are not kept cold and increase in temperature, hence internalisation or recycling occurs during washing or staining.
Solution: Use a prechilled centrifuge, consumables, and solutions throughout the assay unless specified to prevent internalisation and recycling after the time points have ended.
Problem 4: No recycled molecule detected in recycling assay.
Possible cause: Insufficient or extended time for the internalisation to occur.
Solution: Adjust the duration for internalisation at 37 °C in the way that the gMFI of +INT –M Trec = 0 is a lower signal than the gMFI of –INT –M, yet higher than that of +INT +M Trec = 0.
Acknowledgments
The work was supported by a National Health and Medical Research Council (NHMRC) Ideas Grant (2003192). The internalisation assay detailed in this protocol was adapted from the internalisation method developed by Liu et al. [1,2]. The recycling assay was adapted for flow cytometry from a biochemical assay previously used [12,13]. We acknowledge the pioneering work of these authors.
Competing interests
The authors declare no competing interest.
Ethical considerations
Human blood from healthy human donors was obtained from the Australia Red Cross Blood Service with written and informed consent and with ethics approval from the University of Melbourne Human Research and Ethics Committee (#1035100).
References
Liu, H., Dumont, C., Johnston, A. P. R. and Mintern, J. D. (2016). Analysis of Intracellular Trafficking of Dendritic Cell Receptors for Antigen Targeting. In: Segura, E., Onai, N. (eds) Dendritic Cell Protocols. In Methods in Molecular Biology, vol 1423. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3606-9_15
Liu, H. and Johnston, A. P. (2013). A programmable sensor to probe the internalization of proteins and nanoparticles in live cells. Angew. Chem. Int. Ed. Engl. 52(22): 5744–5748. https://doi.org/10.1002/anie.201301243.
Murphy, K. and Weaver, C. (2017). Janeway's immunobiology. Garland Science/Taylor & Francis Group, LLC. New York, NY. ISBN: 9780815345053.
Lim, H. J., Wubben, J. M., Garcia, C. P., Cruz-Gomez, S., Deng, J., Mak, J. Y. W., Hachani, A., Anderson, R. J., Painter, G. F., Goyette, J., et al. (2022). A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells. J. Cell Biol. 221(12). https://doi.org/10.1083/jcb.202110125.
Liu, H., Jain, R., Guan, J., Vuong, V., Ishido, S., La Gruta, N. L., Gray, D. H., Villadangos, J. A. and Mintern, J. D. (2016). Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection. J. Exp. Med. 213(9): 1695–1703. https://doi.org/10.1084/jem.20160312.
Villadangos, J. A., Schnorrer, P. and Wilson, N. S. (2005). Control of MHC class II antigen presentation in dendritic cells: a balance between creative and destructive forces. Immunol. Rev. 207: 191–205. https://doi.org/10.1111/j.0105-2896.2005.00317.x.
Hernández Pérez, S. and Mattila, P. (2022). A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo. Sci. Rep. 12. https://doi.org/10.1038/s41598-021-04544-6.
Shunji, S., Toru, A. and Katsuo, K. (1987). A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3. J. Immunol. Methods 100(1): 83–90. https://doi.org/https://doi.org/10.1016/0022-1759(87)90175-X.
Meinke, S., Sandgren, P., Mörtberg, A., Karlström, C., Kadri, N., Wikman, A. and Höglund, P. (2016). Platelets made HLA deficient by acid treatment aggregate normally and escape destruction by complement and phagocytes in the presence of HLA antibodies. Transfusion 56(2): 370–382. https://doi.org/https://doi.org/10.1111/trf.13350.
He, W., Gea-Mallorquí, E., Colin-York, H., Fritzsche, M., Gillespie, G. M., Brackenridge, S., Borrow, P. and McMichael, A. J. (2023). Intracellular trafficking of HLA-E and its regulation. J. Exp. Med. 220(8). https://doi.org/10.1084/jem.20221941.
Ma, W., Zhang, Y., Vigneron, N., Stroobant, V., Thielemans, K., van der Bruggen, P. and Van den Eynde, B. J. (2016). Long-Peptide Cross-Presentation by Human Dendritic Cells Occurs in Vacuoles by Peptide Exchange on Nascent MHC Class I Molecules. J. Immunol. 196(4): 1711–1720. https://doi.org/10.4049/jimmunol.1501574.
O’Reilly, M. K., Tian, H. and Paulson, J. C. (2011). CD22 Is a Recycling Receptor That Can Shuttle Cargo between the Cell Surface and Endosomal Compartments of B Cells. J. Immunol. 186(3): 1554–1563. https://doi.org/10.4049/jimmunol.1003005.
Barral, D. C., Cavallari, M., McCormick, P. J., Garg, S., Magee, A. I., Bonifacino, J. S., De Libero, G. and Brenner, M. B. (2008). CD1a and MHC Class I Follow a Similar Endocytic Recycling Pathway. Traffic 9(9): 1446–1457. https://doi.org/10.1111/j.1600-0854.2008.00781.x.
McWilliam, H. E., Eckle, S. B., Theodossis, A., Liu, L., Chen, Z., Wubben, J. M., Fairlie, D. P., Strugnell, R. A., Mintern, J. D., McCluskey, J., et al. (2016). The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1. Nat. Immunol. 17(5): 531–537. https://doi.org/10.1038/ni.3416.
Lim, H. J., Wubben, J. M., Garcia, C. P., Cruz-Gomez, S., Deng, J., Mak, J. Y. W., Hachani, A., Anderson, R. J., Painter, G. F., Goyette, J., et al. (2022). A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells. J. Cell Biol. 221(12). https://doi.org/10.1083/jcb.202110125.
McWilliam, H. E. G., Eckle, S. B. G., Theodossis, A., Liu, L., Chen, Z., Wubben, J. M., Fairlie, D. P., Strugnell, R. A., Mintern, J. D., McCluskey, J., et al. (2016). The intracellular pathway for the presentation of vitamin B–related antigens by the antigen-presenting molecule MR1. Nat. Immunol. 17(5): 531–537. https://doi.org/10.1038/ni.3416.
Howson, L. J., Awad, W., von Borstel, A., Lim, H. J., McWilliam, H. E. G., Sandoval-Romero, M. L., Majumdar, S., Hamzeh, A. R., Andrews, T. D., McDermott, D. H., et al. (2020). Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1. Sci. Immunol. 5(49). https://doi.org/10.1126/sciimmunol.abc9492.
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Simplified Protocol to Demonstrate Gene Expression in Nicotiana benthamiana Using an Agrobacterium-Mediated Transient Assay
SV Satyam Vergish
RW Ryan Wolf
WS Wen-Yuan Song
Published: Vol 14, Iss 10, May 20, 2024
DOI: 10.21769/BioProtoc.4987 Views: 1111
Reviewed by: Samik Bhattacharya Anonymous reviewer(s)
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Abstract
Agrobacterium-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death.
Key features
• The protocol simplifies the widely used Agrobacterium-mediated transient gene expression assay [1] and can be completed within one week when plants are available.
• Rice XB3 gene can induce a dramatic and easily identifiable cell death phenotype in Nicotiana benthamiana.
• Allows identification of cell death–inducing genes and is suitable for teaching.
• Compared to the currently used methods, our protocol omits the use of agroinfiltration buffer, pH meter, temperature-controlled growth chamber, centrifuge, and spectrophotometer.
Keywords: Agrobacterium Nicotiana benthamiana Agroinfiltration Cell death Gene expression Ion leakage
Graphical overview
Agrobacterium infiltration (agroinfiltration) of Nicotiana benthamiana. The photo demonstrates the method of agroinfiltration into the abaxial side of leaves using a needleless syringe.
Background
Agrobacterium-mediated transient transformation of the plant Nicotiana benthamiana is a powerful tool widely used in molecular biology studies [2,3]. This assay is ideal for rapidly demonstrating gene expression, especially when used for the visible cell death phenotype induced by specific plant genes from various species. While holding summer professional development workshops for pre-college educators, we found that the current protocols for Agrobacterium-mediated transient gene expression are difficult to use in pre-college classrooms due to lack of the necessary equipment and reagents.
The rice (Oryza sativa) cell surface innate immune receptor XA21 specifies resistance to bacterial strains of Xanthomonas oryzae pv. oryzae [4]. XA21 interacts with a number of proteins in vitro and in vivo. One protein that XA21 interacts with is called XB3 for XA21 binding protein 3 [5]. XB3 induces highly visible cell death in N. benthamiana when over-expressed [1]. To support public education and quickly identify cell death–inducing genes, we developed a novel protocol to simplify the current Agrobacterium-mediated transient gene expression assay.
Materials and reagents
N. benthamiana seeds (available from numerous plant science research labs)
pC1300S-XB3: Agrobacterium tumefaciens strain EHA105 (Intact Genomics, catalog number: 1084-06) harboring the construct pC1300S-XB3 [1]
EV: A. tumefaciens strain EHA105 harboring pC1300S (empty vector)
P19: A. tumefaciens carrying the p19 construct. The P19 protein from tomato bushy stunt virus is an efficient suppressor of posttranscriptional gene silencing, which enables higher expression of the gene of interest [6]
Luria-Bertani broth (LB Broth) (Fisher Scientific, catalog number: 10855001)
Kanamycin sulfate (Fisher Scientific, catalog number: BP906-5)
Rifampicin (PhytoTechnology Lab, catalog number: R501)
100 mM acetosyringone (PhytoTechnology Lab, catalog number: A1104)
Filtered water
Soilless potting mix (Growing Mix, PRO-LINE HFC/B HydraFiber, from Jolly Gardener)
Solutions
Kanamycin sulfate stock solution (50 mg/mL) (see Recipes)
Rifampicin stock solution (50 mg/mL) (see Recipes)
Infiltration buffer [1] (see Recipes)
Recipes
Kanamycin sulfate stock solution (50 mg/mL)
Dissolve 0.5 g of kanamycin sulfate (powder) in 10 mL of distilled water at room temperature. Filter through a 0.2 μm filter. Aliquot in 1 mL fractions and store in a -20 °C freezer.
Rifampicin stock solution (50 mg/mL)
Dissolve 0.5 g of rifampicin (powder) in 10 mL of 100% methanol at room temperature. Aliquot in 1 mL fractions and store in a -20 °C freezer.
Infiltration buffer [1]
Dissolve 1.95 g of MES [2-(N-Morpholino)ethanesulfonic acid] and 1.96 g of MgCl2 in 1,000 mL of sterile water at room temperature. Adjust pH to 5.6. Aliquot in 1 mL fractions and store in a -20 °C freezer.
Laboratory supplies
9-cm square disposable pots with drainage holes (any manufacturer)
15 mL culture tubes (Carolina Biological Supply, catalog number: 215090)
50 mL conical screw cap tubes (Carolina Biological Supply, catalog number: 215095)
Syringes (1 cc) (Carolina Biological Supply, catalog number: 697765)
10 mm cork borer (Carolina Biological Supply, catalog number: 712202)
Equipment
Forceps for transplanting seedlings (Carolina Biological Supply, catalog number: 624790)
Incubator shaker (New Brunswick Scientific, model: C24KC)
Conductivity meter with electrode (Eutech, model: Cond 6+ ECCON603PLUS)
Deep freezer with -20 °C (any manufacturer)
Bunsen burner (Carolina Biological Supply, catalog number: 706706)
Laminar flow hood (any manufacturer)
Procedure
Growth conditions for N. benthamiana plants
Fill a 9 cm disposable square pot with moist potting soil and sprinkle 20–50 N. benthamiana seeds on the surface of the soil. Cover the pot with plastic wrap or a plastic dome to create a humid environment. Place the pot for two weeks in a warm location with a day length of 16 h (24 °C) and a period of darkness of 8 h (21 °C) each day. Once the seeds have germinated and the seedlings are 1–1.5 cm tall, remove the plastic wrap. Water the plants twice a week (~20 mL water/pot).
After two weeks, transplant the germinated seedlings to new pots containing moist potting soil (one seedling per pot) and grow them in the above-mentioned conditions.
Water the plants regularly (~20 mL water/pot, every 48 h), keeping the soil consistently moist but not waterlogged and growing the plants under the same conditions as described above for another five weeks until they reach the 4–6 true-leaf stage.
Note: Keep an eye on the plants for pests and diseases. Fertilize plants with a balanced liquid fertilizer every 2–3 weeks during the growing season, if required. The optimal stage for Agrobacterium infiltration (agroinfiltration) for the experiment occurs when the plants reach a young vegetative stage, characterized by actively dividing and expanding leaf area.
Preparation of A. tumefaciens culture
Inoculate individual A. tumefaciens EHA105 strains carrying p19, pC1300S-XB3, or pC1300S (EV) (Figure 1) into three separate 15 mL culture tubes each containing 3 mL of LB broth supplemented with kanamycin (100 μg/mL) and rifampicin (30 μg/mL) in a laminar flow hood. Place the culture tubes in a shaker set at 28 °C and a rotation rate of 220 rpm and incubate for 36–40 h in the dark.
Figure 1. Maps of the pC1300S (EV, kindly provided by Dr. Yinong Yang) and pC1300S-XB3 [1] constructs used in this study. P19 is described in Voinnet et al. [6].
Establish secondary cultures by taking 25 μL from the primary culture and inoculating into 50 mL conical screw cap tubes filled with 15 mL of LB broth supplemented with kanamycin (100 μg/mL) and rifampicin (30 μg/mL). Grow the new culture at 28 °C overnight in the dark reaching the logarithmic growth phase of the bacteria.
Dilute the overnight bacterial culture into LB broth supplemented with kanamycin (100 μg/mL) and rifampicin (30 μg/mL) based on the visual guidance provided in Figure 2, which covers a range of dilutions of the control LB broth supplemented with kanamycin (100 μg/mL) and rifampicin (30 μg/mL) to translucent and turbid cultures containing bacterial cells. Add 30 μL of 100 mM acetosyringone into 15 mL of bacterial culture.
Figure 2. Reference for bacterial dilution for agroinfiltration experiments. An overnight Agrobacterium culture containing pC1300S-XB3 in LB broth supplemented with kanamycin (100 μg/mL) and rifampicin (30 μg/mL) is diluted to different bacterial densities (within the range of OD600 0.1 to 1). The visual representation of Agrobacterium at varying densities offers a visual target for dilution without a spectrometer.
Incubate the diluted bacterial suspensions for an additional 3 h at room temperature to activate the virulence machinery and prime the cells for successful genetic transformation.
Note: If a laminar flow hood is not available, bacterial inoculation can be performed using a Bunsen burner. A temperature range of 28–30 °C is optimal for the growth and replication of A. tumefaciens. Overnight growth of Agrobacterium culture often leads to an OD600 value within the range of 0.8–1.0.
Agroinfiltration using pC1300S-XB3 and controls
Two Agrobacterium cultures will be combined: either pC1300 (EV) and p19 or pC1300S-XB3 and p19 in a proportion of 3:2 to prepare samples for infiltration.
Inject the abaxial side (lower surface) of N. benthamiana leaves with the two prepared Agrobacterium mixtures using needleless syringes under controlled pressure. Separate the injection sites of each test mixture.
Incubate the infiltrated plants at room temperature with humidity for a defined period of 12–72 h. This incubation is essential for the plants to acclimatize to the introduced genetic material and to enable the expression of the transferred genes.
Notes:
The volume of bacterial cultures used for infiltration can be scaled accordingly. Always include the empty vector control (pC1300S) as one of the infiltration spots in your experiments to validate the success of the infiltration and to identify any potential issues with the transformation process. We have noticed that healthy plant conditions are crucial for the success of the infiltration experiments. Sometimes, this is not easy to visualize from plant appearance. The empty vector control serves as the “baseline” to phenotype the effect of XB3.
As shown in Figure 3, similar results were obtained when LB broth was used to replace infiltration buffer for diluting bacterial cultures.
Monitor cell death response
The infiltrated leaves are tested for the expression of the XB3 gene by monitoring cell death detected as visible grey patches 12–48 h after agroinfiltration (Figures 3A and 3B).
Figure 3. Leaf collapse induced by expression of XB3. (A) Cell death phenotypes induced by various densities of the agrobacterial culture harboring pC1300S-XB3. Photo taken 48 h after infiltration. EV, empty control. Visible cell death phenotypes were observed when the leaves were infiltrated with the Agrobacterium at densities ranging from OD600 = 0.3 to 1. (B) Cell death phenotypes induced by the Agrobacterium harboring pC1300S-XB3 in LB medium and infiltration buffer at OD600 of 0.5. The figure shows an N. benthamiana leaf 48 h after infiltration by four different solutions. Top-left quadrant of the leaf was infiltrated by Agrobacterium containing the empty vector diluted in infiltration buffer and the bottom-left quadrant was infiltrated with Agrobacterium containing XB3 expression plasmid (pC1300S-XB3) diluted in infiltration (INF) buffer at OD600 of 0.5. Similar cell death was observed when LB was used to dilute the Agrobacterium cultures for infiltration (Right).
Ion leakage assay
Electrolyte leakage of infiltrated leaves can be quantified to assess cellular mortality. Punch out three leaf discs, each 10 mm in diameter, from each region infiltrated by Agrobacterium using a 10-mm cork borer.
Submerge these leaf discs in 10 mL of water. Incubate for 2 h at room temperature with gentle agitation of 160 rpm.
Measure the conductivity of the resulting solution using the COND 6+ conductivity meter according to the manufacturer’s instructions (Figure 4). In order to calibrate the conductivity meter, rinse the electrode with sterile water and calibrate it with water, as in our experiment, water served as standard.
Figure 4. Ion leakage induced by the expression of XB3. The graph represents the ion leakage from N. benthamiana leaf tissues isolated at different time points (12, 20, 48, and 72 h) after infiltration with Agrobacterium cultures containing pC1300-XB3 at OD600 values of 1.0, 0.6, 0.5, and 0.3 along with Agrobacterium culture containing the empty vector (EV) pC1300S. Water, conductivity of water. Control, ion leakage from healthy, non-infiltrated leaves.
Validation of protocol
This protocol was modified from Huang et al. [1] and reproducible (Figures 3 and 4).
General notes and troubleshooting
General notes
Our protocol presents advantages over conventional agroinfiltration methods [1,7–9]. We eliminate the centrifugation step, as it is cost-prohibitive for many school programs, and bypass the use of a spectrophotometer to measure OD at a specific wavelength. Additionally, we replace the agroinfiltration buffer with LB to reduce chemical costs. Target bacterial density is instead assessed using Figure 2. The figures demonstrate the efficiency of LB-based agroinfiltration, and the cell death response induced by XB3 expression.
EHA105 possesses a wide host range and is suitable for delivering genetic material to various plant species. Alternatively, researchers may opt for other Agrobacterium strains with similar capabilities, ensuring compatibility with the specific plant species under investigation and the intended genetic modifications.
A wide range of the concentrations of agrobacterial cultures (OD600 from 0.3 to 1) harboring the XB3 gene can induce cell death response. If an incubator shaker is not available, a flask with a magnetic stir bar can be used to culture agrobacterial cells on a magnetic stir plate.
For the ion leakage assay, if a COND 6+ conductivity meter is not available, a less expensive TDS sensor might be used [10].
Troubleshooting
Reduced transformation efficiency. Overgrown Agrobacterium cultures may lead to a reduction in plant transformation efficiency. Avoid plant leaves with visible signs of disease, stress, or damage. We have noticed that certain types of soil affect the growth of N. benthamiana. If plants grow poorly, we recommend using Growing Mix, PRO-LINE HFC/B HydraFiber.
Acknowledgments
This research was supported by the National Science Foundation (MCB-2114833). This protocol was modified from Huang et al. [1]. We extend our sincere gratitude to Beatriz de Toledo Franceschi, Maggie Paxson, Vincent Newman, Paul Gleason, Lilybeth Moreno, William Bartenslager, Heather Sinclair, and Michael Cartamil for their valuable discussion. We also thank Anita K. Snyder for her critical reading of the manuscript and invaluable comments on the work.
Competing interests
The authors declare no competing interests.
References
Huang, X., Liu, X., Chen, X., Snyder, A. and Song, W. Y. (2013). Members of the XB3 Family from Diverse Plant Species Induce Programmed Cell Death in Nicotiana benthamiana. PLoS One 8(5): e63868.
Jacob, P., Kim, N. H., Wu, F., El-Kasmi, F., Chi, Y., Walton, W. G., Furzer, O. J., Lietzan, A. D., Sunil, S., Kempthorn, K., et al. (2021). Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels. Science 373(6553): 420–425.
Qi, T., Seong, K., Thomazella, D. P. T., Kim, J. R., Pham, J., Seo, E., Cho, M. J., Schultink, A. and Staskawicz, B. J. (2018). NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana. Proc. Natl. Acad. Sci. U.S.A. 115(46): e1814856115.
Song, W. Y., Wang, G. L., Chen, L. L., Kim, H. S., Pi, L. Y., Holsten, T., Gardner, J., Wang, B., Zhai, W. X., Zhu, L. H., et al. (1995). A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21. Science 270(5243): 1804–1806.
Wang, Y. S., Pi, L. Y., Chen, X., Chakrabarty, P. K., Jiang, J., De Leon, A. L., Liu, G. Z., Li, L., Benny, U., Oard, J., et al. (2006). Rice XA21 Binding Protein 3 Is a Ubiquitin Ligase Required for Full Xa21-Mediated Disease Resistance. Plant Cell 18(12): 3635–3646.
Voinnet, O., Pinto, Y. M. and Baulcombe, D. C. (1999). Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. U.S.A. 96(24): 14147–14152.
Bai, S., Liu, J., Chang, C., Zhang, L., Maekawa, T., Wang, Q., Xiao, W., Liu, Y., Chai, J., Takken, F. L. W., et al. (2012). Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance. PLoS Pathog. 8(6): e1002752.
del Pozo, O., Pedley, K. F. and Martin, G. B. (2004). MAPKKKα is a positive regulator of cell death associated with both plant immunity and disease. EMBO J. 23(15): 3072–3082.
Melech-Bonfil, S. and Sessa, G. (2010). Tomato MAPKKKε is a positive regulator of cell-death signaling networks associated with plant immunity. Plant J. 64(3): 379–391.
Jain, N., Khurana, P. and Khurana, J. P. (2022). Overexpression of a rice Tubby–like protein-encoding gene, OsFBT4, confers tolerance to abiotic stresses. Protoplasma 260(4): 1063–1079.
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A Standardized Protocol for Extraction and Homogenization of Ocular Tissues
AH Anam Hammid
Published: Vol 14, Iss 10, May 20, 2024
DOI: 10.21769/BioProtoc.4988 Views: 1679
Reviewed by: Vivien J. Coulson-Thomas Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Pharmaceutics 2021
Abstract
The eye is a complex organ composed of multiple tissues in anterior and posterior eye segments. Malfunctions of any of these tissues can lead to ocular diseases and loss of vision. A detailed understanding of the ocular anatomy and physiology in animal models and humans contributes to the development of ocular drugs by enabling studies on drug delivery and clearance routes, pharmacokinetics, and toxicity. This protocol provides step-by-step instructions for the extraction and homogenization of ocular tissues for enzymatic and proteomics analyses.
Key features
• Suitable protocol for the extraction and isolation of ocular tissue from humans and laboratory animals (rabbit, pig, rat, mouse) while minimizing cross-contamination.
• Hard or soft tissue homogenates can be prepared efficiently using a Bead Ruptor homogenizer.
• Allows to determine the protein contents in prepared homogenates.
Keywords: Eye Ocular tissues Extraction Homogenization Sample preparation
Graphical overview
Graphical overview of ocular tissue extraction starting from eye enucleation from human or laboratory animal samples. Extraction and separation of ocular tissues are shown in tissue dissection. Homogenization of ocular tissues collected using the Bead Ruptor Elite homogenizer.
Background
Ocular diseases lead to vision loss in the aging global population [1,2], decreasing quality of life and increasing costs of health care [3]. Ocular drug development seeks to alleviate eye diseases by developing improved ocular therapeutics [4]. With that intention, a detailed understanding of ocular compartments, tissues in the anterior and posterior segments, and barriers is required [5,6].
The eye encompasses multiple tissues that individually play a significant role in the vision [7]. Anatomically, the eyeball is composed of a smaller anterior and a larger posterior segment [8]. The anterior tissues include the conjunctiva, cornea, aqueous humor, lens, iris, and ciliary body. The posterior segment contains vitreous humor, retinal pigment epithelium (RPE), neural retina, choroid, and sclera [9].
Isolation of contamination-free (devoid of neighboring/cross tissues and foreign-substance contaminants that could interfere with subsequent analysis or experiments) individual ocular tissues is challenging [3] due to the small tissue sizes and complexity. This protocol aims to provide details for (i) the separation and isolation of the ocular tissues, (ii) the preparation of ocular tissue homogenates, and (iii) the quantification of protein contents in prepared homogenates, e.g., enzymatic and proteomic analyses. Minimizing cross-contamination from anatomically adjacent tissues and preserving protein integrity is of fundamental importance.
This optimized protocol allows the preparation of tissue homogenates from fresh or frozen eyeballs. The procedure begins with the aspiration of the aqueous humor. Thereafter, all anterior eye tissues (conjunctiva, cornea, lens, iris, and ciliary body) can be individually separated from each other, leaving the vitreous exposed for collection. Further steps allow the collection of posterior eye tissues (retina, RPE, choroid, and sclera). The differences in tissue hardness influence the homogenization steps, which are described in detail.
Materials and reagents
Biological material
Human or laboratory animal (e.g., rabbit or pig) eyeballs. This protocol is equally suitable for rat and mouse eyes, but dissection and tissue isolation are recommended to be performed under the microscope
Reagents
Dulbecco's phosphate-buffered saline, 10× (DPBS) (Thermo Fisher, catalog number: 14200166)
Potassium phosphate buffer (PBS) (Sigma-Aldrich, catalog number: P3813)
Sterile water (Baxter, catalog number: KKF7113)
Ethanol (Sigma-Aldrich, catalog number: 64-17-5)
Syringe 1 mL (Luer Slip IV Syringes, catalog number: FWC345)
26 G needle 1/2″ (0.45 × 12 mm) (Medoject, catalog number: CH26012)
Scissors: Sharp straight (115 mm 122-010) (Elcon Scissor Tenotomy Stevens Straight, catalog number: 547456), curved pointed scissors (115 mm, curved, pointed St) (Elcon Iris Scissors, catalog number: ELC122011)
Beakers
Scalpel (Swann-Morton sterile disposable) (Sigma-Aldrich, catalog number: S2771-100EA)
Small brushes to scrape RPE; size 4–6 mm (round or flat depending on the size of eye and species; for rats and mice, smaller sizes are more suitable)
Eppendorf tubes, 1.5/2 mL sterile [Reaction tube, 1.5 mL (SARSTEDT, catalog number: 72.690.001); reaction tube, 2 mL (SARSTEDT, catalog number: 72.691)]
50 mL Falcon tubes (Screw cap, 114 × 28 mm) (SARSTEDT, catalog number: 62.547.254)
Pipettes and pipette tips [FinnpipetteTM F2 Good Laboratory Pipetting (GLP) Kit] (Thermo Fisher Scientific, catalog number: 4700880)
Bradford assay kit [10] (Bio-Rad Laboratories, catalog number: 5000205)
2 mL reinforced Polypropylene, 2.8 mm ceramic beads tube (Omni Internationals Kennesaw, catalog number: 19-628D)
Costar® 6-well flat-bottom plate, clear, polystyrene (Costar, catalog number: 38015)
Petri dishes, 92 × 16 mm, transparent (SARSTEDT, catalog number: 82.1472)
Filter papers (Whatman® qualitative filter paper) (Sigma-Aldrich, catalog number: WHA1001929)
Note: Use only sterile and clean equipment during the dissection. Also, tissue isolation should be performed in a sterile environment by cleaning surfaces and equipment thoroughly, followed by disinfection and sterilization.
Equipment
Olympus CK2 inverted phase contrast microscope (Microscope Central, model: ULWCD 0.3 N.A.)
Microcentrifuge (Thermo Fisher Scientific, model: Heraeus Biofuge 24 Place)
Bead Ruptor Elite homogenizer Omni Internationals (Kennesaw, GA, USA)
2 mL reinforced tubes filled with 2.8 mm diameter ceramic beads (Omni Internationals. Kennesaw, GA, USA)
Microplate reader (PerkinElmer Wallac, model: Victor2)
Procedure
Extraction
Obtain the eyeballs from a slaughterhouse (pig) or extract eyeballs by sacrificing the rabbit in an animal lab facility, usually by injecting the lethal dose of 60 mg/mL pentobarbital (Mebunat; Orion Pharma, Finland) into the marginal ear veins. The eyeball can be removed via enucleation, which is a surgical process to remove and detach the eye from the orbit. The outer part of the eyelid is usually cut with the help of sharp scissors. All tissue connections should be separated between the eyeball and the surrounding orbit with the help of sharp scissors. Detach the eyeball and place it on ice in prelabeled plastic bags, named with left/right eye, animal numbers, and further experimental details.
Keep the eyeballs immersed in ice-cold 1× DPBS, pH 7.4, on ice after extraction and during transportation.
Set up the required equipment for dissection, including sharp straight and curved scissors, beakers, Petri dishes, scalpel, filter papers, brushes to scrape RPE, and sterile Eppendorf tubes. In addition, buffer (approximately 5–6 mL of PBS/eye), sterile water, and ethanol for washing purposes should be placed in Falcon tubes (Figure 1).
Figure 1. Equipment and reagents for ocular tissue dissection
Label the collection tubes (1.5–2 mL Eppendorf tubes), weigh each empty tube (conjunctiva, lens, iris-ciliary body, cornea, aqueous humor, vitreous, neural retina, RPE, choroid, sclera), and keep them on ice.
Note the weights of the empty tubes and record them on the provided sheet. This data will be used later to calculate the weight of the tissues. To obtain the tissue weight, subtract the weight of the tube with tissues from the weight of the empty tube (Table 1).
Table 1. Sheet to record the weight of isolated ocular tissues
Date: Eye no:
Organism:
Tissue Empty tube [w] g/mg Tube + tissue [w] g/mg Tissue [w] g/mg Notes
(1) Conjunctiva
(2) Cornea
(3) Aqueous humor
(4) Lens
(5) Iris-ciliary body
(6) Vitreous
(7) Neural retina
(8) RPE
(9) Choroid
(10) Sclera
Start the dissection by removing the eye from the buffer and placing it on filter paper in the Petri dish or on a cooled pad (for thermosensitive samples, to preserve the protein/sample integrity) on filter paper.
First, remove all extraocular parts, as shown in Figures 2 and 3 (A and B).
Figure 2. Removing extraocular muscles and parts during ocular tissue extraction. A. Holding the eyeball with the help of tweezers. B. Cutting extra ocular parts with the help of scissors.
Figure 3. Clean eyeball after removing all extraocular muscles and parts. A. Side of the eyeball. B. Front of the eyeball.
Aspirate the aqueous humor through the limbus using a 1 mL syringe and 26 G needle (Figure 4A) before cutting the eyeball, as it will collapse during the extraction of intraocular tissues. Hold the syringe in one hand and, with the other hand, hold the eyeball to keep it in a stable and fixed position. Insert the syringe near the peripheral cornea from the limbus into the anterior chamber, which is the space between the cornea (the clear surface) and the iris (the colored part). Avoid inserting directly into the center to avoid damage to the lens (Figure 4). The amount of aqueous humor collected can vary depending on species and handling, ranging between 20 and 200 µL.
Figure 4. Aqueous humor aspiration. A. Aspiration aided by a syringe and needle. B. Graphical representation of the aspiration of aqueous humor.
After aqueous humor collection, the conjunctiva can be collected from the outer interior part with the help of a scalpel or scissors (Figure 5A–C). Place the eyeball on the Petri dish and pull up the conjunctival layer 3–5 mm away from the limbus with the help of a small tweezer; make a hole in the layer between the conjunctiva and the connective tissues beneath. Start cutting and moving forward to get a layer of conjunctiva while avoiding the adjacent muscles. Cut and collect pieces of bulbar conjunctiva in a tube and place the tube on ice after collection.
Figure 5. Steps for collecting the bulbar conjunctiva. A. Placing the eyeball on the Petri dish, holding the conjunctival layer. B. Cutting with the help of tweezers and scissors. C. Collection of the bulbar conjunctival layer.
Next, grab the eyeball tightly by holding it with the help of filter paper and mark a cut on the limbus (2 mm from the edge of the cornea). Carefully cut the eye with the help of curved scissors around the iris (Figure 6). The eyecup is now cut into two parts: the anterior and the posterior eye cup. The anterior eye cup contains tissues such as the lens, iris-ciliary body, and cornea, while the posterior contains vitreous, retina, retinal pigment epithelium, choroid, and sclera.
Figure 6. Cutting the eye with the help of scissors to open the eye cup
Open the eye cup and collect all the anterior eye tissues [lens, iris-ciliary body, and cornea (Figure 7)] into labeled tubes on ice. The lens is a crystalline, transparent, gelatinous, and round structure (Figure 7A). The iris and ciliary body are ring-shaped and dark-colored structures next to the lens (Figure 7B), while the cornea is a transparent top layer of the eye cup (Figure 7C). From the anterior eye cup, the lens can be easily pulled out with the help of forceps; then, the iris-ciliary body can be detached by holding it with the help of tweezers while peeling and separating gently away from the back of the cornea. In the end, the cornea is visible, isolated from the rest of the anterior ocular tissues.
Figure 7. Anterior eye tissues. A. Lens B. Iris-ciliary body. C. Cornea.
From the posterior eye cup, collect the vitreous and neural retina first and then the retinal pigment epithelium cells; finally, separate the choroid from the sclera. The vitreous can be aspirated via pipetting and carefully separating from the neural retina. Collection will be smoother when using a broader pipette tip (e.g., 1 mL or bigger) compared with a narrow and small tip because of the gel-like structure of the vitreous, as shown in Figure 8. Collect only the clear and transparent vitreous to avoid contamination by retinal tissues.
Figure 8. Neural retina and vitreous visible on top of the posterior eye cup
After removing the vitreous, the neural retina can be pulled out gently and detached easily from the RPE layer (shown in Figure 8) with the help of forceps or broad-tip tweezers. The isolation of the neural retina from RPE is easier when using a fresh eye.
Now, the eye cup has RPE on the top. The dark-colored pigmented RPE can be easily visually distinguished from the retina (Figure 9). To place the posterior eye cup in a multi-well plate, use sterile forceps or tweezers to grip its edges. Carefully lower the eye cup into the selected well and ensure it is placed in any required well. Adjust the position by moving it to locate it exactly in the middle (Figure 9B). Pipette ~1 mL of 1× PBS on the eye cup while placing it in a multi-well plate. Wait at least 5 min to help RPE cells detach and immerse in the buffer. After 5 min, scrape the RPE layer with the help of a small brush to recover the RPE cells. Repeat twice until all RPE is collected (as shown in Figure 9A–E).
Centrifuge the RPE suspensions at 6,000× g for 5 min at 4 °C to pellet the RPE. The supernatant can be discarded; the pellet contains the desired RPE cells that are to be stored (Figure 9E).
Figure 9. Steps for collecting retinal pigment epithelium (RPE) from the eyecup. A. Take the posterior eye cup. B. Pipette 1× PBS to the eye cup (e.g., 1 mL, 2×) while placing it in a multi-well plate and wait for 5 min to let RPE cells detach. C. Scrape with the help of a small brush to detach RPE cells. D. Remove the eye cup and take the buffer containing RPE cells with the help of a pipette in the Eppendorf tube. E. Centrifuge the RPE suspensions at 6000× g for 5 min at 4 °C to pellet the RPE. Discard the supernatant and keep the pellet containing RPE cells.
The choroid is a sticky tissue that can be scraped with a scalpel to separate it from the sclera. The pigmented choroid will be visually distinguishable from the sclera. Peel off the choroid, starting from one edge from the top of the sclera to separate both layers. The detailed steps are shown in Figure 10.
Figure 10. Steps showing the peeling off of the choroid from the top of the sclera
Wash the sclera with PBS to eliminate traces of choroid. Take a clean beaker and fill it with 1× PBS. Make sure there is enough PBS to completely immerse the sclera and repeat this three times to ensure complete removal of choroid traces.
Weigh the individual tubes containing tissues and subtract the empty tube weight to get the actual tissue weight. Keep a record of tissue weights while placing tissues on ice during and after weighing.
Homogenization using Bead Ruptor Elite homogenizer
Homogenization of ocular tissues can be done using a Bead Ruptor Elite homogenizer and 2.8 mm diameter ceramic beads.
Label 2 mL hard tissue homogenizing reinforced tubes prefilled with four beads per tube (2.8 mm diameter).
Precool the Cryo cooling unit of the homogenizer using liquid nitrogen or dry ice with ethanol according to the manufacturer's instructions to maintain a constant temperature at 4 °C before loading the heat-sensitive samples.
Add tissue pieces (cut the tough tissues into smaller pieces using a scalpel to aid in homogenization) to the tubes and 1× DPBS buffer (3:1, volume/tissue weight or depending on working dilution required).
Unlock the finger plate on top of the tube carriage and remove it by rotating it. Load the 2 mL sample tubes on a precooled (~4 °C) 24-bead raptor tube carriage. Samples must be spaced evenly (it is recommended to load at least four tubes simultaneously with approximately equal weight; for that, weigh the tubes beforehand). Lock the carriage before the run.
For hard tissues such as the cornea and conjunctiva, use a 1-min cycle 4× at a speed of 6 m/s as described earlier [11]. The cycle speed (m/s), run time (s), and dwell time between runs can be adjusted according to the tissue. The vitreous and aqueous humor can be homogenized without adding buffer (or depending upon the dilution required) at a low cycle speed and using less run time, e.g., a 1-min cycle one time at a speed of 6 m/s).
Note: Both aqueous humor and vitreous are homogenized to ensure uniformity during protein quantification, enhance the accuracy of analyses, and achieve consistency in results and subsequent analysis.
Here, the starting temperature of each cycle is very critical for temperature-sensitive samples. If the start temperature of the cycle is at -2 °C, for a 1-min cycle it will not exceed the limit of 4 °C. Before the next cycle, turn on the cooling chamber and bring the temperature of the sample chamber down to -2 °C.
In addition, the sample loading carriage can be placed in a fridge at 4 °C to keep it cold.
Once the sample carriage chamber is cool, load the samples and press Run to start the cycle and Stop to stop the run on the screen.
Check with the pipette tip if the sample is completely homogenized; otherwise, repeat the cycle to get a completely homogenized sample.
Centrifuge homogenates at 10,000× g for 5 min at 4 °C to collect the supernatant. Aliquot and store at -80 °C.
Note: Not all labs have Bead Ruptor Elite homogenizers. In that case, a traditional manual or electric homogenizer or grinder can be used, such as a Dounce homogenizer or Potter-Elvehjem homogenizer.
Protein quantification
We routinely use the Bradford assay [10] using bovine serum albumin (0.25–2 mg/mL) as the standard for protein quantification.
Validation of protocol
This protocol has been validated in the following research article:
Hammid et al. [12]. Carboxylesterase Activities and Protein Expression in Rabbit and Pig Ocular Tissues. Molecular Pharmaceutics (Figure 1, panels A, B, and C).
General notes and troubleshooting
Following this protocol, the extraction of tissues from fresh eye samples is relatively easy. Separation of the ocular tissues from frozen eyeballs, especially posterior tissues, is more variable and challenging: clean isolation of RPE and choroid from frozen human eyes may be impossible, and we have often combined RPE and choroid into a single tissue homogenate to avoid variability.
The extraction protocol does not guarantee 100% purity of each individual tissue, although precautions against cross-contamination of adjacent tissues can be made by washing and visual inspection. It is advised to estimate this by immunoblotting with tissue-specific antibodies against marker proteins [11].
In these preparations, we omitted protease and phosphatase inhibitors to avoid interference with the activity of specific enzymes (particularly esterases) under study. However, protease and phosphatase inhibitor cocktails are essential for cell lysis and protein extraction. They protect proteins from degradation and preserve their phosphorylation status by blocking or neutralizing enzymes released during cell lysis, which could otherwise break down proteins and affect their activation states.
Acknowledgments
This protocol was adapted from the publication of rabbit and pig [12] and human eyes [11]. The author acknowledges Professor Paavo Honkakoski at the University of Eastern Finland for kindly reviewing the manuscript. This work was supported by EU-ITN project OCUTHER (H2020-MSCA-ITN-2016, grant number 722717) and the Doctoral Programme in Drug Research (University of Eastern Finland). The author would like to acknowledge The Maud Kuistila Memorial Foundation, The Finnish Cultural Foundation, and Oskar Öflunds Foundation for financial support.
Competing interests
The author declares no conflict of interest.
Ethical considerations
The study used human and animal eye samples. Human eye samples were provided by a biobank [11] at the University Hospital in Santiago de Compostela, Spain. The use of laboratory animals and human tissue [11,12] was approved by the local animal welfare committee (license # ESAVI/8621/04.10.07/2017).
References
Steinmetz, J. D., Bourne, R. R. A., Briant, P. S., Flaxman, S. R., Taylor, H. R. B., Jonas, J. B., Abdoli, A. A., Abrha, W. A., Abualhasan, A., Abu-Gharbieh, E. G., et al. (2021). Causes of blindness and vision impairment in 2020 and trends over 30 years, and prevalence of avoidable blindness in relation to VISION 2020: the Right to Sight: an analysis for the Global Burden of Disease Study. Lancet Glob. Health 9(2): e144–e160.
Bourne, R., Steinmetz, J. D., Flaxman, S., Briant, P. S., Taylor, H. R., Resnikoff, S., Casson, R. J., Abdoli, A., Abu-Gharbieh, E., Afshin, A., et al. (2021). Trends in prevalence of blindness and distance and near vision impairment over 30 years: an analysis for the Global Burden of Disease Study. Lancet Glob. Health 9(2): e130–e143.
Dumouchel, J. L., Chemuturi, N., Milton, M. N., Camenisch, G., Chastain, J., Walles, M., Sasseville, V., Gunduz, M., Iyer, G. R., Argikar, U. A., et al. (2018). Models and Approaches Describing the Metabolism, Transport, and Toxicity of Drugs Administered by the Ocular Route. Drug Metab. Dispos. 46(11): 1670–1683.
Gote, V., Sikder, S., Sicotte, J. and Pal, D. (2019). Ocular Drug Delivery: Present Innovations and Future Challenges. J. Pharmacol. Exp. Ther. 370(3): 602–624.
Maurice, D. M. and Mishima, S. (1984). Ocular pharmacokinetics. In: Pharmacology of the Eye (pp. 19–116). Berlin, Heidelberg: Springer Berlin Heidelberg.
Gower, N. J. D., Barry, R. J., Edmunds, M. R., Titcomb, L. C. and Denniston, A. K. (2016). Drug discovery in ophthalmology: past success, present challenges, and future opportunities. BMC Ophthalmol. 16: 11.
Sebastian, E. T. (2010). The complexity and origins of the human eye: A brief study on the anatomy, physiology, and origin of the eye. Senior Honors Theses. 116.
Mafee, M. F. (1996). Eye and orbit. In: Head and Neck Imaging. Sore, P. M. (Ed.), St. Louis; Mosby, 1109-I 114.
Malhotra, A., Minja, F. J., Crum, A. and Burrowes, D. (2011). Ocular Anatomy and Cross-Sectional Imaging of the Eye. Semin. Ultrasound CT MRI 32(1): 2–13.
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.
Hammid, A., Fallon, J. K., Lassila, T., Vieiro, P., Balla, A., Gonzalez, F., Urtti, A., Smith, P. C., Tolonen, A., Honkakoski, P., et al. (2022). Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues. Drug Metab. Dispos. 50(12): 1483–1492.
Hammid, A., Fallon, J. K., Lassila, T., Salluce, G., Smith, P. C., Tolonen, A., Sauer, A., Urtti, A. and Honkakoski, P. (2021). Carboxylesterase Activities and Protein Expression in Rabbit and Pig Ocular Tissues. Mol. Pharmaceutics 18(3): 1305–1316.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Cell Biology > Cell isolation and culture > Cell isolation
Biochemistry > Protein
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Original Research Article:
The authors used this protocol in Science Oct 2021
Abstract
Calcium signalling in the endocardium is critical for heart valve development. Calcium ion pulses in the endocardium are generated in response to mechanical forces due to blood flow and can be visualised in the beating zebrafish heart using a genetically encoded calcium indicator such as GCaMP7a. Analysing these pulses is challenging because of the rapid movement of the heart during heartbeat. This protocol outlines an imaging analysis method used to phase-match the cardiac cycle in single z-slice movies of the beating heart, allowing easy measurement of the calcium signal.
Key features
• Software to synchronise and analyse frames from movies of the beating heart corresponding to a user-defined phase of the cardiac cycle.
• Software to measure the fluorescence intensity of the beating heart corresponding to a user-defined region of interest.
Keywords: Software development Cardiac cycle Heart valve development Phase matching Image analysis Zebrafish embryos
Graphical overview
Background
The heart begins to beat early during morphogenesis [1]. While most studies on heart development have been performed in fixed tissue samples or in vitro, the development of the heart is regulated by the heartbeat and blood flow [2], and a full understanding of heart development requires performing studies in the beating heart.
Thanks to its transparency, the zebrafish embryo provides an excellent model to study heart development in vivo [3]. Using the Tg(fli1a:galff);(uas:GCaMP7a) zebrafish line, we recently demonstrated that mechanoresponsive calcium signals in valve endothelial cells are present from the earliest stages of valve formation and persist until functional valve leaflets are formed [4]. Calcium signal analysis in single z-slice movies of the beating heart is challenged by the way cells move both within and outside the image plane during heartbeat and the fact that the heart rate can change over the course of imaging. Since there was no readily available software to analyse calcium signals in the beating heart, we developed our own. This protocol provides step-by-step instructions on how to use the software, which can be used to further study calcium signalling in the developing heart or easily adapted for the analysis of other imaging data containing recurring events.
Our image analysis method involves the use of two MATLAB applications developed in our lab as well as FIJI [5], an open-source image processing package based on ImageJ2. The Phase Matching MATLAB application utilises the structural similarity score [6] to select frames corresponding to a particular phase in the cardiac cycle. The output of the Phase Matching application is a phase-matched movie with minimal cardiac cell movement. The Calcium Analysis MATLAB application reads the phase-matched movie and measures the normalised calcium signal intensity over time in a user-defined region of interest (ROI). FIJI is used to manipulate image stacks and to combine animated graphs of the normalised calcium signal with the phase-matched image data.
Equipment
Operating system: Windows 7 or later version, Mac OS X 10.8 or later, Ubuntu 12.04 LTS or later
RAM: 4 GB (8 GB+ recommended)
Disk: 128 GB
Software and datasets
FIJI 2.7.0 or later (https://fiji.sc/)
MATLAB R2014a or later (https://www.mathworks.com/products/matlab.html)
Note: MATLAB is a licensed software by MathWorks; for those who do not have MATLAB leave/licence, we provide a stand-alone executable for the Windows platform. For other platforms, we recommend using the open-source package Octave (https://octave.org)
All data and codes are available on GitHub (https://github.com/HeartValveLab/CalciumAnalysis)
Procedure
Prepare image datasets
Each channel of the single z-slice movie (see Note 1 in General notes and troubleshooting) should be saved as a single TIFF file in the working directory. If this is not the case, ImageJ can be used to convert most image file formats to TIFF.
Open the z-slice movie using FIJI.
Use Image > Duplicate to split channels.
Select File > Save As > TIFF.
Download and installation
Navigate to the GitHub page above and follow the “Getting Started—For Users” instructions for the application of choice.
Using the Phase Matching application
Select the input files and a reference frame (Figure 1).
Browse for the working directory with the input TIFF files. Note that dataset files will not be visible here.
Browse for the input TIFF files. Up to three channels can be selected.
Select the channel to be used as a reference for phase matching (Main Ch).
Select the frame to be used as a reference for phase matching (Phase).
Click Set to lock in variables. This will generate a pop-up window showing the selected reference frame.
Draw a ROI. If the use of the entire image is desired, click and drag from the top-left corner to the bottom-right corner.
Figure 1. Graphics user interface of Phase Matching application. Arrows indicate steps in the written protocol.
Identify the similarity values (Figure 1).
Select either the spatial or temporal method.
Spatial: Compares the similarity of individual frames; this is usually sufficient and faster.
Temporal: Also evaluates the similarity of neighbouring frames. Possibly more accurate, slower.
Click Run. A pop-up window will show a graph showing the similarity index of each frame relative to the reference frame.
(Optional) Adjust the NumScales parameter if peaks are not very prominent, and press Run again. NumScales controls the number of levels of the image pyramid used for determining similarity.
Determine the location of the peaks (Figure 1).
Click Find Peaks to automate finding most of the peaks. A pop-up window showing the position of the peaks will be displayed.
(Optional) MinPeakHeight and MinPeakProminence can be tuned if the user wishes to detect more or less peaks.
(Optional) Click on points on the figure to add or remove them from the peaks list.
Points already detected as peaks will be deselected.
Points not already detected will be selected.
Right-click on a point and Export Cursor Data to Workspace. Note this only needs to be done once; all the other points are saved alongside it.
Always save it as a cursor_info variable. Note that you will need to remove numbering for subsequent adjustments.
Click Redisplay to set values and visualise output. Note that you can click it again to revert a change.
Click Confirm to set the output to be saved.
Save output (Figure 1). A folder will be created in the path of the input files.
Select the output file type.
Save single phase: Saves frames corresponding to the phase of interest as a single OME file.
Save individual frames: Saves frames corresponding to the phase of interest as a series of TIFF files.
Save with padding: Saves frames corresponding to the phase of interest with neighbouring frames padded on as an OME file (see Note 2 in General notes and troubleshooting).
Save session settings: Allows you to easily rerun code in a script.
Click Save. Check the saved image file by opening it in FIJI (Figure 2). Adjust variables and run again if necessary.
Using the Calcium Signal Analysis application
Prepare image datasets.
Ensure each channel is saved as a separate TIFF in the directory.
Image > Duplicate can be used in FIJI to split channels.
Set up the output files (Figure 2).
Browse for the working directory containing the phase-matched TIFF files. Note that dataset files will not be visible here.
Browse for the phase-matched TIFF files.
Select the range of frames you want to use. Note that an End Frame of 0 will default to the last frame of the data.
Click Set to lock in input variables. A pop-up window will show the first and last frames to be analysed.
Figure 2. Graphics user interface of Calcium Signal Analysis application. Arrows indicate steps in the written protocol.
Perform signal analysis (Figure 2).
Overlay all of the frames in each channel.
Select Draw Region of Interest and trace the cell you wish to analyse (Figure 3B). Users may find using a stylus easier.
Save output (Figure 2). A folder will be created in the path of the input files.
Adjust parameters accordingly.
Exposure Time corresponds to the inverse frame rate and depends on microscope settings.
Cycle Length corresponds to the average number of frames between phase-matched cycles and can be obtained from the Find Peaks figure from Phase Matching software.
Plot the Preliminary Signal to check the signal. Note that it will be saved automatically and may overwrite previous figures.
Plot Normalised Calcium Level to obtain output. Note that it will be saved automatically and may overwrite previous figures.
Normalise against reference normalises the calcium signal against the reference.
Normalise against calcium normalises the calcium signal against its own minimum.
Make Graph Movie to obtain a frame-by-frame output. Note that it will be saved automatically and may overwrite previous figures.
Save output (Figure 2). A folder will be created in the path of the input files.
Choose a name for the Output Folder and Save.
Plots are automatically saved.
Saving session settings is particularly helpful if you want to obtain underlying values.
Putting images and graphs side-by-side
See Note 3 in General notes and troubleshooting.
Create a single TIFF from Graph Movie.
In FIJI, File > Import > Image Sequence.
Browse for the directory where Graph Movie has been saved.
Click OK and wait.
Save the TIFF output.
Combine the graph with the visual data.
Open the phase-matching output alongside the graph movie output from above.
Go to Image > Colour > Channels Tool and select Composite from the drop-down menu.
Select the colours for each channel as you wish.
Go to Image > Type > RGB Color to generate TIFF with both channels combined into one.
Go to Image > Adjust > Size for either open file and adjust to have the same vertical resolution.
Go to Image > Stacks > Tools > Combine to collate the datasets together.
(Optional) Add arrow.
Right-click on the line icon and select Arrow Tool.
Draw an arrow to the cell of interest.
Press Ctrl + D to place the arrow on all frames.
Data analysis
This section provides an example of using the protocol to analyse calcium signals generated by endocardial cells in a beating zebrafish heart. Our raw data consists of a 2-channel, 8000-frame movie of a Tg(fli1a:galff; uas:GCaMP7a);Tg(fl1a:lifeact-mCherry) zebrafish heart, where images were captured at 100 frames per second (Figure 3A). Our cell of interest can be seen clearly in the fifth frame of the 8000-frame movie. We used the Phase Matching application to identify images whose reference channel [the Tg(fli1a:lifeact-mCherry) channel] showed a high degree of similarity to that of the fifth frame. The application identified 247 similar images (peaks in Figure 3B) including the fifth frame itself, corresponding to 247 cardiac cycles captured in the raw data. The application was then used to generate a phase-matched movie containing only those 247 frames (Figure 3C) and a phase-matched movie with padding (Figure 3D). Next, we used the Calcium Analysis application to generate an image overlay of the 247-frame phase-matched movie and selected the ROI by drawing around our cell of interest (Figure 3E, F). The Calcium Analysis application was then used to generate static and moving graphs of the normalised calcium signal in the ROI over time. We combined the moving graphs with the 247-frame phase-matched movie using FIJI (Figure 3G, H) and confirmed that the two signal peaks identifiable in our static graphs related to our cell of interest. The phase-matched movie with padding (Figure 3D) generated earlier was then used to browse the data for additional cells that generated calcium pulses. A video demonstration of this protocol, using the same example dataset, can be found in Supplementary Video 1.
Figure 3. Example of procedure. A. Information about the raw data. B. Pop-up window that appears when the Find Peaks button is clicked in the Phase Matching application; 247 peaks were identified, corresponding to 247 cardiac cycles captured in the raw data. C–D. Outputs of the Phase Matching application as visualised in FIJI. C) Output obtained when the Save single frames option is selected. The first slider (c) allows the user to select the channel; the second slider (z) allows the user to select the cardiac cycle of interest. D) Output obtained when the Save with padding option is selected. The first slider (c) allows the user to select the channel; the second slider (z) allows the user to select the cardiac cycle of interest; the third slider (play symbol) allows the user to select the phase of the cardiac cycle. E. A region of interest (ROI) drawn by the user around a cell (ROI outline shown in yellow) within the Calcium Analysis application. F. Zoom-in box showing ROI. G–H. Final output of procedure. G) The Ca2+ signal (magenta) in the cell of interest is high ~20 s into the movie, and this is seen in the corresponding graph. H) The Ca2+ signal (magenta) in the cell of interest is low at the end of the movie, and this is seen in the corresponding graph.
Validation of protocol
This protocol has been validated in the following research article:
Fukui, H., Chow, R. W., Xie, J., Foo, Y. Y., Yap, C. H., Minc, N., Mochizuki, N., Vermot, J. (2021). Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces. Science, 374 (6565), 351-354. doi: 10.1126/science.abc6229 (Figure 1, Supplementary Movie 2) [4].
General notes and troubleshooting
General notes
For our datasets, we used the Tg(fli1a:galff);(uas:GCaMP7a) line crossed to either the Tg(fli1a:h2b-mCherry)ncv31 [7] line or the Tg(fli1a:lifeact-mCherry)ncv7 line [8]. Images obtained from the following microscopes were tested:
Lightsheet Z.1 (Zeiss) equipped with 20× water immersion objective (Zeiss, 1.0 N.A.) and two cameras (PCO.edge sCMOS).
Inverted microscope (Leica, DMi8) combined with a CSUX1 confocal scanning head (Yokogawa) equipped with a 40× water objective (Leica, 1.1 N.A.) and two cameras (ORCA-Flash4.0 V2, Hamamatsu).
ECLIPSE FN1 upright microscope (Nikon) equipped with 16× water immersion objective (Nikon N16XLWD-PF, 0.80 NA, 3.0 mm WD) and combined with a Dragonfly 200 (Andor) two camera system (Andor Zyla sCMOS).
Excitation of GCaMP7a and mCherry was performed using a 488 nm and 561 nm wavelength laser line, respectively; 8 or 16-bit images with an image resolution of at least 512 × 256 pixels were captured at a frame rate of 100 frames per second.
Phase-matched movies with padding are useful for identifying cells that generate calcium pulses, while phase-matched movies without padding are recommended for measuring the calcium signal over time in a particular cell.
When cells are closely packed together, the fluorescent calcium signal from one cell can bleed into a neighbouring cell. In such cases, it is important to relate signal peaks to the cell of interest.
Troubleshooting
Problem 1: GUI application disappears behind other applications during use.
Possible cause: Application window has low display priority.
Solution: Reopen the app window each time or minimise other interfering applications.
Problem 2: GUI application may return an error in the log file.
Possible cause: User inputted incompatible parameter.
Solution: Double-check that parameters are within the bounds of the dataset and try again.
Problem 3: GUI installation may fail.
Possible cause: MATLAB runtime included is for Windows, not Mac or Linux.
Solution: Download runtime from https://au.mathworks.com/products/compiler/matlab-runtime.html.
Problem 4: Resizing windows may change the aspect ratios of graphics.
Possible cause: MATLAB does not automatically resize accordingly.
Solution: Avoid adjusting window size.
Acknowledgments
We thank Professor Naoki Mochizuki for providing the resources to generate imaging data and for his advice. We thank the Liebling Lab for their advice. We thank Zoe Suckling for testing the software and her feedback on the protocol. Renee Chow was supported by funding from the Fondation Lefoulon-Delalande and the Estate of Donald G. Paech. This project was completed as part of the Commonwealth Scientific and Industrial Research Organisation Undergraduate Research Opportunities Program through the Australian Regenerative Medicine Institute.
Author contributions: Renee Chow wrote the code used in Fukui et al. [4]. Raymond Zhang created the present improved version of the software, which includes a new phase-matching algorithm and a user-assisted manual correction function in the Phase Matching software, as well as graphical user interfaces for both the Phase Matching software and Calcium Analysis software. Hajime Fukui generated all image datasets. Raymond Zhang wrote the protocol. Julien Vermot supervised the early stages of the project. Riccardo Gherardi and Renee Chow supervised the later stages of the project.
References
Farraj, K. L. and Zeltser, R. (2023). Embryology, Heart Tube. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing.
Duchemin, A. L., Vignes, H., Vermot, J. and Chow, R. (2019). Mechanotransduction in cardiovascular morphogenesis and tissue engineering. Curr. Opin. Genet. Dev. 57: 106–116.
Staudt, D. and Stainier, D. (2012). Uncovering the Molecular and Cellular Mechanisms of Heart Development Using the Zebrafish. Annu. Rev. Genet. 46(1): 397–418.
Fukui, H., Chow, R. W., Xie, J., Foo, Y. Y., Yap, C. H., Minc, N., Mochizuki, N. and Vermot, J. (2021). Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces. Science 374(6565): 351–354.
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): 676–682.
Wang, Z., Bovik, A., Sheikh, H. and Simoncelli, E. (2004). Image Quality Assessment: From Error Visibility to Structural Similarity. IEEE Trans. Image Process. 13(4): 600–612.
Yokota, Y., Nakajima, H., Wakayama, Y., Muto, A., Kawakami, K., Fukuhara, S. and Mochizuki, N. (2015). Endothelial Ca2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo. eLife 4: e08817.
Wakayama, Y., Fukuhara, S., Ando, K., Matsuda, M. and Mochizuki, N. (2015). Cdc42 Mediates Bmp-Induced Sprouting Angiogenesis through Fmnl3-Driven Assembly of Endothelial Filopodia in Zebrafish. Dev. Cell 32(1): 109–122.
Supplementary information
The following supporting information can be downloaded here:
Supplementary Video 1.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Developmental Biology > Cell signaling > Intracellular calcium
Cell Biology > Cell imaging > Live-cell imaging
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After official publication in Bio-protocol (https://bio-protocol.org/e4825), we realized there are a few of mistakes in the volumes provided in the Recipe section.
The following corrections have been made to the recipes tables:
Recipes
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Glutamax (Final concentration): instead of 10 mM --> 2 mM
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Article Information
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Original Research Article:
The authors used this protocol in eLIFE Jul 2022
Abstract
Understanding dendritic excitability is essential for a complete and precise characterization of neurons’ input-output relationships. Theoretical and experimental work demonstrates that the electrotonic and nonlinear properties of dendrites can alter the amplitude (e.g., through amplification) and latency of synaptic inputs as viewed in the axosomatic region where spike timing is determined. The gold-standard technique to study dendritic excitability is using dual-patch recordings with a high-resistance electrode used to patch a piece of distal dendrite in addition to a somatic patch electrode. However, this approach is often impractical when distal dendrites are too fine to patch. Therefore, we developed a technique that utilizes the expression of Channelrhodopsin-2 (ChR2) to study dendritic excitability in acute brain slices through the combination of a somatic patch electrode and optogenetic activation. The protocol describes how to prepare acute slices from mice that express ChR2 in specific cell types, and how to use two modes of light stimulation: proximal (which activates the soma and proximal dendrites in a ~100 µm diameter surrounding the soma) with the use of a high-magnification objective and full-field stimulation through a low-magnification objective (which activates the entire somato-dendritic field of the neuron). We use this technique in conjunction with various stimulation protocols to estimate model-based spectral components of dendritic filtering and the impact of dendrites on phase response curves, peri-stimulus time histograms, and entrainment of pacemaking neurons. This technique provides a novel use of optogenetics to study intrinsic dendritic excitability through the use of standard patch-clamp slice physiology.
Key features
• A method for studying the effects of electrotonic and nonlinear dendritic properties on the sub- and suprathreshold responses of pacemaking neurons.
• Combines somatic patch clamp or perforated patch recordings with optogenetic activation in acute brain slices to investigate dendritic linear transformation without patching the dendrite.
• Oscillatory illumination at various frequencies estimates spectral properties of the dendrite using subthreshold voltage-clamp recordings and studies entrainment of pacemakers in current clamp recordings.
• This protocol uses Poisson white noise illumination to estimate dendritic phase response curves and peri-stimulus time histograms.
Keywords: Dendrites Pacemaking neurons Acute brain slice Whole-cell patch clamp Perforated patch Optogenetics Phase resetting Entrainment
Background
Whole-cell voltage and current clamp recordings are the fundamental tools for studying neuronal excitability and synaptic physiology. Using the methodology laid out by Hodgkin & Huxley [1], neurophysiologists measure the membrane currents in voltage-clamp mode to elucidate how they shape the voltage trajectory of a neuron as measured in current-clamp mode. However, unlike studies in primary neuronal cultures [2–5] where neurons can be assumed to be isopotential, studies in acute brain slices include neurons with elaborate and extensive dendritic arbors, whose properties are overlooked. In general, dendritic properties will alter the physiological measurement, particularly when estimating synaptic inputs, where the linear cable properties and the nonlinear conductances in the dendrites reshape the synaptic currents and potentials. One approach to overcome this is to use dual-patch clamp measurements, where the soma and the dendrites are patched simultaneously, allowing for direct interrogation of how the dendrite transforms synaptic voltage perturbations [6–11]. Because synaptic inputs are typically weak, even if the dendrites are endowed with strong nonlinearities (e.g., various voltage-activated currents), the dendritic transformation can be considered a linear transformation. Linear time-invariant (LTI) transformations can be characterized by their impulse response to an instantaneous perturbation in the time domain or by their amplitude and phase responses in the Fourier frequency space [12]. Using the latter approach, one can perturb the dendrite with sinusoidal inputs at various frequencies or with an approximation of white noise that simultaneously elicits (nominally) all frequencies. Indeed, this methodology has been used in dual-patch experiments where the perturbation is introduced via the dendritic electrode and measured via the somatic one [7,8,10,11]. However, such dual-patch recordings are not always possible either because the dendrites are too small to visualize or too small to patch. We therefore developed a method that harnesses optogenetics to interrogate the dendritic LTI transformation using sinusoidal and white noise perturbation. Channelrhodopsin-2 (ChR2) is expressed in the neuronal type being investigated (in our case, we used transgenic mice that express ChR2 in specific cell types). We then visually guide a patch pipette to the soma of one of these neurons in an acute brain slice to record its electrical activity. To characterize the dendritic LTI transformation in the time domain, we use either individual brief pulses, or Poisson barrages of pulses of blue (470 nm) LED light to activate ChR2 currents, as pioneered by Higgs and Wilson [13], or sinusoidally modulated blue LED light. We situate the soma at the center of the objective’s field of view so that all blue light perturbations are applied to a somatodendritic region of the cell surrounding the soma and measure their effect on the soma. This is done either in voltage-clamp mode to measure how the currents are transformed or in current-clamp mode to study how the light perturbations affect the autonomous firing of pacemaking neurons (we study basal ganglia pacemakers [14,15]). We either illuminate the entire field of view via a low-magnification microscope objective or we constrain the localization of the blue light to a small perisomatic region (~100 μm diameter that encompasses the soma even for neurons with large somata) by occluding the blue LED light path via a high-magnification objective. We then compare the results from the full-field illumination with those from the proximal illumination to deduce the contribution to the responses that can be attributed solely to the dendrites. Future applications could use patterned laser stimulation and/or multiphoton activation of the opsins for truly localized dendritic (or somatic) stimulation.
Materials and reagents
Biological materials
Mice that express channelrhodopsin-2 in a population of pacemaking neurons. We used either Thy1-ChR2 mice [B6.Cg-Tg(Thy1-COP4/EYFP) 18Gfng/J] (Jackson laboratory, stock: 007612) or ChAT-ChR2 mice: Ai32 mice [RCL-ChR2(H134R)/EYFP] that express flexed ChR2 and an EYFP fusion protein under the CAG promoter (Jackson laboratory, stock: 012569) cross-bred with ChAT-IRES-Cre (∆neo) that express Cre recombinase under the Chat promoter (Jackson laboratory, stock: 031661)
Reagents
NaCl (Sigma-Aldrich, CAS number: 7647-14-5)
KCl (Sigma-Aldrich, CAS number: 7447-40-7)
NaH2PO4·H2O (Sigma-Aldrich, CAS number: 10049-21-5)
CaCl2·2H2O (Sigma-Aldrich, CAS number: 10035-04-8)
MgSO4 anhydrous (Sigma-Aldrich, CAS number: 7487-88-9)
NaHCO3 (Sigma-Aldrich, CAS number: 144-55-8)
Dextrose (Sigma-Aldrich, CAS number: 50-99-7)
Sucrose (Sigma-Aldrich, CAS umber: 57-50-1)
Ascorbic acid (Sigma-Aldrich, CAS number: 50-81-7)
KCH3SO3 (Sigma-Aldrich, CAS number: 2386-56-3)
EGTA (Sigma-Aldrich, CAS number: 67-42-5)
HEPES (Sigma-Aldrich, CAS number: 7365-45-9)
Na2-phosphocreatine (Sigma-Aldrich, CAS number: 19333-65-4)
ATP magnesium salt (Sigma-Aldrich, CAS number: 74804-12-9)
GTP sodium salt (Sigma-Aldrich, CAS number: 36051-31-7)
Tetrodotoxin citrate (HelloBio, CAS number: 18660-81-6)
ZD 7288 (MedChemExpress, CAS number: 133059-99-1)
Ketamine (as hydrochloride) 1 g/10 mL (Vetoquinol, Clorketam®)
Xylazine (as hydrochloride) 20 mg/mL (euroVet, Sedaxylan®)
Gramicidin (Sigma-Aldrich, catalog number: G5002, CAS number: 1405-97-6).
Note: This is a mixture of Gramicidin A,B,C&D and requires higher concentrations of stock solutions than appear in publications using only Gramicidin B [16].
DNQX disodium salt (HelloBio, catalog number: 2379-57-9)
D-AP5 (HelloBio, catalog number: 79055-68-8)
SR 95531 hydrobromide (gabazine) (HelloBio, catalog number: 104104-50-9)
CGP55845 hydrochloride (HelloBio, catalog number: 149184-22-5)
Dimethyl sulfoxide (DMSO), anhydrous (Sigma-Aldrich, catalog number: 276855, CAS number: 67-68-5)
Solutions
10× artificial cerebrospinal fluid (aCSF) stock solution (see Recipes)
10× modified aCSF (Sucrose) stock solution (see Recipes)
K internal solution (see Recipes)
Recipes
10× aCSF stock solution in ddH2O (1,000 mL)
Note: The stock solution is prepared without the sodium bicarbonate. For the final solution, dilute 1:10 in ddH2O and add 2.18 g of NaHCO3.
Reagent Final concentration Quantity or Volume
NaCl 126 mM 73.63 g
KCl 2.5 mM 1.86 g
NaH2PO4·H2O 1.25 mM 1.724 g
CaCl2·2H2O 2.0 mM 2.94 g
MgSO4 anhydrous 2.0 mM 2.408 g
Dextrose 10 mM 18.02 g
NaHCO3 26 mM see note
10× modified aCSF (sucrose) stock solution in ddH2O (500 mL)
Note: The stock solution is prepared without the sodium bicarbonate. For the final solution, dilute 1:10 in ddH2O and add 1.09 g of NaHCO3. Sucrose is added (36 g/500 mL) to the final solution. The aCSF is modified to have much lower concentrations of sodium and calcium to prevent excitotoxicity while the tissue is being dissected under hypoxic conditions. (Similarly, the concentration of magnesium is increased in order to prevent NMDA receptors from contributing to excitotoxicity.) The sucrose compensates for the reduced osmolarity in this sodium-poor solution [17].
Reagent Final concentration Quantity or Volume
Sucrose 210 mM see note
KCl 2.5 mM 0.932 g
NaH2PO4·H2O 1.25 mM 0.862 g
CaCl2·2H2O 0.5 mM 0.37 g
MgSO4 anhydrous 10 mM 6.02 g
Dextrose 10 mM 9.01 g
Ascorbic acid 0.4 mM 0.35 g
NaHCO3 26 mM see note
K internal solution (90 mL)
Note: All ingredients, except ATP and GTP, are mixed in approximately 75 mL of ice-cold ddH2O. Use pH meter to titer to pH 7.3–7.4 with 1 N KOH (using 50 µL increments with a pipettor); then, top off to 90 mL with ice-cold ddH2O. Measure osmolarity with an osmometer, which should be 280–300 mOsm/kg. If pH and osmolarity are good, add ATP and GTP, readjust the pH with 1N KOH, rapidly aliquot to 1 mL Eppendorf tubes, and freeze (-20˚C). Do the whole procedure on ice.
Reagent Final concentration Quantity or Volume
KCH3SO3 135.5 mM 1.637 g
EGTA 0.2 mM 6.8 mg
KCl 5 mM 33.5 mg
NaCl 2.5 mM 13.15 mg
Na2-phosphocreatine 5 mM [18] 114.8 mg
HEPES 10 mM 238.3 mg
ATP magnesium salt 2.0 mM 100 mg, see note
GTP sodium salt 0.21 mM 10 mg, see note
Laboratory supplies
Single-edge razor blade (for vibratome) (WPI, catalog number: BLADES-2)
Single-edge industrial blades (for blocking brain) (any will do, we use Excel, catalog number: 22609)
1 mL insulin syringe (30 G 5/16” 0.3 × 08 mm) (PiC solution, catalog number: 02 022726 100 150)
25G scalp vein sets (KDL, catalog number: 4193)
Super glue (Brush-On) (Loctite)
Filter paper (Whatman, catalog number: 1002-070)
Dental wax (Electron Microscopy Sciences, catalog number: 72660)
Harp (to hold down slice in recording chamber) (Warner Instruments, catalog number: 641418, or other sizes)
50 mL glass cup
250 mL plastic cups
20 mL Luer-lock syringes
4 mm syringe driven 0.22 μm filter units (Millex® GV, catalog number: SLGVR04NL)
Microloader for Eppendorf pipettes (Eppendorf, catalog number: 5242 956.003)
Needles, 16 G, 19 G, and 27 G
10 mL injectable 0.9% (w/v) saline (NaCl) (Braun 5/12606510/0309)
Kimwipes, 4.4 × 8.5 inch (Kimberly-Clark)
Borosilicate glass pipettes, with filament (fire polished, length: 10 cm, OD: 1.5 mm, ID: 0.86 mm) (Sutter Instruments, catalog number: BF150-86-10)
Microelectrode holder for O.D. 1.5 mm pipettes (Warner Instruments, catalog number: QSW-T15P)
Equipment
Vision Isostation optical workstation (Newport, model: VIS3036-SG4-325A)
XY-shifting table + base plate (Luigs & Neumann, catalog number: 200-100 200 0150, 250-200 200 1000)
Axioskop microscope (Zeiss) with a “hi-mag” 60×/1.0 NA (FN 26.5) water immersion (WI) objective (Olympus, model: LUMPLFLN60XW) and a “lo-mag” 5×/0.1 NA (FN 22) air/dry objective (Olympus, model: MPLN5). The filter slider was fitted with a dichroic mirror that reflects the 470 nm illumination onto the slice (Chroma, catalog number: T660lpxrxt)
Micromanipulator including mounting clamp for headstage (Luigs & Neumann, catalog number: 210-100 000 0010)
Slice chamber (Luigs & Neumann, catalog number: 200-100 500 0150)
Temperature controller (Luigs & Neumann, catalog number: 200-100 500 0145)
Remote control system for XY-shifting table and micromanipulator (Luigs & Neumann, catalog number: 200-100 900 9050, 200-100 900 7911)
Amplifier and headstage (Molecular Devices, model: MultiClamp 700B)
A/D board (CED, model: Power 1401-3)
470 nm LED and analog driver (Mightex, model: LCS-0470-03-22, SLA-1000-2)
Monochrome video camera (iDS, model: UI-1240LE-NIR-GL)
Micropipette puller (Sutter, model: P-1000)
Vibratome (Leica, model: VT1000 S)
Osmometer (Advanced Instruments, model: 3320)
Manometer (Dwyer, model: Series 475 mark III)
Hand blender (any that is appropriate for crushing ice)
Surgical scissors sharp-blunt (for decapitation) (FST, catalog number: 14001-14)
Fine scissors sharp (for cutting skull bone) (FST, catalog number: 14060-09)
Vannas fine spring scissors (for cutting right atrium) (Roboz, catalog number: RS-5620)
Bracken forceps (Roboz, catalog number: RS-5211)
Carbogen (95% O2 + 5% CO2) supply tank
Lab microspatulas with various ends
Software and datasets
Signal 6.05a (CED, 2/14/2018), licensed)
Multiclamp commander (Molecular devices, 2018, licensed)
uEye Cockpit 4.94.000 (iDS, 2020)
MATLAB R2020b update 2 (Mathworks, 11/3/2020, licensed)
Procedure
Acute brain slice preparation
Prepare K internal, 10× sucrose, and 10× aCSF at least one day in advance (K internal is good for approximately two months, 10× aCSF for one month, and sucrose solution for approximately two weeks). Dilute the 10× sucrose solution to the final solution (500 mL) and add bicarbonate and sucrose. Mix the solution with a magnetic stirrer, then check its osmolarity with an osmometer. The osmolarity should be 290–310 mOsm. Oxygenate the solution for at least 15 min and freeze 120 mL (-20 °C) in a 250 mL plastic cup for using on one experiment day. Refrigerate the remainder of sucrose and aCSF solutions.
On the day of dissection, prepare equipment and final solutions:
Thaw the frozen sucrose solution (e.g., by submerging the plastic cup with frozen solution in a container with warm tap water of any kind).
Note: Make sure none of the tap water mixes into the sucrose solution. Let the sucrose thaw a bit and, when possible, insert a carbogen tube into the mixture. While the sucrose continues to thaw, perform the following steps.
Dilute the 10× aCSF solution to the final solution (500 mL), add bicarbonate, and mix (this time the solution can be mixed manually due to the small amount of solute). Check the solution’s osmolarity, which should be 290–310 mOsm. Pour enough of the solution into a slice chamber so that the slices are submerged and insert a carbogen tube into the aCSF. Make sure that there are no air bubbles in the chamber, since they might prevent aCSF solution from reaching brain slices and causing cell death.
Critical: Avoid moving the chamber from now on to prevent air bubble formation.
Fill two large containers with ice. In one (container 1), secure a Styrofoam block (perfusion board) in the ice, preferably tilted downwards on one side. Place all surgical tools in the container so that the parts meant to come in contact with the mouse’s brain are covered in ice (Figure 1A). In the other container (container 2), place a single-edge industrial blade, vibratome plate, and dental wax sheet.
Critical: The vibratome plate should be set on Kimwipes on the ice so that ice does not stick to its bottom when you are later ready to insert the slide into the vibratome.
Figure 1. Mouse dissection and perfusion with sucrose. A. Overview of the dissection station arrangement (top view): ice container, surgery tools, and mouse taped to Styrofoam board. Note the Styrofoam board tilted downwards, and surgery tools submerged in ice. B. First incision: lifting the skin using Bracken forceps and creating a transverse incision across the raised piece of skin (along the white dotted line) using sharp-blunt dissecting scissors. C. Detaching the rib cage: inserting the scissors into the orifice created by the previous step and cutting through both sides of the rib cage (along the dotted arrows). D. Cutting the diaphragm (across dotted line) exposed after lifting the rib cage with forceps. E. View of the exposed thoracic cavity after taping the rib cage to the Styrofoam board. The heart along with the lungs can be seen in the upper part of the aperture, and part of the liver can be seen in the left lower part of the aperture. F. Perfusion steps presented on top of a magnified view of the heart from panel E. First, cut the right atrium (marked RA) with fine spring scissors. Next, insert the scalp vein set needle (connected to the Luer-lock syringe with liquid sucrose) into the left ventricle (marked LV) in the apex area, close to the border with the right ventricle (marked RV). Finally, slowly inject sucrose. G. Detaching the mouse’s head: suspending the mouse by its ears and making a transverse incision under the head (along the dotted line).
Prepare a brain slicing station: place the vibratome chamber, filter paper, and super glue in proximity to each other. Insert a new single-edge razor blade into the vibratome’s blade holder and secure it with its finger-tightening screw to connect later to the vibratome head.
Caution: Position the holder so that the blade is directed away from your hands.
Prepare an anesthesia station: bring the mouse to the dissection area in a cage/cardboard box. While the mouse acclimatizes to its new environment, arrange a surface for mouse fixation and intraperitoneal (i.p.) injection (an Eppendorf tube rack holder, for example). Dilute 1 mL of ketamine and 0.5 mL of xylazine in 8.5 mL of injectable saline (ketamine-xylazine cocktail) and inject 20 μL/g of the cocktail i.p. into the mouse (or slightly more, e.g., for a 20 g mouse, inject at least 0.4 mL cocktail). The dosage per mouse is 200 μg/g ketamine and 20 μg/mg xylazine, or slightly higher, which are terminal dosages.
Critical: Although the cocktail’s dosage is meant to be terminal, be wary of injecting a significantly larger volume than the minimal volume required, since it might cause premature death of the mouse, before the dissection is ready to begin. Lay the syringe so that its needle does not come into contact with any surface and points away from you. Do not recap the insulin syringe!
Finish preparing the sucrose: now that the sucrose block had time to thaw, there should be an accumulation of liquid sucrose around the frozen chunk. Fill the 20 mL Luer-lock syringe with liquid sucrose, release air bubbles if necessary, and connect it to a scalp vein set. Release a small amount of liquid from the syringe without removing the needle protector to ensure proper connection of the set and removal of excess bubbles to prevent an air embolism while perfusing. Submerge the syringe (connected to the scalp vein set) into container 1, making sure it is completely covered in ice (Figure 1A). Note: This step should be performed as close as possible to the dissection since from this point on the sucrose in the syringe is no longer oxygenated. Flip the frozen sucrose block so the part that was submerged in liquid sucrose is now facing upwards so that it will be easier to crush. Using the flat square end of a lab microspatula, crush the frozen sucrose until the mixture is of slushy-like consistency. Use a hand blender to crush remaining pieces of frozen sucrose. Note: It is important that the mixture used for brain slicing is homogenous, since large pieces of frozen sucrose may get in the way of the vibratome’s razor blade. Pour 40 mL of slushy sucrose into a 50 mL glass cup. Submerge both cups with slushy sucrose in container 2 and insert carbogen tubes into both of them so that the mixtures continue to oxygenate until used.
Dissection of the mouse:
Inject the ketamine-xylazine cocktail i.p. and return the mouse immediately back to the cage/box. The first sign of mouse’s insensibility is recumbency (motionless head and body, loss of muscle tone) [19], which will appear in a matter of seconds to minutes, depending on the mouse’s weight and anesthetic cocktail dosage. Monitor the mouse and pay close attention to its breathing rate, which should gradually decrease.
Once the mouse’s breathing rate has significantly decreased (<55 breaths/min) [20], transfer it to the perfusion board and lay it on its back. Prior to initiating the procedure, evaluate anesthetic depth by testing the pedal withdrawal reflex: apply alternating hind paw toe pinches every 10 s between the metatarsal and phalanges bone. Appropriate anesthetic depth is determined by the absence of a pain response, measured by three consecutive non-responses to alternating pinches (the mouse does not twitch and withdraw its paws) [19]. Procedure may begin only after the pedal withdrawal reflex disappears. In case pain response persists after multiple tests, inject i.p. up to another third of the initial ketamine-xylazine dosage and repeat the testing process. Procedure should begin when the mouse has no pain response but is still breathing (very slowly).
Stretch the mouse’s limbs to the sides and tape them to the perfusion board with adhesive tape to create a smooth, taut, and fixed surface for dissection. Using Bracken forceps, elevate a large piece of the mouse’s skin and fur around the tip of the sternum (the xiphoid process appears as a slight protuberance between the two halves of the rib cage) (Figure 1B). Using sharp-blunt dissecting scissors, create a transverse incision across the raised piece of skin. Repeat the same step on the uncovered fascia below the skin. These two incisions should create a small round orifice (Figure 1B).
Insert the scissors vertically into the orifice with the sharp edge facing upwards (this will prevent damage to internal organs). Cut once through both sides of the rib cage by sliding the scissors all the way from the orifice to the base of the neck. These cuts should loosen the rib cage, leaving the diaphragm as a holding point (Figure 1C). Lift the rib cage with Bracken forceps and cut the diaphragm using the same dissection scissors (Figure 1D). Now that the chest flap is detached, flip it on its longitudinal axis towards the neck and tape it to the perfusion board/mouse’s body with adhesive tape. The thoracic cavity should now be exposed and the heart visible (Figure 1E).
Grab the heart with Bracken forceps and pull it up for better visibility. Using fine spring scissors, cut the right atrium (Figure 1F, 1), causing pooling of blood around the heart. Next, remove the needle protector from the scalp vein set and insert the needle vertically into the left ventricle in proximity to the apex (Figure 1F, 2), while simultaneously firmly holding the heart with Bracken forceps. The needle should enter the ventricle slightly, without puncturing it or entering other cavities of the heart. Without releasing the scalp vein set, use your other hand to slowly inject the sucrose solution into the left ventricle (Figure 1F, 3).
Figure 2. Brain extraction and preparation for slicing. A. Cutting a longitudinal incision in the mouse’s skin from between the ears to the mouse’s eyes (along white dotted line). B. Removing the remaining spinal cord and excess fat and skin by cutting along the dotted line. Note the skin flaps pulled to the sides of the skull. C. Making three incisions in the skull using medium straight, sharp-tipped scissors along the dotted lines. Then, lifting the skull flap using Bracken forceps in the direction of the arrow. D. Detaching the brain from the skull by pushing it with a spatula with an arrow head. E. Separating the cerebellum and olfactory bulbs from the cerebrum using the single-edged industrial blade. Then, severing the cerebral hemispheres using the same blade. All incisions are marked with dotted lines. These steps are performed on the dental wax sheet (pink square). F. Side-view of a single cerebral hemisphere on the dental wax sheet after the previous step. Flipping the hemisphere onto its medial side (marked with stripes). G. Transferring the hemisphere to the filter paper on its medial side (the anterior part of the hemisphere is at the top of the panel). H. Final orientation of the cerebral hemispheres, glued to the vibratome plate, relative to the vibratome blade. The black arrow marks the plane and movement direction of the blade.
The flow of sucrose will begin washing out blood from the mouse’s vascular system. The tilted perfusion board will prevent blood aggregation and cause it to flow towards the ice underneath. Escaping blood and liver should transition from being dark-maroon to light-orange as blood is diluted by sucrose. Perfusion should continue until the escaping blood is nearly colorless.
Note: If blood does not escape to the perfusion board, or it does not get lighter as time goes by, it means that the needle was not inserted deep enough into the left ventricle, or the ventricle was punctured. Conversely, if sucrose solution begins to come out of the mouse’s nose or mouth, it means that the needle was inserted too deeply and entered the right ventricle/left atrium. In both cases, slightly change the position of the scalp vein set accordingly and try to inject the sucrose once again. Note: Perfusing additional cold sucrose solution after the blood clears is also okay and may serve to further cool and protect the brain tissue.
Remove the adhesive tape from the mouse’s limbs and chest flap. Hold the mouse by its head/ears, allowing the rest of its body to be pulled down, and elongate the neck. Using the dissection scissors, make a transverse incision along the neck, right under the head, ideally detaching the entire head with one cut (Figure 1G).
Hold the mouse’s head firmly in one hand and create a longitudinal incision in the mouse’s skin from between the ears to the mouse’s eyes (Figure 2A). Separate the pieces of skin and pull them to the sides of the skull. Continue holding the head firmly by the skull (not the skin flaps) and cut the remaining spinal cord along with excess fat and skin, which may be an obstacle to accessing the brain (Figure 2B).
Vertically insert medium straight, sharp-tipped scissors between the skull and the brain. Slide the scissors along the longitudinal fissure whilst pushing them against the skull to avoid injuring the brain. Once the scissors reach the area between the eyes, make one incision. Slide the scissors out, continuously pushing them upwards against the skull. Next, insert the scissors horizontally on one side of the skull between the bone and brain. In a similar fashion, insert the scissors slightly while pushing them against the inside of the skull and make an incision. Repeat the same step on the other side (Figure 2C).
Using Bracken forceps, grab the tip of one skull flap, located in the middle of the ear line close to the longitudinal fissure. Pull it upwards and to the side gently to separate it from the brain (the pulling motion should be in the posteromedial to anterolateral direction). Repeat the same process on the contralateral skull flap (Figure 2C).
Note: The skull tissue is very thin, and there is a high chance of grabbing brain tissue along with it. Thus, it is advised to remove the flaps very slowly and consecutively check that the brain tissue is untouched.
Insert a spatula with an arrow end under the brain from the side of the skull and slide it to the front of the skull close to the eye line (Figure 2D). Detach the brain from the skull by gently pushing it upwards and drop it into the 50 mL glass cup with sucrose.
Wipe the vibratome plate with a Kimwipe, bring it to the brain slicing station, and brush super glue on it.
Using a spatula with a spoon end, scoop the brain out of the sucrose and transfer it to a dental wax sheet on its ventral side. Note: This and the following step describe how to slice sagittal slices of the brain that we use both for striatal and nigral recordings. However, if you are studying another part of the brain, you may block and slice it differently. Nevertheless, some of the principles and pitfalls we describe could still be useful. Hold the single-edge industrial blade horizontally (vertical to the brain) and cut once along the posterior fissure separating the cerebrum from the cerebellum. Use the same blade to remove the olfactory bulbs from the cerebrum. Next, perform a similar cut along the midline, thus severing the cerebral hemispheres (dashed lines in Figure 2E). Note: Make sure to perform the cuts as straight as possible and not at an angle. Use a spatula with an arrow or flat square end to separate brain parts after cutting, if necessary.
Flip the hemispheres onto their medial side on the dental wax (Figure 2F) and load each one individually to a flat square spatula. Use the spatula to place the hemispheres on filter paper. Let the medial surface of the hemispheres dry for a few seconds (Figure 2G). Note: Leaving the brain on the filter paper for too long will make it hard to remove it. Next, transfer each hemisphere using the square spatula from the filter paper onto the glue on the vibratome plate in the same orientation (the medial side of the hemisphere should be glued to the base plate). Critical: Avoid touching the glue with the bottom side of the spatula to prevent it from inadvertently sticking, which will spoil the setting of the brain properly. Using another flat spatula to lightly push the brain onto the glue could help.
Place the vibratome plate into the vibratome chamber and secure the chamber with the designated screw to the vibratome, so that the cut posterior surface faces the blades. Note: Depending on what plate you use, you may need to plan ahead the angle at which you mount the brain so that you are able to fasten the plate inside the slicing chamber without the cut-out part (crescent shape in Figure 2H) facing one of the fastening screws. Use the remaining sucrose solution in the syringe and 50 mL cup to gently cover, wet, and cool the glued brain. Fill the vibratome chamber with slushy sucrose from the 250 mL plastic cup, until both hemispheres are entirely submerged in the solution. Note: Pour sucrose only around the chamber perimeter and not straight onto the hemispheres to avoid injuring them.
Set the vibratome’s speed to 3.6 on the dial, slice thickness to 275 µm, and frequency to (9–10) on the dial. Additionally, set the start and end points of the blade in each cycle. Make sure to set them so the blade will cut through the entire hemisphere each time (consult the VT1000 S manual).
Note: Slice thickness may change depending on which brain region is being studied.
To obtain sagittal slices of the striatum (which is what we are describing in this protocol), discard the first few slices that include only cortical tissue. Once the blade is medial enough to produce slices in which the striatum can be seen, begin collecting them using a Pasteur pipette and transfer them individually to the slice chamber. There are typically six slices until you reach the anterior commissure, at which point you should stop collecting slices. The Pasteur pipette can also be used with gentle presses to move ice chunks away from the space between the blade and the brain.
Wait for at least 60 min from the moment all relevant slices are collected before beginning electrophysiological experiments to allow the tissue to recover from the slicing and to accommodate to the physiological conditions created by aCSF after being submerged in sucrose solution.
Patch-clamp recording
Patch-clamp recordings are used in all the experiments in which we want to test how blue LED activation of the proximal vs. the entire somatodendritic arbors affects the currents recorded in the soma. As we are interested in the effect of dendrites on synaptic inputs, these experiments are conducted while holding the soma at subthreshold voltages (the choice of voltage can affect the results [15]). The relevant blue LED perturbations for these experiments include brief pulses (see E below), Poisson barrages of brief pulses (see G below), or the sequence of sinusoids of various frequencies (see F below).
Flowing solutions into the recording chamber (at a rate that should be between 1 and 5 mL/min; slower than that may not be sufficient to provide the oxygen and nutrients to the slices, and faster than that can make the recording unstable due to movement of the slice):
Prepare the K internal and aCSF (with the necessary synaptic blockers, see Reagents) in advance, adjusting their concentrations as required.
Load the K internal solution into a 1 mL syringe affixed to a 0.22 μm syringe filter and a microloader, maintaining it at a cold temperature on ice.
Initiate a 20 min flow of double-distilled water prior to the experiment to ensure the cleanliness of the entire setup.
Continuously oxygenate the aCSF with carbogen for 20 min preceding the experiment. Then, 5 min prior to commencing the experiment, initiate a flow of oxygenated aCSF into the recording chamber to maintain the physiological conditions.
Float the anti-vibration optical table.
Fire up electrophysiology setup:
Turn on micromanipulator and temperature controllers, A/D board, amplifier, LED, microscope lamp, and PC computer.
Note: Make sure the amplifier and computer are grounded to avoid electrical noise.
Set the temperature controller to 31 °C.
Note: This is a value that we find as a good compromise between keeping the cell healthy for a longer time (the cells deteriorate more rapidly at higher physiological temperatures) and reproducing more physiologically relevant autonomous firing rates than observed at room temperature.
Activate software in the following order: Camera software, Multiclamp commander, and Signal 6.
Figure 3. Patch pipette dimensions. The patch pipette has to be pulled so that it has a shaft (A) that is long enough to fit under the hi-mag objective and has nice taper (B) that ends with a 1–3 µm opening (C).
Prepare microelectrodes with micropipette puller from glass pipettes, adjusting their resistance to the desired range, typically between 4 and 5 MΩ. (Consult puller manual on how to pull microelectrodes; see Figure 3.)
One hour after the incubation period, carefully transfer the sagittal brain slice to a Zeiss Axioskop fixed-stage microscope and secure it using a U-shaped harp.
Locating cell to patch:
Use the lo-mag objective lens (5×) to locate the light source positioned beneath the brain slice, which can be identified visually.
Switch to the hi-mag objective lens (60×) in order to visually identify the ChR2-expressing neuron of interest. Note: We have studied striatal cholinergic interneurons (CINs) in ChAT-ChR2 mice and GABAergic projection neurons in the substantia nigra pars reticulata (SNr) in Thy1-ChR2 mice. Once a healthy neuron has been found, situate it in the center of the screen.
Subsequently, load the microelectrode with the K internal solution and meticulously eliminate any air bubbles, particularly at the tip. Caution: Return to the low-magnification objective lens to prevent any inadvertent damage to the pipette.
Affix the loaded microelectrode securely to the microelectrode holder and take caution to lock the holder in place, thus maintaining a stable setup.
Patching the cell:
Before lowering the microelectrode into the recording chamber, exert slight positive pressure to prevent any potential filth from entering the pipette tip.
Note: Use a manometer to set the correct pressure. The manometer should be connected with a T-junction to the tube used to pressurize the pipette through its connection to the microelectrode holder. It is useful to use a 3-way Luer Lock on the end of the tube used by the experimenter to control, manipulate, and stabilize the pressure.
Precisely position the pipette tip at the center of the field of view using the lo-mag objective lens.
Use the micromanipulators to carefully lower the microelectrode into the recording chamber.
The amplifier should be in voltage clamp (VC) mode. Conduct a seal test to assess the pipette's resistance. Note: If the resistance measures too low, it may indicate a compromised pipette tip, whereas excessively high resistance may suggest blockage, either due to debris or a wrong/faulty puller program.
Switch back to the hi-mag objective lens, submerging it into the bath (usually, by gradually moving upwards) while searching for the pipette tip. Be cautious to prevent inadvertent pipette tip breakage during this process.
When initiating the micropipette descent toward the tissue, after locating its tip, proceed with caution in a downward motion. Caution: Always move the objective lens first, followed by catching up with the micropipette, so you can see the new region before moving the pipette there, so as to prevent inadvertently crashing the pipette into the slice. Commence this procedure at an elevated velocity (using one of the C settings on the L&N manipulator controller), while continuously monitoring the resistance of the micropipette.
Once you visualize the tissue, move to a lower speed (H, M, or L on the L&N controller) to minimize potential tissue damage. Lower only the objective to verify presence of the cell in the same initial location. Subsequently, raise the objective back up to the micropipette and methodically continue the downward progression towards the cell; again, first the objective and then the pipette.
Position the tip of the pipette at the optimal viewing point for observing the cell, ensuring that all boundaries are within view, rendering the cell at its maximal observable size.
Delicately approach the cell surface. When done properly, this will result in an increase of micropipette resistance (by approximately 0.2 MΩ) and the appearance of a discernible "dimple" between the cell and the micropipette, signifying close contact between the micropipette tip and the cell surface.
Critical: At this point, adjust the pipette offset via the MultiClamp 700B commander to minimize the voltage offset.
Subsequently, release the positive pressure and gently apply a continuous negative pressure through oral suction, causing a notable increase in the seal resistance, ultimately reaching >1 GΩ. This is called the cell-attached mode.
While the cell is approaching the gigaseal, adjust the holding voltage to the desired holding membrane potential (which should equal the cells’ expected resting membrane potential or, for pacemaker neurons like CINs or SNr neurons, to a voltage close to their zero current, which is typically at -60 mV) using the holding button in the MultiClamp 700B commander.
To prevent saturating the amplifier, leading to distortions in the measured currents, conduct capacitance compensation (in MultiClamp commander, press the auto button opposite Cp fast and Cp slow, which will compensate for the fast and slow transients. It may be necessary to adjust Cp slow manually).
Following stable seal formation, carefully apply an additional slight negative pressure through the pipette to break-in the cell membrane to obtain a whole-cell recording. After break-in, access resistance should ideally be low (typically < 10 MΩ). If the access resistance is too high, then the membrane is not completely broken, and gentle negative pressure can correct this. For Cp fast and Cp slow, optimal values can vary, but Cp fast should typically be in the 2–5 pF range and Cp slow between 10 and 30 pF (depending on the size of the neuron).
Improve voltage-clamp performance through the compensation of series resistance (Rs). (In MultiClamp commander by activating the Rs compensation button. Strive to reach a correction factor of 75%.)
Note: Rs compensation does not always work.
In whole-cell mode, we use VC mode to measure currents and current clamp mode to measure voltage.
In the experiments described below, we compare proximal to full-field optogenetic stimulation. In the proximal illumination, we strive to activate ChR2 in a small region (approximately 100 µm in diameter, a size that is determined by the smallest pin-hole we could produce in the disk placed near the back focal plane of the objective and used to block out most of the field of view) around the soma, nominally activating the soma and proximal dendrites. In the full-field stimulation, we strive to activate the entire somatodendritic field of the neuron. Note: The optogenetic stimulation could activate neighboring neurons that innervate the patched cell. It is, therefore, critical to include the appropriate synaptic blockers in the recording chamber aCSF solution to ensure that measured effects are postsynaptic. See also General Notes.
Perforated patch recording
The perforated patch configuration is used to study the effect of the blue LED light perturbations on the firing pattern of the pacemaking neuron. Here, we use current clamp in order to record how the input affects the timing of the autonomous action potentials of the pacemaking neuron. The relevant perturbations are: a) long sinusoidal waveforms set to frequencies below, in the vicinity of or above the autonomous firing rate of the pacemaker neuron (see H below), and b) the barrage (see G below) that can be used to estimate both the phase response curve (PRC) of the neuron [21–24] and the peri-stimulus time histogram (PSTH) of the neuron in response to a sudden increase in ChR2 currents
The perforated patch method is a variation of patch clamp that establishes electrical access between the cell and the patch pipette using pore-forming antibiotics (here, we use Gramicidin). The resulting pores selectively permit small monovalent ions while preserving the integrity of many cytoplasmic components and the autonomous spiking of pacemaking neurons.
To prepare the Gramicidin internal, first add 5 mg of Gramicidin into 500 μL of anhydrous Gramicidin in DMSO. This stock solution can be stored at room temperature and used for three days, after which it should be discarded.
Prefilter 1 mL of K internal solution by drawing it into a syringe affixed to a syringe filter. Combine 1–2 μL of the stock solution with 1 mL of internal solution, resulting in a final concentration of 10–20 μg/mL. Note: This quantity has to be determined individually based on experience and practice attaining a seal. Front-fill the recording pipette with regular K internal by connecting the pipette's back to a syringe using soft tubing, immersing the tip in K internal solution, and applying negative pressure with the syringe. Note: We prefer to use pipettes without a filament (to reduce capillary action), but other groups do not. Fill 2/3 of the tip with regular K internal solution. Use a microloader to backfill the pipette with Gramicidin internal to the standard level.
For successful perforated patch recordings, it is crucial to establish a gigaseal while the pipette tip still has clean K internal solution. This will prevent Gramicidin from spewing onto the cell surface during the patching process. To prevent expelling all clean K internal solution before reaching the cell, which can interfere with the formation of a seal, apply very low positive pressure (<0.5 psi) until the pipette is just above the slice, increasing it immediately before entering the slice. Speed is also crucial for achieving this goal, with the pipette tip ideally positioned above the cell within one minute of entering the bath. Note: The final concentration of Gramicidin can also be titrated on the fly during the experimental day by adjusting the volume of backfilled Gramicidin internal to speed up or slow down the perforation.
While speed is essential, it is equally vital to be gentle; minimal dimpling of the cell membrane before releasing pressure to create the gigaseal typically yields more stable recordings.
After a gigaseal has been established, perforation usually takes 10–30 min to complete (at 31 °C). We judge the extent of the perforation by the size of the action currents in voltage clamp mode that should be >300 pA in amplitude or the size of the action potentials in current clamp that should overshoot -20 mV (but preferably 0 mV).
Switching apertures for proximal vs. full-field optogenetic illumination
Since at this time point, after completion of the patching, you are using the hi-mag objective lens, you begin with arranging the proximal 470 nm LED illumination (Figure 4A, red region) by sliding in the IRDIC analyzer fitted with the disk with the small hole into the analyzer slot just behind the back of the objective, and closing the diaphragm of the incident-light pathway (Figure 5). Later, after the protocols are run in the proximal configuration, we then open the diaphragm, pull out the analyzer with the disk, pull the water immersion high-magnification objective out, and swing in the low-magnification air/dry objective for the full-field illumination (Figure 4A, green region).
Figure 4. Optogenetic activation of proximal vs. full-field dendritic arbor. A. Schematic of the proximal (red) vs. full-field regions of illumination. Inset: decay time of current response to a brief pulse of blue light illumination is longer for full-field (green) than proximal (red) illumination. B. Concatenated sinusoidal waveforms of blue light illumination (top) are used to elicit current responses to either full-field (green) or proximal (red) illuminations (bottom). C. The input waveforms (red or green) are cross-correlated with the current (black) response (top) to generate the respective cross-correlograms (bottom). The latency of the peak (in seconds) is translated into a phase by multiplying it by the driving frequency.
Figure 5. Proximal illumination. A. A disk, with a central pinhole, is machined to fit into the Axioskop analyzer. B. The disk is fitted into the analyzer. C. The analyzer slot in the incident 470 nm light pathway is situated near the back focal plane of the objective lens. D. The analyzer with the disk is gently inserted into the slot. E. The disk with the central pinhole is in place near the back focal plane of the objective. Note: Disregard the lo-mag air objective in the images; proximal illumination is done via the high-mag water-immersion objective.
Brief 470 nm pulse perturbation
In voltage clamp mode, while holding at a hyperpolarized voltage (e.g., -70 mV), apply a brief 1-ms-long blue light pulse to elicit an inward current. Repeat several times to extract the average response (Figure 4A, inset).
The ChR2 is expressed in the postsynaptic neuron that you are interrogating, and you are therefore stimulating it directly. Nevertheless, you could be activating synaptic inputs into the neuron (e.g., collaterals of the neuron you are stimulating or of other neuronal types that express ChR2 in the slice). Thus, it is pertinent to include synaptic blockers in the aCSF that are relevant in the neuronal type you are studying [we commonly use blockers of ionotropic glutamate (10 mM DNQX & 50 mM D-AP5), GABAA (10 mM gabazine), and GABAB (2 mM CGP55845) receptor blockers].
Later, after completing all the proximal measurements, repeat in the full-field illumination configuration. Critical: The amplitude of the LED voltage should be chosen (by trial and error) so that the currents generated in the somatic recording pipette will be on the order of approximately 10 pA. This parameter needs to be set separately for each of the illumination configurations. The amplitudes required for the proximal illumination are typically larger by an order of magnitude, because most of the light is blocked by the disk making the effective illumination small.
Sinusoidal 470 nm perturbations
Program (in advance) a whole-cell voltage clamp protocol in Signal 6 in which the soma is held at a subthreshold voltage and in which the 470 nm LED’s analog driver is fed a waveform that is composed of concatenated sinusoids (Figure 4B). Critical: The amplitude of the LED voltage sinusoidal should be chosen (by trial and error) so that the currents generated in the somatic recording pipette will be on the order of a few tens of picoamperes. The frequencies should include both sub- and supra-Hertz values and should be chosen so that each frequency is represented by an integer multiple of cycles so that the overall waveform will be continuous. We used the following frequencies (durations indicated in parentheses): 0.2 Hz (5 s), 0.4 Hz (5 s), 0.6 Hz (5 s), 0.8 Hz (5 s), 1 Hz (5 s), 1.2 Hz (5 s), 1.4 Hz (5 s), 1.6 Hz (5 s), 1.8 Hz (5 s), 2 Hz (3 s), 3 Hz (3 s), 4 Hz (3 s), 5 Hz (3 s), 6 Hz (3 s), 8 Hz (3 s), 10 Hz (3 s), 12 Hz (3 s), 14 Hz (3 s), 16 Hz (3 s), 18 Hz (3 s), and 20 Hz (3 s). Note: In Signal 6, you can actually program this long waveform using their built-in protocols. Alternatively, you can program the waveform with another software and upload it into a Signal 6 protocol. The sampling frequency of the voltage signal needs to not be too high because these are low-frequency sinusoidals (we used 10 kHz).
Optional: Repeat the stimulation several times to average the response and reduce noise. The waveform can be inverted in time and fed into LED to control history dependence.
In order to study the dependence of the results on holding potential, steps F1–2 can be repeated at various holding potentials (e.g., -70, -60 & -50 mV).
Later, after completing all the proximal measurements, repeat in the full-field illumination configuration.
Barrage 470 nm stimulation
Program (in advance) 25 individual realizations of a Poisson barrage of brief (0.5–1 ms) 470 nm LED pulses using interpulse intervals (IPIs) that are distributed exponentially (Figure 6C) [13]. Note: The barrage waveforms will need to be generated with another software (e.g., MATLAB) and uploaded into a Signal 6 protocol. The mean IPI is a parameter to be chosen depending on the neuron’s spontaneous firing rates (the idea is to get several dozen pulses between each spike). The amplitude of the pulses needs to be set to affect the spike timing without dramatically altering the ongoing firing rate. This intensity will be different for proximal vs. full-field illumination.
Figure 6. Poisson Barrage of blue light pulses is used to estimate the neuron’s peristimulus time histogram (PSTH) and dendritic phase response curve (dPRC). A. On the large time scale, the barrage acts as a current step function, which drives an increase in the pacemaker neuron’s firing rate. B. The PSTHs for proximal (red) and distal illuminations (green) are estimated by binning the spiking response over multiple trials. C. At a finer time scale, dozens of individual pulses are visible between spikes. D. The sequences of pulses leading up to each spike are used to estimate the dPRC via regression analysis.
After patching the cell, allow the perforation to advance (this can be done either while holding the cell in voltage clamp and watching the action currents gradually increase, or in current clamp—the spike amplitude increases to approximately 0 mV).
Note: This experiment can also be conducted in cell-attached mode. If so, we suggest holding the cell in current clamp, because in voltage clamp you will inadvertently be clamping the cell as it perforates.
Once perforated, run the 25 realizations of the Poisson process and record the spiking.
As above (step E2), include synaptic blockers.
Later, after completing all the proximal measurements, repeat in the full-field illumination configuration, with the required changes in barrage parameters (mostly intensity).
Oscillatory entrainment in perforated patch
After establishing a gigaseal, allow the perforation to advance (as described in step C7 above).
Estimate the autonomous firing rate of the neuron.
Program 30–60 s long protocols on Signal 6 (on the fly) of 470 nm LED sinusoids with frequencies that are below, above, and near the autonomous rate (you can also try integer multiples of the autonomous rate) and run these (Figure 7A). Note: The aim is to use 470 nm LED intensities (that will vary for proximal vs. full-field illuminations) that will slightly perturb. Trial and error is inevitable. Additionally, the frequency of the pacemaking neuron may change during the course of the experiment, so estimate it frequently and adapt.
Figure 7. Estimation of the degree and phase of entrainment of a pacemaker neuron to oscillatory inputs. A. A long sinusoidally modulated blue light waveform, at a frequency (f) near the autonomous frequency (fp) of the pacemaker, is used to entrain the pacemaker neuron (recorded in the perforated patch configuration) either via full-field (left) or proximal (right) illumination. B. Estimation of the phase of the input 𝜓 at which the spike occurs (we defined the phase of the trough as phase 0 and the peak as phase 0.5). C. Histogram of the phases 𝜓 at which the neuron fires, exhibiting a narrow peak indicative of entrainment. D. Histogram used as a probability distribution function P(𝜓) from which the circular variance vector is calculated using the displayed formula. The modulus of the resulting complex number is used to estimate the strength of the circular variance vector, which is used, in turn, to determine whether entrainment is significant (as compared to threshold determined by bootstrapping). The phase of entrainment is extracted from the argument of the complex number (divided by 2𝜋). E. An example of the distribution of significant circular variance vectors estimated for full-field (green) vs. proximal (red) illumination.
Data analysis
Dendritic phase and amplitude estimation and model fitting
Estimate phase and amplitude response at each driving frequency f.
Parse the long current trace to segments corresponding to the LED driving at each frequency f (Figure 4C).
Calculate the cross-correlation function (CCF) between each segment and the sinusoidal waveform used to drive the LED using the MATLAB function xcov with the parameter SCALEOPT set to ‘biased’(Figure 4C).
Locate the peak of the CCF using the MATLAB function findpeaks with the parameter MinPeakHeight set to 0 (Figure 4C). Note: You may need to visually inspect the CCF to verify the identification of the peak as the one closest to the zero. Convert the location of this peak to units of seconds and then multiply time by the driving frequency f to convert to phase.
Find the maximal value of the CCF function.
Note: Normally, this corresponds to the value of the CCF at the peak you found in c above. However, in some cases the maximal deflection from the zero of the CCF amplitude is a negative peak, and then the amplitude should be taken from that. Divide this value by the amplitude of the voltage sinusoidal used to drive the LED to get a value in units of pA.
You will typically need to repeat this experiment on at least three animals and get a sufficient quantity of cells (in the order of 10 or so), in order to extract a mean and standard error of the mean (SEM) for the phase and amplitude. From our experience, the error on the estimation of the phase is very small, whereas the error on amplitude is very large.
There are qualitative findings you can extract from the data at this point. You might see a change in the amplitude of the response in the presence of a drug or at a different holding potential [15], or a difference in the phase response between proximal and wide-field stimulation [14,15]. These findings are more robust in the sense that they are model-independent.
Fitting a model to the data enables you first to fit specific parameters to the data and, perhaps more importantly, to rule out models when the points cannot be fit by them. The best example of this is the appearance of a negative peak in the phase response, which can only be explained by the presence of a restorative current such as the hyperpolarization-activated cyclic nucleotide-gated (HCN) current.
Note: The derivation of the various models is beyond the scope of this bio-protocol. You are referred to the original papers in which we used these protocols [14,15] for the various models, which are based on the assumption that the amplitude and phase responses are the concatenation of two filters: one arising from the filtering properties of the ChR2 current response and the other arising from the contribution of dendritic and somatic passive membrane properties and nonlinear conductances. The ChR2 current filters because it deactivates with particular time constants. The somatodendritic arbor filters because of the passive and semi-active membrane properties that arise from its specific capacitance, leak channels, and nonlinear ionic channels. We developed equations for these filters in previous publications (under the assumption of a very simple geometry of a dendrite). You are encouraged to refer to those [9,14,15] to understand the derivations and the assumptions they are based on.
Models are fit with the lsqcurvefit function in MATLAB. Note: In principle, you should be able to fit the parameters by simultaneously fitting the mean amplitude values and the mean phase values. However, on a practical level, we found that first fitting the parameters to the phase model (whose error bars are much tighter) and then using the extracted parameters to fit the additional parameter of the overall single scale of the amplitude response worked more robustly.
Dendritic peristimulus time histogram (PSTH) estimation
The PSTH we refer to is measuring how the neuron’s firing rate changes in response to a step function increase in current and should be estimated for neurons recorded either in perforated patch or cell-attached mode (which does not disturb the neurons intracellular milieu). While you can add a separate protocol for this purpose, this will require many more measurements. Instead, we found that using the barrage stimulus suffices, because on the time scale of changes in firing rate, the barrages behave like a step-function increase in activation (Figure 6A).
Generating a PSTH requires many action potentials. Depending on the rate of the neuron, this will typically require many trials per neuron and pooling many neurons (for SNr neurons, it was on the order of 20 neurons, see Figure 4 in Tiroshi et al [14]).
Extract the spike times used in units of seconds for each of the trials.
The PSTH can be calculated using the MATLAB command histc, which returns a count of spikes that fall within each bin. To convert this to an instantaneous firing rate, you need to divide this number by the number of trials and by the width of the bin used in the histc command (Figure 6B).
Note: You will probably need to try several bin widths until you find one that gives the best looking rendition.
Dendritic phase response curve (dPRC) estimation
The dPRC depicts how the pacemaker neuron’s phase is influenced by small voltage perturbations delivered to the dendrite as a function of the timing of the perturbation [9]. dPRCs are estimated based on slight modifications to previously published methods [13,25].
Detect spike times using the MATLAB function findpeaks with the parameter MinPeakHeight set to the average of the minimal and maximal values.
Divide each interspike interval (ISI) into 50 equally sized bins (i.e., for each ISI, the bin size scales with the duration of the ISI), where the jth bin corresponds to the phase ϕj = (j-0.5)/50.
Count the pulses delivered in each bin and denote the number of pulses delivered in the αth ISI and jth bin by npα,j. Calculate the mean number of pulses per bin, averaged across all bins, and subtract it from pα,j resulting in Δpα,j.
Perform a multiple regression analysis with Δpα,j values as the independent variables and ISI durations as the dependent variables. If we denote the PRC as Z(ϕ), then the regression coefficients Z(ϕj) provide a unique solution for the PRC (Figure 6D, Netoff et al., 2011).
Measuring entrainment
The degree of entrainment of the cell’s spiking activity to rhythmic inputs is evaluated based on previously described methods [13]. Throughout the analysis, we consider the effective phase of each spike, which is its phase with respect to the period T of the rhythmic stimulation (Figure 7B).
To quantify the strength of entrainment:
For each cell and each stimulation frequency, plot the distribution of effective phases estimating Pr(ψ = ψj) for each phase ψj (Figure 7C).
Use this estimation to generate a circular variance vector, which provides a measure of the variation of effective phases:
The amplitude of the circular variance vector represents the extent to which the firing of the neuron was entrained by the oscillatory input, and its phase indicates the effective phase of entrainment, which corresponds to the peak of the distribution of effective phases (Figure 7D,E).
In order to investigate phases of entrainment, only include trials where the spiking of the neuron was successfully entrained:
Use bootstrapping to generate a distribution of the amplitude of circular variances. In each bootstrapping iteration, the surrogate data should consist of a series of N uniformly distributed random numbers (between 0 and 1), where N is the typical number of ISIs in a trial.
Only trials where the amplitude of the circular variance is larger than the 95th percentile of the bootstrapping distribution, calculated as described previously [14], should be included in the analysis.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Tiroshi L et al. (2019). Population dynamics and entrainment of basal ganglia pacemakers are shaped by their dendritic arbors. PLoS Computational Biology (Figure 2–6).
Oz O et al. (2022). Non-uniform distribution of dendritic nonlinearities differentially engages thalamostriatal and corticostriatal inputs onto cholinergic interneurons. eLife (Figure 1–3)
General notes and troubleshooting
General notes
Some laboratories perfuse intracardially with aCSF (i.e., not with modified aCSF), while other laboratories skip the perfusion altogether and decapitate the deeply anesthetized animal. In our hands, and particularly when working with neurons we study (e.g., striatal cholinergic interneurons, basal ganglia pacemaker neurons) the perfusion with sucrose yields slices that are healthier for longer.
Optogenetic stimulation, particularly in the full-field condition, might elicit activation of nearby neurons, some of which may project to the patched neuron. To ensure that the measured effects are generated post-synaptically, the aCSF solution should include antagonists for the channels expressed by the neuron. For our recordings in the striatum and substantia nigra, we supplemented the aCSF with DNQX to block AMPA receptors, D-AP5 to block NMDA receptors, SR to block GABAA receptors, and CGP to block GABAB receptors. Different blockers may be necessary for experiments conducted in other brain regions.
The 470 nm waveforms used throughout the protocol must have a minimal value at the threshold voltage of the LED, i.e., approximately 40 mV.
Acknowledgments
This work was funded by an Israel Science Foundation (ISF) Grant (no. 1959/22), and a US-Israel Binational Science Foundation (BSF) grant (no. 2021212) to J.A.G.
Competing interests
The authors declare no competing financial interests.
Ethical considerations
All experimental protocols were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and with the Hebrew University guidelines for the use and care of laboratory animals in research. The experiments adhered to, received prior written approval from, and were supervised by the Institutional Animal Care and Use Committee of the Faculty of Medicine, under protocol: MD-18-15657-3.
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A Detailed Protocol for the Induction of Anemia and RBC Transfusion–associated Necrotizing Enterocolitis in Neonatal Mice
BR Balamurugan Ramatchandirin
MB Marie Amalie Balamurugan
SD Suneetha Desiraju
YC Yerin Chung
KM Krishnan MohanKumar
Published: Vol 14, Iss 10, May 20, 2024
DOI: 10.21769/BioProtoc.4993 Views: 1089
Reviewed by: Nafisa M. JadavjiDushyant MehraBassam A. Elgamoudi
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Original Research Article:
The authors used this protocol in Nature Communications Aug 2019
Abstract
Anemia is a common and serious health problem, nearly universally diagnosed in preterm infants, and is associated with increased morbidity and mortality worldwide. Red blood cell (RBC) transfusion is a lifesaving and mainstay therapy; however, it has critical adverse effects. One consequence is necrotizing enterocolitis (NEC), an inflammatory bowel necrosis disease in preterm infants. The murine model of phlebotomy-induced anemia and RBC transfusion–associated NEC enables a detailed study of the molecular mechanisms underlying these morbidities and the evaluation of potential new therapeutic strategies. This protocol describes a detailed procedure for obtaining murine pups with phlebotomy-induced anemia and delivering an RBC transfusion that develops NEC.
Keywords: Phlebotomy-induced anemia RBC transfusion Necrotizing enterocolitis Neonatal mice Intestinal injury
Graphical overview
Schematic diagram of murine model of anemic and red blood cell (RBC) transfusion-associated necrotizing enterocolitis (NEC)
Background
Red blood cell (RBC) transfusions are necessary and essential therapeutic interventions frequently utilized in the neonatal intensive care unit and to treat critically ill infants who experience severe anemia due to physiologic and iatrogenic factors [1–4]. Nearly 80% of all infants born at <27 weeks of gestational age receive one or more RBC transfusions during their birth hospitalization [5–8]. Though repletion with packed RBC has obvious benefits (such as increased oxygen-carrying capacity), transfusions have also been implicated in the subsequent development of necrotizing enterocolitis (NEC) within 48–72 h after transfusion [7–15]. NEC is the most common acquired gastrointestinal disease of premature neonates, affecting 5%–15% of infants born with <1,500 g [5–8,16–18] and, despite technological advancement, remains a leading cause of death in neonates born between 22 and 28 weeks gestation [9–12,19,20]. NEC is also associated with multiple immediate serious complications, such as death due to sepsis, and long-term complications, including intestinal failure, growth delay, and adverse neurodevelopmental outcomes [21,22]. The causes of NEC are diverse; however, our recent findings documented that phlebotomy-induced anemia resulted in a disproportionate and persistent increase in intestinal permeability in pre-weaned mice because of the disruption of epithelial adherens junctions [23], developing a low-grade inflammatory state in the intestine with prominent macrophage precursor infiltration. Subsequent RBC transfusions activate these macrophages and thus cause NEC-like injury [24]. To examine the pathogenesis of NEC, various murine models have been established. This study will describe an improvement upon the previous version and a detailed protocol of our murine model of RBC transfusion–associated NEC.
Materials and reagents
Biological materials
Serratia marcescens (American Type Culture Collection, catalog number: 13880)
C57BL/6J mice (The Jackson Laboratory, catalog number: 000664)
Reagents
Citrate phosphate dextrose adenine (CPDA-1) (Sigma, catalog number: C4431)
Luria-Bertani (LB) broth (Quality Biological, catalog number: 340004101)
Normal saline (0.9% sodium chloride) (Sigma-Aldrich, catalog number: 7647145)
Trypan blue (Thermo Fisher, catalog number: 15250061)
0.5% Proparacaine hydrochloride ophthalmic solution (Alcon Laboratories, Inc., catalog number: NDC0998-0016-15)
Phosphate-buffered saline (PBS) (Gibco, catalog number: 10010023)
Intestinal fatty acid binding protein ELISA kit (MyBioSource, catalog number: MBS035456)
Laboratory supplies
Cell pack (Sysmex America, model: DCL 300A)
Sterile Acrodisc WBC syringe filter (Cytiva, catalog number: AP-4952)
Magnifier lens
1.5 mL Eppendorf tubes (Eppendorf, catalog number: 022363204)
14 mL culture tube (Corning, catalog number: 352057)
1 mL syringe (Thermo Fisher, catalog number: 309625)
0.3 mL insulin syringe (Becton, Dickinson and Co., model: BD Ultra-Fine II)
Umbili-cath polyurethane UVC catheter, single lumen, 3.5 French (Utah Medical, catalog number: 4183505)
Equipment
Weighing balance (Mettler-Toledo, catalog number: ME104TE)
Sysmex XN-1000TM hematology analyzer (Sysmex Corp, IL)
Spectrophotometer (Molecular Devices, model: SpectraMax Plus)
Procedure
Preparing packed stored RBCs for transfusion
Time duration: 30 min
One week before transfusion, anesthetize FVB/NJ adult donor mouse with isoflurane (1.5%–2.5%) in an induction box until the mouse is non-responsive and apply 0.5% proparacaine ophthalmic solution 5 min before whole blood retro-orbital bleeding.
Prepare a 1.5 sterile microliter centrifugal tube with a final concentration of 14% (140 μL) of CPDA-1 solution and 860 μL of whole blood collected from each adult mouse to make a combined volume of 1 mL.
Standard heparinized or nonheparinized microhematocrit capillary tubes can be used. Hold the animal by the back of the neck and tighten the loose skin of the head with the thumb and middle finger to keep the animal stable. Place the tip of the capillary tube at the medial canthus of the eye under the nictitating membrane.
With a gentle thrust and rotation motion past the eyeball, the tube will enter the slightly resistant sinus membrane. The eyeball itself remains uninjured. As soon as the sinus is punctured, blood enters the tubing by capillary action. When the desired amount of blood is collected, withdraw the tube and apply slight pressure to the eye with a clean gauze pad to ensure hemostasis. Take care not to scratch the cornea with the gauze pad.
Euthanize the mice in accordance with IACUC and institutional policies.
Immediately after collection, leukoreduce the whole blood using a sterile Acrodisc WBC syringe filter. Then, centrifuge the leukoreduced blood at 295× g for 10 min and partially remove the supernatant to obtain a hematocrit of approximately 75%. Transfer the blood to 1.5 mL centrifugal tubes to create multiple aliquots of 500 μL, leaving a small residual air space, and store in the dark at 4 °C until use.
Preparing Serratia marcescens
Considering the potential importance of enteric Gammaproteobacteria in NEC pathogenesis, we introduced a well-characterized strain of Serratia marcescens in our mice on postnatal day 7 (P7) to achieve fecal Gammaproteobacteria abundance similar to what is seen in premature infants. Serratia sp. has been previously used as prototypical Gram-negative bacteria in rodent models of NEC based on several characteristics: (a) translational relevance, as they were originally isolated from a premature infant with NEC; (b) non-pathogenicity in mice, as mice colonized with these bacteria in our laboratory have remained asymptomatic for several months with normal body growth and no histological evidence of intestinal inflammation; and (c) natural red pigmentation of Serratia colonies, which facilitates detection in fecal/tissue cultures.
Time duration: 10 min
Three days before transfusion, use a sterile pipette tip to scrape approximately 10 μL of an enteric bacterial glycerol stock from a frozen cryovial.
Place the sterile pipette tip containing the bacterial aliquot into a 15 mL culture tube containing 10 mL of LB broth. Culture the bacteria overnight (16 h) in an orbital shaker at 37 °C with agitation speed at 150 rpm. A control culture tube with 10 mL of sterile LB broth should also be cultured simultaneously in the orbital shaker to ensure there is no concern for bacterial contamination of the LB broth.
Use a spectrophotometer to measure the culture density at 600 nm (OD600nm). Add 1 mL of sterile LB broth into a 1 cm cuvette and measure the OD600nm to serve as the blank. In a separate 1 cm cuvette, add 1 mL of the S. marcescens culture and measure the OD600nm.
Note: The OD600nm value should be 0.6 ± 0.02, corresponding with the exponential bacterial growth phase. If the OD600nm is greater than 0.6, use LB broth to dilute the culture until the diluted culture exhibits the targeted OD600nm value. If the OD600nm is less than 0.6, continue to culture the inoculum until the OD600nm reaches 0.6.
Once the S. marcescens culture has achieved the targeted OD600nm, transfer 2 mL of the culture to 2 mL centrifuge tubes and centrifuge at 3,000× g for 10 min. Discard the supernatant.
Resuspend the bacterial pellets each in 1 mL sterile PBS for oral gavage to mouse pups.
Phlebotomy-induced anemia
We have improved our previously published protocol by retrieving a smaller blood volume (10 μL per gram of body weight) daily rather than 20 μL on alternate days. This change reduced animal stress and anxiety and prevented acute losses of plasma volume while continuing to achieve effective hematocrits of 18%–23%.
Time duration: 10 days
On the first day of the experiment, in the animal facility, weigh the P1 mouse pups and randomly assign the animal into one of two experimental groups: anemic or control groups.
Anemic pups: Gently hold the mouse pups by grasping the loose skin at the base of the neck and perform facial vein phlebotomy to remove 10 μL of blood per gram of body weight (Figure 1).
Note: Restrain the mouse gently. Figure 1 shows a detailed enlarged representation of the approximate area of the facial vein by measuring the length of the eye below the lateral canthus and the width of the eye caudally.
Figure 1. Facial-vein phlebotomy in postnatal day 1 (P1) and P10. Hematocrit levels will be measured in blood specimens using Sysmex XN-1000TM hematology analyzer according to their standard procedures. To maintain plasma volume, an aliquot of normal saline (0.9% sodium chloride) equal to the amount of blood removed was administered intraperitoneally by carefully inserting a 30 G needle fixed to a 0.5 mL syringe.
Control groups: Prick the control pups with a needle through the scruff of the neck while ensuring that they do not bleed in order to subject all mice to similar handling and stress.
Note: The facial vein in murine neonates is well visible during P1–4 days; after P5 fur has developed, which reduces the visibility of the facial vein. Even so, facial vein phlebotomy in P5–10 animals is possible by keeping the mouse pups under warm lights for 5 min to dilate the veins for phlebotomy.
Figure 2. Visible pallor in anemic mice.
Repeat the above steps for both anemic and control group animals every day until P10. In addition to measuring the hematocrit during each phlebotomy, thus confirming the level of anemic condition, visually monitor the anemic mouse pups in the anemic group for the gradual development of pallor. Figure 2 shows the anemic mice visibly pale in the face and toes compared with the control mice following a reduction in hematocrit.
Note: The size and weight of each experimental mouse pup are monitored and recorded during phlebotomy and after RBC transfusion.
On P7, administer oral gavage feedings of S. marcescens to the mouse pups of both the control and anemic groups.
Fill a 1 mL syringe with S. marcescens culture suspension and attach it to a 3.5 French umbili-cath polyurethane UVC catheter.
Gently hold the pup by grasping the loose skin at the base of the neck and use forceps to grasp the distal end of the catheter. Gently introduce 2 cm of the catheter into the oropharynx and esophagus (Figure 3). There should be no significant resistance with the insertion of the catheter.
Figure 3. Oral gavage of Serratia marcescens using 3.5 French catheter.
Slowly dispense 50 μL (104 CFU) into the stomach.
Slowly withdraw the catheter from the oral cavity.
Monitor the animal for increased respiratory effort or emesis associated with a mispositioned catheter.
Transfusion of stored RBC transfusion by orbital-sinus injection
On P11, randomly assign a few mouse pups from the above anemic and control groups again into two experimental groups: anemia–RBC transfusion and control–RBC transfusion groups. Both group animals are transfused as below:
Bring the stored RBCs from the refrigerator and gently resuspend them by rotating tubes in the rotator at room temperature.
To administer retro-orbital injections in pups, use a 31 G, 0.3125 in needle attached to a 0.3 mL insulin syringe. Do not inject more than 50 μL of liquid in each orbital sinus. The pups are not anesthetized for this procedure, because they can be adequately manually restrained without being anesthetized.
For neonatal mice, right-handed lab personnel will find it easiest to place the pup in left lateral recumbency, with their head facing right, and administer the injection into the right retro-orbital sinus. This procedure can be reversed to accommodate left-handed personnel to inject into the left side of retro-orbital sinus (Figure 4).
Figure 4. Orbital sinus injection of stored red blood cells (RBCs)
To test the efficiency of orbital sinus injection, extra mouse pups may be injected with Trypan blue and monitored for body color change to confirm systemic circulation (Figure 5).
Figure 5. Effectiveness of orbital sinus injection evaluated with Trypan blue.
Note: Tryan blue injection helps to confirm the efficiency of our intravenous injection method via orbital sinus in neonatal mice and it does not vary in either experimental group.
Gently restrain the pup’s head with the tip of the thumb and forefinger. Lab personnel must be careful not to place pressure on the trachea or impede venous flow. Nestle the rest of the pup’s body between the thumb and forefinger. In our experience, once the mouse is comfortably restrained, there is minimal struggle, and the mouse does not emit audible vocalizations.
Note: Use sterile saline and a cotton-tipped applicator to gently clean the area above the eye. This helps to remove any skin flakes that may get in the way of the injection and helps to make the skin slightly more transparent.
Care must be taken not to overly wet the pup because this could increase the risk of hypothermia. We do not use alcohol or a topical ophthalmic anesthetic. The ophthalmic anesthetic will not penetrate the skin, and we think that alcohol might irritate the pup’s facial skin.
Insert the needle, bevel down, at the 3 o’clock position into the eye socket (the area that will become the medial canthus) at an angle of approximately 30°. Mentally visualize the back of the socket and advance the needle to the area of the retro-orbital sinus.
Make the injection in a gentle, smooth, fluid motion. If the injection is successful, the lab personnel might observe blanching of the superficial temporal vein, but this does not always occur. Regardless of whether blanching is noted, we have seen the injectate in the target organs.
Withdraw the needle slowly, allowing the injectate to redistribute. We sometimes see a small drop of blood at the injection site, which can be gently cleaned with a cotton-tipped applicator.
Place the pup in the second prepared nest. When all the pups in a group have received injections, check each one for any additional bleeding and clean the blood, if necessary. Return the pups to their mother in the home cage.
Development of NEC after RBC transfusion
Check hematocrit values immediately after each phlebotomy and after RBC transfusion. Figure 6 shows a steady reduction from 51% ± 1.54 to 22.5% ± 0.67 from P1 to P11 in anemic mice groups. After RBC transfusion on P11 in a typical experiment, the hematocrit increased to 40.33% ± 1.28 in anemic groups versus 58.67% ± 1.11 in control groups.
After the RBC transfusion (on P11), sacrifice pups on P12 according to the institutional ethical guidelines, collecting blood and intestinal tissues for further analyses.
Figure 6. Hematocrit values in experimental groups: Line chart (mean ± SE) demonstrating serial reduction of hematocrit (Hct in %) during the sequence of daily phlebotomy in P1–10 mouse pups and a significant increase in hematocrit after red blood cell (RBC) transfusion in both anemic and control mouse pups. N = 6 mice per group. *p < 0.05; Šídák's multiple comparisons test. **p < 0.01, #p < 0.001 vs. P1 baseline; $p < 0.001 vs. pre-transfused.
Intestinal injury is marked by measuring intestinal fatty acid binding protein (i-FABP2) concentrations in the plasma of all four groups using a commercially available ELISA kit per the manufacturer’s protocol. The assay has a linear range of 78–5,000 pg/mL. As depicted in Figure 7, i-FABP2 levels were significantly increased in anemic-transfused groups compared with others.
Figure 7. Intestinal injury marker of plasma fatty acid binding protein (FABP2) level in experimental groups. Bar diagram (mean ± SE) summarizes plasma intestinal fatty acid-binding protein 2 (iFABP2) concentrations in naïve control, anemic control, transfusion control, and anemic-transfused mice. N = 8 mice per group. ***p < 0.001; Šídák's multiple comparisons test.
Consistent with FABP2 levels, histopathology analyses show that anemic mouse pups that received RBC transfusion developed intestinal injury in the ileocecal region with complete disruption of the crypt-villus axis, severe separation of the lamina propria and transmural necrosis (Figure 8).
Figure 8. Hematoxylin–eosin staining of the ileum (left) and colon (right) shows necrotizing enterocolitis (NEC) injury in anemic-transfused mice.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
MohanKumar, K. et al. (2019). A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions. Nature Communication.
General notes and troubleshooting
Troubleshooting
Problem 1
The experimental model solely relies on neonates; therefore, handling plays a crucial role.
Potential solution
Pups need to be held in appropriate positions by giving them maximum comfort. Make sure to reduce the stress while performing phlebotomy to the maximum. Do not disturb the nest while taking the pups away from the cage. Use soft facial tissues to wipe off the blood during phlebotomy. Wait until the dam moves away and gives some space to handle the pups. Making sure to clean the blood from the pups after bleeding improves their survival, because if the dam senses the blood smell, it may ignore the pups while nursing.
Problem 2
Identification of facial veins in neonatal mouse pups for phlebotomy.
Potential solution
The facial vein in murine neonates is well visible during P1–4 days; then, once fur has developed, it reduces the visibility of the facial vein. P5–10 mouse pups are kept in a box under warm lights to dilate the veins, which helps to visualize the dilated “skin dune” for phlebotomy.
Problem 3
Pups develop respiratory distress during oral gavage of S. marcescens using a 3.5 French catheter.
Potential solution
If the catheter is inserted into the trachea, it should be immediately withdrawn and reinserted back slowly. Ensure that there is no significant resistance to the insertion of the catheter.
Problem 4
Administration of stored RBCs may cause circulatory overload.
Potential solution
Acute administration of stored RBCs to neonatal mouse pups may cause circulatory overload, characterized by acute respiratory distress, tachycardia, increased blood pressure, and acute pulmonary edema. To reduce the risk of these iatrogenic conditions, it is important to carefully monitor the neonatal mouse pups after injection and maintain a warm temperature using warm lights. It is also important to train personnel on careful handling and intravenous injection of pups to avoid unintentional death. During the procedure of phlebotomy and/or RBC transfusion, if any of the experimental mouse pups display signs of distress, pain, or discomfort, they should be euthanized and autopsied to determine the cause of death.
Acknowledgments
We acknowledge Sysmex America Inc. for loaning the XN-1000TM hematology analyzer. The authors would like to thank Dr. Matthew Sandbulte, PhD, of the Child Health Research Institute for manuscript review and editing assistance. The research was supported by National Institutes of Health awards HL163043, HL133022, and HD105880 (to M.K.K). This protocol has been used in Nature Communications [24].
Competing interests
We have no competing interests to declare.
Ethic consideration
Institutional permissions: All mice were bred, maintained, and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under a study proposal approved by the IACUC (#23-004-04 FC). All experiments were performed in accordance with procedures approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC), and the Institutional Biosafety Committee (IBC).
References
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Holzapfel, L. F., Rysavy, M. A. and Bell, E. F. (2023). Red Blood Cell Transfusion Thresholds for Anemia of Prematurity. Neoreviews 24(6): e370-e376. https://doi.org/10.1542/neo.24-6-e370.
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Elabiad, M. T., Harsono, M., Talati, A. J. and Dhanireddy, R. (2013). Effect of birth weight on the association between necrotising enterocolitis and red blood cell transfusions in <=1500 g infants. BMJ Open 3(11): e003823. https://doi.org/10.1136/bmjopen-2013-003823.
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Saroha, V., Josephson, C. D. and Patel, R. M. (2019). Epidemiology of Necrotizing Enterocolitis: New Considerations Regarding the Influence of Red Blood Cell Transfusions and Anemia. Clin. Perinatol. 46(1): 101–117. https://doi.org/10.1016/j.clp.2018.09.006.
Christensen, R. D., Lambert, D. K., Henry, E., Wiedmeier, S. E., Snow, G. L., Baer, V. L., Gerday, E., Ilstrup, S. and Pysher, T. J. (2010). Is "transfusion-associated necrotizing enterocolitis" an authentic pathogenic entity? Transfusion 50(5): 1106–1112. https://doi.org/10.1111/j.1537-2995.2009.02542.x.
Cunningham, K. E., Okolo, F. C., Baker, R., Mollen, K. P. and Good, M. (2017). Red blood cell transfusion in premature infants leads to worse necrotizing enterocolitis outcomes. J. Surg. Res. 213: 158–165. https://doi.org/10.1016/j.jss.2017.02.029.
Killion, E. (2021). Feeding Practices and Effects on Transfusion-Associated Necrotizing Enterocolitis in Premature Neonates. Adv. Neonatal. Care. 21(5): 356–364. https://doi.org/10.1097/ANC.0000000000000872.
Marin, T. and Strickland, O. L. (2013). Transfusion-related necrotizing enterocolitis: a conceptual framework. Adv. Neonatal. Care. 13(3): 166–174. https://doi.org/10.1097/ANC.0b013e318285f901.
Nair, J. and Lakshminrusimha, S. (2019). Anemia, transfusion, feeding, and racial factors in the pathogenesis of transfusion-associated necrotizing enterocolitis. J. Perinatol. 39(7): 1016–1017. https://doi.org/10.1038/s41372-019-0389-7.
Rai, S. E., Sidhu, A. K. and Krishnan, R. J. (2018). Transfusion-associated necrotizing enterocolitis re-evaluated: a systematic review and meta-analysis. J. Perinat. Med. 46(6): 665–676. https://doi.org/10.1515/jpm-2017-0048.
Rose, A. T., Saroha, V. and Patel, R. M. (2020). Transfusion-related Gut Injury and Necrotizing Enterocolitis. Clin. Perinatol. 47(2): 399–412. https://doi.org/10.1016/j.clp.2020.02.002.
Cibulskis, C. C., Maheshwari, A., Rao, R. and Mathur, A. M. (2021). Anemia of prematurity: how low is too low? J. Perinatol. 41(6): 1244–1257. https://doi.org/10.1038/s41372-021-00992-0.
Rosebraugh, M. R., Widness, J. A., Nalbant, D. and Veng-Pedersen, P. (2013). A mathematical modeling approach to quantify the role of phlebotomy losses and need for transfusions in neonatal anemia. Transfusion 53(6): 1353–1360. https://doi.org/10.1111/j.1537-2995.2012.03908.x.
Maheshwari, A., Patel, R. M. and Christensen, R. D. (2018). Anemia, red blood cell transfusions, and necrotizing enterocolitis. Semin. Pediatr. Surg. 27(1): 47–51. https://doi.org/10.1053/j.sempedsurg.2017.11.009.
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Bazacliu, C. and Neu, J. (2019). Necrotizing Enterocolitis: Long Term Complications. Curr. Pediatr. Rev. 15(2): 115–124. https://doi.org/10.2174/1573396315666190312093119.
Bazacliu, C. and Neu, J. (2019). Pathophysiology of Necrotizing Enterocolitis: An Update. Curr. Pediatr. Rev. 15(2): 68–87. https://doi.org/10.2174/1573396314666181102123030.
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Fluorescent Labeling and Imaging of IL-22 mRNA-Loaded Lipid Nanoparticles
RM Rabeya Jafrin Mow
AS Anand Srinivasan
EB Eunice Bolay
DM Didier Merlin
CY Chunhua Yang
Published: Vol 14, Iss 10, May 20, 2024
DOI: 10.21769/BioProtoc.4994 Views: 1409
Reviewed by: Agnieszka ZienkiewiczRiddhi Atul Jani Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Biomaterials Sep 2022
Abstract
Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA. Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles. The ability to track LNPs in vivo is crucial for understanding their fate within biological systems. Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies. Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis. This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum. This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest.
Key features
• This protocol is developed to assess the delivery of IL-22 mRNA to ulcerative colitis sites using lipid nanoparticles.
• This protocol uses fluorescent DiR dye for imaging of IL-22 mRNA-loaded lipid nanoparticles in the gastrointestinal tract of mice.
• This protocol employs the IVIS spectrum for imaging.
Keywords: Oral gene delivery Biodistribution Gastrointestinal tract Ulcerative colitis
Background
In ulcerative colitis, interleukin-22 (IL-22) plays a crucial role by promoting mucosal healing and regulating the inflammatory response. Lipid nanoparticles (LNPs) offer a targeted delivery platform for IL-22 in this context, effectively harnessing the cytokine's therapeutic potential to address mucosal healing and inflammation precisely at the site of injury [1]. Despite the increasing interest in utilizing LNPs for targeted delivery of therapeutic agents in ulcerative colitis, our understanding of their in vivo behavior is limited, hindering the clinical translation of LNP-based therapies. The tracking of LNPs in vivo can provide crucial insights into their biodistribution, migration abilities, and mechanism of action [2]. Therefore, the development of efficient and sensitive techniques for labeling LNPs is highly desired.
To date, several methods have been developed to unravel the in vivo dynamics of LNPs. Notably, the use of fluorescent dyes to label LNPs stands out as an effective approach for confirming successful therapeutic delivery. This highly sensitive and selective technique enables real-time monitoring and visualization of nanoparticle behavior and distribution in biological systems. However, a potential limitation of fluorescent labeling is the risk of dye leakage from nanoparticles in vivo, resulting in diminished brightness over time and the development of a background signal that may hinder accurate nanoparticle localization [3].
In a prior study, we engineered LNPs loaded with IL-22 mRNA for treating ulcerative colitis, evaluating the biodistribution of DiR-labeled IL-22/LNP [4]. In this protocol, we will describe the detailed process of labeling and imaging IL-22/LNP in the gastrointestinal (GI) tract using a fluorescent dye via the IVIS spectrum. In our previously published study [4], LNPs loaded with mRNA as described in this protocol displayed a distinct signal in the targeted organ (colon) and exhibited therapeutic efficacy.
Materials and reagents
Biological materials
C57BL/6J mice (Jackson Laboratory, female, 6–7 weeks of age)
Reagents
Curved feeding needles (Kent Scientific, catalog number: FNC-20-1.5-2)
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DiR') (Thermo Scientific, InvitrogenTM, catalog number: D12731)
Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100)
Phosphate-buffered saline (PBS) (Corning, catalog number: 21-040-CV)
Laboratory supplies
15 mL conical centrifuge tube (Thermo Scientific, NuncTM 15 mL, catalog number: 339650)
Amicon® Ultra 15 mL centrifugal filter (Millipore, Ultracel®-100k, catalog number: UFC910024)
5 mL Eppendorf tube (Eppendorf, catalog number: 0030119401)
1 mL syringe PP/PE without needle (Sigma-Aldrich, catalog number: Z683531-100A)
Petri dish (CELLTREAT, catalog number: 229620)
Equipment
Micropipettes, 10–100 µL (Eppendorf, catalog number: 13-684-251)
Orbital shaker (CORNING, model: LSETM)
Centrifuge (Thermo Scientific, model: SORVALL ST-16R)
In vivo imaging system (PerkinElmer, model: IVIS Spectrum CT)
Software and datasets
Living Image software (PerkinElmer, IVIS® version: 4.7.4)
Procedure
Fluorescent labeling of IL-22 mRNA-loaded LNPs
Prepare 5 mL of IL-22 mRNA-loaded LNPs in a 15 mL conical centrifuge tube following the previously published protocol [5].
Dissolve 5 mg of DiR' powder in 1 mL of DMSO to make a DiR'/DMSO solution at a concentration of 5 mg/mL.
Add 10 µL of DiR'/DMSO solution to 5 mL of IL-22/LNPs in a 15 mL conical centrifuge tube (Figure 1).
Figure 1. Spiking a 10 µL DiR' solution into the IL-22/LNPs suspension
Cover the tube with aluminum foil and incubate the suspension mix at room temperature (RT) for 15 min (Figure 2, left) shaking at 100 rpm on an orbital shaker.
Figure 2. Incubating the lipid nanoparticles (LNPs) DiR' mix on an orbital shaker
Transfer the LNP suspension to 15 mL centrifugal filters, each containing 2.5 mL of LNPs (Figure 3).
Figure 3. Transfer the lipid nanoparticles (LNP) suspension to a centrifugal filter
Centrifuge at 4,696× g for 10 min at 10 °C.
Take out the filter (Figure 4).
Figure 4. DiR'-labeled lipid nanoparticles (LNPs) in the filter after centrifugation
Add 1 mL of PBS to each filter and reconstitute the suspension by repeated pipetting (> 100 times). Then, transfer the suspension to a 5 mL Eppendorf tube.
Oral gavaging of DiR'-labeled IL-22/LNPs
Take healthy mice and divide them into two groups (Control group and Treated group).
Fast the mice for 4 h before gavage.
Fill DiR'-labeled IL-22/LNPs suspension into a 1 mL syringe equipped with an animal feeding needle and remove all air bubbles (Figure 5).
Figure 5. Filling of DiR'-labeled IL-22/LNPs suspension into syringe
Restrain each mouse by manually grasping it and carefully insert the feeding needle into its mouth and esophagus to administer 200 µL of DiR'-labeled IL-22/LNPs suspension directly into the stomach of the treated group. To the control group, administer 200 µL of free LNPs suspension (without labeling) (Figure 6).
Figure 6. Gavaging of DiR'-labeled IL-22/LNPs suspension to mouse
Note: If any resistance is experienced, it may indicate incorrect positioning of the feeding needle; in such cases, retract the feeding needle and reposition it.
After gavage, fast the mice for 1 h.
Euthanize mice 24 h after being gavaged with mRNA-loaded LNPs.
Dissect the mice and collect their GI tract.
Imaging of DiR-labeled IL-22/LNPs biodistribution in mouse GI tract
Turn on the IVIS Spectrum CT instrument 45 min prior to the test.
Start Living Image software, click the initializing button, and wait until the temperature button turns green (Figure 7; CCD camera reaches -90 °C).
Place the GI tract (with and without DiR-labeled IL-22/LNPs) in the imaging chamber.
Set the imaging mode to fluorescence, excitation filter to 745 nm, and emission filter to 800 nm in the IVIS acquisition control panel (Figure 7).
Take imaging by selecting Acquire in the IVIS acquisition control panel and save the acquired pictures to the appropriate path (Figure 8).
Figure 7. Parameter for acquiring imaging of fluorescently labeled IL-22/LNPs
Figure 8. Living Image software. Left side: GI tract of the control mouse without IL-22/LNPs treatment. Right side: DiR-labeled IL-22/LNPs deposition in the gastrointestinal (GI) tract of the treated mouse.
Validation of protocol
Sung et al. [4]. Oral delivery of IL-22 mRNA-loaded lipid nanoparticles targeting the injured intestinal mucosa: A novel therapeutic solution to treat ulcerative colitis. Biomaterials (Supplementary Figure 10, panel A).
Acknowledgments
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-116306 to D.M.) and the Department of Veterans Affairs (Merit Award BX002526 to D.M.). D.M. is a recipient of a Senior Research Career Scientist Award (BX004476) from the Department of Veterans Affairs.
Competing interests
The authors declare no conflicts of interest within the work.
References
Yan, J., Yu, J., Liu, K., Liu, Y., Mao, C. and Gao, W. (2021). The Pathogenic Roles of IL-22 in Colitis: Its Transcription Regulation by Musculin in T Helper Subsets and Innate Lymphoid Cells. Front Immunol. 12: e758730.
Lindner, J. R. and Link, J. (2018). Molecular Imaging in Drug Discovery and Development. Circ: Cardiovasc Imaging. 11(2): e005355.
Abdel-Mottaleb, M. M., Beduneau, A., Pellequer, Y. and Lamprecht, A. (2015). Stability of fluorescent labels in PLGA polymeric nanoparticles: Quantum dots versus organic dyes. Int J Pharm. 494(1): 471–478.
Sung, J., Alghoul, Z., Long, D., Yang, C. and Merlin, D. (2022). Oral delivery of IL-22 mRNA-loaded lipid nanoparticles targeting the injured intestinal mucosa: A novel therapeutic solution to treat ulcerative colitis. Biomaterials. 288: 121707.
Alghoul, Z., Sung, J., Wu, K., Alpini, G., Glaser, S., Yang, C. and Merlin, D. (2023). Preparation and Characterization of IL-22 mRNA-Loaded Lipid Nanoparticles. Bio Protoc. 13(7): e4647.
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Reversible Photoregulation of Cell–Cell Adhesions With Opto-E-cadherin
CR Christopher A. Raab
SW Seraphine V. Wegner
Published: Vol 14, Iss 10, May 20, 2024
DOI: 10.21769/BioProtoc.4995 Views: 1300
Reviewed by: Munenori IshibashiJamie A. Davies Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Communications Oct 2023
Abstract
The cell–cell adhesion molecule E-cadherin has been intensively studied due to its prevalence in tissue function and its spatiotemporal regulation during epithelial-to-mesenchymal cell transition. Nonetheless, regulating and studying the dynamics of it has proven challenging. We developed a photoswitchable version of E-cadherin, named opto-E-cadherin, which can be toggled OFF with blue light illumination and back ON in the dark. Herein, we describe easy-to-use methods to test and characterise opto-E-cadherin cell clones for downstream experiments.
Key features
• This protocol describes how to implement optogenetic cell–cell adhesion molecules effectively (described here on the basis of opto-E-cadherin), while highlighting possible pitfalls.
• Utilises equipment commonly found in most laboratories with high ease of use.
• Phenotype screening is easy and done within a few hours (comparison of cell clusters in the dark vs. blue light in an aggregation assay).
• Three different functionality assay systems are described.
• After the cell line is established, all experiments can be performed within three days.
Keywords: Photoswitchable Cell–cell adhesions E-cadherin Reversibility Extracellular optogenetics 3D clustering Bottom-up tissue engineering
Graphical overview
Analysis and characterisation of light-dependent opto-E-cadherin expressing cells
Background
In multicellular organisms, many essential biological processes such as embryonic development, tissue integrity, collective cell behaviour, and migration, are regulated by cell–cell adhesions [1], while aberrations play a key factor in diseases such as arthritis, neuronal dystrophy, and tumour progression [2–4]. Epithelial cadherin (E-cadherin) is a well-known cell-adhesion protein. Upon binding of two E-cadherin molecules on neighbouring cells, intracellular proteins are recruited to the cytoplasmic tail and connect directly to the cytoskeleton, affecting transcriptional regulation [5]. Its loss of expression or change in function may lead to epithelial-to-mesenchymal transition (EMT), tumour development, and metastasis [6,7]. In healthy tissues, EMT of cells is dynamically and locally regulated, which may be reversed by mesenchymal-to-epithelial transition (MET). At the same time, EMT plays a central role in gastrulation and wound healing in adults [8]. Attempts to study the underlying mechanisms have relied on gain- or loss-of-function experiments, inhibition with antibodies, or the removal of Ca2+ ions, necessary for the homophilic interaction between E-cadherins [9–11]. However, none of these methods fully captures the spatio-temporal regulation in cell–cell adhesion during EMT. Attempts to mimic cell–cell adhesions through chemical modifications of the cell surface with synthetic cell adhesion molecules were neither sustainable nor dynamic [12,13]. To gain spatiotemporal control over cell–cell adhesions, various chemo-optogenetic [14,15] and optogenetic tools that utilise light to dissociate cell adhesions [16] and create artificial cell–cell adhesions have been successfully developed [17–21].
More recently, we developed a blue light switchable E-cadherin molecule (opto-E-cad), which allows us to reversibly and dynamically switch between ON and OFF states of E-cadherin [22]. In opto-E-cad, a light-oxygen-voltage sensing domain (LOV2) is embedded in the extracellular domain of E-cadherin such that, in the dark, cell–cell adhesion can form. Upon blue light absorption by its cofactor FMN or FAD, the LOV2 domain undergoes a conformational change, disturbing a critical Ca2+ binding site and turning OFF the opto-E-cad-based adhesions. In the dark, these conformational changes reverse, and the cell–cell adhesions can form again. Overall, opto-E-cad is fully genetically codable and provides bidirectional control over cell–cell adhesions with high spatiotemporal precision. At the same time, opto-E-cad allows us to link optogenetics to cellular signalling associated with E-cadherin-based cell–cell adhesions, providing a unique tool to suitably study EMT and MET processes and their impact on development and disease progression. As proof of concept, we have demonstrated the feasibility and ease-of-use of opto-E-cad in several invasion assays either with single cells and spheroids in vitro and in vivo, as well as its influence on transcriptional regulation similar to observed changes in gene expression during EMT via q-RT PCR [23]. When considering E-cadherin, varying expression levels and adhesion strengths are a determining factor in its function [24,25]. Thus, straightforward methods to characterise cells with different levels of opto-E-cad expression are necessary, which we describe here.
Materials and reagents
Biological materials
MDA-MB-231 human breast adenocarcinoma epithelial cells (American Type Culture Cells, catalog number: HTB-26)
L929 mouse fibroblast cells derivative of strain L (American Type Culture Cells, catalog number: CCL-1)
HeLa human cervix adenocarcinoma epithelial cells (American Type Culture Cells, catalog number: CCL-2)
A431D (Magaret J. Wheelock, origin: University of Toledo, Ohio [26])
Opto-E-cad-GFP plasmid (Addgene, catalog number: 203327)
Reagents
Dulbecco’s modified Eagle medium (DMEM) (PAN Biotech, catalog number: 04-03591)
Fetal bovine serum (FBS) (PAN Biotech, catalog number: P30-3031)
Penicillin-streptomycin (P/S) (Gibco, catalog number: 15140122)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4304)
Lipofectamine 3000 (Thermo Fisher, catalog number: L300001)
Geneticin (G418) (Roche, catalog number: 4727878001)
Phosphate-buffered saline (PBS) (Gibco, catalog number 18912014)
StemPro Accutase (Thermo Fisher, catalog number: A1110501)
Flavin adenine dinucleotide (FAD) (Carl Roth, catalog number: 6833)
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127)
Hank’s buffered salt solution (HBSS) (Gibco, catalog number: 12549069)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030)
Calcium chloride (CaCl2) (Carl Roth, catalog number: A119.1)
Ethylenediaminetetraacetic acid (EDTA) (Carl Roth, catalog number: 80423)
Triton X-100 (PanReac AppliChem, catalog number: not available)
Hoechst 3342 (Invitrogen, catalog number: H3570)
Phalloidin iFluor-594 (Abcam, catalog number: ab176757)
Cell tracker Green BODIPY dye (Invitrogen, catalog number: C2102)
Fluoromount GTM (Invitrogen, catalog number: 00595802)
Methyl cellulose (viscosity 15cP) (Sigma-Aldrich, catalog number: M7027)
Recipes
Growth medium
Reagent Final concentration Quantity or Volume
DMEM n/a 435 mL
FBS 10% 50 mL
P/S (10,000 U/mL) 1% 5 mL
HEPES (625 mM) 12.5 mM 10 mL
Total n/a 500 mL
Spheroid medium
Reagent Final concentration Quantity or Volume
DMEM n/a 470 mL
FBS 3% 15 mL
P/S (10,000 U/mL) 1% 5 mL
HEPES (625 mM) 12.5 mM 10 mL
Methyl cellulose (viscosity 15cP) 0.66% 3.3 g
Total n/a 500 mL
Note: Filter FBS through a 0.22 μm filter before adding it to DMEM for growth and spheroid medium.
Laboratory supplies
T-25 flasks (Sarstedt, catalog number: 83.3910.002)
6-well microplates (Greiner Bio-One, catalog number: 657160)
12-well microplates (Greiner Bio-One, catalog number: 665180)
96-well F-bottom microplates (Greiner Bio-One, catalog number: 655087)
96-well U-bottom suspension microplates (Greiner Bio-One, catalog number: 650185)
DNA LoBind microfuge tubes (Eppendorf, catalog number: 022431021)
24 mm × 24 mm glass coverslips (Roth, catalog number: HKE8.1)
Microscope slides (Thermo Scientific, catalog number: 15457544)
10 μL TipOne® pipette tips (Starlab, catalog number: S1111-3700-C)
200 μL TipOne® pipette tips (Starlab, catalog number: S1113-1706-C)
1,000 μL TipOne® pipette tips (Starlab, catalog number: S1111-6701-C)
Equipment
10 μL NeoLab pipette (Eppendorf, catalog number: A-0861)
100 μL NeoLab pipette (Eppendorf, catalog number: A-0864)
1,000 μL NeoLab pipette (Eppendorf, catalog number: A-0867)
CO2 incubator (PHCbi, model: MCO-170AICUVD-PE)
CO2 incubator with custom drilled cable inlets for LED panels (Thermo Fisher, model: Midi CO2 40L 3403)
Herasafe cabinet (Thermo Fisher, model: Herasafe 2030i)
Ultra centrifuge (Eppendorf, model: 5804)
Automated cell counter (Bio-Rad, model: TC20)
Cell sorter (BD Biosciences, model: FACSAria Fusion)
CLF flora LED (CLF Plant Climatics GmbH, model: not available)
Custom made LED panels and control modules. Equivalent self-made products can be found here [27]
Power meter (Thorlabs, catalog number: PM16-120)
3D orbital shaker (Grant-Bio, model: PSM3D)
Dmi8 brightfield microscope (Leica, model: Dmi8 automated)
Software and datasets
LAS X v.3.7.1.21655 (Leica, 25/01/2024)
ImageJ v.1.53f51 (ImageJ, 02/03/2023)
Procedure
Cell culture and generation of monoclonal opto-E-cad cell lines
The breast cancer cell lines MDA-MB-231, the skin cancer cell line A431D (A431, E-cadherin-deficient), HeLa, and the fibroblast cell line L929 were successfully tested and used with the opto-E-cad construct. The cells were cultured in DMEM without phenol red, supplemented with 10% FBS, 1% P/S (10,000 U/mL), and 12.5 mM HEPES at 37 °C and 5% CO2.
Transfect cells in a 24-well plate with 0.5 µg per well of opto-E-cad-GFP plasmid and Lipofectamine 3000 according to the manufacturer’s protocol.
Note: Transfection efficiency depends on the amount of DNA and transfection agent used and varies from cell line to cell line. Optimisation is recommended and efficiency can be visualised by fluorescent microscopy.
Two days after transfection, begin selecting cells with 1,800 μg/mL of G418 and maintain in the presence of selection medium for all further experiments.
Sort transfected cells into 96-well F-bottom plates as single clones after one week of cultivation with selection medium based on the fluorescent strength of GFP.
Note: When lifting cells for single-cell sorting, it is important to be gentle. Using Trypsin/EDTA is not recommended, as the E-cadherin molecules with the GFP-tag will be targets of proteolytic cleavage. Instead, Accutase as described below or 5 mM EDTA in PBS should be employed for lifting the cells.
Cultivate cells until monoclonal lines are established for characterisation [22]. This procedure may take 4–6 weeks.
Note: Monoclonal lines express different levels of Opto-E-cad-GFP on the cell membrane. The experiments described in Procedure B–D help identify clones that are suitable for downstream applications where adhesion strength (summary of E-cadherin adhesions) and/or fold change in dark vs. blue light states are of importance.
Critical: For reproducible results, it is imperative that presentation of Opto-E-cad-GFP on the outer membrane of cells is the same, as the amount is a determining factor in its function. Thus, attempting experiments with polyclonal variants is less likely to succeed.
Light-controlled cell–cell adhesions in 2D culture
Prior to the experiment, monoclonal stable opto-E-cad cell lines were seeded into T25 flasks at 20%–30% confluence and cultivated for three days.
Wash the cells briefly with 1 mL of PBS, discard, and add 0.5 mL of Accutase diluted 1:4 in HBSS. It takes approximately 10 min for the cells to detach.
Use cell growth medium to suspend and collect cells in 15 mL tubes. Subsequently, centrifuge cells at 600× g for 5 min at room temperature. Resuspend the cells in growth medium.
Place 24 mm × 24 mm glass coverslips to the bottom of a 6-well plate. Wash twice briefly with 70% ethanol, remove the liquid, and let the well dry. Count the cells and seed 8,600/cm2 onto the coverslips in a total volume of 2 mL of growth medium supplemented with 0.5 μM FAD either under blue light (20.4 μW/cm2) or in the dark for 4 h in the incubator with 5% CO2 at 37 °C (see Figure 1A).
Notes:
The addition of the co-factor FAD is necessary for Opto-E-cadherin to work.
Distance between the light source and sample and general setup affects light intensity. A power meter is useful to determine absolute power and irradiance values if there are concerns about opto-E-cad activation or phototoxicity.
Wash the cells briefly with 1 mL of PBS three times and fix in 500 μL of 4% PFA in PBS for 10 min at room temperature.
Wash the cell briefly three times with 1 mL of PBS before permeabilizing the cell membrane with 0.1% Triton X-100 in PBS for 5 min.
Wash twice with 1 mL of PBS and stain the cells with 1 µg/mL in nuclear dye (Hoechst) and 0.1 µg/mL actin cytoskeleton (Phalloidin iFluor-594) dyes in 500 μL of PBS for 1 h at room temperature.
Mount the cells with 15 µL of Fluoromount GTM onto microscope slides and acquire fluorescence images at 10× for an area of 1 cm2 using a motorised stage on the Dmi8 microscope (see Figure 1B).
Determine the number of cells using the nucleus staining and cell cluster area by Phalloidin iFluor staining using the particle analysis tool in ImageJ. Consider areas larger than 500–1,500 μm2 as cells and areas larger than 10,000 μm2 as clusters. The average MDA-opto-E-cad cell size in 2D is 950 ± 390 μm2. The image analysis tool has been previously established [17].
Figure 1. 2D cluster experiment setup. A. Light panel setup in the incubator. The panels are set on top of a 7 cm high frame above the cell samples, which are either kept under blue light or wrapped in aluminium foil. B. Example images of MDA opto-E-cad cells in the dark or under blue light for 4 h. Fixed and stained with Phalloidin iFluor-594. Scale bar is 500 µm.
3D clustering: Photoswitchable cell–cell adhesions in suspension culture
Prior to the experiment, cells were seeded into T25 flasks at 20%–30% confluence and cultivated for three days.
Remove growth medium from adherent cells in a T25 flask and wash twice briefly with 1 mL of PBS.
Add 0.5 mL of 5 mM EDTA and keep the flask in the incubator until cells are lifted. Depending on the cell confluency, this takes 5–10 min.
Add 5 mL of growth medium and subsequently transfer the cell suspension into a 15 mL Falcon tube.
Centrifuge the cells at 600× g for 5 min at room temperature.
Remove the supernatant and gently resuspend the cells in 3 mL of HBSS supplemented with 1% BSA and 2 mM Ca2+.
Determine the cell density in the suspension and verify if cells are indeed present as a single-cell suspension; otherwise, gently resuspend the cells some more.
Adjust the concentration to 5 × 104 cells/mL in HBSS supplemented with 1% BSA and 2 mM CaCl2 and add 0.5 µM (final conc.) of the FAD cofactor.
Aliquot the cell suspension to 1 mL per low-binding 1.5 mL microfuge tube and run every condition in duplicates.
On a 3D orbital shaker, place the tubes laterally and run the experiment for 1–6 h at room temperature (see Figure 2A). Use approximately 270 μW/cm light (light sample) or protect the dark sample from the light by wrapping in aluminium foil. Light conditions are typically changed every 30 min when performing experiments that rely on reversibility and bidirectional switching.
Note: The clustering results are affected by the orbital shaker settings; generally, an angle of 5° and 30 rpm works best in our hands.
Pipette 500 μL of 4% PFA into a 12-well cell culture plate per well. Use a cut 1,000 μL pipette tip to carefully transfer the cluster suspension from the tube into the PFA.
Note: The aim is to slightly increase the pipette tip opening to prevent potential clusters from being disrupted during the transfer into the 12-well plate.
Critical: Remove any bubbles from the wells with a pipette to ensure good image quality and easy analysis.
Acquire brightfield images of the entire well. Ideally, use a microscope with a scanning platform and subsequently use a stitching tool to merge single image files. Typically, low magnification objectives (4×) are sufficient to retain the sharp contrast between cell cluster and background during analysis, while keeping the number of acquired images to a minimum (see Figure 2C).
Analyse the clusters with ImageJ. As the cell clusters are 3D objects, the two-dimensional projected area of 5,000 μm equals approximately 20 MDA-MB-231 cells. Anything equal or larger is considered a cell cluster. A step-by-step analysis has been previously described [19].
Figure 2. 3D cluster experiment setup. A. Cell samples are placed on an orbital shaker that rotates 25× per minute at a 5° angle, under blue light or wrapped in aluminium foil. B. Schematic of opto-E-cad reversibility. C. MDA opto-E-cad cells alternating between dark conditions and blue light incubation every 30 min. Scale bar is 500 µm.
Light-controlled spheroid formation
Grow cells to 80% confluence in a T25 flask. Remove growth medium and wash cells twice briefly with 1 mL of PBS.
Dilute Cell Tracker Green BODIPY to a final concentration of 5 µM in 5 mL of serum-free medium and add to the cells. Incubate for 45 min in the incubator.
Wash cells twice briefly with 1 mL of PBS to remove excess dye. Detach the cells using 0.5 mL of Accutase diluted 1:4 in HBSS. Keep the cells in the incubator at 37 °C and 5% CO2. The cells lift from the flask within 5–10 min. If necessary, tapping the flask will facilitate the process.
Harvest cells with 5 mL of growth medium by pipetting, centrifuge at 600× g for 5 min at room temperature, remove the supernatant, and resuspend cells in 1 mL of growth medium.
Determine the concentration of the cell suspension and add 2 × 104 cells/mL to working spheroid medium (see Recipe 2) supplemented with 0.5 µM FAD.
Seed 100 µL of medium (2 × 103 cells) into 96-well suspension U-bottom plates.
Centrifuge the plates at 200× g for 3 min at room temperature for the cells to collect at the centre of the well.
Incubate the cells at 37 °C with 5% CO2 under blue light (20.4 μW/cm2) or in dark conditions up to three days.
Acquire images of every well with a brightfield microscope at 10× magnification (see Figure 3).
Determine the volume of the spheroids with the MATLAB code SpheroidSizer1_0 [28].
Figure 3. Representative images of cells in 3D culture. MCF7 cells and MDA opto-E-cad cells in the dark, form of compact spheroids overnight. MCF7 is present here to depict a spheroid formed from cells with high levels of native E-cadherin. MDA-MB-231 wild type and MDA opto-E-cad cells under blue light, form loose, ramified structures. Scale bar is 250 µm.
Data analysis
After image acquisition of 2D or 3D opto-E-cad cell clusters, ImageJ is a useful tool to quickly analyse differences in cell aggregation between dark and blue light conditions and reversibility. Below is a useful list of commands in IJ1 Macro language, which can be directly run as a macro for image analysis.
run("Sharpen");
run("8-bit");
run("Gaussian Blur...", "sigma=2");
run("Find Edges");
setAutoThreshold("Default dark");
waitForUser("adjust Threshold and press OK.");
//adjusting the threshold manually may be necessary to make sure that cluster edges are accurately distinguished from the background.
setOption("BlackBackground", true);
run("Convert to Mask");
run("Fill Holes");
//double-check image quality and manually remove stitching artefacts or dirt, which may be falsely recognised as a cell cluster.
run("Set Scale...", "distance=xxxx known=xxxx unit=um");
//set the scale to um if images are saved in a different unit from the microscope
waitForUser("Check Cluster accuracy and press OK.");
run("Analyze Particles...", "size=10000-Infinity display clear include add");
//the same commands can be used when analysing 3D clusters by changing the particle size detection to ≥ 5,000.
The experiments are run with two technical replicates and three biological replicates per condition. All comparisons are performed using Fisher’s one-way ANOVA test, and p < 0.05 was treated as the significance threshold.
The analysis method for the spheroid size in MATLAB has been described in detail [28]. The spheroids experiments are run with 60 technical replicates and three biological replicates per condition. All comparisons are performed using a two-tailed Student’s t-test, and p < 0.05 was treated as the significance threshold.
Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:
Mueller et al. [19]. The Importance of Cell-Cell Interaction Dynamics in Bottom-Up Tissue Engineering: Concepts of Colloidal Self-Assembly in the Fabrication of Multicellular Architectures. Nano Letters (Figures 1–3).
Rasoulinejad et al. [20]. Orthogonal Blue and Red Light Controlled Cell-Cell Adhesions Enable Sorting-out in Multicellular Structures. ACS Synthetic Biology (Figures 1–5).
Nzigou Mombo et al. [21]. Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell-Cell Interactions. Advanced Biology (Figures 1–3).
Nzigou Mombo et al. [22]. Reversible photoregulation of cell-cell adhesions with opto-E-cadherin. Nature Communications (Figures 1–3, 6). Source data can be found here [22].
General notes and troubleshooting
General notes
In the context of tissue formation, sorting, or cell invasion, it is important that cells express homogenous levels of cell adhesion molecules for reproducible results. The methods described here were primarily used to find and characterise opto-E-cad clones with good switching behaviour, i.e., large differences in cell clustering in the dark and under blue light. However, the experiments can be expanded to provide further valuable information.
2D clustering: Immunostaining of proteins involved in cell adhesion (e.g., p120 catenin) gives insights on how opto-E-cad influences signal transduction and is connected to the cytoskeleton in a spatio-temporal manner. This can be further elucidated with subsequent western blot and RNA expression analysis.
Spheroid formation: Spheroids formed from opto-E-cad cells can be used to study tumour invasion in collagen or by chorioallantoic membrane assay in vivo.
Troubleshooting
Problem 1: Cells do not cluster in 2D or 3D.
Possible causes: The expression level of opto-E-cad in the clone is not sufficient. / Cells were lifted with trypsin or incubated with Accutase for too long.
Solution: Prior to performing experiments, determine which clones have relative high expression levels of opto-E-cad by flow cytometry or fluorescent microscopy. If low levels of opto-E-cad expression are desired, reducing the rotation speed of the orbital shaker during 3D clustering allows weaker cell–cell adhesions to be sufficient for clustering. / Lift cells with EDTA or Accutase and avoid too-long incubation with proteases.
Problem 2: Cells cluster in 2D or 3D regardless of light condition.
Possible cause: The expression level of opto-E-cad in the clone is too high. Even though the binding affinity under blue light is greatly reduced, high expression levels may lead to sufficient cell–cell adhesions, resulting in cell clustering. Another possible cause can be that light intensities are too low for effective photoswitching.
Solution: Perform the experiments with opto-E-cad clones that have lower expression levels. Increase the light intensity but check for light toxicity with the untransfected cell line under the same conditions. Use a power meter to determine the absolute intensity values.
Problem 3: Opto-E-cad cells do not form compact spheroids, and rather loose and ramified structures persist.
Possible cause: FBS and insoluble components influence spheroid structure.
Solution: Filter FBS with a 0.2 µm filter before adding it to the spheroid medium. Decrease the concentration of FBS in the spheroid medium.
Acknowledgments
This work was funded by the European Research Council ERC Starting Grant ARTIST (# 757593, S.V.W) and published as Nzigou Mombo et al. [22].
Competing interests
The authors declare no financial or non-financial competing interests.
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4,996 | https://bio-protocol.org/en/bpdetail?id=4996&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Rearing and Shipping of Uranotaenia lowii, a Frog-Biting Mosquito
RS Richa Singh
NS Neil D. Sanscrainte
AE Alden S. Estep
KG Katherine González
XB Ximena E. Bernal
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.4996 Views: 228
Reviewed by: Luis Alberto Sánchez VargasEmily Jane DennisRubikah Vimonish
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Original Research Article:
The authors used this protocol in Journal Of Experimental Biology Dec 2023
Abstract
Many studies on mosquito biology rely on laboratory-reared colonies, emphasizing the need for standardized protocols to investigate critical aspects such as disease biology, mosquito behavior, and vector control methods. While much knowledge is derived from anthropophilic species from genera like Anopheles, Aedes, and Culex, there is a growing interest in studying mosquitoes that feed on non-human hosts. This interest stems from the desire to gain a deeper understanding of the evolution of diverse host range use and host specificity. However, there is currently a limited number of comprehensive protocols for studying such species. Considering this gap, we present a protocol for rearing Uranotaenia lowii, a mosquito species specialized in feeding on anuran amphibians by eavesdropping on host-emitted sound cues. Additionally, we provide instructions for successfully shipping live specimens to promote research on this species and similar ones. This protocol helps fill the current gap in comprehensive guidelines for rearing and maintaining colonies of anuran host–biting mosquitoes. It serves as a valuable resource for researchers seeking to establish colonies of mosquito species from the Uranotaeniini tribe. Ultimately, this protocol may facilitate research on the evolutionary ecology of Culicidae, as this family has recently been proposed to have originated from a frog-feeding ancestor.
Key features
• Rearing and maintenance of colonies of non-human host-biting mosquitoes that feed on frogs using host-emitted acoustic cues.
• Provides shipping guidelines aimed to enhance the establishment of colonies by new research groups and specimen exchanges between labs.
Keywords: Non-human host-biting mosquitoes Egg Eavesdropper Larva Pupa Adult Colony Blood feeding Uranotaeniini Uranotaenia lowii Husbandry
Background
An essential component in the comprehensive study of mosquitoes involves sustaining laboratory populations that allow experimental manipulations. Mosquito colonization enables in-depth exploration of life history traits, behavior, and the physiological mechanisms underlying them. Colonization of mosquitoes of medical importance, such as Anopheles gambiae, Aedes aegypti, and Culex pipiens, has, for instance, accelerated the rate of understanding of the biology of these anthropophilic species [1–4]. Establishing and maintaining robust and standardized mosquito colonies requires the development and application of well-established protocols as well as a good understanding of the key factors influencing mosquito fitness in captivity. Recent interest in mosquitoes that feed on non-human hosts has increased due to the increased recognition of the implications that mosquitoes feeding on other hosts can have in the ecosystem [5,6]. Until now, however, little was known regarding the rearing techniques and successful establishment of laboratory populations of mosquitoes with non-human hosts. Among those, mosquitoes that feed on frogs provide a valuable opportunity to understand the evolutionary ecology of Culicidae, as this family has recently been proposed to have originated from a frog-feeding ancestor [7]. While some mosquitoes are generalists and opportunistically feed on frogs as well as other hosts, other mosquitoes are specialized in feeding exclusively on anurans. The use of host-emitted acoustic signals, such as the mating calls of frogs for instance, is an adaptation that has evolved independently multiple times across flies and mosquitoes to detect and localize anuran hosts [8]. Among such frog-biting mosquitoes, Uranotaenia lowii is characterized by its preference for anuran hosts [9] and the use of auditory cues to locate them [10,11]. To date, no detailed documented studies have outlined protocols for maintaining laboratory colonies for Ur. lowii (but see Chapman [12]) or any other frog-biting mosquito species. Here, we present a standardized protocol for the rearing and colony maintenance of Ur. lowii mosquitoes. We also include a protocol for effectively shipping Ur. lowii specimens to promote specimen exchange between research groups.
Materials and reagents
Biological materials
Hosts for blood feeding
Uranotaenia lowii feed from anuran hosts in nature [9]. In contrast to mosquito species from genera like Aedes and Anopheles, which can often be raised using artificial blood diets [13], it is recommended to feed frog blood for the rearing of Ur. lowii. Field host surveys show that this species feeds on frogs from different families [9], so a variety of anuran species are likely to support a colony of Ur. lowii. We recommend, however, the following hosts to be used for blood feeding, as they successfully promote robust egg production: barking tree frog (Dryophytes gratiosus), Cuban tree frog (Osteopilus septentrionalis), cane toad (Rhinella marina), and southern toad (Anaxyrus terrestris) [12,11]. These anuran species can be procured from local shops or online stores (e.g., https://joshsfrogs.com/ or https://www.backwaterreptiles.com/).
Note: Protocols for using anurans for blood feeding must be approved by the appropriate Institutional Animal Care and Use Committee.
Uranotaenia lowii
This protocol describes the rearing and maintenance of Ur. lowii strain MFRU-FL (NCBI BioSample: SAMN33601576). It is to be expected, however, that these guidelines are transferable to other strains of this species. This protocol also provides a foundation for developing similar approaches to maintaining colonies of other Uranotaenia frog-biting species (i.e., de Silva et al. [14]). The colonies were originally established from egg rafts obtained from the USDA-ARS-CMAVE Mosquito and Fly Research Unit (Gainesville, FL, USA).
Reagents
Deionized water
Potbelly pig chow powder finely sieved (i.e., Mazuri® Mini Pig Youth); grind using a porcelain mortar and pestle (Letoyi, catalog number: B08LB3TJL4) and sieve through a fine mesh strainer (Clscea, catalog number: B0CBDFW92V)
Bovine liver powder (i.e., MP Biomedicals, catalog number: 290039601)
Brewer’s yeast (i.e., MP Biomedicals, catalog number: 290331205)
White sugar (e.g., Domino Cane Granulated Sugar, catalog number: 83FF)
Bleach (Clorox, catalog number: 30966, concentrated regular bleach)
70% ethanol (sterile 70% denatured ethanol) (Texwipe, catalog number: TX3265)
Solutions
Liver powder suspension (larval food) (see Recipes)
Sucrose solution [10% (w/v) adult food] (see Recipes)
Recipes
Liver powder suspension (larval food)
Reagents Quantity or Volume
Bovine liver powder 3 g
Brewer’s yeast 2 g
Deionized H2O 100 mL
Note: Shake well before each use. Can be stored at 4 °C for a week.
Sucrose solution (adult food)
Reagents Quantity or Volume
White sugar (e.g., Domino Cane Granulated Sugar) 10 g
Deionized H2O 100 mL
Note: Mix the reagents well before use.
Laboratory supplies
While we provide manufacturers’ names and models for some items, we are not endorsing or promoting those specific products. Instead, we offer them as examples and, when possible, suggest low-cost alternatives to facilitate accessibility for a wider range of laboratories.
Cotton balls, size: 5 cm, prepared using any cotton roll (e.g., Intrinsics, catalog number: 227200)
Transfer pipettes [Fisherbrand Transfer Pipette, 4.6 mL (Fisher Scientific, catalog number: 13-711-274MD)], or wood applicator sticks (i.e., Fisher Scientific, catalog number: 22-363-158), or plastic spoons obtained from stores for transferring egg rafts
Scissors (8-inch titanium scissors) (Westcott, catalog number: 13901)
Brown, non-white, seed germination paper (38-lb regular weight creped seed germination paper) (Anchor Paper Company, catalog number: NC1466201). Prepare strips of 17.5 cm length × 8 cm width using scissors
Labeling tape (Write-On label tape, white, 1/2" roll) (Research Products International Corp, catalog number: 560172)
Manual insect aspirator (Forestry Suppliers, catalog number: 53758)
Digital thermometer and hygrometer (i.e., ThermoPro TP49 Digital Hygrometer; temperature range: 14 °C–70 °C; humidity range: 10%–99%)
Oviposition and emergence cups: plastic cup (Rubbermaid 2-Cup, catalog number: 7J60)
Larval rearing trays; plastic trays, size: 38 cm length × 30 cm width × 7 cm depth (any polypropylene plastic tray can be used, e.g., United Scientific Supplies, catalog number: 81702)
Shipping supplies
Cardboard box (small to medium to fit the Styrofoam box; Uline outer shipping carton with insulated Foam Shipping Kit, catalog number: S-12682)
Styrofoam box with a lid (Uline Insulated Foam Shipping Kit, catalog number: S-12682)
Bubble wrap (Uline Air Bubble Wrap Roll, catalog number: S-5120)
Small Petri dishes (i.e., one 75 mm diameter petri dish can contain around 10 egg rafts) (non-sterile transparent Polypropylene Petri dish: United States Plastic Corp., catalog number: 89824)
Germination paper cutout in circles (diameter determined by Petri dish diameter) (Anchor Paper Company, 38 lb regular weight creped seed germination paper, catalog number: NC1466201)
Ice packs (two minimum) (Uline cold packs, catalog number: S-7890)
Industrial tape 2 Mil (Uline, catalog number: S-423)
Equipment
Environmental chamber
The use of environmentally controlled chambers (insectaries) dedicated to mosquito rearing is encouraged (e.g., Percival WE-35VL, Boone, IA). However, it is possible to maintain the colony in a small room in which temperature and humidity are maintained close to target values (temperature: 22–28 °C, humidity 50%–80%, 12:12 h light/dark photoperiod).
Note: If there is no environmental chamber, the relative humidity within the cage can be increased by placing a humidifier (e.g., Vick humidifier, catalog number: V745A/V745-JUV; not restricted to any specific manufacturer) near the rearing cage and covering the entire setup with a plastic shelf cover (e.g., Formosa covers premium wire shelf cover, heavy-duty storage solution for wire shelving rack, #shelf 4919 pvc off white). The humidifier must be regularly filled with deionized water to regulate the relative humidity in the enclosure.
Frog restraining cage
A cylindrical cage (height: 8 cm; diameter 12 cm) made entirely of mesh. The top mesh can be replaced by an acrylic circular sheet (i.e., PET Thick Plexiglass sheet; 3 mm, Uline Acrylic sheet, catalog number: S-22486) to observe blood-feeding behavior during experiments. The frog restraining cage should be made of a robust mesh that has holes big enough (1 cm) for the mosquitoes to enter but not too large for the frog to escape (e.g., Home Intuition Heavy Duty Plastic Gutter Guards Mesh, catalog number: B07D91Y8BC).
Note: The use of a frog restraining cage is essential for limiting the movement of the frogs to increase the likelihood of blood feeding. Limited host movement promotes feeding opportunities and reduces frog defensive behaviors such as swatting, a common strategy used by anurans to repel attacking mosquitoes and midges (i.e., de Silva et al. [15]). Additionally, restraining the frogs prevents them from consuming mosquitoes. It is also advisable to provide a substantial meal for the frog before placing it in the restraining cage, so they are less motivated to eat the mosquitoes.
Mosquito rearing cage
Mosquito cages that provide access through a sleeve (e.g., BioQuip lightweight aluminum collapsible cages; 46 cm × 46 cm × 46 cm; 2 mm × 2 mm mesh size; Mosquito Rearing Cage, catalog number: 4S4545).
Procedure
Environmental conditions for rearing Ur. lowii
Rear Ur. lowii (sex ratio 1:1) in an insect cage inside an insectary room with controlled environmental conditions (temperature: 22–28 °C, humidity 50%–80%, 12:12 h light/dark photoperiod). Mosquitoes require constant access to moisture, and they perform best in humid environments.
Blood feeding and oviposition
Egg production starts with adult females blood feeding on a frog. As in other species of mosquitoes, only female mosquitoes feed on blood, which provides the nutrients necessary for egg development. To promote feeding, place an adult frog in a frog restraining cage inside the rearing cage for 3 h (Figure 1A).
Place oviposition cups half-filled with deionized water (250 mL) and semi-submerged strips of brown seed germination paper for egg laying inside the mosquito cage immediately after removing the frog (Figure 1B). Females take two days to start laying eggs after feeding on their anuran host.
Note: When placing the frog in the restraining cage, it is important to include moist paper towels at the base to create a humid and comfortable environment for the frog. After 3 h of feeding, remove the frog from the restraining cage and place them back in the housing tank. Additionally, ensure abundant food and water are provided after each use to support their well-being. To reduce stress on the frogs, refrain from using the same individual repeatedly, and instead rotate the use of different individual frogs. Increasing the time between feedings using the same individual frogs as much as possible is advisable. Allowing a recovery time of a month for the same individual frog has worked well in promoting good health. For routine maintenance, mosquito colonies should be fed with blood every other week. Before blood feeding, remove the cotton balls containing the 10% sucrose solution. The age at which Ur. lowii mosquitoes first blood feed has not been investigated. Based on our observations, female Ur. lowii mosquitoes successfully feed from a frog after one week of age. Hence, we propose this timeframe for the initial blood meal offered to adult mosquitoes.
Figure 1. Blood feeding setup for Ur. lowii colony. A. Cane toad placed in a frog restraining cage built with mesh and topped with a transparent plexiglass lid. B. Frog restraining cage placed inside the mosquito rearing cage with adult Ur. lowii. Oviposition cups half-filled with water and brown germination paper are placed along the walls of the rearing cage. No cotton balls containing sucrose solution are present to promote frog-feeding behavior.
Egg collection and hatching
Like other species from the subfamily Culicinae, Ur. lowii also produces egg rafts [16]. Blood-engorged females lay egg rafts in oviposition cups, which should then be moved using a transfer pipette to rearing trays filled with 1.5 L of deionized water (water temperature 20–26 °C) (Figure 2A). To facilitate the uptake of the egg rafts without damaging them, we suggest using a transfer pipette after cutting the tip to increase the opening (5 mm wide) and a plastic spoon or wood applicator sticks to gently lift the rafts out of the water without breaking apart.
Once the egg rafts are transferred to the larval trays, add a pinch (0.16 g) of potbelly pig chow powder finely sieved to promote hatching. Label the tray with the hatching date, experiment type, or other relevant information. A maximum of 10–15 egg rafts should be placed in each tray marked at the level equivalent to 1.5 L to avoid overcrowding.
Place fresh oviposition cups half filled with deionized water daily in the adult mosquito cage to promote oviposition of new egg rafts. After exposure to a frog in the restraining cage, females lay eggs for approximately a week, so regular monitoring and transferring of egg rafts into rearing trays is repeated daily until no additional rafts are laid.
Note: Egg rafts of Ur. lowii are oviposited directly into the water [17] and die if they desiccate. Unlike the eggs of other mosquitoes, such as Ae. aegypti whose eggs can be stored for several months [3], the egg rafts of Ur. lowii cannot be stored long term. Hatching can be delayed by 1–2 days by storage at 4–8 °C, but their viability under these conditions rapidly decreases.
Figure 2. Life cycle of Ur. lowii and colony care at each developmental stage. A. Egg raft stage: rafts are laid inside oviposition cups half-filled with deionized water. B. Larval stage: larval development involves four instar stages that take place in the rearing trays with deionized water feed with liver powder suspension (see details under larval rearing). C. Pupal stage: manually picked pupae placed in half-filled emergence cups. D. Adult stage: adult rearing cage equipped with half-filled water cups with germination paper, adult food in cotton balls containing 10% sucrose solution and a digital thermometer and hygrometer to record environmental conditions.
Larval rearing
Egg rafts take two days to hatch into larvae in a rearing tray filled with 1.5 L deionized water when maintained at a density of 1.3–2 larvae per cm2 of surface area at approximately 20 °C water temperature. Larvae can be counted manually. Maintain the water level for each tray during the entire rearing process.
Provide larval food as liver powder suspension every other day. Feed each tray with 30 mL of liver powder suspension (Figure 2B). Feed the larvae with a transfer pipette by placing the food on the bottom of the tray, then mix the food well until the water turns light brown. Ensure that the food is evenly distributed across the tray.
Note: Water in rearing trays might dry out, so it is necessary to refill the tray with deionized water until reaching the mark of 1.5 L. When feeding the larvae, check the color and scent of the water in the trays. If the water appears dark brown, emits a strong odor, or if dead larvae are noticed near the bottom, it is advisable to promptly move the mosquito larvae to a clean container filled with clean deionized water and recently mixed food.
Pupation
Upon reaching the fourth instar stage, larvae take approximately three days to pupate. Pick pupae manually from the larvae trays using a transferring pipette (cut off the tip by 2 cm to increase the opening so it is 5 mm wide) to move them into half-filled emergence cups.
Place the emergence cups back into the adult cage (Figure 2C). No food is to be provided as pupae do not feed. Repeat the above procedure daily for pupae collection until all larvae have reached this developmental stage.
Note: It is necessary to regularly remove the pupae from the larval trays and place them into an adult cage at least once a day to ensure that no recently emerged adult mosquitoes escape. In our experience, the pupal phase is the most sensitive period in Ur. lowii development. Therefore, it is also important to ensure that the cups containing pupae are not overcrowded. It is recommended to put 50 pupae per cup.
Adult
Pupae emerge into adults after two days in the pupal stage. Provide constant access to adult food of 10% (w/v) sucrose solution in cotton balls and water wicks in the adult insect rearing cage (Figure 2D). To make sure mosquitoes can land safely to feed without being trapped by loose threads, compact the cotton by rolling each ball with the palm of your hands before soaking them with sugar solution (10% g/mL or one tablespoon of sugar in 100 mL of deionized water).
Place the containers with pupae inside the rearing cage in an evenly distributed pattern to make sure food is easily accessible for all individuals. Replace food and water on alternate days to prevent mold growth or fermentation of the sugary solution. While changing food and water, mosquitoes may try to escape, so shake the containers checking that there are no adults perched on them before removing the containers from the cage. Use a manual aspirator to catch any mosquitoes that may try to escape and place them back in the cage.
Note: Both male and female mosquitoes need access to water and sugar sources for reproduction and survival. Although adult mosquitoes require substantially less careful monitoring than the larval and pupal phases, it is necessary to perform daily checks of the cages containing adult mosquitoes to ensure that they have continuous access to the sugary solution and water.
Maintenance
Maintain and regularly monitor the required temperature, humidity, and light conditions. We recommend daily checks of the condition of the colony.
If humidifiers are used, they should be filled with deionized water as often as necessary to maintain the target relative humidity relatively constant. A 1.5-gallon humidifier (see Equipment for details) needs to be filled daily.
Ensuring proper larval and adult feeding can help in maintaining healthy colonies. Blood feeding every other week can lead to longer lifespans of mosquitoes. To avoid overcrowding, it is recommended to have a colony size of around 2,000 mosquitoes in the aforementioned rearing cage.
Clean and disinfect the frog restraining cage, emergence and oviposition cups, aspirator collection tubes, etc., by rinsing them in hot water or soaking them for at least 15 min in 10% bleach after every use. Rinse thoroughly and avoid using soap.
Regularly maintain mosquito cages by replacing the deionized water and cotton balls containing sucrose solution and aspirating dead mosquitoes every other day. Clean cages with 70% ethanol every six months.
The eggs of Ur. lowii mosquitoes cannot be stored. To dispose of eggs, leave them overnight in a cup with 10% bleach before discarding them into the sink. Similarly, excess larvae or pupae can be transferred to a container with 10% bleach. Check the container for larvae before discarding it into the sink and run hot water for several minutes afterward. The excess adults should be kept in a freezer in a container overnight before disposal.
Shipping egg rafts
The egg raft stage is recommended for shipping, as adults are prone to higher mortality during shipping conditions. As elevated mortality has been observed during the larval stage in laboratory conditions, we discourage shipping specimens at this stage.
Preparation
In a small plastic Petri dish (diameter: 75 mm), up to 10 egg rafts can be shipped on germination paper. First, prepare by cutting the germination paper into approximately 50 small circles of 75 mm in diameter using scissors, so they fit tightly within the Petri dish. To create a depression that is safe for the egg rafts to travel, use these circular germination papers to have two types: (i) unmodified circles, and (ii) donut-shaped circles where there is a single, central small hole (20 mm diameter) in each piece. At the bottom of the Petri dish, add a base layer of unmodified circles of germination paper until it reaches approximately 1/3 of the height of the dish. Then, place donut-type germination paper circles to create a depression that is approximately 10 mm deep. Add deionized water to completely dampen the germination paper and create a shallow water pool for the egg rafts (Figure 3). Use a transfer pipette or a wooden applicator stick to carefully transfer up to 10 egg rafts, so they are floating on the water. Check that the egg rafts are not upside down when placing them in the pool. Once the egg rafts are placed, add one unmodified circle of germination paper to cover the egg rafts.
Note: Plan to feed the colony 3–4 days before the expected shipment day, so you can obtain egg rafts as close to that day as possible. Shipping should occur as soon as the mosquitoes lay the eggs to avoid hatching during shipment. Consider the weather during shipping, as extreme, low, or high temperatures can expose the package to deadly conditions for the mosquitoes. When possible, shipping the mosquitoes during the spring and fall when conditions are mild should be favored. It is important to note that the volume of water for the egg rafts should be large enough to allow the egg rafts to float on the surface but not too high to result in turbulence that can displace the egg rafts out of the pool where they can desiccate.
Packing, shipping, and receiving egg rafts
Place the Petri dish lid and, using tape, secure it to the bottom, sealing it tightly to minimize leaking. Once the Petri dish is ready, place it in the middle of a Styrofoam box and add ice packs to compress the Petri dish(es) (Figure 3). Fill the space of the box with bubble wrap to avoid movement of the Petri dish inside the box during transportation. The Styrofoam should be tightly closed, securing the lid with abundant tape and placed in a cardboard box. Before shipping, add large visible arrows indicating which side goes up and place the shipping label. Since the eggs take 48 hours to mature, shipping overnight is recommended.
Figure 3. Packing of egg rafts for shipping. A. Egg rafts floating in the water pool made in the germination paper. B. Sealed Petri dishes with egg rafts inside placed in a Styrofoam box equipped with ice packs. Fill empty places with bubble wrap for shipping.
On the receiving end, carefully take out the Petri dishes and transfer the eggs to larval trays by gently moving the germination paper into the tray and adding deionized water, so the eggs continue to float as the water level increases. The germination paper can be removed from the trays once the water level has elevated the egg rafts.
Data analysis
This protocol does not directly involve data analysis, but it is the foundation for producing specimens that allow the implementation of diverse experimental approaches to address questions using frog-biting mosquitoes. For example, this protocol facilitated the data acquisition for neurophysiological and behavioral experiments conducted on mosquitoes reared using this method [11].
Validation of protocol
This protocol has been validated by implementing it in four different laboratories across different institutions: 1) Fly and Mosquito Research Unit, Center for Medical, Agricultural & Veterinary Entomology, Agricultura Research Service, USDA, Gainesville, FL 32608; 2) Laboratory of Neurogenetics and Behavior, HHMI – The Rockefeller University, New York, NY 10065; 3) Texas Tech University, 2500 Broadway W, Lubbock, TX 79409; 4) Sensory and Behavioral Ecology Lab, Purdue University, West Lafayette, IN 47907. During this process, it was validated using different anuran hosts and improved and refined to produce this cost and time-efficient approach to rearing a sustainable colony of frog-biting mosquitoes [11].
Acknowledgments
The authors would like to thank Kelly Anderson, Catherine (McDermott) Lee, Lucy Li, Lettie Cronin, and Chanakya Bhosale for helping with establishing and maintaining the original Uranotaenia lowii colony at USDA-ARS-CMAVE. We are also grateful to Hoover Pantoja-Sanchez and Shilpi Singh who provided support in moving the colony to Purdue University. We appreciate the help from many undergraduate researchers who have been involved in maintaining the colony over the years. Richa Singh was supported by a Fulbright-Nehru grant (Award No. 2821 FNPDR/2022). The Ur. lowii colony at Purdue University is supported by the National Science Foundation (IOS-2054636 to X.E.B.).
Competing interests
The authors declare no competing or financial interests.
Ethical considerations
The protocol for using anurans for blood feeding was approved by the Institutional Animal Care and Use Committee at Purdue University (Protocol no. 2101002102A001).
References
Leite, L. N., Bascuñán, P., Dotson, E. M. and Benedict, M. Q. (2023). Considerations for Rearing and MaintainingAnophelesin the Laboratory. Cold Spring Harb Protoc. 2024(3): pdb.top107802.
Kauffman, E., Payne, A., Franke, M., Schmid, M., Harris, E. and Kramer, L. (2017). Rearing of Culex spp. and Aedes spp. Mosquitoes. Bio Protoc. 7(17): e2542.
Clemons, A., Mori, A., Haugen, M., Severson, D. W. and Duman-Scheel, M. (2010). Culturing and Egg Collection of Aedes aegypti. Cold Spring Harb Protoc. 2010(10): pdb.prot5507.
Meuti, M. E., Siperstein, A. and Wolkoff, M. (2023). Rearing and Maintaining a Culex Colony in the Laboratory. Cold Spring Harb Protoc. 2023(8): pdb.prot108080.
Lehane, M. J. (2005). The biology of blood-sucking in insects. Cambridge University Press.
Trillo, P. A., Bernal, X. E. and Hall, R. J. (2023). Mixed-species assemblages and disease: the importance of differential vector and parasite attraction in transmission dynamics. Philos Trans R Soc Lond B Biol Sci. 378(1878): e0109.
Soghigian, J., Sither, C., Justi, S. A., Morinaga, G., Cassel, B. K., Vitek, C. J., Livdahl, T., Xia, S., Gloria-Soria, A., Powell, J. R., et al. (2023). Phylogenomics reveals the history of host use in mosquitoes. Nat Commun. 14(1): 6252.
Campos, L., Oliveira, S., Kvifte G. and Bernal, X. E. (In review). The diverse and intricate strategies of flies interacting with amphibians: Host-use patterns, mechanisms, and opportunities. Annu Rev Entomol.
Reeves, L. E., Holderman, C. J., Blosser, E. M., Gillett-Kaufman, J. L., Kawahara, A. Y., Kaufman, P. E. and Burkett-Cadena, N. D. (2018). Identification of Uranotaenia sapphirina as a specialist of annelids broadens known mosquito host use patterns. Commun Biol. 1(1): 92.
Borkent, A. and Belton, P. (2006). Attraction of female Uranotaenia lowii (Diptera: Culicidae) to frog calls in Costa Rica. Can Entomol. 138(1): 91–94.
Pantoja-Sánchez, H., Leavell, B. C., Rendon, B., de-Silva, W. A. P. P., Singh, R., Zhou, J., Menda, G., Hoy, R. R., Miles, R. N., Sanscrainte, N. D., et al. (2023). Tiny spies: mosquito antennae are sensitive sensors for eavesdropping on frog calls. J Exp Biol. 226(24): e245359.
Chapman, H. C. (1970). Colonization of Uranotaenia lowii Theobald (Díptera: Culicidac). Mosquito News, 30(2).
Baughman, T., Peterson, C., Ortega, C., Preston, S. R., Paton, C., Williams, J., Guy, A., Omodei, G., Johnson, B., Williams, H., et al. (2017). A highly stable blood meal alternative for rearing Aedes and Anopheles mosquitoes. PLoS NeglTrop Dis. 11(12): e0006142.
de Silva, W. P. P., Bernal, X. E., Chathuranga, W. D., Herath, B. P., Ekanayake, C., Abeysundara, H. T. K. and Karunaratne, S. H. P. P. (2020). Feeding patterns revealed host partitioning in a community of frog‐biting mosquitoes. Ecol Entomol. 45(5): 988–996.
de Silva, P., Jaramillo, C. and Bernal, X. E. (2014). Feeding Site Selection by Frog-Biting Midges (Diptera: Corethrellidae) on Anuran Hosts. J Insect Behav. 27(3): 302–316.
Clements, A. N. (1992). The biology of mosquitoes. Volume 1: Development, Nutrition and Reproduction. Chapman & Hall.
Hinton, H. (1968). Structure and protective devices of the egg of the mosquito Culex pipiens. J Insect Physiol. 14(2): 145–161.
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Visualising Neutrophil Actin Dynamics in Zebrafish in Response to Laser Wounding Using Two-Photon Microscopy
IW Ivanna Williantarra
AG Antonios Georgantzoglou
MS Milka Sarris
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.4997 Views: 581
Reviewed by: Alberto RissoneIvonne Sehring Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Jun 2022
Abstract
Cells need to migrate along gradients of chemicals (chemotaxis) in the course of development, wound healing, or immune responses. Neutrophils are prototypical migratory cells that are rapidly recruited to injured or infected tissues from the bloodstream. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein. Neutrophil chemotaxis has been largely studied in vitro; few assays have been developed to monitor gradient responses in complex living tissues. Here, we describe a laser-wound assay to generate focal injury in zebrafish larvae and monitor changes in behaviour and cytoskeletal dynamics. The first step is to cross adult fish and collect and rear embryos expressing a relevant fluorescent reporter (for example, Lifeact-mRuby, which labels dynamic actin) to an early larval stage. Subsequently, larvae are mounted and prepared for live imaging and wounding under a two-photon microscope. Finally, the resulting data are processed and used for cell segmentation and quantification of actin dynamics. Altogether, this assay allows the visualisation of cellular dynamics in response to acute injury at high resolution and can be combined with other manipulations, such as genetic or chemical perturbations.
Key features
• This protocol is designed to trigger laser wound in zebrafish larvae using two-photon intravital microscopy.
• The ability to wound while imaging makes it possible to monitor the behaviour and actin changes of the cells immediately after gradient exposure.
• The protocol requires a two-photon microscope for best results. Compared with one-photon laser wounding, the injury is more precise and has better tissue penetration.
• The focal nature of the wounds is suitable for studies of neutrophil swarming/aggregation and can be further adapted to infectious settings.
Keywords: Cell migration Neutrophil Two-photon imaging Laser wounding Actin dynamics Chemotaxis
Background
Directed migration along gradients of chemicals (chemotaxis) is fundamental to many developmental and physiological processes. Immune cells represent prototypical migratory cells that rely on active motion to search tissues, find pathogens, and interact with other cells in order to launch immune responses. Among these cells, neutrophils are the first to be recruited to injured or infected tissues from the bloodstream [1]. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein, including primary chemoattractants produced by microbes or damaged cells (e.g., formyl peptides) or secondary attractants (i.e., attractants produced by inflamed tissue cells or by neutrophils themselves, such as chemokines and leukotriene B4) [1,2]. Understanding neutrophil chemotaxis mechanisms is important for ultimately controlling the accumulation of these cells in tissues in inflammatory disease settings, but also for providing mechanistic paradigms for understanding how other types of cells undergo directional motion in general.
Neutrophil motility and gradient sensing have been studied in vitro in settings where gradients can be administered in a controlled fashion. For example, assays where micropipettes are introduced to cells during imaging are very useful for inferring causal effects of gradients on cell behaviour. In addition, a variety of microfluidic setups can be used to generate long-lived gradients on 1D, 2D, or 3D migration devices, where the movement of the cells can be monitored over time [3–6]. Relatively few assays have been developed to profile neutrophil chemotaxis and actin dynamics in response to gradients in vivo. For example, injection of chemoattractants is difficult to perform in vivo during imaging.
Here, we provide a protocol for implementing laser-assisted tissue injury to profile the behaviour and actin dynamics of neutrophils in response to gradients. Wounds introduce a cocktail of damage-induced chemoattractants, and the behaviour of cells can be followed in response to such endogenous tissue-derived gradients [7,8]. Whilst the profile of gradients is relatively complex and difficult to determine, the advantage of this approach is that cells can be profiled in an intact organism. To compare with unspecific motion dynamics before gradient exposure, wounds can be performed in an anatomical site where neutrophils are already present and motile (such as the cephalic mesenchyme region of the zebrafish larva), or cell motion can be pre-stimulated in the tissue of interest [9]. This enables the determination of changes in the same moving cell before and after gradient exposure.
The approach has certain advantages over other intravital methods. Two-photon laser ablation is advantageous over one-photon ablation, offering more precise wounding and less background photodamage, as only the focal plane is exposed to a high number of photons [10,11]. Furthermore, the use of near-infrared light allows the performance of wounding deeper into tissues [10]. The use of the transparent zebrafish larva, as opposed to a mouse, simplifies the experimental approach and enables higher-resolution imaging of sub-cellular scale dynamics. In terms of further applications, the focal nature of the laser wound makes the assay suitable for studies of neutrophil swarming, notably when performing these wounds near or on the caudal hematopoietic tissue, where neutrophil density is highest [12]. In addition, the assay can be adapted to visualise host–pathogen interactions during wound healing, by the inclusion of microbes in the medium [12]. Genetic or chemical inhibition experiments can be used in conjunction with this assay to investigate signalling mechanisms [9,12].
Materials and reagents
Biological materials
Transgenic zebrafish: Tg(mpx:Lifeact-Ruby)
Promoter: Lysozyme neutrophil-specific promoter
Specificity: Lifeact (17 amino acids of yeast Abp140) fused to Ruby, which detects all F-actin, as described in Riedl et al. [13].
Reagents
4.53% NaOCl (Cleanline, catalog number: CL3013)
Methylene blue (Sigma-Aldrich, catalog number: M9140-25G)
1-phenyl-2-thiourea (PTU) (Sigma-Aldrich, catalog number: P7629-25G)
3-amino benzoic acid ethyl ester (MS-222 or Tricaine) (Sigma-Aldrich, catalog number: E10521-50G)
Low-melting point (LMP) agarose (Invitrogen, catalog number: 16520)
Leukotriene B4 (LTB4) (Sigma-Aldrich, catalog number: L0517)
Sodium chloride (NaCl), certified AR for analysis (Fisher Chemical, catalog number: S/3160/60)
Potassium chloride (KCl) (Honeywell Riedel-de. Haën, catalog number: 31248)
Calcium chloride 2-hydrate (CaCl2·2H2O) (AnalaR, catalog number: 100703H)
Magnesium sulphate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773)
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), bioPerformance certified (Sigma-Aldrich, catalog number: H4034)
Trizma base (Sigma-Aldrich, catalog number: T6066)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 06203)
Solutions
E3 embryo medium (see Recipes)
MS-222 solution (see Recipes)
PTU stock solution (see Recipes)
LMP agarose solution (see Recipes)
Recipes
E3 embryo medium solution
Reagent Final concentration (for 1×) Quantity (for 60×)
NaCl 5 mM 17.2 g
KCl 0.17 mM 0.76 g
CaCl2.2H2O 0.33 mM 2.9 g
MgSO4.7H2O 0.33 mM 4.9 g
ddH2O n/a Up to 1 L
Total n/a 1 L
E3 can be made up as a 60× stock and stored at room temperature. To prepare 60×, weigh each salt as per the table above and include 10 g of HEPES per 1 L as buffering agent. Add ~800 mL of ddH2O, adjust the pH to 7.8, and then top up with ddH2O to a final volume of 1 L. To make a 1× solution, dilute 16.7 mL of 60× solution and 1 mL of 0.01% methylene blue into a final volume of 1 L of ddH2O.
MS-222 solution
Reagent Final concentration (for 1×) Quantity (for 25×)
MS-222 0.16 mg/mL 400 mg
ddH2O n/a Up to 100 mL
Total n/a 100 mL
MS-222 can be made up as a 25× stock and stored at -20 °C for long-term storage or at 4 °C for short-term storage. To make 25× stock, weigh the corresponding amount of MS-222 as per the table above, add ~80 mL of ddH2O, adjust the pH to 7.5 using 1 M Tris pH 9, and then top up with ddH2O to a final volume of 100 mL.
PTU solution
Reagent Final concentration (for 1×) Quantity (for 50×)
PTU 0.003% (w/v) 1.5 g
ddH2O n/a 1 L
Total n/a 1 L
PTU can be made up as a 50× stock and stored in the dark at room temperature. PTU is not very soluble at room temperature. Therefore, stirring and mild heating at 60 °C is recommended to dissolve.
LMP agarose solution
Reagent Final concentration Quantity
LMP agarose 1.2% (w/v) 0.24 g
1× E3 n/a 20 mL
Total n/a 20 mL
Use fresh LMP agarose to ensure polymerisation.
Laboratory supplies
1.5 mL TubeOne microcentrifuge tubes (Starlab, catalog number: S1615-5500)
Aquafine shader sable watercolour brush size 4/0 (Daler-Rowney, catalog number: 282034040)
Nunc glass-bottom dishes (Thermo Scientific, catalog number: 150680)
Fine point, non-sterile disposable transfer (Pasteur) pipettes (Liquipette, catalog number: 127-P406-000)
3 mL non-sterile disposable transfer (Pasteur) pipettes (Liquipette, catalog number: 127-P503-000)
50 mL skirted, sterile conical tubes (CellStar, catalog number: 210261)
15 mL sterile conical tubes (CellStar, catalog number: 188271)
Equipment
Incubator LMS series 2 (Wolflabs, catalog number: 220)
Techne FDB02AD DB-2A Analogue Dri-Block® heater (Techne, catalog number: 36620-12)
Analytical balance (Sartorius, catalog number: TE412)
pH meter with InLab® Routine Pro-OSM pH electrode (Mettler Toledo, catalog number: 51344055)
Olympus MVX10 Macro zoom fluorescence upright microscope (Olympus Life Science, catalog number: MVX10) equipped with pE-300 white LED microscope illuminator (CoolLED, catalog number: pE-300 white) to screen embryos for transgenic expression
Zeiss Stemi 2000-C stereomicroscope to remove unfertilised eggs and unhealthy or dead embryos (Carl Zeiss, catalog number: Stemi 2000-C)
TriM Scope II Multiphoton microscope (LaVision BioTec TriM Scope II) for laser wounding and live-cell imaging
Software and datasets
ImSpector Pro software (5.0.284.0, LaVision Biotec, © 1998-2016) for image acquisition on TriM Scope II – Multiphoton microscope
Fiji (ImageJ 1.52p, June 2019, publicly available [14]) for generation of maximum intensity z-projection of the 3D image datasets and generation of individual timelapse video for each neutrophil we track
MATLAB (R2018b, Sept 2018, requires a license) for image analysis and data plotting
Procedure
Crossing and rearing
Figure 1 illustrates the crossing and rearing process used in this experiment.
Maintain zebrafish under standard conditions at 28.5 °C ± 0.5 on a 14:10 h light/dark cycle until the desired stage.
Set up breeding pairs four days before laser wounding (Figure 1A).
Collect eggs as soon as possible post-fertilisation to prevent mould growth and avoid debris buildup (Figure 1B).
Note: Eggs are fertilised ~30 min after light stimulation [15] and ideally collected within 3 h from laying.
Remove the non-fertilised eggs shortly after collection (Figure 1C). Clean the selected eggs by washing them in 1× E3 embryo media three times. To further prevent mould or bacterial infection, embryos can be treated with 0.003% bleach for 5 min, followed by 3× washing in E3 media (Figure 1D).
Transfer the eggs to growth media (E3 medium supplemented with methylene blue and 1× PTU to prevent pigmentation) (Figure 1E). Grow the embryos in a 10 cm Petri dish (100 embryos/50 mL E3 medium) at 28 °C (Figure 1F).
Note: PTU should be added before 24 h post-fertilisation (hpf) to prevent pigment formation.
Figure 1. Diagram of the general workflow for the crossing and rearing procedures described in this paper. Adult zebrafish are set up for crossings in tanks and fit with a spawning mesh and a barrier separating the male from the female (A). For the crossing event, the barrier is removed in the morning. Eggs are collected at a maximum of 3 h later (B), followed by sorting of fertilised (arrowed) from non-fertilised eggs (C). To prevent mould or bacterial infection, embryos may be treated with a bleach solution, followed by three times washing with E3 medium (D). Finally, eggs are incubated at 28 °C in E3 supplemented with methylene blue to help prevent fungal and bacterial growth and PTU to prevent pigmentation (E, F). dpf = days post-fertilisation; PTU = 1-phenyl-2-thiourea.
Mounting of zebrafish larvae
At three days post-fertilisation (dpf), anaesthetise larvae with 1× MS-222 in E3 (Figure 2A).
Figure 2. Schematic diagram and representative images of the mounting process for laser wounding. Low-melting point (LMP) agarose is used to keep live embryos in place for an extended period of time. Larvae at 3 dpf with the strongest signal are anaesthetised with 1× MS-222 (A), screened for red (Lifeact-Ruby) fluorescent signal (B), and mounted onto (D–G) a glass-bottom plate in 1.2% LMP agarose. Once the larvae have been pipetted into the agarose (E), the larvae must be quickly (F) positioned and aligned, as the agarose will start to solidify within 1–2 min. After 10 min, agarose-embedded embryos are covered with E3 medium supplemented with MS-222 and any optional molecules, such as LTB4, and (G) imaged using a two-photon microscope. MS-222 = 3-amino benzoic acid ethyl ester; LTB4 = Leukotriene B4.
Screen larvae for those that are positive for a transgene under a fluorescent stereomicroscope (Figure 2B).
Note: If the clutch contains a mix of heterozygous and homozygous larvae for the transgene, the level of expression may vary. If the brightness of the sample is important for the imaging, select the brightest larvae.
Place a 35 mm glass bottom dish with 14 mm micro-well #1.5 on the stereoscope stage and pipette 500 μL fresh 1.2% LMP agarose to the centre of the dish (Figure 2C, D).
Note: The type of imaging dish can be customised to the confocal stage requirements. The agarose has to be made fresh and kept at 37–39 °C before usage. If the agarose solidifies too quickly, the temperature at which the agarose is kept can be incrementally increased (the actual temperature of the agarose cools down quickly upon placing it on the Petri dish). Another option is to reduce the percentage of agarose to 1%.
Use a transfer pipette to transfer a few (2–3) 3 dpf larvae to the agarose with minimum E3 medium to prevent dehydration (Figure 2E). Using a fine brush, gently swirl the larvae to mix them into the agarose.
Note: It is helpful to mount as many larvae as possible to have extra fish in case of technical issues (see troubleshooting section). However, it is important to minimise the amount of E3 added into the agarose when adding the embryos, as this may dilute the agarose and create delays in the setting or stability of the embryo during imaging. We find it easier to place embryos into the agarose rather than the embryos onto the Petri dish first because it is harder to eliminate excess E3 medium in the latter scenario.
CRITICAL: The most time-sensitive step is aligning and orienting all the zebrafish larvae quickly so that they are lying flat in the agarose before it begins to solidify. The agarose usually takes 1–2 min to start solidifying after pipetting to the dish. Use a fine brush to orient each one of the larvae quickly (Figure 2F). Take caution not to injure the larvae.
Once the larvae are correctly oriented, remove as much agarose as possible using a fine-tip pipette so that the larvae are embedded in a thin agarose layer.
CRITICAL: When using an upright microscope, the goal is to have the larvae embedded in a thin agarose layer to minimise the distance between the objective and the sample. If an inverted microscope is used, the larvae need to be placed as close as possible to the bottom of the dish.
Wait for agarose around the larvae to solidify (~10 min).
Add 3 mL of E3 supplemented with 1× MS-222 to cover the agarose to prevent dehydration and keep the larvae healthy during microscopy. Mounted larvae are ready for imaging (Figure 2G).
Optional: At 20–30 min before laser wounding, add 30 μL of LTB4 solution to the E3 to attract the exit of neutrophils from the CHT (caudal hematopoietic tissue) to the ventral fin after wounding. This step is useful if the experiment requires neutrophils to be already motile or present in the tissue at the time of wounding.
Imaging and laser wounding
Laser wounding is performed on a multiphoton microscope (LaVision TriM Scope), equipped with a fast power modulation unit (electro-optic modulator) for fast microsecond laser power switching that allows specified regions of the scanned image plane to be treated with sufficient energy density to ablate through tissue. An Insight DeepSee dual-line laser was tuned to 900 nm and the laser power adjusted to approximately 500 mW.
Turn on all microscope hardware components, turn on the laser device, and start the ImSpector imaging software.
Place the dish on the microscope stage and bring the 25×/NA 1.05 water dipping objective lens down until it touches the media. Adjust the microscope stage so that the specimen is at the centre of the view.
Using the brightfield, focus on the area of the CHT ventral fin and determine the field of view that is going to be acquired containing the intended wounding site through the eyepiece (Figure 3A).
Figure 3. Schematic of a 3 dpf zebrafish larva showing the area of a two-photon laser wound and its representative field of view. Shown are a schematic of the anatomical area imaged (A) and the image projections before (B), during (C), and after (D) laser wounding. Neutrophils in a Tg(lyz:Lifeact Ruby) larva are shown (red fluorescence channel). CHT = caudal hematopoietic tissue; LW = laser wound; VF = ventral fin. Scale bar represents 50 μm. Note that upon laser wounding, the tissue becomes autofluorescent.
Set the laser wavelength to 900 nm. Activate the main shutter for imaging and the 1,040 nm shutter for ablation. For the used Spectra-Physics DeepSee, the laser output power is 1.2–1.3 W.
Note: The laser power here is given for guidance but has to be optimised in pilot experiments as the values will be instrument specific. The precise choice of wavelength depends on the range available in the specific instrument and the spectral properties of the proteins imaged. The excitation maximum for Ruby is 558 nm [16].
Move the focal plane to 10 μm below the larval top surface.
Define the wounding site by selecting an ROI (wound ROI). For a field of view of 168 μm × 168 μm, a circular region of interest of 40 μm in diameter was defined in one focal plane. The pixel size was 240 nm × 240 nm with a dwell time of 15 μs.
Note: The wound size can be trialled experimentally to achieve a certain objective. In our case, we found that adjusting the wound at a diameter of 40 μm was optimal for two reasons: a) it does not cause excessive damage to the fish, so it can stay healthy and alive, and b) neutrophils could respond to the wound without any visible impairments. When locating the wound ROI, avoid pigment blasting.
After defining the wound site, determine the imaging field of view as an ROI of 300 μm × 300 μm around the wound ROI, trying to position the wound ROI at the centre of the field of view and including as many neutrophils in the image as possible. Figure 3 shows a representative field of view and laser wound ROI.
Define the z-stack with a step size of 2 μm covering roughly a 35 μm stack thickness. This yields 17–18 z-planes to acquire for every time point. To determine the top and bottom positions, the neutrophil fluorescence signal can be used as a criterion, as we want to include as many neutrophils as possible.
Note: The more parallel the larva is to the plate surface/objective, the smaller the stack thickness. The thinner the stack, the smaller the interval between elapsed stacks can be. This can be optimised during mounting to maximise the temporal resolution of the time-lapse movie.
Set the interval of image acquisition at 20 s.
Acquire a test stack to confirm that the number of defined z-planes can be acquired within the desired 20 s interval, allowing for 1–2 s before the next acquisition starts. If the stack takes longer than 20 s, adjust the z-stack size accordingly.
Define the duration of total imaging time and the time point to trigger the wounding. For example, you can acquire 30 min pre-wounding and 30 min post-wounding to image the behaviour of neutrophils shortly before and after.
Start the acquisition. This setting will automatically perform laser wounding during imaging. Alternatively, wounding can also be triggered manually at the desired time point.
Check that the tissue stays in focus during the imaging. Adjust focus as needed.
Data analysis
The image analysis scripts described below are available on GitHub, using this link: https://github.com/LeukocyteMotionAndDynamics/ActinDynamics.
To generate the maximum z-projection for analysis
Open Fiji.
Click on Plugins/Bioformats/Bio-Formats Importer.
Choose the 3D file (if the microscope generates a sequence of images and not just a single file, choose the first file of the sequence as this usually contains the metadata).
Choose the following options: Hyperstack in View stack with: and click on Use virtual stack.
When the file opens, click Image/Stacks/Z-Project.
Select top and bottom slice, Max Intensity in the Projection Type, and click on All time frames.
Save the file as .tiff.
Click Image/Properties and write down the pixel size in µm/pixel.
For each neutrophil to analyse, generate a new .tiff file
Select the first and last frame that the first neutrophil can be analysed and duplicate the .tiff file by right-clicking on the image, clicking Duplicate, defining the range of time-points (first to last image), and unselecting Duplicate stack.
Save the new file with the same experiment name but add the number of the neutrophil index.
Write down the index of the frame where the laser wound occurs (this is needed to synchronise all neutrophils later in the analysis).
Write down the centroid of the neutrophil by putting the mouse on the centre of the neutrophil at the first timeframe and reading the x-y coordinates at the bottom space of Fiji.
Define the perimeter of the laser wound
For each imaged zebrafish larva, create a maximum z-projection of the already z-projected file generated before (this file can be called time-projection; there you can see the maximum extent of the wound with maximum auto-fluorescence).
Open MATLAB.
Create a folder with the time-lapse images of the individual neutrophils and the time-projection.
Create a script to read and display the time-projection file; then, add the function to let the user choose the points on the image (script wound_select.m in GitHub).
Click sequentially at the perimeter of the wound until all the wound area has been enclosed.
Double-click to finish the selection.
Write down the x-y coordinates of the wound perimeter as they appear in the variable window of MATLAB.
Create another script in which you store, for each individual neutrophil (script cell_data.m in GitHub):
Neutrophil ID (arbitrary number starting from 1 for each neutrophil larva).
Pixel size of the image (from section Data analysis, step A8).
Time interval in seconds (i.e., 20 s) (from section Imaging and laser wounding, step 9).
x-y coordinates of the neutrophil centroid (from section Data analysis, step B4).
x-y coordinates of the perimeter of the laser wound (from section Data analysis, steps C5–C7).
The timeframe where the laser wound occurs (from section Data analysis, step B3).
To segment and analyse the neutrophils
Create a new script (script cell_analysis.m in GitHub) and define:
The number of cells to analyse.
A loop over all cells.
Reading of frames sequentially for each cell image file.
Segmenting the cell based on active contours technique [17] using MATLAB function activecontour (Figure 4A).
Figure 4. Analysis of neutrophils and calculation of Lifeact polarity with time. A. Outline (red line) of an example of a segmented neutrophil within a Tg(mpx:Lifeact-Ruby) transgenic larva. Scale bar = 10 μm. B. Neutrophil velocity vector (arrow) determines the direction of movement. C. Automated separation of the front (F) and rear (R) part of the cell; in the left image, Lifeact is concentrated at the front part (t0) while in the right image, Lifeact is concentrated at the rear part (t1). Arrows indicate the direction of motion. Scale bar = 10 μm. D. Neutrophil Lifeact polarity, in relation to time; time sequence was synchronised based on the time of the laser wounding. Adapted from Figures 2 and 3 in Georgantzoglou et al. [9].
Finding the centroid of the cell.
Defining a line that separates the cell into the front and rear parts (script define_line.m in GitHub).
Separating a cell into front and rear parts based on the direction of motility of the cell (script divide_cell.m in GitHub) (Figure 4B, C).
Calculating the mean intensity at the front and rear part of the cell.
Saving the values in a MATLAB data file format.
To plot the actin polarity value as ‘mean front Lifeact’/‘mean rear Lifeact’, we create a script (script plot_actin_polarity_vs_time.m)
Create a loop over all cells.
For each cell, load the MATLAB file with the intensities (script get_cell_data.m in GitHub).
Obtain and smooth the front and rear Lifeact values for pre-wounding data.
Calculate the ratio Lifeact polarity = Lifeact front/Lifeact rear.
Synchronise data based on the timeframe of laser wound.
Repeat steps 1–5 for post-wounding data.
Calculate the mean and standard error of mean (SEM) of Lifeact polarity for each timeframe.
Plot Lifeact polarity vs. time and the SEM as a shaded area using the function boundedline.m [18] (Figure 4D).
Save the plot.
Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:
Georgantzoglou et al. [9]. A two-step search and run response to gradients shapes leukocyte navigation in vivo. DOI: 10.1083/jcb.202103207.
Poplimont et al. [12]. Neutrophil Swarming in Damaged Tissue Is Orchestrated by Connexins and Cooperative Calcium Alarm Signals. DOI: 10.1016/j.cub.2020.05.030.
General notes and troubleshooting
General notes
Although related, two-photon absorption spectra are different than doubled one-photon spectra. Absorption cross-sections can also be quite different. It is therefore important to carefully choose the right fluorescent proteins and appropriate combinations [11].
A key benefit of two-photon microscopy is its property to image a thin focal volume in thick samples. The objective focal plane is the only space with a sufficient photon density to ensure the excitation of the fluorophore by two simultaneous photons [10]. To create high-resolution 3D images, the sample needs to be scanned through the focal plane of the objective. Collected over time, these volumes can be used to visualise the migration of motile cells. Consequently, the temporal resolution is limited by the scanning speed and the pixel dwell time to collect sufficient fluorescent signal. One approach to maximising the temporal resolution is to ensure the larva is positioned as parallel as possible to the objective, to minimise the size of the stack required to scan the region of interest.
Although laser wound using multiphoton imaging has been described in other organisms, we found that the application in zebrafish confers many practical advantages including (but not limited to):
Easier administration of agents and general manipulation.
Optically transparent embryos allowing high-resolution imaging of sub-cellular dynamics.
Other than the laser wound itself, the imaging procedure is non-invasive.
The possibility of continuous imaging as there is no placental barrier or influence of the maternal compartment.
It is a more refined choice of model organism, particularly in early larval stages.
There are technical aspects that can contribute to variabilities in the recruitment of neutrophils to the wound. The variabilities can be attributed to equipment settings (laser power can vary over time) and the mounting process (see troubleshooting). To this, it is important to factor in the biological variations across samples. Sufficient optimisation of protocol and biological replicates are needed to ensure reproducible results. In addition, we recommend mounting more than one larva for imaging, to have the choice to select a well-mounted larva for imaging. For more details on how to reduce variabilities, see the troubleshooting section.
Troubleshooting
Problem Probable cause Solution/comments
Sample moves during imaging Not enough agarose Removal of excess agarose is important to minimise the working distance of the lens (if using an upright microscope). However, excess removal may compromise the stability of the specimen. Repeat mounting, ensuring a thin layer of agarose covers the sample.
Agarose has not solidified properly Always use fresh agarose. We have noticed that agarose older than two weeks does not solidify well.
No laser wound No evidence of laser wound Confirm whether laser wound has occurred. One thing to check is whether autofluorescence increases locally after wounding, which is a sign of tissue damage (Figure 3). In addition, one can also confirm with brightfield illumination if there is a wound, as evidenced by local opaque/discoloration of the tissue. If wound has not occurred, it is likely due to hardware or software issues. Check the hardware settings. If low laser power is observed, ask for technical support and consider system maintenance.
No or low neutrophil response Laser wound is not strong enough to trigger migration Mounting issues. Repeat mounting and ensure the sample is as parallel as possible to the bottom of the dish to ensure the entire wound is on the same focal plane.
Vary the wound size to find the setting that recruits cell migrations without compromising the sample’s health. If the wound is too large, it may not trigger recruitment due to overheating of the overall tissue. If the wound is too small, it may also not cause recruitment.
Wound location can also determine recruitment. For our case, we found that wounding at the region of the ventral fin or the boundary of the ventral fin and caudal hematopoietic tissue triggers more recruitment than in the head region, possibly due to the number of neutrophils present.
Sample is unwell Sometimes mounting process can accidentally hurt the fish and cause a downward trend in heartbeat rate and blood flow velocity. Before mounting, pick larvae with good heartbeat rate, blood flow velocity, and normal morphology in addition to being positive for a transgene. Assess the heartbeat rate and blood flow velocity again before image acquisition.
Acknowledgments
We thank Kevin O’Holleran and Martin Lenz of the Cambridge Advanced Imaging Centre, for their support and assistance in this work with two-photon microscopy; fish facility staff for assistance with zebrafish husbandry.
M. Sarris, A. Georgantzoglou, I. Williantarra and the relevant research were supported by a Medical Research Council Career Development Award (MR/L019523/1); Wellcome Trust (204845/Z/16/Z); Isaac Newton Trust (12.21 [a]i and 19.23 [n]), a Physiological Society research grant, and a Leverhulme Trust grant (RPG-2021-226). We also acknowledge all contributors to prior work [9,12] in which this protocol is based.
Competing interests
The authors declare no competing interests.
Ethical considerations
Zebrafish were maintained in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act 1986. Adult zebrafish were maintained under project licenses 70/8255 and P533F2314, which were reviewed by the University Biomedical Services Committee. Animals were maintained according to ARRIVE guidelines.
References
Kolaczkowska, E. and Kubes, P. (2013). Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 13(3): 159–175.
Sarris, M. and Sixt, M. (2015). Navigating in tissue mazes: chemoattractant interpretation in complex environments. Curr Opin Cell Biol. 36: 93–102.
Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B. and Devreotes, P. N. (1998). G Protein Signaling Events Are Activated at the Leading Edge of Chemotactic Cells. Cell. 95(1): 81–91.
Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W. and Bourne, H. R. (2000). Polarization of Chemoattractant Receptor Signaling During Neutrophil Chemotaxis. Science. 287(5455): 1037–1040.
Vargas, P., Terriac, E., Lennon-Duménil, A. M. and Piel, M. (2014). Study of Cell Migration in Microfabricated Channels. J Visualized Exp. 84: e51099.
Zhao, W., Zhao, H., Li, M. and Huang, C. (2020). Microfluidic devices for neutrophil chemotaxis studies. J Transl Med. 18(1): 168.
Eming, S. A., Krieg, T. and Davidson, J. M. (2007). Inflammation in Wound Repair: Molecular and Cellular Mechanisms. J Invest Dermatol. 127(3): 514–525.
Metzemaekers, M., Gouwy, M. and Proost, P. (2020). Neutrophil chemoattractant receptors in health and disease: double-edged swords. Cell Mol Immunol. 17(5): 433–450.
Georgantzoglou, A., Poplimont, H., Walker, H. A., Lämmermann, T. and Sarris, M. (2022). A two-step search and run response to gradients shapes leukocyte navigation in vivo. J Cell Biol. 221(8): e202103207.
Denk, W., Strickler, J. H. and Webb, W. W. (1990). Two-Photon Laser Scanning Fluorescence Microscopy. Science. 248(4951): 73–76.
Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E. and Rebane, A. (2011). Two-photon absorption properties of fluorescent proteins. Nat Methods. 8(5): 393–399.
Poplimont, H., Georgantzoglou, A., Boulch, M., Walker, H. A., Coombs, C., Papaleonidopoulou, F. and Sarris, M. (2020). Neutrophil Swarming in Damaged Tissue Is Orchestrated by Connexins and Cooperative Calcium Alarm Signals. Curr Biol. 30(14): 2761–2776.e7.
Riedl, J., Crevenna, A. H., Kessenbrock, K., Yu, J. H., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T. A., Werb, Z., et al. (2008). Lifeact: a versatile marker to visualize F-actin. Nat Methods. 5(7): 605–607.
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): 676–682.
Westerfield, M. (2007). The zebrafish book. A guide for the laboratory use of zebrafish (Brachydanio rerio). Eugene: Univ. Oregon Press.
Kredel, S., Oswald, F., Nienhaus, K., Deuschle, K., Röcker, C., Wolff, M., Heilker, R., Nienhaus, G. U. and Wiedenmann, J. (2009). mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures. PLoS One. 4(2): e4391.
Chan, T. and Vese, L. (2001). Active contours without edges. IEEE Trans Image Process. 10(2): 266–277.
Kearney, K. (2022). boundedline.m [WWW Document]. URL https://github.com/kakearney/boundedline-pkg, GitHub.
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4,998 | https://bio-protocol.org/en/bpdetail?id=4998&type=0 | # Bio-Protocol Content
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Peer-reviewed
Linearly Amplified Single-Stranded RNA-Derived Transcriptome Sequencing (LAST-seq)
JL Jun Lyu
CC Chongyi Chen
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.4998 Views: 447
Reviewed by: Alka MehraClara Morral Martinez Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Genome Biology Aug 2023
Abstract
Single-cell RNA sequencing (scRNA-seq) stands as a cutting-edge technology widely used in biological and biomedical research. Existing scRNA-seq methods rely on reverse transcription (RT) and second-strand synthesis (SSS) to convert RNA to cDNA before amplification. However, these methods often suffer from limited RT/SSS efficiency, which compromises the sensitivity of RNA detection. Here, we develop a new method, linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq), which directly amplifies the original single-stranded RNA without prior RT and SSS and offers high-sensitivity RNA detection and a low level of technical noise in single-cell transcriptome analysis. LAST-seq has been applied to quantify transcriptional bursting kinetics in human cells, advancing our understanding of chromatin organization’s role in regulating gene expression.
Key features
• An RNase H/DNA polymerase-based strategy to attach the T7 promoter to single-stranded RNA.
• T7 promoter mediated IVT on single stranded RNA template at single cell level.
Keywords: In vitro transcription Linear amplification Single-stranded RNA template Single-cell RNA sequencing Gene expression noise Transcriptional bursting
Graphical overview
Figure 1. Scheme of linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq). A. LAST primer design and preparation. B. LAST-seq workflow.
Background
Single-cell transcriptome analyses have been fueled by the advancement in scRNA-seq methods. Recent technical progress has focused on enhancing digital counting through unique molecular identifiers (UMIs) [1–3], increasing cellular throughput while reducing the cost [4–10], optimizing individual protocol steps [1,2,11,12], and miniaturization [11,13,14].
Despite these improvements, the fundamental chemistry involving reverse transcription (RT) and second-strand synthesis (SSS) before single-stranded RNA amplification remains unchanged. While RT relies on reverse transcriptase, current scRNA-seq methods utilize various SSS strategies with limited efficiency, including terminal transferase [15,16] or template switching [1,5,6,9,11,12,17,18], to create cDNA priming sites for subsequent PCR, conversion from RNA/cDNA hybrid to double-stranded DNA by RNase H and DNA Pol for in vitro transcription [8,14,19], random annealing to the single-stranded cDNA for extension [2], and direct Tn5 tagmentation of the RNA/cDNA hybrid molecules [20]. As a result, the inherent limitations of RT/SSS efficiency in existing scRNA-seq methods compromise the single-molecule capture efficiency of the original RNA molecules in single cells, leading to reduced measurement accuracy and increased technical noise.
To address this challenge, we developed a novel scRNA-seq method, linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq). Unlike previous methods reliant on inefficient RT/SSS before RNA amplification, LAST-seq directly amplifies original ssRNA molecules in single cells in a linear fashion without prior RT/SSS. This approach achieves a high single-molecule capture efficiency and reduced technical noise compared with existing scRNA-seq methods [21]. Using LAST-seq, we characterized gene expression noise and transcriptional bursting kinetics and investigated the role of chromatin organization in regulating gene expression [21].
Materials and reagents
Reagents
E. coli DNA ligase (New England BioLabs, catalog number: M0205S)
10× TBE (Tris/boric acid/EDTA) buffer (Bio-Rad, catalog number: 1610770)
TEMED (Bio-Rad, catalog number: 1610800)
Ammonium persulfate (APS) (Bio-Rad, catalog number: 1610700)
40% Acrylamide/Bis Solution, 29:1 (Bio-Rad, catalog number: 1610146)
Urea (Bio-Rad, catalog number: 1610731)
Sodium acetate (NaOAc) (3 M) pH 5.5 (Thermo Fisher Scientific, catalog number: AM9740)
NovexTM TBE-urea sample buffer (Thermo Fisher Scientific, catalog number: LC6876)
Formamide (MilliporeSigma, catalog number: 11814320001)
Tris (1 M), pH 8.0 (Thermo Fisher Scientific, catalog number: AM9855G)
GenEluteTM-LPA (MilliporeSigma, catalog number: 56575)
SYBRTM Gold nucleic acid gel stain (Thermo Fisher Scientific, catalog number: S11494)
Trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056)
DMEM (Thermo Fisher Scientific, catalog number: 10564011)
Fetal bovine serum (MilliporeSigma, catalog number: F2442)
Phosphate-buffered saline (PBS), 1× without calcium and magnesium (Corning, catalog number: 21-040-CV)
TritonTM X-100 (MilliporeSigma, catalog number: T8787)
SUPERase•InTM RNase inhibitor (Thermo Scientific, catalog number: AM2694)
Deoxynucleotide (dNTP) solution mix (New England BioLabs, catalog number: N0447S)
RNase H (New England BioLabs, catalog number: M0297S)
Klenow fragment (3'→5' exo-) (New England BioLabs, catalog number: M0212L)
Ribonucleotide solution mix (New England BioLabs, catalog number: N0466S)
T7 RNA polymerase (New England BioLabs, catalog number: M0658S)
DL-dithiothreitol solution (DTT) (MilliporeSigma, catalog number: 646563)
MgCl2 (Thermo Fisher Scientific, catalog number: AM9530G)
RNA MagClean DX (Aline Bioscience, catalog number: C-1005-5/50)
Nuclease-free water (Thermo Fisher Scientific, catalog number: AM9937)
SuperScriptTM IV reverse transcriptase (Thermo Fisher Scientific, catalog number: 18090050)
Q5® high-fidelity 2× Master Mix (New England BioLabs, catalog number: M0492S)
PCRClean DX (Aline Bioscience, catalog number: C-1003-5)
QubitTM dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog number: Q32854)
QubitTM assay tubes (Thermo Fisher Scientific, catalog number: Q32856)
Agilent High Sensitivity DNA kit (Agilent, catalog number: 5067-4626)
Oligos (Integrated DNA Technologies)
(Optional) High Output v2.5 reagent kit (Illumina, catalog number: 20024906)
Solutions
MIX solution for 10% TBE-Urea acrylamide gel (see Recipes)
Recipes
MIX solution for 10% TBE-Urea acrylamide gel
Components Volume
40% Acrylamide 29:1 125 mL
Urea 210 g
Formamide 100 mL
10× TBE 50 mL
Nuclease-free water X
Total 500 mL
Note: The MIX can be stored at 4 °C for at least six months.
Laboratory supplies
Pipette tips (Neptune Scientific, model: S3)
AlumaSeal® CS films for cold storage (MilliporeSigma, catalog number: Z722634-100EA)
Hard-Shell® 96-well PCR plates (Bio-Rad, catalog number: HSP9601)
Axygen® 0.2 mL Maxymum Recovery® thin-wall PCR tubes (Corning, catalog number: PCR-02-L-C)
DNA LoBind® tubes (Eppendorf, catalog number: 022431021)
QubitTM assay tubes (Thermo Fisher Scientific, catalog number: Q32856)
(Optional) x-tracta gel extraction tool (MilliporeSigma, catalog number: Z722390-100EA)
Notes:
Pipette tips used in this protocol should be low-retention, RNase-free, and DNase-free, with an aerosol filter.
PCR tubes and plates used in this protocol should be low-retention, RNase-free, and DNase-free.
Equipment
GILSON® Pipetman (GILSON®, model: D10-D1000)
Freezer (VWR, model: VWR ULT Freezer 528 Eco Premium)
Countess 3 (Thermo Fisher Scientific, model: FL model)
Centrifuge 5418 R (Eppendorf, model: 5418 R)
PCR Cooler (Eppendorf, catalog number: 022510509)
VWR® Mini Centrifuge (VWR, catalog number: 76269-064)
Vortex-Genie® 2 mixer (Fisher Scientific, catalog number: SI-0236)
Vacufuge plus, Centrifuge Concentrator (Eppendorf, model: basic with A-2-VC rotor)
Eppendorf 5810R (Eppendorf, model: 5810R)
ThermoMixer C (Eppendorf, model: 5382)
Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad, model: 1851148)
AirClean Systems 600 PCR workstation (AirClean Systems, model: 600)
Mini-PROTEAN® Tetra Cell (Bio-Rad, catalog number: 1658004)
Mini-PROTEAN® Tetra Cell Casting Module (Bio-Rad, catalog number: 1658021)
UViewTM Mini Transilluminator (Bio-Rad, catalog number: 1660531)
NanoDrop (Thermo Fisher Scientific, catalog number: ND-ONE-W)
PowerPacTM Basic Power Supply (Bio-Rad, catalog number: 1645050)
Qubit 4 Fluorometer (Thermo Fisher Scientific, catalog number: Q33238)
2100 Bioanalyzer Instrument (Agilent, model: G2939A)
High-performance computing cluster (NIH, Biowulf, model: NA)
BD FACSAria IIu (BD Bioscience, model: NA)
Magnetic rack (Diagenode, catalog number: B04000001)
Roto mini plus (Benchmark, catalog number: R2020)
Software and datasets
bcl2fastq, v2.20, free: https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.htmL
cutadapt, v 1.15, free: https://cutadapt.readthedocs.io/en/stable/
zUMIs, v.2.9.7, free: https://github.com/sdparekh/zUMIs
The datasets are available at https://github.com/lyuj2022/LAST-seq.
Procedure
LAST-seq primer preparation
Cast 10% TBE-Urea acrylamide gel.
Assemble the 1.5 mm gel-casting module according to the manufacturer’s manual.
Prepare the gel mixture in a 50 mL conical tube as below (Table 1).
Table 1. 10% TBE-Urea acrylamide gel solution
Components Volume
MIX 10 mL
30% APS 33.2 µL
TEMED 4 µL
Slowly pour the gel solution into the gel-casting module filling all the space between the short plate and spacer plate and avoiding introducing bubbles. Then, insert a 10-well comb. Wait for 30 min for gel polymerization.
Note: The gel solution is prepared for casting one TBE-Urea acrylamide gel, with 10 wells for 10 LAST primers. Alternatively, one can use a commercially available 10% precast TBE-urea gel that fit the Mini-PROTEAN® Tetra Cell.
Assemble the mixture in a 96-well plate for each hairpin primer (BC1-BC80, see Table S1 for hairpin primers) as below (Table 2).
Table 2. Annealing mixture
Components Volume Working concentration
100 µM hairpin primer 25 µL 50 µM
10× E. coli DNA ligase reaction buffer 5 µL 1×
Nuclease-free water 20 µL
Seal the plate with AlumaSeal® CS film and place the plate in a thermocycler with a lid temperature set to 70 °C. Then, heat the plate to 65 °C for 5 min and slowly cool down to 25 °C (-0.5 °C/cycle).
Notes:
The hairpin primer contains a 5' end docking site (20 nt) for loop anchoring and dTrU tail annealing, a T7 promoter (20 nt) for IVT and partial loop anchoring, a PCR handle (21 nt of TruSeq Read2) to dock index primers, a UMI/barcode (8 nt/6 nt) to tag mRNA copies and individual cells, and a 3' end docking site (11 nt) for loop anchoring. After annealing, the primer will form a loop and leave a 5' end overhang (Figure 1A). The hairpin primer can be stored at -20 °C for at least one year.
A higher lid temperature than 75 °C will detach the AlumaSeal® CS films from the plate.
Assemble the ligating mixture in a new 96-well plate as below (Table 3), seal the plate, and incubate at 16 °C for 30 min.
Table 3. Ligating mixture
Components Volume Working concentration
50 µM hairpin primer 2 µL 5 µM
100 µM dTrU tail primer 1 µL 5 µM
10× E. coli DNA ligase reaction buffer 2 µL 1×
E. coli DNA Ligase 1 µL 0.5 U/µL
Nuclease-free water 14 µL
Note: The dTrU primer contains a linker (17 nt) to join the hairpin primer, a poly-dT-rU region (7 nt–19 nt) to capture mRNA, and a 3' end blocker to prevent primer extension (Figure 1A).
Set up the electrophoresis apparatus and carefully remove the comb, avoiding damaging the wells. Pre-run the gel in 1× TBE buffer at 300 V for 10 min, then rinse wells with buffer using a P200 pipette.
Mix 20 µL of ligated mixture and 20 µL of 2× TBE-Urea sample buffer. Load 40 µL sample per well. Run the gel at 150 V for 45 min.
Purify the ligated LAST primer by ethanol precipitation.
Carefully open the glass chamber and take out the gel. Soak the gel in 20 mL of 1× TBE containing 1× SYBR Gold for 20 min.
Place the gel on the UV transilluminator. While the oligo is illuminated with UV, use a clean razor blade to cut the oligo band of interest (~130 nt in length) of each well, keeping the size of the gel slice as small as possible. Then, transfer the gel slice to 1.5 mL Eppendorf tubes.
Note: Alternatively, one can use x-tracta gel extraction tool to extract the band of interest.
Crush the gel slice with disposable tips, add 400 µL of 0.3 M sodium acetate per gel slice, and incubate the tubes on dry ice for 15 min.
Note: Incubating gel slices on dry ice for 15 min will freeze the buffer and gel, which will enlarge the meshes of the acrylamide gel, facilitating primer diffusion from the gel while the buffer and gel are unfrozen.
Rotate the tubes at 10× rpm at room temperature for 4 h.
Centrifuge the tube at 20,000× g for 15 min and transfer the supernatant to a 1.5 mL tube.
Add 2.5-fold volume of cold 100% ethanol and 3 µL of 5 µg/µL GenElute-PLA. Place tubes at -20 °C overnight.
Note: GenElute-PLA is short for Linear PolyAcrylamide, which is a neutral carrier for precipitating nucleic acids with ethanol.
Centrifuge the tube at 20,000× g for 30 min at 4 °C and discard the supernatant.
Wash the white pellet twice with 75% cold ethanol and centrifuge at 20,000× g for 10 min at 4 °C.
Dry the pellet in a clean PCR workstation for 20 min and resuspend the pellet (invisible) with 10 µL of TE.
Quantify the LAST primer using NanoDrop and adjust the concentration to 40 ng/µL (equal to ~1 µM).
Note: The UV absorbance at 260 nm is proportional to the nucleic acid concentration. The primer consists of 19 ribonucleotides and 111 deoxyribonucleotides, and thus we used 34 as the conversion factor for the LAST primer. The ultimate yield is approximately 0.6 µg.
Transfer primers to a 96-well plate, seal the plate with AlumaSeal® CS films, and store the plate at -80 °C for up to one year.
Preparation of lysis buffer
Add 2 µL of 1 µM LAST primers (BC1-BC80) into wells (each well for a unique LAST primer) of a new 96-well plate containing 48 µL of 10 mM Tris buffer and 0.5% RNase inhibitor, resulting in 40 nM LAST primer per well.
Assemble the lysis buffer in a new 96-well plate with the components as below (Table 4), resulting in 20 µL of lysis buffer per well. Then, use a P10 multi-pipette to aliquot 2 µL of lysis buffer per well to new 96-well plates. Seal plates with AlumaSeal® CS films and store at -80 °C.
Table 4. Lysis buffer
Components Volume Working concentration
Nuclease-free water 10 µL
1% Triton X-100 2 µL 0.1 %
40 nM LAST primer 1 µL 2 nM/µL
2 U/µL RNase inhibitor 1 µL 0.1 U/µL
2 mM dNTP 5 µL 0.5 mM
600 pg RNA carrier 1 µL 30 pg/µL
Notes:
Each 96-well plate contains 80 LAST primers in 80 wells and thus can barcode up to 80 single cells.
The working volume of lysis buffer per well is 2 µL. Here, we assembled 20 µL of lysis buffer per well (per LAST primer) and aliquoted them to multiple plates for future use.
Preparation of single-cell suspension
Remove the medium of HEK293T cells in a 10 cm Petri dish and quickly rinse cells twice with 5 mL of 37 °C prewarmed PBS.
Immediately add 1 mL of 0.25% Trypsin-EDTA to a 10 mm Petri dish, and then incubate in a 5% CO2 humidified incubator at 37 °C for 2 min.
Neutralize the Trypsin-EDTA by adding 2 mL of DMEM containing 10% fetal bovine serum and rinse the dish by pipetting to collect all cells. Transfer 3 mL of cell suspension to a 15 mL conical tube.
Spin down cells at 300× g for 3 min at 4 °C.
Aspirate the supernatant, resuspend cells with 1 mL of cold PBS, and place the tube on ice.
Count cells using an automated cell counter and adjust the concentration with cold PBS to 1 × 107 cells/mL.
Pass cells through a 40 µm cell strainer, keep cells on ice, and proceed with single-cell sorting.
Single-cell sorting
Take out 96-well plates containing lysis buffer, keep them sealed, and place them on ice until loading them into the sorter.
Use a small portion of your samples or flow cytometry compensation beads as a pilot sample to align the sorter to an empty 96-well plate sealed with film. Make sure, by visual inspection, that the droplets have been ejected in the center of each well.
Replace the plate and the pilot sample with an unsealed plate containing lysis buffer and your sample of interest, respectively.
Sort single cells into individual wells. Seal the plates with AlumaSeal® CS films and centrifuge them at 1,000× g for a few seconds at 4 °C.
Note: Store plates at -80 °C if you do not proceed immediately.
Lysis buffer evaporation
Place the 96-plate in the thermal cycler and set the lid to 70 °C. Heat the plate at 65 °C for 5 min and immediately put it on a PCR cooler for 2 min.
Peel off the film carefully and vacuum-centrifuge the plates at 1,400 rpm for 25 min at 30 °C in a PCR workstation. Prepare the tagging buffer per well as below (Table 5), while waiting for the evaporation, and keep the buffer on ice.
Table 5. Tagging buffer
Components Volume Working concentration
Nuclease-free water 1.875 µL
10× NEB2 0.25 µL 1×
20 U/µL RNase inhibitor 0.125 µL 1 U/µL
0.5 U/µL RNase H 0.1 µL 0.02 U/µL
5 U/µL Klenow fragment (3'→5' exo-) 0.15 µL 0.3 U/µL
Notes:
Place the vacuum-centrifuge in a UV-treated PCR workstation to minimize the impact of environmental RNase contamination.
After evaporation, the lysis buffer is invisible. Make sure the lysis buffer is completely evaporated. The purpose of using vacuum-centrifugation is to increase the primer concentration by evaporating the lysis buffer, thereby achieving high annealing efficiency with a small quantity of LAST primer.
The tagging buffer recipe is for one well of a 96-well plate. When preparing the tagging mixture, scale up the volume according to the number of wells.
T7 promoter attachment and single-stranded RNA in vitro transcription (IVT)
Add 2.5 µL of tagging buffer to each well and seal the plate with film. Spin the plate for seconds and vortex the plate using a ThermoMixer at 1,000× rpm for 1 min.
Spin the plate for a few seconds and place it in a thermal cycler at 37 °C for 30 min. Meanwhile, prewarm RNA beads (RNA MagClean DX) and RNA beads buffer (spin down 550 µL of RNA beads to obtain 520 µL of buffer) to room temperature for at least 30 min. Prepare 2.5 µL of rinsing buffer per well as below (Table 6):
Table 6. Rinsing buffer
Components Volume Working concentration
Nuclease-free water 2.125 µL
10× NEB2 0.25 µL 1×
20 U/µL RNase Inhibitor 0.125 µL 1 U/µL
Notes:
In this process, RNase H nicks the RNA polyA tail at the DNA–RNA hybrid region formed by annealed polyA and poly-dT-rU. Subsequently, DNA polymerase (Klenow exo-) extends the remaining polyA end of this RNA using the LAST primer as a template, thereby attaching T7 promoter, UMI/barcode to each RNA molecule.
The attachment efficiency of T7 promoter is approximately 65%, which is measured by assembling the LAST-seq primer and FAM-labeled RNA template at the molar ratio of 1.5:1, followed by nicking and extension reaction. The original RNA template would be nicked and elongated to ~150 nt after T7 promoter attachment. The FAM intensity is linearly and positively correlated to the number of molecules and thus could be used to estimate the percentage of molecules with T7 promoter attached (Figure 2).
The purpose of rinsing buffer is to rinse wells, therefore minimizing the residual IVT template in wells.
Figure 2. Strategy to measure the efficiency of T7 promotor attachment
After incubation, quickly spin the plate, place it on a PCR cooler, and carefully peel off the film.
Transfer the tagging buffer from each well to a 1.5 mL Eppendorf tube.
Add 2.5 µL of rinsing buffer to each well and quickly spin the plate. Transfer the rinsing buffer to the same 1.5 mL Eppendorf tube.
Add 200 µL of beads and 520 µL of beads buffer to the tube and mix well by pipetting; then, incubate at room temperature for 15 min. Meanwhile, thaw all reagents needed for IVT (see Reagents in Table 7).
Table 7. IVT buffer
Components Volume Working concentration
Nuclease-free water 26 µL
10× RNA polymerase buffer 4 µL 1×
25 mM NTP 2 µL 1.25 mM
20 U/µL RNase inhibitor 2 µL 1 U/µL
100 mM DTT 2 µL 5 mM
100 mM MgCl2 2 µL 5 mM
50 U/µL T7 RNA polymerase 2 µL 2.5 U/µL
After incubation, place the tube on the magnet for 2 min or until the solution clears. Then, discard the supernatant without disturbing beads.
Add 1 mL of freshly prepared 80% ethanol and incubate for 30 s, then discard the supernatant.
Repeat step F8.
Remove all residual liquid using a P10 pipette. Dry beads for up to 15 min until the pellet is not shiny. Meanwhile, assemble the IVT buffer as below and keep it on ice:
Resuspend beads with 40 µL of IVT buffer and transfer the mixture to a 200 µL PCR tube.
Incubate the tube in a thermal cycler at 37 °C for 14 h, then keep it at 4 °C.
Notes:
IVT efficiency of assembled single-stranded RNA template could reach a few 100-fold. Detailed information can be found in the original research article [21] (Supplementary Figure 2).
Before proceeding to step F13, prewarm RNA beads and beads buffer at room temperature for at least 30 min.
Place the tube on a magnetic stand for 2 min, then transfer the supernatant to a new PCR tube.
Add 32 µL of RNA beads and 40 µL of beads buffer, mix well, and incubate at room temperature for 15 min.
Place the tube on a magnetic stand for at least 5 min until the liquid clears. Then, discard the supernatant without disturbing beads.
Add 200 µL of freshly prepared 80% ethanol and incubate for 30 s, then discard the supernatant.
Repeat step F16.
Dry the beads for 3–5 min until the pellet is not shiny.
Resuspend beads with 7 μL of nuclease-free water. Pipette the entire volume up and down 10 times to mix thoroughly. Incubate at room temperature for 2 min.
Place the tube on a magnetic stand for 2 min. Then, transfer 6 µL of supernatant to a new PCR tube and keep it on ice.
Library preparation and sequencing
Reverse transcription
Mix 6 µL of RNA elution above with 1 µL of annealing buffer as below (Table 8). Incubate the tube in a thermal cycler at 70 °C for 2 min, then transfer it to the PCR cooler immediately.
Table 8. Annealing buffer
Components Volume Working concentration
25 mM random primer 0.5 µL 1.25 µM
10 mM dNTP 0.5 µL 0.5 mM
Note: Random primer consists of a PCR handle (21 nt of TruSeq Read1) and a random hexamer.
Add 3 µL of RT buffer as below (Table 9) to 7 µL of the mixture above, mix well, and spin down.
Table 9. RT buffer
Components Volume Working concentration
5× SSIV Buffer 2 µL 1×
20 U/µL RNase inhibitor 0.25 µL 0.5 U/µL
0.4 M DTT 0.25 µL 10 mM
200 U/µL SuperScriptTM IV reverse transcriptase 0.5 µL 10 U/µL
Incubate the tube at a thermal cycler and run the program as below.
Program: 23 °C for 10 min; 50 °C for 15 min; 80 °C for 10 min; hold at 4 °C
PCR for adaptor addition
Add the 10 µL of reaction product above to the PCR mixture as below (Table 10), mix well, and run the PCR program below. While waiting for the PCR, prewarm the PCRClean beads at room temperature for 30 min.
Table 10. PCR mixture
Components Volume Working concentration
Q5 high-fidelity 2× Master Mix 50 µL 1×
10 µM universal primer 5 µL 0.5 µM
10 µM index primer 5 µL 0.5 µM
Nuclease-free water 30 µL
Program:
Step 1: 98 °C for 30 s;
Step 2 for 8 cycles: 98 °C for 10 s; 69 °C for 15 s; 72 °C for 20 s;
Step 3: 72 °C for 2 min;
Hold at 4 °C.
Note: Eighty barcoded LAST primers can only barcode 80 cells in a 96-well plate. In cases where users want to profile more than 80 cells, they can assign different index primers to cDNAs from different 96-well plates, thereby further scaling up the cellular throughput. As a result, for less than 80 cells, users can use the cellular barcode of LAST to distinguish cells; for more than 80 cells, users can distinguish each cell through the combination of barcodes from LAST primers and index primers. See Table S1 for index primers.
Add 90 µL of PCRClean beads to the PCR product, mix well, and incubate at room temperature for 5 min.
Place the tube on the magnetic stand for 5 min. Discard the supernatant without disturbing the beads.
Add 200 µL of freshly prepared 80% ethanol, incubate beads for 30 s, and then discard the supernatant.
Repeat step G2d.
Spin briefly and place the tube on the magnetic stand. Remove any remaining ethanol using a P10 pipette.
Resuspend beads with 51.5 µL of nuclease-free water and incubate for 2 min.
Place the tube on a magnetic stand for 2 min. Then, transfer 50 µL of supernatant to a new PCR tube.
Add 45 µL of beads to the PCR product, mix well, and incubate at room temperature for 5 min.
Repeat steps G2c–f.
Resuspend beads with 31.5 µL of nuclease-free water and incubate for 2 min.
Place the tube on a magnetic stand for 2 min. Then, transfer 30 µL of supernatant to a new PCR tube.
Add 27 µL of beads to the PCR product, mix well, and incubate at room temperature for 5 min.
Repeat steps G2c–f.
Resuspend beads with 11.5 µL of nuclease-free water and incubate at room temperature for 2 min.
Place the tube on a magnetic stand for 2 min. Then, transfer 10 µL of supernatant to a new PCR tube.
PCR for library enrichment.
Add the 10 µL product above to the PCR mixture as below (Table 11), mix well, and run the PCR program below.
Table 11. PCR mixture
Components Volume Working concentration
Q5 High-Fidelity 2× Master Mix 12.5 µL 1×
10 µM P5 primer 1.25 µL 0.5 µM
10 µM P7 primer 1.25 µL 0.5 µM
Program:
Step 1: 98 °C for 30 s;
Step 2 for 8 cycles: 98 °C for 10 s; 65 °C for 15 s; 72 °C for 20 s;
Step 3: 72 °C for 2 min;
Hold at 4 °C.
Notes:
P5 and P7 primers are adaptors for Illumina sequencing. They were used to amplify and enrich our final library. Their sequences are provided in Table S1.
PCR cycles could vary when using different cell lines or pooling various numbers of cells. We suggest using 10–12 cycles of PCR for 80 cells.
Add 90 µL of beads to the PCR product, mix well, and incubate at room temperature for 5 min.
Place the tube on the magnetic stand for 5 min. Discard the supernatant without disturbing the beads.
Add 200 µL of freshly prepared 80% ethanol and incubate beads for 30 s, then discard the supernatant.
Repeat step G3d.
Spin briefly and place the tube on the magnetic stand. Remove any remaining ethanol and dry beads for 3 min or until the pellet is not shiny.
Resuspend beads with 11.5 µL of nuclease-free water and incubate for 2 min.
Place the tube on a magnetic stand for 2 min. Then, transfer 10 µL of supernatant to a new PCR tube.
Quantify the library using the QubitTM dsDNA HS Assay Kit.
Note: The library concentration is approximately 1 ng/µL for eight cycles of PCR, which is sufficient for sequencing on NEXTseq550.
Check library distribution using 2100 Bioanalyzer Instrument (Figure 3) and sequence library using a High Output v2.5 reagent kit (Illumina) on NEXTseq550, with Read1 sequenced for 50 cycles and Read2 sequenced for 14 cycles.
Figure 3. Distribution of linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq) library. The bioanalyzer data showing a library distribution for the transcriptome of 10 pooling HEK293T cells.
Notes:
To check library distribution, load 1 µL (~1.0 ng/µL) library to the high-sensitivity DNA chip and run the bioanalyzer according to its manual.
Read1 contains RNA information. Read2 contains the UMI (8 nt) and barcode (6 nt) information.
Data analysis
Convert BCL files to fastq files using bcl2fastq v2.20.
Trim reads to remove bases of low quality at 3' end and discard reads with a length of Read1 and Read2 less than 22 nt and 14 nt, respectively, using cutadapt v 1.15 with parameters ‘--nextseq-trim=20 -m 22:14’.
Use zUMIs v.2.9.7 [22] for reads QC, read mapping, downsampling, and UMI counting. The resultant dataset is an expression matrix containing reads and UMI counts for genes (in rows) in each cell (in columns).
Check batch effect using principal component analysis or correct batch effect using sva R package (optional).
The related scripts (Rawdataprocess01 and 02) are available at https://github.com/lyuj2022/LAST-seq and are implemented in a Linux system.
Note: After reads trimming, more than 95% of Read1 and Read2 are in length of 50 nt and 14 nt, respectively. LAST-seq requires paired-end sequencing to obtain both RNA sequence (Read1) and UMI/barcode sequence (Read2).
Validation of protocol
This protocol was validated in:
Lyu, J., Chen, C. LAST-seq: single-cell RNA sequencing by direct amplification of single-stranded RNA without prior reverse transcription and second-strand synthesis. Genome Biol 24, 184 (2023) [21].
General notes and troubleshooting
Troubleshooting
Problems Solutions
Low yield of library Perform additional rounds of PCR on your library with P5/P7 primers.
Small peaks dominate the library distribution Make sure the LAST primer concentration is approximately 40 ng/µL before dilution. A higher concentration of LAST primer could lead to a large portion of smaller peak in the library.
Acknowledgments
We thank Ferenc Livak, Subhadra Banerjee, and Caiyi Li from the CCR Flow Cytometry Core for single-cell sorting, and other members of the Chen lab for discussions. The HEK293T cell line is a gift from Dr. Peter D. Aplan. This study used the Biowulf Linux cluster at the National Institutes of Health. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (1ZIABC011884).
Competing interests
The authors declare no competing interests.
References
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Supplementary information
The following supporting information can be downloaded here:
Table S1.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Update Notice: TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs
HX Hengyi Xu
RN Ryan M. Nottingham
AL Alan M. Lambowitz
Published: May 20, 2024
DOI: 10.21769/BioProtoc.4999 Views: 310
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After official publication in Bio-protocol (https://bio-protocol.org/e4239), we noticed that the R1 sequencing adapter in this protocol, which was derived from the NEBNext small RNA library preparation kit, is not compatible with the i5 sequencing primer used for demultiplexing reads by unique dual indices (UDIs) in newer versions of Illumina sequencing kits, particularly for sequencing on the Illumina NovaSeq platform. The adapter sequences described in the published protocol still allow demultiplexing by using the i7 index and overall read quality is unaffected. We are adding this note to update the R1 adapter sequence for use with the Illumina NovaSeq platform, enabling UDI demultiplexing for high-depth sequencing on this instrument.
The following information was replaced in the updated published Bio-Protocol article:
For applications requiring Illumina Unique Dual Indices (UDI), the protocol for steps c, d, and e should be as follows:
c. R1R DNA 5’/5Phos/AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG TGT/3SpC3/3’
Note: The Read 1 (R1) sequence corresponds to the adapter sequences used in the Illumina TruSeq RNA Library Prep Kit v2 for Illumina sequencing.
d. 6N unique molecular identifier (UMI) R1R DNA
5’/5Phos/NNN NNN AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG TGT/3SpC3/3’
Note: UMI nucleotides (machine-mixed equimolar A, C, G, and T residues, denoted N) are added at the 5’ end of the R1R sequence. The number of N nucleotides can be changed to suit the complexity of the samples being sequenced and the number of duplicates expected after PCR.
e. Illumina barcode PCR primer (P5)
5’-AAT GAT ACG GCG ACC ACC GAG ATC TAC AC BARCODE ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT
Note: All the sequences in (a-e) should be updated to correspond to any changes in Illumina sequencing kits in the future.
Article Information
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Xu et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
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Immunophenotyping and Intracellular Staining of Fixed Whole Blood for Mass Cytometry (CyTOF)
SK Sangeeta Kowli
Holden Maecker
Published: Sep 5, 2020
DOI: 10.21769/BioProtoc.5004 Views: 5137
Reviewed by: PRASHANT SHARMAAnna SloutskinBalaji Olety Amaranath
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Abstract
In this report, we present the implementation of mass cytometry for intracellular staining using fixed whole blood. In our assay described here, 250 µl of whole blood, is stimulated in vitro with PMA/ionomycin (or left unstimulated), in the presence of secretion inhibitors (brefeldin A and monensin), lysed-fixed using SMART TUBE buffers, barcoded (optional), surface stained, fixed, stained for intracellular markers, fixed and DNA stained. Using 250 µl of whole blood from a healthy donor, we show that the expression of major lineage populations such as T cells, B cells, NK cells and monocytes, as well as cytokines such as CD4+ and CD8+ IFNγ and TNFα across multiple batches (n = 27) is consistent, with the co-efficient of variation (CVs) ≤ 21%, implying minimum inter-variability. For each major cell type, the percentage is reported as a percent of singlets. The percentage of cytokine expression in response to stimulation is reported as a percent of the immediate parent cell type. This protocol has a number of benefits: from a biological perspective, it can be applied to clinical studies especially where blood draw volumes are limiting. Technically, the protocol can be adapted for barcoding, which adds the benefits of more uniform sample staining as well as antibody conservation especially for large study cohorts. Finally, for studies involving infectious diseases including the current global COVID-19 pandemic, this protocol permits infectious samples to be fixed prior to processing and staining, thereby reducing biosafety risks.
Keywords: CyTOF Mass cytometry Flow cytometry Barcoding Fixed whole blood Immune profiling
Background
Non-invasive methods to obtain biological samples and single cell technologies are much sought after, as they provide an opportunity to comprehensively study human diseases. Blood not only provides for a minimally-invasive, cost effective and readily accessible source of immunological sample, but whole blood stimulation also serves as the closest mimic of the in vivo condition. Single-cell Mass Cytometry (or Cytometry by Time of Flight mass spectrometry, CyTOF) is ideally suited to broad profiling of the immune system, since it allows for > 40 parameter panels, with little to no spillover between channels, which is a significant advancement over the procedural limitations of fluorescence flow cytometry (Leipold et al., 2015). Conversely, cell acquisition speed is significantly lower, and cell loss significantly higher, for CyTOF compared to fluorescence flow cytometry. In this report, we show the establishment of a reference panel of 39-anti human antibodies for mass cytometry that broadly identifies the major immune cell types, well established T and B cell subpopulations, activation markers, cytolytic markers and cytokines. To characterize these immune cell lineages and their functional states, the panel of anti-human heavy metal-conjugated monoclonal antibodies was selected to target the epitopes shown in Table 3, and to be compatible with fixed whole blood samples (Fernandez and Maecker, 2015 and our unpublished data). The panel described here, comprises an almost equal number of pre-made and in-house conjugates. To build this panel, we implemented the recommended panel design guidelines for mass cytometry. Low-abundance targets were allocated to higher-sensitivity channels and antibodies were designated to channels to minimize potential spectral overlap (Takahashi et al., 2017). All antibodies in the designed panel were then titrated to best discriminate the positive population from the negative. This panel can be further customized for specific hypothesis driven studies. In addition, by integrating barcoding (Behbehani et al., 2014) within this framework, we also demonstrate the compatibility of this panel with large clinical studies, to minimize technical variability. The ability to freeze fixed blood samples is also convenient for assembly of sample sets for retrospective batched analysis, with decreased biosafety risks in the setting of infectious disease. With regard to SARS-CoV-2 or other infectious agents that might be present, whole blood fixation by the method described here has not been proven to be fully inactivating. But given the known effects of fixation on viral infectivity (Möller et al., 2015), it provides an increased level of safety.
Materials and Reagents
Parafilm (PARAFILM, catalog number: H32207017002 )
BD VacutainerTM Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper (Fisher Scientific, catalog number: 367874 )
96 Well, Square V-bottom deep well plate (Costar/Corning, catalog number: 3960 )
Universal Lids for deep well plates (Corning, catalog number: 3099 )
5 ml polystyrene round-bottom tube with cell strainer cap (Falcon Coring, catalog number: 352235 )
1.8 ml cryotubes (NUNC, catalog number: 375418 )
50 ml polystyrene reservoir (Costar/Corning, catalog number: 4870 )
15 ml and 50 ml polypropylene conical tubes (Falcon, catalog numbers: 352096 and 352070 , respectively)
0.5 ml PCR tubes (Corning, catalog number: 3750 )
1.7 ml Microcentrifuge tubes (GeneMate, catalog number: C-3260-1 )
10 ml serological pipettes (Corning, catalog number: 4488 )
25 ml serological pipettes (Falcon, catalog number: 357525 )
Freshly drawn whole blood in heparin green top tubes, from healthy donors or patients
EQTM Four Element Calibration Beads (Fluidigm, catalog number: 201078 )
Erythrocyte Lysis Buffer (Qiagen, catalog number: 79217 )
Brefeldin A (Sigma-Aldrich, catalog number: B7651 , stock concentration 5 mg/ml)
Monensin (Sigma-Aldrich, catalog number: M5273 , stock concentration 5 mg/ml)
Phorbol 12-myriate 12 acetate (PMA) (Sigma-Aldrich, catalog number: P8139 , stock concentration 1 mg/ml)
Ionomycin (Sigma-Aldrich, catalog number: I0634 , stock concentration 1 mg/ml)
Stable Lyse-V2 (SMART TUBE Inc., catalog number: 3L7080 )
Stable Store-V2 (SMART TUBE Inc., catalog number: 3S1988 )
BD FACSTM Lysing Solution (BD, catalog number: 349202 )
Cell-IDTM 20-Plex Pd Barcoding Kit (Fluidigm, catalog number: 201060 )
16% paraformaldehyde (PFA) (Alfa Aesar, catalog number: 43368 )
Phenotypic and intracellular antibodies (filtered through 0.1 µm spin filters) (Millipore, catalog number: UFC30VV00 )
10x phosphate-buffered saline (PBS) (ROCKLAND, catalog number: MB-008 )
MilliQ water
Note: Beakers or bottles used to store MilliQ water are not washed with soap, due to the barium content of most commercial soaps.
BSA (Sigma-Aldrich, catalog number: A7284 )
10% Sodium Azide (Teknova, catalog number: S0209 )
0.5 M EDTA (Gibco, catalog number: 15575-038 )
Cell-IDTM Intercalator Ir (Fluidigm, catalog number: 201192A )
10x saponin-based permeabilization buffer (eBioscience, catalog number: 00-8333-56 )
CyPBS (see Recipes)
CyFACS buffer (see Recipes)
Equipment
-80 °C freezer
Incubator at 37 °C, 5% CO2
Biosafety Cabinet (BSL-2 rated) or appropriate biosafety cabinet recommended for the pathogen being tested
Centrifuge (Eppendorf Centrifuge 5810R)
p2, p10, p20, p100, p200, p1000 single-channel calibrated micropipettes (Rainin)
p200 and p1200 multi-channel calibrated micropipettes (Rainin)
12 pin aspirator (V&P Scientific, Inc., catalog number: VP 187PC-3S )
Procedure
Whole Blood Stimulation and Fixation
Use a 96-well deep well plate and label the wells with the unstimulated (US) and stimulated conditions (PMA/ionomycin). Keep PMA/ionomycin stimulated wells spatially separated (at least one well apart) from unstimulated or antigen-stimulated wells, to avoid cross-contamination and false positive signals.
Invert the heparin green top tube a few times to mix the freshly drawn whole blood.
Using a p1000 single-channel pipette, transfer 250 μl of whole blood (WB) into the labeled deep well plate.
Add secretion inhibitors Brefeldin A and Monensin to both unstimulated and stimulated conditions. Add PMA/ionomycin stimulants to the wells containing whole blood to be stimulated (Tables 1 and 2). Pipette up and down to mix well.
Table 1. Protein secretion inhibitors
Table 2. Activators
Incubate the deep well plate for 4 h in an incubator at 37 °C, 5% CO2.
At the end of the incubation, add 350 μl Stable-Lyse V2 (at room temperature) per 250 μl whole blood. Add the Stable-Lyse V2 in the same order as the addition of the secretion inhibitors and stimuli to the whole blood to maintain a consistent incubation time. Pipette up and down to mix the contents of the well. Incubate at room temperature (RT) for 15 min.
At the end of the 15 min incubation, immediately add 1,000 μl Stable-Store V2 (at room temperature) per 250 μl whole blood. Add the Stable-Store V2 in the same order to the wells as maintained in Steps A4 and A6 to maintain a consistent incubation time. Pipette up and down to mix well. Incubate at RT for 15 min. Total volume per well will be 1.6 ml (250 μl WB + 350 μl stable-lyse V2 + 1,000 μl stable-store V2).
At the end of the incubation, transfer 1.6 ml of the unstimulated and stimulated fixed whole blood sample to cryo-labeled tubes and place in -80 °C freezer until the samples are ready to be thawed and stained.
Note: Samples are transferred to cryovials for freezing, to facilitate quick sample-thaw. Depending on the study design, samples, may be frozen in the deep well plate.
Thaw Fixed Whole Blood Samples
Remove samples from -80 °C freezer and thaw in cold water for about 15 min.
Transfer the WB samples (1.6 ml) to a deep well plate.
Centrifuge cells at 974 x g for 10 min at 4 °C.
Aspirate the supernatant using the 8-12 pin aspirator.
(Optional step) If red blood cells (RBCs) are observed upon thawing the samples, add 1 ml of Erythrocyte Lysis Buffer (does not contain fixative). Incubate at RT up to a maximum of 10 min (watch for lysis). Stop the lysis by adding 0.8 ml of CyFACS buffer and proceed to Step B7.
If no RBCs are observed, add 1 ml CyFACS buffer.
Centrifuge cells at 974 x g for 10 min at 4 °C.
Repeat wash with 1 ml CyFACS buffer.
Centrifuge cells at 974 x g for 10 min at 4 °C.
Note: Cell count/sample post 2 CyFACS washes should be ~1 x 106 cells.
Cells are now ready to be barcoded or surface stained as required.
Note: If samples are not barcoded, skip Procedure C and proceed to Procedure D of surface staining.
Barcoding
Barcode Perm Buffer: Prepare 4 ml for each sample to barcode by mixing 1 part Maxpar 10x Barcode Perm with 9 parts Maxpar PBS; store at 4 °C for up to one week.
Wash each sample with 1 ml Barcode Perm Buffer. Centrifuge cells at 974 x g for 10 min. Aspirate supernatant from the cells.
Repeat washes 2x with 1 ml Barcode Perm Buffer.
Resuspend each sample to be barcoded completely in 800 μl Barcode Perm Buffer.
Resuspend each barcode tube containing 10 μl of the pre-mixed barcode completely in 100 μl Barcode Perm Buffer.
Transfer 110 μl of barcodes to the appropriate samples.
Mix the sample with the barcodes immediately and completely by pipetting.
Incubate the samples with the barcodes for 30 min at RT.
Wash cells thrice with 2 ml of Maxpar Cell Staining Buffer. Centrifuge cells at 974 x g for 10 min. Aspirate supernatant from the cells.
Resuspend in 100 μl Maxpar Cell Staining Buffer.
Combine all barcoded samples into one well.
Centrifuge cells at 974 x g for 10 min. Aspirate supernatant from the cells.
Surface staining
Make the surface antibody cocktail in CyFACS buffer of metal-chelating polymer-labeled surface antibodies according to previously determined titration. Prepare sufficient volume of the antibody cocktail (Table 3) to add 200 μl of the cocktail for a barcoded pool of 10 samples or 70 μl of the antibody cocktail/ sample for non-barcoded samples.
Table 3. CyTOF panel for the phenotypic and functional analysis of immune cell subsets in fixed whole blood
Transfer the surface antibody cocktail into a 0.1 μm spin filter and centrifuge using a tabletop microcentrifuge (RCF = 14,000 or max speed) for 2 min at room temperature.
For a barcoded sample pool (x 10 conditions) add 200 μl surface antibody cocktail.
Add 70 μl surface antibody cocktail to each non-barcoded sample.
Incubate the cells at RT for 30 min.
Wash cells with 1 ml CyFACS buffer. Centrifuge cells at 974 x g for 10 min. Discard supernatant by aspiration.
Repeat CyFACS wash. Centrifuge cells at 974 x g for 10 min. Aspirate the supernatant.
Fix: Add 1.8 ml of BD FACS Lysing solution (stock diluted 1x with MilliQ water) to all samples.
Incubate cells overnight at 4 °C.
Intracellular staining
Centrifuge cells at 974 x g for 10 min at 4 °C. Discard supernatant by aspiration.
Prepare 1x Perm (eBioscience permeabilization) Buffer (1:10 dilution with MilliQ water). Add 1 ml Perm buffer.
Centrifuge cells at 974 x g for 10 min at 4 °C.
Repeat wash with Perm buffer.
Centrifuge cells at 974 x g for 10 min at 4 °C.
Make the intracellular antibody cocktail (Table 3) in 1x Perm buffer of metal-chelating polymer-labeled surface antibodies according to previously determined titration. Prepare sufficient volume of the antibody cocktail to add 200 μl of the cocktail for a barcoded sample pool of 10 samples and 70 μl of the antibody cocktail/sample for non-barcoded samples.
Transfer the intracellular antibody cocktail into a 0.1 µm spin filter and centrifuge using a tabletop microcentrifuge (RCF = 14,000 or max speed) for 2 min at room temperature.
For a barcoded sample pool (x 10 conditions) add 200 μl of intracellular antibody cocktail.
Add 70 μl intracellular antibody cocktail to each non-barcoded sample.
Incubate the cells at RT for 30 min.
Wash cells with 1 ml CyFACS buffer. Centrifuge cells at 974 x g for 10 min. Discard supernatant by aspiration.
Repeat 2 x CyFACS washes. Centrifuge cells at 974 x g for 10 min. Discard supernatant by aspiration.
Fix: Prepare 2% PFA from 16% PFA in CyPBS.
Add 200 μl of 2% PFA in CyPBS to each sample.
Incubate at 4 °C overnight.
DNA staining
Add 1 ml CyFACS buffer to each sample.
Centrifuge cells at 974 x g for 10 min at 4 °C. Discard supernatant by aspiration.
Prepare 1:1,000 dilution in 2% PFA (in CyPBS) of Ir-intercalator.
Add 300 μl of diluted Ir-intercalator to each sample and pipet to mix thoroughly.
Incubate at RT for 20 min.
Wash 2 x in CyFACS buffer. Centrifuge cells at 974 x g for 10 min at 4 °C. Discard supernatant by aspiration.
Wash 2 x with MilliQ water. Centrifuge cells at 974 x g for 10 min at 4 °C. Discard supernatant by aspiration.
Using a cell strainer, dilute cells with EQTM Four Element Calibration Beads (1:10 dilution with MilliQ water). Aim for a cell concentration of 0.6-1.0 million cells/ml.
Acquire samples (250k events per sample) on the CyTOF machine, after standard instrument set up.
Figure 1. Schematic of the steps described in the procedure
Data analysis
Identification of Cell Lineages and Functional Subsets
After acquisition on the Helios instrument, FCS files are obtained for downstream analysis. If EQ beads (Fluidigm) are added, the files can be normalized based on EQ bead intensity, using the Nolan Lab MATLAB normalizer available freely on github.com (https://github.com/nolanlab/bead-normalization/releases). The normalized files can then be uploaded to Cytobank (www.cytobank.org) or analyzed in another software such as FlowJo (BD Biosciences) for manual gating. A gating schematic for both barcoded and non-barcoded samples (Figures 2A, 2B, respectively) is shown to demonstrate the application of the panel to both approaches. Hierarchical gating is performed using 191Ir and 193Ir DNA intercalator, 140Ce beads and the event length parameter to discern intact singlets from debris and cell aggregates. All other major immune cell populations are sequentially identified using the lineage surface protein markers as indicated in the respective lineage plots (Figures 2A and 2B). Our panel also enables the identification of immune cell subpopulations such as memory T subsets (CD45RA, CD27 on CD4+ and CD8+ T cells), Tregs (CD4+CD25hiCD127low), activation markers (HLA-DR and CD38 on CD4+ and CD8+ T cells), plasmablasts (CD27hiCD38hi on CD19+CD20+ B cells) and stages of isotype-switched naïve and memory B cells (CD27, IgD on CD19+CD20+ B cells) as indicated in the respective defined plots (Figures 2A and 2B). Figure 2C shows the cytokine expression of TNFα and IFNγ upon PMA/ionomycin stimulation on CD4+ and CD8+ T cells.
Data Reproducibility across Different Batches
To test the reproducibility of the panel, we analyzed data from replicate frozen vials of a fixed control blood sample, previously unstimulated or stimulated with PMA+ionomycin. These control sample replicates were stained and run in 27 separate batches. For each batch, we processed the unstimulated sample replicate in the context of 10 barcoded samples that included other donors and stimulations (Figure 2A), whereas the PMA+ionomycin stimulated replicate was stained and run independently in the same batch (without barcoding). This was to prevent false positive signals due to potential contamination of barcoded samples with the highly-stimulated PMA+ionomycin sample (Figure 2B). We calculated the frequency of the lineage populations using singlets as the parent population (Figure 3A) as well as frequency of TNFα and IFNγ for both CD4+ and CD8+ T cells (Figure 3C) (Leipold et al., 2018). The percent co-efficient of variation (CV) was calculated to determine the degree of variability of expression across batches (Leipold et al., 2018). Our analysis showed that the frequency of parent is comparable (CV ≤ 21%) across the 27 batches for both the lineage populations (Figure 3B) and cytokine expression (Figure 3D), suggesting minimum technical variability.
In summary, this study demonstrates the establishment and assessment of an ICS antibody panel for fixed whole blood that can be adapted to a barcoding approach.
Representative Data
Figure 2. Schematic gating of fixed whole blood. The gating scheme is shown for fixed whole blood from a healthy donor processed in the same batch. The unstimulated condition (A) was included in a barcoded pool of 10 samples, while the PMA/ionomycin stimulated sample (B) was treated independently. Panel C shows the expression of two cytokines IFNγ and TNFα for CD4 and CD8 T cells.
Figure 3. Expression of cell populations and cytokines across batches. A and C show the frequencies of cell lineages and functional subsets, respectively, across 27 batches of healthy control sample replicates. Frequencies are expressed as a percentage of singlets (3A) or of CD4+ or CD8+ T cells (3C). B and D show the percent coefficient of variation calculated across the 27 batches for the cell lineages (3B) and functional subsets (3D).
Recipes
CyPBS
1x PBS without heavy metal contaminants, such as 10x PBS
Made in MilliQ water
Sterile filter before use
CyFACS buffer
1x CyPBS with 0.1% BSA
2 mM EDTA (from 0.5 M EDTA stock)
0.05% sodium azide (from 10% stock)
Made in MilliQ water
Sterile filter before use
Acknowledgments
This work was supported by the NIH grant - R01AI102918. We are grateful to Sheena Gupta and Dongxia Lin (Lin et al., 2015) and Rosemary Fernandez (Fernandez and Maecker, 2015) for development of related protocols. We also thank Natalia Sigal for testing of fixation-resistant antibody clones.
Competing interests
The authors declare that they have no conflicts or competing interests.
References
Behbehani, G. K., Thom, C., Zunder, E. R., Finck, R., Gaudilliere, B., Fragiadakis, G. K., Fantl, W. J. and Nolan, G. P. (2014). Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining. Cytometry A 85(12): 1011-1019.
Fernandez, R. and Maecker, H. (2015). Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry. Bio-protocol 6(11): e1495.
Leipold, M. D., Newell, E. W. and Maecker, H. T. (2015). Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry. Methods Mol Biol 1343: 81-95.
Leipold, M. D., Obermoser, G., Fenwick, C., Kleinstuber, K., Rashidi, N., McNevin, J. P., Nau, A. N., Wagar, L. E., Rozot, V., Davis, M. M., DeRosa, S., Pantaleo, G., Scriba, T. J., Walker, B. D., Olsen, L. R. and Maecker, H. T. (2018). Comparison of CyTOF assays across sites: Results of a six-center pilot study. J Immunol Methods 453: 37-43.
Lin, D., Gupta, S. and Maecker, H. T. (2015). Intracellular cytokine staining on PBMCs using CyTOFTM mass cytometry. Bio-protocol 5(1): e1370.
Möller, L., Schünadel, L., Nitsche, A., Schwebke, I., Hanisch, M. and Laue, M. (2015). Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy. Viruses 7(2): 666-679.
Takahashi, C., Au-Yeung, A., Fuh, F., Ramirez-Montagut, T., Bolen, C., Mathews, W. and O'Gorman, W. E. (2017). Mass cytometry panel optimization through the designed distribution of signal interference. Cytometry A 91(1): 39-47.
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Cell Biology > Cell imaging > Fixed-cell imaging
Molecular Biology > Protein > Detection
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5,005 | https://bio-protocol.org/en/bpdetail?id=5005&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Heterologous Expression and Purification of SARS-CoV2 Nucleocapsid Protein
AG Ankur Garg
LL Lihong Liu
DH David D. Ho
LJ Leemor Joshua-Tor
Published: Aug 5, 2020
DOI: 10.21769/BioProtoc.5005 Views: 4405
Reviewed by: Molly M. LeungZhenying Liu Timo A Lehti
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Abstract
This protocol describes a step by step method for heterologous expression of SARS-CoV2 Nucleocapsid (N) protein in Escherichia coli. Moreover, this protocol includes steps to purify the N protein to high purity and homogeneity. Thus, purified protein can be used for ligand binding assays and other biochemical experiments.
Keywords: COVID-19 SARS CoV2 Nucleocapsid protein Recombinant expression Protein purification
Background
Since its detection in late 2019 in Wuhan, China, SARS-CoV2 infections have been rampant around the world (Wu et al., 2020). In an effort to follow the course of early infections, the nucleocapsid protein (N) was used along with the spike protein (S) in serological assays to monitor the course of early infections in the New York area. N is one of the most highly expressed viral protein, and therefore a good target to follow. To facilitate these assays, a purification protocol was developed for N and used successfully in these studies.
Materials and Reagents
Syringe (VWR International, catalog number: BD309653 )
0.2 μm Syringe filters (VWR International, catalog number: 28196-368 )
Econo-Column 2.5 x 10 cm (Bio-Rad, catalog number: 737-4251 )
HiTrap Heparin HP column 5 ml (GE Healthcare, catalog number: 17040703 )
Amicon Ultra-15 Centrifugal Filter Units 30 kD MWCO (Millipore, catalog number: UFC803096 )
HiLoad 16/600 Superdex200 pg column (GE Healthcare, catalog number: 17106901 )
96-well Uniplate (Fisher Scientific, catalog number: 09-003-36 )
Culture plates (Thermo Fisher Scientific, catalog number: 0 8-757-100D )
Culture flasks (Thermo Fisher Scientific, catalog number: 09-552-70 )
E. coli Rosetta 2 (DE3) chemically competent cells (Novagen, catalog number: 71400-3 )
pET28a(+)_Nucleocapsid plasmid [pET28a(+) vector (Novagen, catalog number: 69864-3 ) with SARS CoV2 Nucleocapsid encoding gene fused to an in frame C-terminal AAALE linker and a 6xHis tag]
Note: pET28a(+)_nucleocapsid plasmid is available upon request from the corresponding author.
Agar (Fisher Scientific, catalog number: BP1423-500 )
Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
Tryptone (Fisher Scientific, catalog number: BP1421-2 )
Di-potassium hydrogen phosphate (K2HPO4) (Sigma-Aldrich, catalog number: 60353 )
Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: 92214 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
1,000x Kanamycin stock solution (50 mg/ml) (Gold Biotechnology, catalog number: K-120-25 )
1,000x Chloramphenicol stock solution (34 mg/ml) (Gold Biotechnology, catalog number: C-105-5 )
1 M IPTG (IsoPropyl-1-Thio-B-D-Galactopyranoside) (Gold Biotechnology, catalog number: I2481C50 )
Tris (Gold Biotechnology, catalog number: T-095-1 )
Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
β-Mercaptoethanol (β-Me) (Sigma-Aldrich, catalog number: M6250-250ML )
100% Glycerol (Fisher Scientific, catalog number: G33-4 )
TurboNuclease (Accelagen, catalog number: N0103M )
Protease inhibitor cocktail (2 μM Pepstatin, 6 μM Leupeptin, 1 μM PMSF, and 2 mM Benzamidine) (Sigma-Aldrich, catalog numbers: 11524488001 , 11017128001 , p7626-25 , B6506-25 )
Polyethylenimine (PEI) (Acros Organics, catalog number: AC178571000 )
Ni-NTA Agarose resin (Qiagen, catalog number: 30250 )
Bradford assay dye reagent concentrate (Bio-Rad, catalog number: 500-0006 )
Imidazole (Affymetrix, catalog number: 17525 )
ATP (P212121, catalog number: CI-00015-25G )
12% Mini-PROTEAN TGX Precast protein gel, 15-well (Bio-Rad, catalog number: 4561046 )
4x Laemmli protein sample buffer for SDS-PAGE (Bio-Rad, catalog number: 1610747 )
InstantBlue Ultrafast protein gel stain (VWR Scientific, catalog number: 95045-070 )
Liquid Nitrogen
Luria-Bertani (LB) medium (see Recipes)
Terrific Broth (TB) medium (see Recipes)
Resuspension buffer (see Recipes)
Ni-NTA buffers
Wash buffer (see Recipes)
Elution buffer 1 (see Recipes)
Elution buffer 2 (see Recipes)
Heparin buffers
Heparin buffer A (see Recipes)
Heparin buffer B (see Recipes)
Heparin dilution buffer (see Recipes)
Size exclusion (SEC) buffer (see Recipes)
10x SDS-PAGE running buffer (see Recipes)
Equipment
Nalgene PPCO Centrifuge bottles (1 L) (Fisher Scientific, catalog number: 05-562-25 )
Ultracentrifuge tubes (Polycarbonate bottle, 38 x 102 mm, 70 ml) (Beckman Coulter Life Sciences, catalog number: 355655 )
Beaker (Sigma-Aldrich)
Magnetic stirrer (Sigma-Aldrich)
Rocker (IBI Scientific)
Sonicator (12.7 mm tip) (QSonica LLC)
Incubator (at 37 °C)
Incubator shaker (Eppendorf New Brunswick Innova)
Centrifuge (Beckman Coulter Life Sciences, model: J6-MI )
Rotor (Beckman Coulter Life Sciences, model: JS 4.3 )
Ultracentrifuge (Beckman Coulter Life Sciences)
Ultracentrifuge Rotor (Beckman Coulter Life Sciences, model: Ti45 )
Mini-PROTEAN® Tetra Vertical Electrophoresis Cell system (Bio-Rad, catalog number: 165-8005 )
AKTA pure FPLC system (GE Healthcare)
Cold room
UV-VIS spectrophotometer (Thermo Fisher Scientific, model: NanoDrop1000 )
Procedure
SARS CoV2 N-protein was cloned into pET28a(+) with an in-frame AAALE linker and 6xHis tag at C-terminal.
Transform 50 μl of chemically competent E. coli Rosetta 2 (DE3) cells with the 50-100 ng pET28a(+)_Nucleocapsid plasmid according to manufactures instructions. Plate the revival mix onto LB agar plate with Kanamycin (Kan) (50 µg/ml) and Chloramphenicol (Cam) (34 µg/ml) antibiotics, and leave it for incubation overnight in 37 °C incubator.
Select a single colony from the LB agar plate and inoculate into 100 ml LB media (supplemented with Kan [50 µg/ml] and Cam [34 µg/ml]) at 37 °C at 220 rpm shaking.
Inoculate 5-10 L TB media (supplemented with 50 µg/ml Kan and 34 µg/ml Cam) with 1% overnight culture and shake at 200 rpm at 37 °C till the cells grow to an OD of 1.5.
Induce protein expression by adding IPTG to final concentration of 0.5 mM, and reduce the culture temperature to 18 °C. Let the culture shake at 180 rpm for next 16-18 h.
Harvest the cells by centrifuging culture at 4,000 x g for 20 min at 4 °C, followed by resuspending the pellet in 20 ml resuspension buffer per liter culture. Pellet can be stored frozen in -80 °C for long term storage. Further purification steps were performed with samples either on ice or in cold room. Where otherwise, temperature is mentioned.
Thaw out the pellet in water bath (at 30 °C) and add 2 U/ml TurboNuclease in the lysate, and lyse the cells by sonication at 100% amplitude for 4-5 min (3 s pulse on and 6 s pulse off). For large volumes (> 150 ml), divide the lysate into two beakers and sonicate separately.
Add PEI (final 0.2%) to the lysate while mixing with a magnetic stirrer for 5 min and ultracentrifuge the lysate for 1 h at 95,000 x g.
Filter the cleared lysate through a 0.2 μm syringe filter. Equilibrate the Ni-NTA agarose beads (0.5 ml beads per liter culture) in Wash buffer and add it to the cleared lysate. Let the Ni-NTA beads incubate on a rocker for 1 h in cold room. [Total volume of Ni-NTA beads = 1 column volume (CV)]
Separate the Ni-NTA beads from the lysate under gravity flow using Econo-column.
Wash the beads with 20 column volumes (CV) of Wash buffer and then elute the protein in Elution buffer 1 (4 fractions of 1 CV each). Most of the protein elutes out from the Ni-NTA beads with maximum protein in fraction 3 (Figure 1).
Elute the remaining protein with Elution buffer 2 (2 fractions of 2 CV each).
Measure the protein amount with Bradford method according to manufacturer’s instructions.
Dilute the 10 ul of collected fractions with the 4x Laemmli protein sample buffer, heat for 5 min at 95 °C and resolve the samples on SDS-PAGE at 200 V to analyze the purity of protein (Figure 1).
Figure 1. A representative SDS-PAGE for Ni-NTA affinity chromatography for SARS CoV2 N-protein. Different samples collected during Ni-NTA were analyzed on 12% SDS-PAGE. Elution fractions are numbered on top of the gel. Fractions 1-4 and 5-6 show protein eluted with Elution buffers 1 and 2 respectively. Some protein stays bound to Ni-beads after elution with 500 mM imidazole buffer.
Pool the elution fractions and dilute 1:1 using Heparin dilution buffer to reduce the NaCl concentration to 100 mM.
Connect HiTrap Heparin-HP column to AKTA pure system and equilibrate the column with 5 CV Heparin buffer A before loading the diluted protein on column using the sample pump. A constant flow rate of 4 ml/min was used for Heparin chromatography.
Wash the Heparin HP column with 8 CV Heparin buffer A, followed by protein elution using a linear gradient from 10 to 100% Heparin buffer B over 150 ml. Protein will elute between 60-70% Heparin buffer B concentration.
Collect 1-2 ml fractions in clean tubes and run the peak fractions on SDS-PAGE for analyzing the purity of the protein. A typical Heparin-HP elution chromatogram for N-protein is shown in Figure 2.
Figure 2. A representative SDS-PAGE and chromatogram for Heparin HP (5 ml) chromatography for SARS CoV2 N-protein. N-proteins eluted out between 60-70% Heparin buffer B and the fractions covering whole peak (marked with red arrow on top of the peak) were analyzed on 12% SDS-PAGE. Fractions having pure protein were further purified on size exclusion chromatography.
Pool the pure fractions from HiTrap Heparin HP elutions and concentrate it to ~2 ml final volume using 30 kD MWCO Amicon Ultra-15 Centrifugal Filter Units as per manufacturer’s instructions.
Equilibrate the HiLoad 16/600 Superdex200 pg column with SEC buffer and inject the concentrated protein using capillary loop followed by eluting it out with SEC buffer at 1 ml/min flow rate. A single peak for the homogenous protein will be observed.
Collect 1 ml fractions in clean tubes and run the peak fractions on SDS-PAGE for analyzing the protein purity (Figure 3).
Figure 3. A representative SDS-PAGE and chromatogram for the size exclusion chromatography for SARS CoV2 N-protein. Concentrated protein was loaded on HiLoad 16/600 Superdex200 pg column and fractions covering whole peak (marked with red arrow in chromatogram) were analyzed on a 12% SDS-PAGE. Only pure protein containing fractions (marked on top of the gel) were pooled, concentrated and stored.
Pool the pure protein containing fractions and concentrate using 30 kD MWCO Amicon Ultra-15 Centrifugal Filter Units to desired concentration.
Protein aliquot can be flash frozen in liquid nitrogen and stored at -80 °C for long term storage.
Data analysis
This section explains how to determine the yield and purity of protein after size exclusion chromatography.
a.A Spectrophotometer/NanoDrop can be used to measure the absorbance at 280 nm and 260 nm.
b.The 280 nm absorbance and the correction factor of 0.932 was used to determine the real concentration of N protein (Real cons = A280/0.932). Purified protein shows a 260/280 ratio between 0.55-0.60, which represents that there is no nucleic acid contamination in the protein.
The UNICORN 7.4 software (GE Healthcare) was used to visualize the chromatograms and preparing chromatogram figures.
Recipes
LB medium
Weigh out 10 g tryptone, 5 g yeast extract, and 10 g NaCl
Add up to 900 ml deionized water
Adjust pH to 7.5 with 5 N NaOH
Adjust the final volume to 1 L with deionized water
Sterilize by autoclaving at 121 °C for 30 min
Store at room temperature
TB medium
Add 900 ml water to 20 g tryptone, 24 g yeast extract and 4 ml glycerol
Stir until the solutes have dissolved and sterilize by autoclaving
Prepare 100 ml Phosphate buffer (0.17 M KH2PO4 + 0.72 M K2HPO4) and sterilize by autoclaving
When at room temperature mix both the solutions and use immediately
Resuspension buffer
Tris 25 mM (pH 8.0)
NaCl 500 mM
β-Me 2 mM
Glycerol 5%
Imidazole 10 mM
Protease inhibitor cocktail
Ni-NTA buffers
Wash buffer
Tris 25 mM (pH 7.4)
NaCl 500 mM
β-Me 2 mM
Glycerol 5%
Imidazole 50 mM
ATP 1 mM
Elution buffer 1
Tris 25 mM (pH 7.4)
NaCl 200 mM
β-Me 2 mM
Glycerol 5%
Imidazole 250 mM
Elution buffer 2
Tris 25 mM (pH 7.4)
NaCl 200 mM
β-Me 2 mM
Glycerol 5%
Imidazole 500 mM
Heparin buffers
Heparin buffer A
Tris 25 mM (pH 7.4)
NaCl 100 mM
β-Me 2 mM
Heparin buffer B
Tris 25 mM (pH 7.4)
NaCl 1 M
β-Me 2 mM
Heparin dilution buffer
Tris 25 mM (pH 7.4)
β-Me 2 mM
Size exclusion (SEC) buffer
Tris 25 mM (pH 8.0)
NaCl 500 mM
β-Me 2 mM
10x SDS-PAGE running buffer
Dissolve 30 g Tris base, 144 g glycine and 10 g SDS in 1000 ml of deionized water
pH of solution should be 8.3 and no pH adjustment is required
Store the buffer at room temperature and dilute to 1x with deionized water before use
Acknowledgments
This work was supported by the Meier & Linnartz Family Foundation. L.J. is an Investigator of the Howard Hughes Medical Institute.
Competing interests: Authors declare no competing interest.
References
Wu, F., Zhao, S., Yu, B., Chen, Y. M., Wang, W., Song, Z. G., Hu, Y., Tao, Z. W., Tian, J. H., Pei, Y. Y., Yuan, M. L., Zhang, Y. L., Dai, F. H., Liu, Y., Wang, Q. M., Zheng, J. J., Xu, L., Holmes, E. C. and Zhang, Y. Z. (2020). A new coronavirus associated with human respiratory disease in China. Nature 579(7798): 265-269.
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Molecular Biology > Protein > Expression
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Extraction of Orthologs from Genome-Sequencing Data for Phylogenetic Analysis
GP Guan Pang
FC Feng M. Cai
Published: Jun 5, 2024
DOI: 10.21769/BioProtoc.5008 Views: 84
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Abstract
Homologs, including paralogs and orthologs, are genes that share sequence homologies within or between species. Determination of single-copy orthologs for phylogenomic analysis is the first step in all comparative genomic research. The current protocol provides a detailed bioinformatic pipeline from sequence data acquisition to phylogenetic reconstruction with the use of two commonly adopted tools: OrthoFinder and IQ-TREE. The protocol is demonstrated using genomic data from five fungi, including four Trichoderma spp. and an Escovopsis weberi, which served as the outgroup in the current case. Additionally, we also demonstrate a partitioned analysis for concatenated multi-locus datasets. The protocol is simple, does not require extensive bioinformatic training or special equipment, and can be easily reproduced for genome-sequencing data from other taxonomic groups.
Keywords: Gene tree Maximum likelihood phylogeny Molecular evolution Orthogroup Phylogenetics Species tree Substitution model
Background
With huge advances both in evolutionary theories and sequencing technologies, phylogenetic analysis is entering a new era—phylogenomics. Current methods for phylogenomic inference can generally be categorized into two types: supertree and supermatrix methods [1–3]. The former approach obtains one supertree by combining inferred individual gene trees, each containing information from partially overlapped sets of taxa. Alternatively, the supermatrix approach analyzes the concatenated alignment of individual genes. Unavailable genes/loci are coded as missing data in the supermatrix [2,4]. Likelihood-based reconstruction methods are particularly suited for the analysis of supermatrices. These methods consider the heterogeneity across genes referring to evolutionary rates by using partitioned-likelihood models, which allow each gene to evolve under a different substitution model. According to the total evidence principle of using all the relevant available data, it is somewhat more popular as a strategy to adopt the supermatrix method, which is also used in the current pipeline of demonstrated examples.
The two crucial steps of standard phylogenetic inference are the identification of homologous sequences and tree reconstruction. Therefore, besides the accuracy of the tree-building method, the reliability of a phylogenomic tree also largely depends on the quality of homology, that is, the determination of paralogs and orthologs within and between genomes [5]. In contrast to paralogs, which are derived from gene duplication and should thus be excluded from phylogenetic analyses, orthologs are genes that are derived from speciation events; orthology, in this case, refers to the relationship between the corresponding genes in different species. So far, the most widely used methods for orthology inference can be classified into two groups [6]. One group infers pairwise relationships between genes in two species and then to multiple species, while the other identifies complete orthogroups (OGs), which are identified as the set of genes descended from a single gene in the last common ancestor of all of the species considered [5,7].
In the current pipeline, we use OrthoFinder, a popular method for inferring OGs of protein-coding genes. Starting from gene sequences (the input files), an advantage of using this program is that, by default, it infers OGs, orthologs, the complete set of gene trees for all OGs, the rooted species tree, and all possible gene duplication events. Furthermore, it also provides extensive comparative genomics statistics [5,7]. Despite the fact that OrthoFinder generates individual gene trees and the species tree, for customized tree building we recommend IQ-TREE (IQ-TREE 2 here) for subsequent analyses. In our laboratory, when working with multiple fungal genomes, IQ-TREE runs fast and provides automatic model selection, which also includes data partitioning, an efficient search algorithm for ML trees, ultrafast bootstrapping, and more [8]. With this protocol, we aim to demonstrate effective examples of orthology inference, ortholog extraction, paralog exclusion, individual gene tree reconstruction with the ML method, and data partitioning. This is done using five fungal genomes, with the four main members belonging to the genus Trichoderma. Trichoderma spp. are among the best studied groups of filamentous fungi due to their high value in applications from agriculture to industrial enzyme production [9]. The present protocol is simple and can also be easily adopted for genomic data from other organisms.
Equipment
We explicitly assume that the user has some basic skills in working in a Linux-based operating system.
Linux cluster
In the present study, we used the AuthenticAMD supercomputer, which has two nodes, each containing 32 cores (model name: AMD EPYC 7452 32-Core Processor) and 256 GB of memory in total
Personal computer (PC)
We recommend using a PC with an Intel Core i7-10510U CPU or higher and at least 16 GB of RAM for sufficient post data processing
Software
OrthoFinder ([5,7], v2.5.4, https://github.com/davidemms/OrthoFinder)
IQ-TREE (8,10, v2.2.0.3, https://github.com/iqtree/iqtree2)
Note: The required software and its dependents (including IQ-TREE, which is also integrated in OrthoFinder) should be installed properly according to the tutorials mentioned above before the analysis.
Procedure
The individual steps in this protocol are summarized in Figure 1.
Figure 1. Bioinformatic workflow for orthology inference and maximum likelihood (ML) tree reconstruction based on genome-sequencing data. Three sections are illustrated: (A) orthology inference using OrthoFinder, (B) single-copy ortholog extraction, and (C) ML tree reconstruction (including model test and data partitioning) using IQ-TREE.
Orthology inference
Prepare input data
For given genomes on which annotation has been previously performed, download the coding sequences (CDSs) or protein sequences from the appropriate database. The current protocol uses genome datasets from five fungal strains [9,11,12] deposited in MycoCosm of the DOE Joint Genome Institute (JGI) [13]. The data resource of each fungal strain used in this protocol is shown below, which allows downloading after registration. For customized data, a minimum set of three samples (genomes) is required for such an analysis. We also recommend a minimum sequencing depth of 10× for each sample in order to obtain a sufficient OG set.
Escovopsis weberi CC031208-10:
https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Escweb1
Trichoderma atroviride IMI 206040:
https://genome.jgi.doe.gov/portal/Triat2/Triat2.download.ftp.html
Trichoderma harzianum CBS 226.95:
https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Triha1
Trichoderma reesei QM6a:
https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Trire_Chr
Trichoderma virens Gv29-8:
https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=TriviGv29_8_2
An example of the designated sequence files is illustrated with the CDS file from Escovopsis weberi (Figure 2A). Once the compressed files of all strains are downloaded in one folder (“test” in this protocol), use the gunzip command to obtain the FASTA files for each strain. Rename each FASTA file as shown in Figure 2B.
gunzip *.gz
Figure 2. An overview of the architecture of genomic data deposited in MycoCosm. (A) Escovopsis weberi CC031208-10 (the CDS file is highlighted by a red frame). (B) List of renamed FASTA files for the five fungal genome examples in this protocol.
Find OGs
In this protocol, we adopted OrthoFinder to infer the orthology of protein-coding genes across multiple species. OrthoFinder does not only infer OGs; it also automatically produces several output files, such as individual gene trees, a rooted species tree, or gene duplication event results (see more in Emms and Kelly [7]). A full picture of the standard outputs can be found in Figure 3. A detailed guided tour of the other result files can be found at the following link: https://github.com/davidemms/OrthoFinder#orthofinder-results-files. In the current pipeline, we focus on the identification of the orthologous genes from a certain group of fungal species. To this end, use the following command:
orthofinder -f test/ -d -M msa -t 40 -a 40
Figure 3. List of typical output files generated by running the command shown above via OrthoFinder
Note: -f is an option to specify the directory containing the working FASTA files. -d specifies the input as DNA sequences [Default = protein, p]. OrthoFinder uses MAFFT for the alignment and FastTree for the tree inference by default. In order to obtain a more accurate result, such as inference of ML trees from multiple sequence alignments (MSAs), it is also possible to use an alternative alignment or tree inference program in OrthoFinder, which usually requires a more computationally costly resource. For example, to call MUSCLE and IQ-TREE, the commands to add are -M msa -A muscle -T iqtree. The option -M msa in this case allows the inference of the species tree from a concatenated MSA of single-copy orthologous genes. However, in the case of a first analysis, FastTree is highly recommended.
Optionally, while OrthoFinder is designed to require minimal computation, it can be tailed by using the -t and -a options to suit the computational and data sources. -t indicates the number of threads for sequence search, MSA, and tree inference [Default is the number of cores on the machine], and -a indicates the number of parallel analysis threads for internal RAM-intensive tasks [Default = 1]. Note that an extra step of adding a [Speciesidentifier] at the head of each FASTA sequence is required if partitioning tree reconstruction is subsequently needed for the phylogenetic analysis (Section C). To do this, write the following command before running OrthoFinder.
For file in *.fasta; do sed -i 's/>/>Speciesidentifier_/g' $file; done
Single-copy orthologous gene extraction
Orthologs are genes that derive from speciation events, in contrast to paralogs, which derive from gene duplication and should thus be excluded from phylogenetic analyses [6], as mentioned above. In the current pipeline, to obtain the orthologous gene sequences from multiple genomes for subsequent analyses such as gene tree reconstruction (Section C), only the results exported in the folders of [Single_Copy_Orthologue_Sequences] and [MultipleSequenceAlignments] are needed.
Extract the IDs of single-copy OGs
The [Single_Copy_Orthologue_Sequences] folder contains FASTA files of each OG. These OGs ideally contain one gene per species at most, from which paralogs have been excluded. Here is an example of extracting the IDs of single-copy OGs from the designated FASTA files to a txt file as an output (namely [singlecopyOG_idlist.txt].
ls -l Single_Copy_Orthologue_Sequences | awk '{print $9}' | awk NF > singlecopyOG_idlist.txt
Retrieve multiple sequence alignments of single-copy OGs
In the current protocol, we take advantage of ready MSAs of each OG generated in Section A. Thus, with the ID list of single-copy OGs obtained in the last step, the aligned and trimmed FASTA files of each single-copy OG can be easily retrieved from the results sorted in the folder of [MultipleSequenceAlignments]. To do so, use the following command:
mkdir singlecopyOG_seqs
length=`awk 'END{print NR}' ./singlecopyOG_idlist.txt`; for x in $(seq 1 $length); do y=`awk "{if (NR == $x) print }" ./singlecopyOG_idlist.txt`; cp ./MultipleSequenceAlignments/$y* ./singlecopyOG_seqs; done
Note: An example (OG0000819) comparing the data in the folder of [Single_Copy_Orthologue_Sequences] and the output files (see [singlecopyOG_seqs] generated in this step) is given in Figure 4. Importantly, although the majority of species tree inferences are recommended to be restricted to one-to-one orthologous sequences that are present in all species in the analysis, realistically, such groups of sequences are rare in real biological datasets, especially for distant taxa. Thus, such cases are only available if gene duplication or loss has not occurred during the divergence of that gene family [7]. To address this challenge, the updated OrthoFinder2 has been designed to leverage the sequence data from all genes via a new algorithm: Species Tree from All Genes (STAG). STAG was developed to allow species tree inference for sample sets with few or no complete sets of one-to-one orthologs present in all species of interest using the most closely related genes within single-copy or multi-copy OGs [7].
Figure 4. Example of sequence data (OG0000819). Sequence data before (from the folder [Single_Copy_Orthologue_Sequences] (A) and after alignment ([singlecopyOG_seqs] (B).
Reconstruction of individual gene trees with ML method
If a single gene tree for some specific gene(s) of interest is required, ML tree reconstruction via IQ-TREE is recommended in this protocol. An example for using the single-copy OG MSAs (OG0000819) obtained in Section B is given below.
Select substitution model and start ML tree reconstruction
Phylogenetic tree reconstruction methods such as the ML method usually start with model selection, in which the program searches for the best-fit model of sequence evolution of the available data [14]. IQ-TREE has been developed to support a wide range of substitution models for DNA, protein, codon, and also morphological data by integrating ModelFinder in it [8]. Start the tree reconstruction by entering:
iqtree2 -s OG0000819.fa -B 1000
With this command, IQ-TREE calls ModelFinder by default. If only the best-fit model is required without doing the tree reconstruction, then run:
iqtree2 -s OG0000819.fa -m MF
For multiple MSAs, such as all of the single-copy OG MSAs obtained in [singlecopyOG_seqs] of Section B, write a loop as follows:
for file in *.fa; do iqtree2 -s $file -B 1000; done
Note: -s is used to specify the name of the MSA file. IQ-TREE also supports other input file formats such as NEXUS, CLUSTALW, and PHYLIP. -m is used to specify the name of the model if known beforehand (for example, -m TIM+F). -B is used to specify the number of bootstrap replicates, and 1000 is recommended. IQ-TREE writes several output files, such as .iqtree, .treefile, and .log files (see more at http://www.iqtree.org/doc/). In the current protocol, the .treefile is the designated file for tree visualization. The .log file records the entire run, including the best-fit model used in the tree reconstruction. ModelFinder integrated in IQ-TREE computes the loglikelihoods of an initial parsimony tree for different models and automatically chooses the model that minimizes the Bayesian Information Criterion (BIC) score if no customized command is given. Otherwise, -AIC or -AICc can be used to change model selection according to the Akaike Information Criterion (AIC) or the corrected Akaike Information Criterion (AICc), respectively. Note that IQ-TREE prevents loss of data by overwriting [8]. If a rerun is needed, add the -redo option at the end of the command line.
Run partitioned analysis for multi-locus alignments
IQ-TREE also allows combining sub-alignments from different MSAs (e.g., the MSAs in the folder of [singlecopyOG_seqs] generated in Section B). Here, an example is given below using three of the single-copy OG MSAs prepared in a NEXUS input file (i.e., threeOG.nex). As every gene within each OG has its own unique ID, it is essential to rename all of the gene IDs for each taxon (refer to the [Speciesidentifier]).
for file in singlecopyOG_seqs/*.fa; do awk -F "_Speciesidentifier" '{print $1}' ${file##/*} > ${file##/*}.renamed; done
iqtree2 -s threeOG.nex -p threeOG.nex -B 1000
Note: -p allows each partition to have its own evolution rate. Note that with the use of this command, ModelFinder implements a greedy strategy that starts with the full partition model and then merges every two loci until the model fit does not increase further [15]. This causes considerable computational burden. Therefore, partitioning tree reconstruction should be used as a precaution for large datasets. For preparing concatenated multi-locus datasets in a NEXUS file, programs such as PhyloSuite [16], SequenceMatrix [17], or some other software providing similar functions are recommended.
Data analysis
The resulting ML tree (in NEWICK format) from IQ-TREE can be visualized by any supported tree viewers on a PC, such as Figtree (v1.4.4, https://github.com/rambaut/figtree/releases) and iTOL (https://itol.embl.de/). Figure 5 shows an example of an annotated tree. The results obtained using the current protocol showed a similarly robust phylogenomic relationship for the fungi tested as the ones previously published [9,12].
Figure 5. Maximum likelihood (ML) phylogenetic tree constructed based on the concatenated sequence matrix of three randomly selected single-copy orthologs in [singlecopyOG_seqs]. Individual models TN+F+G4, TIM3+F+G4, and TPM3u+F+G4 were selected according to BIC for the three orthologs OG0000545, OG0002657, and OG0002676, respectively. The IQ-TREE ultrafast bootstrap values are represented by the nodes (ultrafast bootstrap, N = 1000).
Acknowledgments
This work was supported by the National Natural Science Foundation of China (32102473) and the Fundamental Research Funds for the Central Universities (KYQN2022046).
Competing interests
The authors declare no competing financial interest.
References
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Delsuc, F., Brinkmann, H. and Philippe, H. (2005). Phylogenomics and the reconstruction of the tree of life. Nat Rev Genet. 6(5): 361–375. https://doi.org/10.1038/nrg1603
Sanderson, M. J., McMahon, M. M. and Steel, M. (2011). Terraces in Phylogenetic Tree Space. Science (1979) 333(6041): 448–450. https://doi.org/10.1126/science.1206357
Chernomor, O., von Haeseler, A. and Minh, B. Q. (2016). Terrace Aware Data Structure for Phylogenomic Inference from Supermatrices. Syst Biol. 65(6): 997–1008. https://doi.org/10.1093/sysbio/syw037
Emms, D. M. and Kelly, S. (2015). OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 16(1): e1186/s13059–015–0721–2. https://doi.org/10.1186/s13059-015-0721-2
Debray, K., Marie-Magdelaine, J., Ruttink, T., Clotault, J., Foucher, F. and Malécot, V. (2019). Identification and assessment of variable single-copy orthologous (SCO) nuclear loci for low-level phylogenomics: a case study in the genus Rosa (Rosaceae). BMC Evol Biol. 19(1): e1186/s12862–019–1479–z. https://doi.org/10.1186/s12862-019-1479-z
Emms, D. M. and Kelly, S. (2018). OrthoFinder: phylogenetic orthology inference for comparative genomics: Genome Biol. 238 https://doi.org/10.1101/466201
Nguyen, L. T., Schmidt, H. A., von Haeseler, A. and Minh, B. Q. (2014). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol Biol Evol. 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
Kubicek, C. P., Steindorff, A. S., Chenthamara, K., Manganiello, G., Henrissat, B., Zhang, J., Cai, F., Kopchinskiy, A. G., Kubicek, E. M., Kuo, A., et al. (2019). Evolution and comparative genomics of the most common Trichoderma species. BMC Genomics. 20(1): 485. https://doi.org/10.1186/s12864-019-5680-7
Minh, B. Q., Schmidt, H., Chernomor, O., Schrempf, D., Woodhams, M., von Haeseler, A. and Lanfear, R. (2019). IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 37(5):1530–1534. https://doi.org/10.1101/849372
de Man, T. J. B., Stajich, J. E., Kubicek, C. P., Teiling, C., Chenthamara, K., Atanasova, L., Druzhinina, I. S., Levenkova, N., Birnbaum, S. S. L., Barribeau, S. M., et al. (2016). Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture. Proc Natl Acad Sci U S A. 113(13): 3567–3572. https://doi.org/10.1073/pnas.1518501113
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Article Information
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Systems Biology > Genomics > Phylogenetics
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A Multi-Color Immunofluorescence Assay to Distinguish Intracellular From External Leishmania Parasites
AD Arani Datta
UB Umaru Barrie
DW Dawn M. Wetzel
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.5009 Views: 506
Reviewed by: Chiara AmbrogioRamu Kakumanu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of Cell Science Jun 2023
Abstract
Leishmaniasis, a neglected tropical disease, is caused by the intracellular protozoan parasite Leishmania. Upon its transmission through a sandfly bite, Leishmania binds and enters host phagocytic cells, ultimately resulting in a cutaneous or visceral form of the disease. The limited therapeutics available for leishmaniasis, in combination with this parasite’s techniques to evade the host immune system, call for exploring various methods to target this infection. To this end, our laboratory has been characterizing how Leishmania is internalized by phagocytic cells through the activation of multiple host cell signaling pathways. This protocol, which we use routinely for our experiments, delineates how to infect mammalian macrophages with either promastigote or amastigote forms of the Leishmania parasite. Subsequently, the number of intracellular parasites, external parasites, and macrophages can be quantified using immunofluorescence microscopy and semi-automated analysis protocols. Studying the pathways that underlie Leishmania uptake by phagocytes will not only improve our understanding of these host–pathogen interactions but may also provide a foundation for discovering additional treatments for leishmaniasis.
Key features
• This protocol visualizes and quantifies multiple intracellular forms of Leishmania.
• It offers flexibility at various points for researchers to introduce modifications according to their study needs.
Keywords: Leishmania Amastigote Macrophage Confocal microscopy Fluorescent labeling Phagocytic index
Graphical overview
Background
Leishmaniasis wreaks havoc in a population of approximately 2 million annually across 90 endemic countries, being caused by < 20 different species of the parasite Leishmania [1]. Depending on the infective species and host immune system function, the resulting pathology can include cutaneous, mucocutaneous, or visceral disease. A need for new, effective drugs is demonstrated by the inadequacies of existing treatments since current drugs for leishmaniasis suffer serious limitations such as poor efficacy, toxicity, and resistance [2,3].
In their sand fly vector, Leishmania exist as free, motile promastigotes with an elongated cell shape as well as a long flagellum. After transmission into the mammalian host, they multiply, undergo transformation, and subsequently survive as intracellular amastigotes in the phagolysosomal compartment of macrophages (MΦ) and other phagocytes [4]. Amastigotes exhibit significantly lower cell surface-to-volume ratio, and the flagellum is reduced to a tiny distended tip [5]. Although both forms are capable of infecting mammalian cells, only amastigotes can spread and eventually sustain the infection in the host [6,7]. Conversely, for many (although not all) Leishmania species, only promastigotes can be cultured without mammalian cells (axenically). As such, although the process of Leishmania uptake by human MΦ has been studied using confocal microscopy, the relative intractability of amastigotes for most species has resulted in a predominant focus on promastigotes, which in turn overlooks many of the intricacies involved in Leishmania’s interactions with host MΦ [8]. Since the host cell’s response to promastigotes and amastigotes varies significantly, a more comprehensive understanding of the entire life cycle is required for effective pathogenesis studies and drug discovery strategies.
Here, we provide a multi-color immunofluorescence protocol for studying the uptake of Leishmania by MΦ with microscopy. This protocol has been adapted from a number of prior studies in the parasitology field. However, in combination with various culture techniques, our protocol, encompassing both promastigotes and amastigotes in the experimental setup, has further expanded approaches to studies characterizing host–pathogen interactions. Furthermore, our protocol allows many ways in which it can be adapted to the situation at hand, including characterization of higher-order subcellular structures during parasite internalization or high-throughput screening and analysis for drug discovery. Our expanded methodology provides a foundation for a better understanding of the pathogenesis of leishmaniasis and the development of more efficacious and targeted therapeutic interventions.
Materials and reagents
Biological materials
RAW 264.7 cells (ATCC, catalog number: TIB-71)
.Leishmania (L.) amazonensis promastigotes (strain IFLA/BR/67/PH8) (Norma W. Andrews, University of Maryland, College Park, MD)
L. amazonensis-mNeon, expressing bright monomeric green fluorescent protein, mNeonGreen [9]
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), with 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma-Aldrich, catalog number: D6429), store at 4 °C
Benchmark fetal bovine serum (FBS), heat inactivated (GeminiBio, catalog number: 100-106-500-HI), store at -20 °C
Penicillin/streptomycin solution (GeminiBio, catalog number: 400-109-100), store at -20 °C
Hygromycin B solution (50 mg/mL) (GeminiBio, catalog number: 400-123-020), store at 4 °C
Schneider’s Drosophila medium (Sigma-Aldrich, catalog number: S9895), store at 4 °C
Hemin (Sigma-Aldrich, catalog number: H9039), store at 4 °C
Sodium hydroxide solution, 10 M in water (Sigma-Aldrich, catalog number: 72068)
Phosphate buffered saline (PBS), pH 7.4, liquid, sterile-filtered, suitable for cell culture (Sigma-Aldrich, catalog number: 806552), store at 4 °C
Formaldehyde, 37% in water (Sigma-Aldrich, catalog number: F1635)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number A7906), store at 4 °C
Triton X-100, molecular biology grade (Sigma-Aldrich, catalog number: T8787)
Mouse anti-p8 antibody, generated from hybridoma cell line generously donated from McMahon-Pratt laboratory, Yale University; available upon request, store at -20 °C
Anti-mouse IgG (H+L), F(ab')2 fragment, CF-488A-conjugate antibody produced in goat (Sigma-Aldrich, catalog number: SAB4600388), store at -20 °C
Anti-mouse IgG (H+L), highly cross-adsorbed, CF-568-conjugate antibody produced in donkey (Sigma-Aldrich, catalog number: SAB4600075), store at -20 °C
Hoechst 33342 solution, 20 mM (Thermo Fisher, catalog number: 62249), store at 4 °C
Alexa FluorTM 647 Phalloidin (Thermo Fisher, catalog number: A22287), store at -20 °C
Solutions
Complete RAW 264.7 cell media (see Recipes)
50 mM sodium hydroxide (see Recipes)
Hemin solution (see Recipes)
Complete promastigote media (see Recipes)
Complete amastigote media (see Recipes)
Fixing solution (see Recipes)
Blocking solution (see Recipes)
Permeabilization solution (see Recipes)
Recipes
Complete RAW 264.7 cell media, final pH 7.4 (4 °C storage)
Reagent Final concentration Quantity
DMEM n/a 440 mL
FBS 10% 50 mL
Penicillin/streptomycin solution 1× 5 mL
Hygromycin 1× 5 mL
Total n/a 500 mL
50 mM sodium hydroxide
Reagent Final concentration Quantity
10 M sodium hydroxide 50 mM 0.5 mL
Water n/a 99.5 mL
Total n/a 100 mL
Hemin solution (4 °C storage)
Reagent Final concentration Quantity
Hemin 2.5 mg/mL 25 mg
50 mM sodium hydroxide n/a 10 mL
Total n/a 10 mL
Complete promastigote media, final pH 7.4 (4 °C storage)
Reagent Final concentration Quantity
Schneider’s Drosophila medium n/a 835 mL
FBS 15% 150 mL
Penicillin/streptomycin solution 10% 10 mL
Hygromycin 1× 5 mL
Total n/a 1000 mL
Complete amastigote media, final pH 5.4 (4 °C storage)
Reagent Final concentration Quantity
Schneider’s Drosophila medium n/a 785 mL
FBS 20% 200 mL
Penicillin/streptomycin solution 1× 10 mL
Hemin solution (Recipe 3) 0.5% 5 mL
Total n/a 1,000 mL
Adjust final pH to 5.4.
Fixing solution (-20 °C storage)
Reagent Final concentration Quantity
Formaldehyde, 37% in water 4% 5 mL
1× PBS n/a 45 mL
Total n/a 50 mL
Blocking solution (4 °C storage)
Reagent Final concentration Quantity
BSA 5% 2.5 g
1× PBS n/a 50 mL
Total n/a 50 mL
Permeabilization solution
Reagent Final concentration Quantity
Triton X-100 5% 0.5 mL
Blocking solution n/a 9.5 mL
Total n/a 10 mL
Laboratory supplies
Sterile 15 mL polypropylene centrifuge tubes (any vendor)
Sterile 50 mL polypropylene centrifuge tubes (any vendor)
Nalgene rapid-flow sterile disposable filter units 1 L (Thermo Scientific, catalog number: 5670020)
Sterile nuclease-free filter tips (10, 200, and 1,000 μL) (any vendor)
Vented flask surface area 25 cm2 (any vendor)
Sterile cell culture dish, 100 mm (Greiner Bio-One, catalog number: 664160)
Sterile cell scraper (Falcon, catalog number: 353089)
PhenoPlate 96-well, black, optically clear flat-bottom, tissue-culture treated microplate (PerkinElmer, catalog number: 6055300)
Disposable plastic serological pipettes, sterile, individually wrapped, 5 mL, 10 mL, 25 mL capacity (any vendor)
Hemocytometer (Hausser Scientific, catalog number: 3200)
Microcentrifuge 1.7 mL tubes (Corning, Axygen®, catalog number: MCT-175-C-S)
Bleach (Clorox)
50 mL reagent reservoir (Corning, catalog number: 4870)
PoseidonTM 12 channel pipettor P200, adjustable volume, 20–200 µL (Genesee Scientific, catalog number: 33-GSC-12P200)
Plastic container for waste collection, 1 L size (any vendor)
Equipment
Centrifuge 5810R (Eppendorf, catalog number: 022628187)
Inverted confocal microscope (Zeiss, model: LSM 800)
Software and datasets
Zen 2.3 SP1 FP3 (black) 64-bit (Carl Zeiss), licensed
GraphPad Prism Version 10.1.2 (324), licensed
ImageJ 1.53t (NIH, https://imagej.net/ij/download.html)
Microsoft Excel 2016 (16.0.5422.1000) MSO 32-bit, licensed
Adobe Illustrator 28.1 64-bit, licensed
Procedure
Preparation of amastigotes from promastigotes
Filter-sterilize both complete promastigote media (see Recipes) and complete amastigote media (see Recipes) using the Nalgene rapid-flow sterile disposable filter units prior to any cell culture.
Culture L. amazonensis promastigotes in 10 mL of complete promastigote media in vented 25 cm2 tissue culture flasks in an incubator at 26 °C for a week.
Caution: The incubator containing the culture should not contain any fungal cells or bacteria to avoid potential cross-contamination issues. Regular cleaning of the incubator (at least once every two weeks) is recommended. All species of Leishmania that can infect humans are Biosafety Level 2 organisms and appropriate precautions should be taken in this regard.
Remove 5 mL of confluent L. amazonensis promastigotes from the flask and add 5 mL of complete amastigote media to the same flask. Keep in an incubator at 32 °C overnight with 5% CO2.
Without discarding the flask, pipette 10 mL of this mixed culture into a sterile 15 mL tube and spin in a centrifuge at 3,000× g for 5 min at room temperature. Discard the supernatant into a container with 10% bleach. Carefully resuspend the parasite cell pellet in 10 mL of complete amastigote media in the same flask and keep in the incubator at 32 °C with 5% CO2 for five days to grow healthy amastigote cells.
Simultaneous preparation of RAW 264.7 cells
Filter-sterilize complete RAW 264.7 cell media (see Recipes) using the Nalgene rapid-flow sterile disposable filter unit prior to any cell culture.
Place 10 mL of complete RAW 264.7 cell media with RAW 264.7 cells in a new 100 mm cell culture dish for 3–4 days.
At ~50% confluency, discard the media using a pipette into a bleach container and rinse carefully with 5 mL of sterile PBS. Pipette 5 mL of fresh DMEM media without serum into the plate. Carefully scrape the base of the plate until the cells are off the base, resuspend, and pipette them into a sterile 15 mL tube prior to infection.
Set up infection plate
Using a 1.7 mL microcentrifuge tube, prepare 100 µL of 1:5 cell dilution from the 5 mL culture of RAW 264.7 cells from section B, and use 10 µL on hemocytometer for cell counting.
Prepare a suspension of ~50,000 cells per milliliter in 10 mL of DMEM media without serum and pipette the suspension into a 50 mL reagent reservoir (see note after section D for important considerations).
Using a 12-channel pipettor, pipette 100 µL of cell suspension in each well of a microscopy-grade, black, optically clear flat-bottom, tissue-culture treated, 96-well microplate (coverslips can also be used).
(Optional) Carefully transfer the microplate into a plate adapter before placing in the centrifuge and spin at 500× g for 1 min at room temperature to ensure the settling of the cells at the base of the wells.
Place plate in an incubator at 37 °C overnight with CO2.
Set up internalization
Next morning (~12–18 h after section C), carefully pipette out 10 mL of amastigotes from step A4 into a 15 mL tube and centrifuge at 3,000× g for 5 min at room temperature to remove any trace of complete amastigote media. Discard the supernatant into a container with 10% bleach. Carefully resuspend cell pellet in 10 mL of complete RAW 264.7 cell media in a new 15 mL tube. Repeat two times.
Using a 1.7 mL microcentrifuge tube, prepare 1,000 µL of 1:100 cell dilution from this culture. Subsequently, use 10 µL of this cell dilution on hemocytometer for cell counting.
Calculate and prepare a suspension of 106 amastigotes per milliliter in 10 mL of complete RAW 264.7 cell media and pipette the suspension into a 50 mL reagent reservoir. Using a 12-channel pipettor, pipette 100 µL of amastigote suspension to the existing 100 µL of RAW 264.7 cells in each well of the microscopy grade 96-well plate set up in step C3 (see note below for important considerations at this step).
Carefully transfer the microplate into a plate adapter before placing in the centrifuge and spin at 500× g for 1 minute at room temperature to ensure that parasites settle at the base of the wells.
Incubate at 37 °C with 5% CO2 for 30 min to 2 h (see note below).
Note: To ensure optimal cell density and parasite burden for immunofluorescence analysis, a strategic approach to dilutions is recommended. The parasite and cell concentration should be determined by systematic dilutions to find the most suitable concentration range for achieving robust unsaturated signal strength from sufficient cells and parasites. This optimization step addresses potential issues related to overcrowding or sparse distribution of parasites on the imaging surface, attains appropriate signal-to-noise ratios, reduces background fluorescence, and enhances the clarity of cellular structures. Similarly, incubation time should be systematically tested to allow adaptation to the experimental question being addressed. Finally, when initiating this protocol, it is helpful to include uninfected MΦ as controls, as well as control samples with parasites and MΦ that were incubated at 4 °C (so parasites should be bound by MΦ, but not internalized).
Fixing
Thaw the fixing solution (see Recipes) at 4 °C.
Pour 50 mL of PBS in a reagent reservoir.
Pour 50 mL of fixing solution in a reagent reservoir.
Take the plate out and carefully pipette out the 200 µL of media from each well into a bleach container.
Using the 12-channel pipettor, pipette 50 µL of PBS in each well of the microplate and carefully aspirate it out into the bleach container, ensuring that no damage is inflicted on the cells. This also ensures removal of excess uninternalized parasites.
Using the 12-channel pipettor, pipette 100 µL of fixing solution in each well of the microplate and incubate at room temperature for 15 min.
Aspirate the fixing solution from each well into the bleach container.
Wash the wells of microplate three times with 50 µL of PBS.
Pause point: After fixing, you can add 50 µL of PBS in each well of the microplate and it can be stored at 4 °C, ideally for a maximum of 48 h, before proceeding with the next steps.
Immunolabeling for external parasites
Using the 12-channel pipettor, pipette 100 µL of blocking solution in each well of the microplate and incubate at room temperature for 30 min.
Aspirate the blocking solution from each well into the bleach container.
Incubate for 45 min with mouse anti-p8 primary antibody diluted at 1:100 in PBS.
Wash each well three times with PBS.
Incubate for 45 min with anti-mouse IgG (H+L), highly cross-adsorbed, CF-568-conjugate antibody diluted to 1:1,000 in PBS.
Wash each well three times with PBS.
Immunolabeling for internalized parasites
Pour 10 mL of permeabilizing solution in a reagent reservoir.
Aspirate PBS from each well of microplate from section F into the bleach container.
Using the 12-channel pipettor, pipette 50 µL of permeabilizing solution in each well of the microplate and incubate at room temperature for 20 min.
Aspirate the permeabilizing solution from each well into the bleach container.
Wash each well three times with PBS.
Incubate for 45 min with mouse anti-p8 primary antibody diluted to 1:100 in PBS.
Wash each well three times with PBS.
Incubate for 45 min with anti-mouse IgG (H+L), F(ab')2 fragment, CF-488A-conjugate antibody diluted to 1:1,000 in PBS.
Wash each well three times with PBS.
Caution: The cell membrane of RAW 264.7 cells acts as a natural barrier during the antibody labeling steps. Following the outlined sequence of first labeling the external parasites, permeabilization, and finally antibody-labeling all parasites is critical to avoid any anomalous labeling of parasites.
Nuclei staining
Add 50 µL of Hoechst 33258 solution to 50 mL of PBS and mix thoroughly in a 50 mL sterile tube.
Pour the contents into a 50 mL reagent reservoir.
Aspirate PBS from each well of microplate from section F into the bleach container.
Using the 12-channel pipettor, pipette 100 µL of the Hoechst 33258–PBS mix in each well of the microplate and prepare for microscopy.
Confocal microscopy
Locate the RAW 264.7 cells by adjusting the height, using a low-magnification objective on brightfield setting.
Using Zen 2.3 SP1 FP3 (black) 64-bit software, switch to a 63× oil immersion objective lens (Plan-Apochromat 63×/1.4 Oil DIC M27) and select an imaging area that contains multiple distinguishable RAW 264.7 cells.
Set up fluorescence through three separate channels using the settings outlined in Table 1.
Note: The settings can be saved by the user for future use. However, the values may need adjustment depending on laser conditions, microscope type, and fluorescence signal intensity.
Table 1. Parameters set for confocal microscopy
Parameters External parasites (red) Internalized parasites (green) Nuclei (blue)
Contrast method Fluorescence Fluorescence Fluorescence
Excitation wavelength (ex.) 561 nm 488 nm 405 nm
Emission wavelength (em.) 588 nm 521 nm 432 nm
Pinhole 0.68 AU 0.59 AU 0.78 AU
Scan mode Frame Frame Frame
Scan zoom 1.0 1.0 1.0
Rotation 0° 0° 0°
Pixel time 2.05 µs 2.05 µs 2.05 µs
Line time 30.00 µs 30.00 µs 30.00 µs
Frame time 15.10 s 15.10 s 15.10 s
Scan direction Unidirectional Unidirectional Unidirectional
Averaging 16 16 16
For imaging, proceed with the settings from Table 2.
Note: Imaging may be optimized by adjusting the laser power, gain, and offset, and saturated pixels (if any) can be eliminated by reducing laser power.
Table 2. Parameters set for imaging
Parameters Values
Image size 1,024 × 1,024 pixels
Speed 8
Averaging 4 lines
Bit depth 16
Pixel size 0.1 μm × 0.1 μm × 1 μm
Z-stack 5 slice (4 μm)
Scan to generate the representative image(s). See Figure 1 for a sample image.
Figure 1. Representative image of immunofluorescence assay for amastigote uptake by RAW 264.7 cells. A. External parasites labeled red. In this image, two extracellular Leishmania amastigotes and one partly internalized amastigote can be seen. B. All parasites are labeled green. In this image, four total Leishmania amastigotes are visualized. Faint autofluorescence of RAW 264.7 cells can be seen in the green channel, but its intensity is well below that of the labeled parasites, and the mammalian cells are also a different size than the parasites. C. RAW 264.7 cell nuclei labeled blue with Hoechst 33342. In this image, one can see six MΦ nuclei. D. Overlay of the three images. The fully internalized amastigote is green. The two external amastigotes are both green and red (orange in the merged image). The amastigote that is in the process of being taken up by the macrophage is orange (extracellular) on the right side and green (intracellular) on the left lower side. Scale bar represents 20 μm.
Data analysis
Using Zen 2.3 SP1 FP3 (black) 64-bit software, open the representative image and go to the Split tab for isolating the individual channels showing the three separate colors and the overlay.
Tabulate the content of each channel into GraphPad Prism and perform the following calculations:
Total parasites = internalized green + external red parasites
Phagocytic index (%) = (internalized green parasites/blue RAW 264.7 cell nuclei) × 100
Adhesion index (%) = (total parasites/blue RAW 264.7 cell nuclei) × 100
Variations
Further expansion of this protocol to incorporate different study items includes:
Immunostaining of cells in the wells involves multiple washes and exposure to multiple antibodies, which may lead to loss of quantifiable cell-parasite population after fixing, thereby introducing potential errors. A stable clone of L. amazonensis expressing bright monomeric green fluorescent protein, mNeonGreen, can be used, which eliminates the need for section G (Immunolabeling for internalized parasites). The parasites remain fluorescent even after they have been internalized by MΦ.
For experiments involving drug treatments on either parasites or RAW 264.7 cells, one can add requisite drug volumes into the media during section D (Set up internalization). The phagocytic indices of the untreated internalizations can be compared with the drug-treated groups, generating a quantitative analysis. Relevant statistical analysis can be further pursued through GraphPad Prism.
In the absence of confocal microscopy, visualization of multi-color immunofluorescence may be performed using alternative instruments, e.g., an epifluorescence microscope or more high-throughput approaches such as a BioTek Cytation 5 (Agilent) or the InCell 6000 (GE Healthcare). The BioTek Cytation 5 machine has emerged in our lab as a viable alternative for visualizing multi-color immunofluorescence, offering distinct advantages and features. One primary advantage lies in its ability to accurately visualize fluorescence signals, provided that the excitation and emission wavelengths of individual channels are precisely configured. A notable strength of the BioTek Cytation 5 is its capacity for automated analysis, streamlining the labor-intensive process of quantifying immunofluorescent signals. Researchers can establish specific signal intensity and size thresholds, enabling the machine to automatically calculate critical parameters such as the number of external and internalized parasites and the quantification of RAW 264.7 cell nuclei. Furthermore, the BioTek Cytation 5 allows users to save conditions or template protocols for data analyses, facilitating consistency across experiments and enabling seamless replication of methodologies.
Caution: Despite its merits, the BioTek Cytation 5 also has its limitations. Factors such as the need for accurate configuration of excitation and emission wavelengths, magnification limits, and potential variations in signal intensities may impact the precision of results. Additionally, the machine’s capabilities may be influenced by the quality and specificity of the antibodies used in the immunofluorescence assays. Users should compare the automated analyses with representative images at all relevant times.
We have also visualized the process of internalization between various treatment groups through labeling of phagocytic cups by labeling with Alexa FluorTM 647 phalloidin (1:1,000) in step G8 alongside the secondary antibody for the internalized parasites. Subsequently, we add a fourth channel on the confocal microscope, setting the following wavelengths: ex. 633 nm., em. 669 nm. We typically observe the actin filaments in the color white (Figure 2). Fluorescence intensity of the phagocytic cups is quantified in ImageJ through the following workflow:
Identify phagocytic cup area on the channel for actin.
Create region of interest (ROI) around it using polygon selection tool in ImageJ.
Open ROI Manager (Analyze > Tools > ROI Manager).
Add to list (can also tap t on keyboard).
Measure all ROIs.
Export to Microsoft Excel.
Calculate mean fluorescence intensity or size.
Compare the data between treatment groups and perform statistical analyses on GraphPad Prism.
Note: As an alternative to ImageJ, Adobe Illustrator can be used for creating ROI for phagocytic cups and measuring fluorescence intensity.
Figure 2. Representative image of immunofluorescence assay to visualize phagocytic cup formation upon amastigote uptake by RAW 264.7 cells. A. External parasites labeled red. B. All parasites labeled green. C. Nuclei labeled blue with Hoechst 33342. D. Actin filaments labeled white. Phagocytic cups (n = 8) are identified by ROIs (marked with purple circles), where we have delineated all parasites that are fully in focus and could be undergoing the process of entering mammalian cells. E. Overlay of the four images. Scale bar represents 20 μm.
Validation of protocol
The concepts of amastigogenesis and Leishmania internalization by phagocytic cells has been studied and replicated for some time [6,10], but only limited studies have employed quantitative analyses through image-based approaches [11] or characterized the uptake of amastigotes. This protocol, along with its variations, has been thoroughly peer-reviewed and validated independently by multiple researchers before publication [9,12,13]. Similar techniques have been used by our group and others for Leishmania and other microbes (e.g., the parasites Toxoplasma and Cryptosporidium) [14,15] for a number of years.
General notes and troubleshooting
Phagocytic cells, i.e., MΦs in this case, can display a lack of uniformity and consistency in the number of phagocytosed parasites. A general benchmark of RAW 264.7 cells-to-amastigotes ratio of 1:15 is a good starting point for setting up infections in 96-well plates. As described above (section D), this ratio can be adjusted to improve parasite visualization in a single pane. However, assessing the total numbers of cells and parasites should always be performed during microscopy to ensure quality results. We have found that there can be some variation between biological replicates of this assay. Under these circumstances, normalization of experimental values against a specific control group can be performed, which still maintains the integrity of the results. If this technique is used, one must either adjust corresponding statistical analysis accordingly (e.g., use one-sample two tailed t-tests to analyze for deviations from an expected result) or perform analysis before normalization. We also note that our flexible protocol allows incorporation of various fluorophores simultaneously to suit the objectives of the experiment. Naturally, it is imperative that fluorophores’ excitation and emission spectra are checked for potential overlap through spectrum viewers available on various product websites, such as https://www.thermofisher.com/order/fluorescence-spectraviewer.
Acknowledgments
We appreciate Bio-protocol’s invitation to submit this manuscript and the ongoing interest of the cell biology and microbiology communities in our multi-color immunofluorescence internalization assay. In particular, Drs. I. George Miller and Anthony J. Koleske, both from Yale University, have previously advocated for our submitting this assay as a protocol-based manuscript. We must acknowledge the generations of parasitologists who have been conducting similar studies to ours for inspiring the experimental variations that we have described, but whom we could not cite due to space constraints. We have adapted and modified our protocol on the basis of their previous work. We thank additional members of the Wetzel lab who have characterized host–pathogen interactions, including Gina Aloisio, Francis T. H. Khuong, and Imran Ullah, for their dedication to this ongoing project. We appreciate the assistance of Hanspeter Niederstrasser and Bruce A. Posner at the UT Southwestern High Throughput Screening Core with designing our automated analysis. We also thank the Biochemistry Department for access to the departmental Cytation 5 and Catherine Trice for their help with editing this manuscript. Work conducted over the years as a result of the protocol described here has been supported by NIH F32 AI094905, NIH K08 AI103036, NIH R01 AI146349, a Pediatric Infectious Diseases Society of America Fellowship, a Yale Center for Molecular Discovery Pilot Project Grant, Children’s Clinical Research Advisory Committee (CCRAC) Junior Investigator and Early Investigator Awards, a Welch Grant for Chemistry (I-2086) and funds from the UT Southwestern Department of Pediatrics (all to Dr. Wetzel). U. B. was supported by a National Institutes of Health Supplement to Promote Diversity in Health-Related Research (R01 AI146349-S1) and Medical Scientist Training (MD/PhD) Grant NIH T32GM008014. The Zeiss LSM880 with Airyscan was purchased with a Shared Instrumentation grant from NIH (1S10OD021684-01 to Katherine Luby-Phelps). We have also occasionally used an InCell Analyzer 6000 in the High Throughput Screening Core for visualizing our samples, which was purchased through NIH S10 OD018005. The funders did not play a role in the writing of this manuscript.
Competing interests
The authors declare no conflict of interest.
Ethical considerations
Use of Leishmania amazonensis, which is a Biosafety Level (BSL) 2 organism, was approved by the Biosafety Committee (Protocol # RDSR-2023-044) of The University of Texas Southwestern Medical Center, Dallas, TX.
References
WHO. (2023). Retrieved from https://www.who.int/news-room/fact-sheets/detail/leishmaniasis.
Dorlo, T. P. C., Rijal, S., Ostyn, B., de Vries, P. J., Singh, R., Bhattarai, N., Uranw, S., Dujardin, J. C., Boelaert, M., Beijnen, J. H., et al. (2014). Failure of miltefosine in visceral leishmaniasis is associated with low drug exposure. J Infect Dis. 210(1): 146–153.
Mohapatra, S. (2014). Drug resistance in leishmaniasis: Newer developments. Trop Parasitol. 4(1): 4–9.
Handman, E. (1999). Cell biology of Leishmania. In: Baker, J. R., Muller, R. and Rollinson, D. (Eds.). Advances in Parasitology (Vol. 44, pp. 1–39): Academic Press.
Sunter, J. and Gull, K. (2017). Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding. Open Biol. 7(9): 170165.
Mandell, M. A. and Beverley, S. M. (2017). Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts. Proc Natl Acad Sci USA. 114(5): e1619265114.
Serafim, T. D., Coutinho-Abreu, I. V., Oliveira, F., Meneses, C., Kamhawi, S. and Valenzuela, J. G. (2018). Sequential blood meals promote Leishmania replication and reverse metacyclogenesis augmenting vector infectivity. Nat Microbiol. 3(5): 548–555.
Paixão, A. R., Dias, B. R. S., Palma, L. C., Tavares, N. M., Brodskyn, C. I., de Menezes, J. P. B. and Veras, P. S. T. (2021). Investigating the phagocytosis of Leishmania using confocal microscopy. J Visualized Exp.: (173). doi:10.3791/62459.
Ullah, I., Barrie, U., Kernen, R. M., Mamula, E. T., Khuong, F. T. H., Booshehri, L. M., Rhodes, E. L., Bradford, J. M., Datta, A., Wetzel, D. M., et al. (2023). Src- and Abl-family kinases activate spleen tyrosine kinase to maximize phagocytosis and Leishmania infection. J Cell Sci. 136(14): e260809.
Mosser, D. M. and Edelson, P. J. (1985). The mouse macrophage receptor for C3bi (CR3) is a major mechanism in the phagocytosis of Leishmania promastigotes. J Immunol. 135(4): 2785–2789.
Siqueira-Neto, J. L., Moon, S., Jang, J., Yang, G., Lee, C., Moon, H. K., Chatelain, E., Genovesio, A., Cechetto, J., Freitas-Junior, L. H., et al. (2012). An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages. PLoS Negl Trop Dis. 6(6): e1671.
Wetzel, D. M., McMahon-Pratt, D. and Koleske, A. J. (2012). The Abl and Arg kinases mediate distinct modes of phagocytosis and are required for maximal Leishmania infection. Mol Cell Biol. 32(15): 3176–3186.
Wetzel, D. M., Rhodes, E. L., Li, S., McMahon-Pratt, D. and Koleske, A. J. (2016). The Src kinases Hck, Fgr, and Lyn activate Abl2/Arg to facilitate IgG-mediated phagocytosis and Leishmania infection. J Cell Sci.: e185595.
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A New Approach for Assessment of Neutrophil Extracellular Traps Through Immunofluorescence Staining in Whole Blood Smears
SB Sakshi Bansal
Vinit Sharma
RG Rajesh Gupta
HS Harjeet Singh
AA Anjali Aggarwal
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.5010 Views: 745
Reviewed by: Pilar Villacampa AlcubierreSaskia F. Erttmann Anonymous reviewer(s)
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Abstract
Neutrophils, constituting 50%–70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies.
Key features
• Characterization of neutrophil extracellular traps in whole blood smears through immunofluorescence staining.
• Affordable and quantitative approach to neutrophil extracellular trap detection.
Keywords: NETosis Smear assay Immunofluorescence Neutrophils Neutrophil extracellular traps
Graphical overview
Graphical representation of the immunofluorescence-based blood smear assay to characterize neutrophil extracellular traps.
Background
Cancer is a serious global public health issue, and an upsurge in advanced-stage disease and mortality may result from delays in diagnosis and treatment [1]. Pancreatic ductal adenocarcinoma (PDAC), the most prevalent histologic variant of pancreatic cancer (PC), is considered one of the most aggressive solid tumors, having a high metastatic potential with a 5-year survival rate of less than 5% [2]. According to estimates, PDAC will overtake the principal cause of cancer mortality by 2030, ranking third globally in terms of cancer-associated fatalities [2,3]. It is currently widely acknowledged that immunological dysfunction and carcinogenesis are intimately related [4]. The most prevalent type of immune cell, neutrophils, comprising approximately 50%–70% of all circulating leukocytes, are essential for both the inflammatory and immune responses to solid tumors [5]. Neutrophils are responsible for maintaining the immune system of the host and provide not only the defense against foreign antigens but also anti-tumorigenic properties. The behavior of neutrophils can vary depending on the various stimuli: N1 neutrophils show pro-inflammatory and anti-tumorigenic properties, while N2 neutrophils show anti-inflammatory and pro-tumorigenic properties. According to recent research, a high peripheral blood neutrophil-to-lymphocyte ratio is associated with a poorer probability of survival for cancer patients [6]. Neutrophils, upon activation by cytokines or a variety of stimuli in the tumor microenvironment, undergo a cellular response called neutrophil extracellular trap (NET) formation (NETosis), in which DNA, along with histone and granule proteins, are extruded into the tissue surroundings and ultimately come into circulation [7]. A crucial enzyme in the production of NETs is peptidyl arginine deiminase 4 (PADI4), which catalyzes the deamination of arginine on histone H3 to induce chromatin decondensation and subsequent DNA extrusion [8]. The proteins that constitute NETs vary depending on the stimulus; nevertheless, the NET core signature proteins are histones, neutrophil elastase (NE), and myeloperoxidase (MPO), which are present regardless of the stimulus [9].
NETosis is a physiological phenomenon in which NETs are formed from neutrophils. Physiologically, NETs were found to be involved in host immune defense against fungi, parasites, and viruses [7]. However, studies on a variety of malignancies have revealed that NETs are crucial to the development of tumors [8]. Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies [10,11]. Through the promotion of the epithelial-to-mesenchymal transition (EMT), NETs enhanced the ability of cancer cells to migrate and invade [12]. According to recent research, NETs trap circulating tumor cells, which promotes the growth and metastasis of the primary tumor, being directly correlated with the burden of metastatic disease [13,14]. Through the trapping of cancer cells and the reawakening of dormant cells through ECM remodeling, NETs may conspire to promote cancer recurrence [15]. NETs have also been observed to aid in the advancement of PC patients' tumor angiogenesis, venous thrombosis, and inhibition of apoptosis [16,17]. Overall, these findings indicate that NET detection could be significant for cancer diagnosis and prognosis.
Clinical evidence suggests that tumor-associated neutrophils (TANs) correlate with poor prognosis, and the tumor microenvironment plays a crucial role in controlling neutrophil recruitment. However, the extent of infiltration of neutrophils and their differentiation into NETs in the tumor microenvironment in a variety of cancers including PDAC remains unexplored [9]. Therefore, the need of the hour is to gain insight into the efficient characterization of NETs, so that their role in tumor progression can be deciphered. The purpose of this work was to identify NETs in the peripheral blood of PDAC patients. Detecting NETs in peripheral blood may indicate underlying pathological conditions. Thus, using a non-invasive approach to identify NETs, alongside other diagnostics, holds promise for identifying inflammatory disorders, assessing disease severity, and determining suitability for surgical resection. To that end, a unique smear immunofluorescence assay was established, which can identify NETs in only as little as 2 μL of blood. This study could aid in developing a diagnostic biomarker for various diseases and a therapeutic approach to boost immunotherapy following curative cancer excision.
Materials and reagents
APC anti-human CD15 (SSEA-1) (BioLegend, catalog number: 323008)
Mouse monoclonal antibody [2C7] to myeloperoxidase (MPO) (Abcam, catalog number: 25989)
Rabbit polyclonal antibody to histone H3 (anti-Cit H3 citrulline R2 + R8 + R17) (Abcam, catalog number: 5103)
Goat polyclonal antibody to rabbit IgG Alexa Fluor 488 (2 mg/mL) (Abcam, catalog number: ab150077)
Goat polyclonal antibody to mouse IgG Alexa Fluor 405 (2 mg/mL) (Abcam, catalog number: ab175660)
Sytox Orange nucleic acid stain, 5 mM solution in DMSO (Invitrogen, catalog number: S11368)
10× phosphate buffered saline (PBS), pH 7.2 (HiMedia, catalog number: TL1032-500mL)
Tween 20 (Sigma, catalog number: 9005-64-5)
Triton X-100 (HiMedia, catalog number: MB031-500mL)
Bovine serum albumin (BSA) (HiMedia, catalog number: MB083-100g)
Paraformaldehyde (PFA) (Nice Chemicals, catalog number: P64929)
Glycerol (≥ 99.5%) (HiMedia, catalog number: MB060)
Ultra-pure distilled water
Solutions
Wash buffer (see Recipes)
Fixing buffer (see Recipes)
Dilution buffer (see Recipes)
Permeabilization buffer (see Recipes)
Blocking buffer (see Recipes)
70% glycerol (see Recipes)
Recipes
Wash buffer (1× PBS)
Reagent Final concentration Quantity or Volume
10× PBS 1× PBS 5 mL
Distilled water ~ 45 mL
HCl 1 M
Mix 5 mL of 10× PBS with 40 mL of distilled water. Adjust to pH 7.2 with 1 M HCl. Adjust the final volume up to 50 mL with distilled water. Store wash buffer at 4 °C.
Fixing buffer
Reagent Final concentration Quantity or Volume
PFA 4% 10 g
1× PBS NA ~250 mL
NaOH 1 M
Dissolve 10 g of PFA in 200 mL of 1× PBS in a heating and stirring block (60 °C) and cool down upon complete dissolving of PFA. Add 1 M NaOH solution (4 g of NaOH pellets dissolved in 100 mL of distilled water) dropwise to adjust to pH 6.9. Adjust the final volume up to 250 mL with 1× PBS. Store aliquots at 4 °C (see General note 1).
Dilution buffer
Reagent Final concentration Quantity or Volume
BSA 1% 0.5 g
1× PBS NA 50 mL
Gently mix the solution by inverting the reaction tube. Sterile filter with 0.22 µm pore microfilter. Store at 4 °C.
Permeabilization buffer
Reagent Final concentration Quantity or Volume
1× PBS NA 100 mL
Tween 20 0.1% 100 µL
Triton X-100 0.5% 500 µL
Prepare this solution in the dark and mix well. Store at 4 °C in air-tight amber bottles, as Tween 20 and Triton X-100 are light-sensitive.
Blocking buffer
Reagent Final concentration Quantity or Volume
BSA 5% 0.5 g
1× PBS NA 10 mL
Gently mix the solution by inverting the reaction tube. Sterile filter with 0.22 µm pore microfilter. Store at 4 °C.
70% glycerol
Reagent Final concentration Quantity or Volume
Glycerol 70% 35 mL
1× PBS NA 15 mL
Prepare 70% glycerol solution by mixing 35 mL of glycerol with 15 mL of 1× PBS. Use pH paper to ensure a pH of ~7.4. Acidic glycerol will cause rapid fading of fluorochromes. Store at 4 °C in air-tight amber bottles, as glycerol is light sensitive.
Laboratory supplies
15 mL conical centrifuge tubes (Tarsons, catalog number: 546021)
50 mL conical centrifuge tubes (Tarsons, catalog number: 546041)
BD Vacutainer K2EDTA vials (BD, catalog number: 454020)
BD EmeraldTM single-use syringe, 5 mL (BD, catalog number: 307725)
LifeLongTM 24 G needle, 0.55 mm × 25 mm (Lifelong, catalog number: 021824-H)
Microscope slides, 75 mm long × 25 mm wide, thickness 1.35 mm (Blue Star, catalog number: PIC-1)
Microscopic cover glass, rectangular (Special) 22 mm × 60 mm (Blue Star, catalog number: 5128789)
PierceTM microcentrifuge tubes, 1.5 mL (Thermo Scientific, catalog number: 69715)
PierceTM microcentrifuge tubes, 2.0 mL (Thermo Scientific, catalog number: 69720)
Syringe-driven filters, filter pore size 0.22 µm, diameter 30 mm (HiMedia, catalog number: SF137-100NO)
Advanced PAP Pen, 5 mm tip width (Sigma, catalog number: Z377821)
Borosilicate glass graduated round reagent bottles with screw caps (Borosil, catalog number: 1519)
Graduated cylinder (Borosil, catalog number: 3021)
Humidifier slide chamber (we used customized humidifier slide chamber of dimensions 35 cm × 30 cm × 6 cm from Seven Star Scientific Instruments. Researchers can also use Evergreen Scientific slide moisture chamber of dimensions 81/4×7×11/4" from VWR, catalog number: 76278-832) (see General note 4)
Qualigens Labolene Neutral pH 5 L (Thermo Fisher Scientific, catalog number: Q42218)
Sterile pipette tips 0.5–10 µL, 1–200 µL, 100–1,000 µL volume range (Tarsons)
Equipment
Micro-pipettes 0.5–10 µL, 10–100 µL, 20–200 µL, 100–1,000 µL (Eppendorf)
Class II biological safety cabinet (ESCO Lifesciences, model: AC2-4S8-NS)
SPINOTTM Digital Magnetic Stirrer Hot Plate (Tarsons, model: MC 02, Catalogue number: 6040)
Olympus Fluoview confocal laser scanning microscope (Olympus, model: FV3000)
RO-DI Ultra (Rions Labpure water solutions, model: ASTM Type I)
Software and datasets
ImageJ; Java-based program (1.5.4) (https://imagej.nih.gov/ij/download.html)
Microsoft Office Professional Plus 2019
Procedure
Collect pre-operative blood samples of PDAC patients and healthy controls in EDTA vials (all the patients/participants provided written, informed consent for their participation in this study).
With the help of a micro-pipette, put a sample of 2 μL of whole blood on a glass slide cleaned with 90% ethanol. Place the blood sample 1 cm away from one edge of the glass slide as illustrated in Figure 1.
Figure 1. Stepwise protocol for whole-blood smear preparation. a. Clean the slides with 90% alcohol. b. Place 2 μL EDTA-treated blood on a slide with pipette. c. Position the spreader at a 45° angle on the blood drop and through a capillary mechanism, blood will spread along the edge in contact with the spreader. Move the spreader forward quickly, straight, and smoothly along the length of the slide. d. Make the boundary with advanced PAP pen around the smear to prevent the buffers spill out from the slide. e. Permit the smear to air-dry for 15 min. f. Position the slide above the racks of humidifier slide chamber.
Position the spreader at a 45° angle in front of the blood drop. Push it back until it makes contact with the blood. Through a capillary mechanism, blood will spread along the edge in contact with the spreader. Move the spreader forward quickly, straight, and smoothly along the length of the slide (see General note 2).
Create a perimeter using the hydrophobic advanced PAP pen around the smear to prevent the spilling out of buffers from the slide. Allow the smear to air-dry naturally for 15 min at room temperature. Be cautious of excessive air-drying, as it can damage proteins and cellular structures, potentially affecting immunodetection outcomes.
Apply 200 μL of fixing buffer onto the smear using a micro-pipette to fix the samples and place them in a humidifier slide chamber for 10 min (see General note 3).
Tilt the slide gently to allow the excess PFA liquid to drain into the waste container. Following this, wash the slide three times with wash buffer (1 mL each time) and dispose of these washes in the same waste container to ensure proper disposal of toxic PFA residues.
Apply 200 μL of APC-labeled CD15 antibody diluted with dilution buffer and incubate in the dark for 30 min at room temperature in a humidifier slide chamber. Remove excess antibody by rinsing the slides three times with 1 mL of wash buffer and gently tilt the slides to discard the wash liquid into the waste container (see General note 4).
Apply 200 μL of permeabilization buffer to permeabilize the slides for 30 min at room temperature in the dark and wash the slides with 1 mL of wash buffer three times.
Apply 200 μL of blocking buffer on the smear for 30 min at room temperature in the dark to block the sections and then rinse the slides three times with 1 mL of wash buffer.
Apply 200 μL of diluted (as listed in Table 1) anti-MPO and anti-Cit H3 primary antibodies using the dilution buffer. Let the slides incubate in the dark for 3 h at room temperature in a humidifier slide chamber to avoid the tissue drying out and then wash the slides three times with 1 mL of wash buffer.
Table 1. Stock and working volume of antibodies
Antibody Stock solution Working solution
APC-labeled CD15 1 mg/mL 1:200
Cit H3 1 mg/mL 1:500
MPO 1 mg/mL 1:500
Goat anti-rabbit Alexa Fluor 488 2 mg/mL 1:1,000
Goat anti-mouse Alexa Fluor 405 2 mg/mL 1:1,000
Sytox Orange 5 mM 1:1,500
Apply 200 μL of goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 405 secondary antibodies diluted 1:1,000 in dilution buffer. Let the slides incubate in the dark for 1 h at room temperature in a humidifier slide chamber and wash the slides three times with 1 mL of wash buffer.
Stain slides with 5 mg/mL of Sytox Orange diluted 1:1500 in wash buffer for 5 min in the dark at room temperature. After staining, wash three times using 1 mL of wash buffer.
Apply a drop (20–30 μL) of 70% glycerol to the edge of the smear in the dark and then gently lower the coverslip onto the slide from that side, making sure there are no bubbles (see General note 5).
Let the slides dry for at least 15 min in a dark environment. Then, proceed with imaging of the slides using the Olympus Fluoview FV3000 confocal microscope.
Controls
Two types of negative controls can be utilized for quantifying positive staining of NETs:
1. Isotype control antibodies (mouse IgG and rabbit IgG) as a negative control in place of anti-MPO, and anti-citrullinated Histone 3 for primary staining at similar doses. As an alternative, incubate the slides in the host-specific serum in place of the primary antibodies (such as mouse and rabbit sera against MPO and citrullinated Histone 3 antibodies). The secondary staining is performed as described above.
2. Stain secondary antibodies in a manner akin to this, but without the addition of primary antibodies.
Data analysis
Image acquisition
Acquire images with a confocal microscope (Olympus Fluoview FV3000 in this case). Choose the suitable lasers; in this instance, 405 nm (MPO), 488 nm (Cit H3), 650 nm (CD15), and 555 nm (Sytox Orange). For identification of stained structures, adjust the voltages of the laser. Make sure staining is specific by comparing positive samples to control samples. After the lasers have been adjusted, utilize the same parameters for each slide you need to compare and examine. Extracellular structures that stain positively for CD15, MPO, and Cit H3 are referred to as NETs, illustrated in Figures 2 and 3.
Figure 2. Representative images of neutrophil extracellular traps in peripheral blood smears of healthy controls. Individual images represent the positive staining for nuclear binding dye Sytox Orange (555 nm, red), CD15 (650 nm, magenta), MPO (405 nm, blue), Cit H3 (488 nm, green), and other combinations of CD15, Cit H3, and MPO, along with nuclear dye. Neutrophil extracellular traps are identified as extracellular structures where nuclear binding dye Sytox Orange (555 nm, red), colocalized with CD15 (650 nm, magenta), MPO (405 nm, blue), and Cit H3 (488 nm, green) in the merged image as indicated by the white arrows. Images acquired at 40× magnification, scale bars = 60 µm.
Figure 3. Representative images of neutrophil extracellular traps in peripheral blood smears of pancreatic cancer patients. Individual images represent the positive staining for nuclear binding dye Sytox Orange (555 nm, red), CD15 (650 nm, magenta), MPO (405 nm, blue), Cit H3 (488 nm, green), and other combinations of CD15, Cit H3, and MPO, along with nuclear dye. Neutrophil extracellular traps are identified as extracellular structures where nuclear binding dye Sytox Orange (555 nm, red), colocalized with CD15 (650 nm, magenta), MPO (405 nm, blue), and Cit H3 (488 nm, green) in the merged image as indicated by the white arrows. Images acquired at 40× magnification, scale bars = 60 µm.
Quantitation of NETs
To further quantify the NETs, analyze the slides through confocal microscopy. Visualize the slides and take images of each slide at three high-power random fields, i.e., 40× magnification. Open the images in ImageJ and split the image into channels. Visualize the mentioned channels, i.e., blue (MPO), green (Cit H3), magenta (CD15), and red (Sytox Orange). The overlaid channels, which are positive for all markers (MPO, Cit H3, CD15, and Sytox Orange) represent the NETs. Count the NET-positive cells (CD15+, MPO+, and Cit H3+) and their subtypes (CD15+ MPO+ and CD15+ Cit H3+) in the peripheral blood smear images of both control and PC patients. Calculate the relative percentage of NETs and their subtypes as shown in Figure 4.
Note: Alternatively, researchers can use different immunofluorescence-based reporting methods available in literature such as fluorescence intensity to report the presence of markers.
Figure 4. Comparison of neutrophil extracellular trap markers in peripheral blood smears. The bar graph indicates the relative percentage of neutrophils (CD15+), neutrophil extracellular traps (CD15+, MPO+, and Cit H3+), and their subtypes (CD15+ MPO+ and CD15+ Cit H3+) in relation with total Sytox positive cells in the peripheral blood smears of both control and pancreatic cancer patients. The x-axis represents the markers of neutrophil extracellular traps in the peripheral blood smears of both control and pancreatic cancer patients, whereas y-axis represents the relative percentage of various markers of neutrophil extracellular traps in relation with total Sytox positive cells. Data in bar graphs is represented in the form of mean ± standard error of mean, n = 5. PC, pancreatic cancer.
Validation of protocol
This protocol is a part of our ongoing research work on the role of neutrophil extracellular traps in promoting epithelial–mesenchymal transition and immunomodulation in pancreatic cancer. We have followed a standardized methodology to identify and characterize NETs in whole blood smears and provided every minute details regarding the same. The protocol will be cited accordingly in future publications. For related protocols, researchers can also refer to other immunofluorescence-based protocols for NETs detection performed in plasma samples (Matta, B., Battaglia, J. and Barnes, B. J., 2022. Detection of neutrophil extracellular traps in patient plasma: method development and validation in systemic lupus erythematosus and healthy donors that carry IRF5 genetic risk. Frontiers in immunology, 13, 951254. https://doi.org/10.3389/fimmu.2022.951254 [18]).
General notes and troubleshooting
General notes
Safety precautions must be taken when handling PFA, which is toxic and carcinogenic, and for NaOH pellets, which are highly corrosive. Prepare these solutions in a well-ventilated environment, wear a lab coat, gloves, safety glasses, and other protective gear, and avoid direct contact with these chemicals.
A spreader should be thinner than a glass slide when it comes to spreading blood on slides. The spreader's edge ought to be polished, smooth, and slim. The smear ought to extend halfway over the slide. It must be devoid of gaps, cracks, and bubbles. The smear should be smooth, leveled, and slightly curved in appearance. It must be sufficiently thin to produce a low power field of at least 10× in areas without the RBCs overlapping. Avoid applying too much pressure when spreading, and let the smear air-dry fully before staining in order to prevent RBC overlap. During staining, drying aids in preventing RBC overlapping.
To maintain high humidity, add 10 mL of water to the humidifier slide chamber. Slides should be placed atop the racks of the humidifier slide chamber. To preserve the moisture inside the container, close the lid of the humidifier slide chamber.
For washing, rinse the slides three times with 1 mL of wash buffer (each time gently without shaking) and gently tilt the slides to remove the liquid into the waste container.
Commercially available anti-fade mounting media can also be used instead of 70% glycerol. A refractive index (RI) of approximately 1.53 is suggested for the mounting medium. The brightness and clarity of the image increase with the proximity of the sample's and mounting medium's RI values to this value. With an RI of 1.47, glycerol boosts the sample's RI and helps in the emergence of a brighter, better-resolution image.
Troubleshooting
Problem Possible causes Possible solutions
Sample signal
a. No signal
i. The fluorescence microscope might not be operating correctly.
ii. Antigen detached from the sample during preparation.
iii. PBS wash done too vigorously.
iv. The antigen substrate will be degraded by microbial contamination of the specimen.
v. Unclean glassware or not completely rinsed.
vi. Loss of antigen adherence to slide may result from an excessively acidic or alkaline pH of PBS.
vii. Improper storage of slides; prolonged exposure of fluorophores to light may cause the signal to fade.
viii. Prolonged storage of slides.
ix. Improper fixation.
x. Improper dilution of the antibody (too diluted).
xi. Not giving the specified incubation period.
xii. Inappropriate utilization of the secondary antibody.
xiii. Incorrect wavelength of excitation.
i. Use previously stained slides to examine under a microscope. If nothing is still visible, make sure the filters are set to the right wavelength, change the bulb, or realign it.
ii. Avoid ever touching an antigen surface with your hands or a pipette.
iii. Carefully follow the washing instructions.
iv. Be cautious to prevent contamination when handling and collecting specimens.
v. Properly clean glassware using Labolene cleaning agent.
vi. Before using any buffer, always check the pH and adjust if needed.
vii. Carry out incubations and keep slides in a dark place. For best outcomes, sections should be imaged right away after mounting.
viii. To prevent antigenicity from being lost, use recently prepared slides.
ix. Immediately incubate thoroughly in fixative. Use at least 4% formaldehyde to suppress endogenous phosphatases when using phospho-specific antibodies.
x. Use low to high series dilutions or reference the antibody datasheet to determine the optimal antibody dilution.
xi. Incubate slides with primary antibodies following a thoroughly standardized methodology to start from at least 3 h incubation to overnight incubation to obtain a consistent outcome.
xii. Choose the suggested concentration and ensure that the secondary antibody matches the primary antibody's host species.
xiii. Verify that the fluorophore(s)' excitation wavelength is matched by the illumination and detection (laser, excitation, emission filter).
b. Too low signal Insufficient expression of the target protein.
i. Raise the antibody concentrations.
ii. Adjust the method of detection by binding with a fluorophore that is more luminous.
c. Too high signal
i. Not enough blocking.
ii. Improper dilution of the antibody (too concentrated primary or secondary antibody).
i. A normal serum that matches the same species as the secondary antibody should be utilized.
ii. To find the suggested antibody dilution, go to the datasheet for the antibody product.
High background
i. Autofluorescence in samples.
ii. Not enough blocking.
iii. Improper dilution of the antibody (too concentrated primary or secondary antibody).
iv. The samples became dry.
v. Not enough washing.
vi. Secondary cross-reactivity.
i. To ensure the levels of autofluorescence, use unstained samples as a control. Replace outdated formaldehyde stocks and make new solutions, because outdated fixatives have the potential to autofluoresce and may lead to high background staining (non-specific signals). For targets with low abundance, select channels with longer wavelengths.
ii. Apply normal serum that belongs to the same species as the secondary antibody.
iii. To find the suggested antibody dilution, go to the datasheet for the antibody product.
iv. The sample must be kept immersed in liquid throughout the whole staining process.
v. Wash to get rid of extra secondary antibodies, excess fixative, and weakly bound, non-specific antibody connections.
vi. To find out if your secondary antibody is cross-reacting, employ isotype control antibodies.
Distorted morphology
i. Blood drop size.
ii. Spreader sliding angle.
iii. Spreading speed.
iv. Viscosity of blood.
i. An extremely small drop might not allow for a lengthy enough smear for a sufficient diagnostic assessment; a large drop might cause the smear to stretch past the edge of the slide, losing cell clumping and preventing the development of the feathered edge.
ii. A spreader sliding angle of more than 30° produces a thicker and smaller smear, while a smaller angle produces a thinner and lengthier smear.
iii. While slower spreading can produce a lengthy or thicker smear, faster spreading can provide a narrower or thinner blood smear.
iv. A patient with a higher hematocrit will have blood that is more viscous, which could lead to an excessively thick smear. In these situations, reducing the spreader slide's angle can aid in creating a thinner smear that is better suited for microscopic inspection. Anemia also causes a drop in blood viscosity, which leads to a thin smear preparation. In such scenarios, thickening the smear preparation will be achieved by angling the spreader slide more.
Acknowledgments
We appreciate the engagement of every patient and volunteer. A Ph.D. scholarship from the Department of Biotechnology, Government of India, to Sakshi Bansal, helped with this work during the protocol's optimization (Award letter Number DBTHRDPMU/JRF/BET-21/I/2011-22/251). We express our gratitude to Ms. Meenakshi Sinha for her help with confocal microscopy, the Central Sophisticated Instrumentation Core (CSIC), and PGIMER, Chandigarh for their infrastructure support.
Competing interests
There are no competing interests to declare.
Ethical considerations
All described procedures for sample collection were consented to by the Institutional Ethics Committee of the Post Graduate Institute of Medical Education and Research, Chandigarh on February 27, 2023 (reference number IEC-INT/2023/PhD-885). All the patients/participants provided written, informed consent for their participation in this study.
References
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Cancer Biology > Tumor immunology > Immunological assays
Immunology > Immune cell staining > Immunodetection
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5,011 | https://bio-protocol.org/en/bpdetail?id=5011&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue
AA Ankita Arora
Vinit Sharma
RG Rajesh Gupta
AA Anjali Aggarwal
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.5011 Views: 576
Reviewed by: Xinlei Li Anonymous reviewer(s)
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Abstract
Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions.
Key features
• Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue.
• Microscopic visualization of extracellular vesicles’ morphology without dehydration steps, with minimum effect on their shape.
• Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.
Keywords: Extracellular vesicles Tissue-derived EVs Visceral adipose tissue Transmission electron microscopy Western blotting Flow cytometry
Background
Extracellular vesicles (EVs) are nanoparticles possessing a lipid bilayer membrane that participate in intercellular communication by transferring encoded information and cargo molecules in the form of lipids, cytokines, RNA, DNA, proteins, peptides, and other biomolecules to the recipient cells [1]. Despite considerable progress, the challenges and complexity associated with EVs remain substantial. The isolation of EVs is frequently complicated by the presence of other extracellular macromolecules with similar features [2]. There are three main subtypes of EVs distinguished by their respective sources: body fluids, cell culture, and tissues [3].
Most of the studies have been accomplished on EVs derived from cell culture supernatants or body fluids, such as serum, plasma, whole blood, milk, saliva, and urine [3]. However, several drawbacks are associated with cell culture and body fluid–derived EVs related studies.
Cell culture supernatant-derived EVs can be considered as a reliable source for mechanistic studies due to the continuous availability and reproducibility of results. Cultured cells lose their unique features after long-term subcultures, affecting the quality and composition of the EVs released, thereby altering the interpretation of EVs biological function [4]. Moreover, cells are mostly cultured in a two-dimensional environment with single-cell type and no heterogenic cell–cell interactions that exist in the tissue microenvironment [4,5]. Therefore, cell culture–derived EVs cannot reflect the exact dynamic progression of the disease. Body fluid–derived EVs provide a minimally invasive method to imitate the dynamic development of diseases [6,7]. However, mixtures of EVs from various sources are included in body fluids, comprising serum-derived proteins [8]. Also, it is difficult to identify the exact tissue of origin from where the EVs are isolated [9]. Therefore, it is required to focus on the studies involving tissue-derived EVs from whole tissues so as to generate a better understanding; unfortunately, the number of related studies are limited, with existing isolation and characterization protocols primarily tailored to the former subtypes.
Tissue-derived EVs, residing in the extracellular interstitium, serve as integral mediators of intercellular signal transduction, providing the exact pathophysiological state and interactions between different types of cells, demonstrating the 3D structure and cell properties [5,10]. Notably, tissue-derived EVs overcome challenges associated with contaminants found in body fluid–derived EVs [4]. Analyzing EVs from tumor and adjacent non-tumor tissues provides valuable insights into their common biological properties [8].
Recognizing this gap, we present a robust and reproducible method for isolating and characterizing tissue-derived EVs, specifically from adipose tissue comprising of a heterogeneous population of cells that cooperate to perform multiple physiological roles. Adipose tissue dysfunction, usually observed in obesity, is linked to changes in the adipose secretome comprised of cytokines, free fatty acids, hormones, lipids, and, more lately, extracellular vesicles, which subsequently contribute to the disease pathology [11]. We have developed a simple, reliable, and reproducible method to isolate and characterize tissue-derived extracellular vesicles. We highly recommend further studies on tissue-derived EVs, as it can support a better understanding of the tissue microenvironment in normal and in diseased conditions.
Materials and reagents
Biological materials
Human visceral adipose tissue
Note: Peri-pancreatic adipose tissue (adipose tissue in the retroperitoneum region surrounding the pancreas) is used as biological experimental material in this protocol. Peri-pancreatic adipose tissue has been taken from human subjects undergoing pancreatic cancer surgery after written informed consent. However, the protocol can be used with any type of tissue.
The study has been approved by the Institutional Ethics Committee (INT/IEC/2023/SPL-287, 21st March, 2023) of Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Reagents
Distilled water
10× Dulbecco’s phosphate buffered saline (DPBS) (HIMEDIA, catalog number: TL1022). Store at 15–30 °C
Antibiotic–Antimycotic cocktail (GibcoTM, catalog number: 15240-062)
Exosome-depleted FBS (GibcoTM, catalo number: A2720801)
HiAdipo XLTM adipocyte differentiation media (HIMEDIA, catalog number: AL521)
Part A: Basal medium (store at 2–8 °C)
Part B: Differentiation supplement (store at -5 to -20 °C)
For complete medium: Add the contents of Part B to Part A under aseptic conditions with 1 mL of antibiotic cocktail solution and 10% exosome-depleted FBS
PierceTM BCA Protein Assay kit (Thermo Scientific, catalog number: 23227)
RIPA (EMD Millipore, catalog number: 20-188)
Protease inhibitor cocktail (Sigma, catalog number: P8340)
Sodium dihydro orthophosphate anhydrous (Sigma, catalog number: 5438400100)
Sucrose (Sigma, catalog number: S0389)
Sodium hydroxide (Sigma, catalog number: 1.06498)
Uranyl acetate dihydrate (SRL laboratories, catalog number: 81405)
25% glutaraldehyde EM grade (Electron Microscopy Sciences, catalog number: 16220)
30% Acrylamide-bisacrylamide solution (HIMEDIA, catalogue number: ML037)
Tris base (HIMEDIA, catalog number: TC072M)
Sodium do-decyl sulphate (SDS) (HIMEDIA, catalog number: GRM205)
Glycine (HIMEDIA, catalog number: MB013)
Sodium chloride (NaCl) (HIMEDIA, catalog number: GRM3954)
Bovine serum albumin (BSA) (HIMEDIA, catalog number: MB083)
Tween 20 (Sigma, catalog number: P9416)
Ponceau S (HIMEDIA, catalog number: ML045)
Glacial acetic acid (Central drug house, product code: 027017)
2- propanol (HIMEDIA, catalog number: MB063)
Glycerol (HIMEDIA, catalog number: MB060)
Bromophenol blue (Sigma, catalog number: 114391)
β-mercaptoethanol (Sigma, catalog number: 6250)
Methanol LR (SDFCL limited, product code: 39192)
Ammonium persulphate (APS) (HIMEDIA, catalog number: MB003)
N, N, N’, N’,-Tetramethyl ethylenediamine (TEMED) (Sigma, catalog number: T9281)
Clarity Western ECL substrate (Bio-Rad, catalog number: 170-5061)
Pre-stained protein ladder (HIMEDIA, catalog number: MBT092)
Coomassie® brilliant blue (HIMEDIA, catalog number: MB092)
Primary and secondary antibodies for Western blotting (Table 1)
Table 1. Primary and secondary antibodies
Antibody Host Company Catalog number
CD 63 (clone: E1W3T) Rabbit mAb Cell Signaling Technology 52090
CD 9 (clone: D8O1A) Rabbit mAb Cell Signaling Technology 13174
TSG 101 (clone: E6V1X) Rabbit mAb Cell Signaling Technology 72312
Calnexin Rabbit pAb Cell Signaling Technology 2433
Anti-Rabbit-HRP Goat IgG Abcam ab97051
CD 9 exosome capture beads (Abcam, catalog number: ab239685)
CD 63 exosome capture beads (Abcam, catalog number: ab239686)
Conjugated antibodies for flow cytometry (Table 2)
Table 2. Conjugated antibodies
Antibody-Fluorochrome Host Company Catalog number
CD 63-FITC (clone: H5C6) Mouse mAb BioLegend 353005
CD 9-PE (clone: HI9A) Mouse mAb BioLegend 312105
Solutions
1× Dulbecco’s phosphate buffered saline (DPBS) (see Recipes)
1× RIPA buffer (see Recipes)
BCA working solution (see Recipes)
Sorensen’s phosphate buffer (see Recipes)
3% Glutaraldehyde (see Recipes)
5% Uranyl acetate (see Recipes)
10× Electrophoresis running buffer (see Recipes)
1× Electrophoresis running buffer (see Recipes)
Transfer buffer (see Recipes)
1.0 M Tris-HCl, pH 6.8 (see Recipes)
1.5 M Tris-HCl, pH 8.8 (see Recipes)
10% SDS (see Recipes)
10% APS (see Recipes)
10% SDS-PAGE resolving gel (see Recipes)
12% SDS-PAGE resolving gel (see Recipes)
15% SDS-PAGE stacking gel (see Recipes)
6× Loading dye (Laemmli buffer) (see Recipes)
Working loading dye (reducing) (see Recipes)
CBB staining solution (see Recipes)
10× TBS (see Recipes)
1× TBST (see Recipes)
5% BSA Blocking Buffer (see Recipes)
Recipes
Note: All the solutions are kept under room temperature unless described. Always refer to the manufacturer’s instructions for the storage temperature and shelf life of reagent.
1× DPBS
Reagent Final concentration Amount
10× DPBS 10× 5 mL
Distilled water n/a 45 mL
1× RIPA Buffer
Reagent Final concentration Amount
10× RIPA n/a 100 μL
Distilled water n/a 900 μL
Protease inhibitor n/a 2 μL
BCA working solution
Reagent Final concentration Amount
Part A n/a 50 mL
Part B n/a 1 mL
This solution is stable for 1 day only. Prepare just before use. Working solution quantity will vary upon the requirement and the number of samples. The ratio of part A and part B should be 50:1.
Sorensen’s phosphate buffer
Reagent Final concentration Amount
Sodium dihydro orthophosphate n/a 1.9 g
Sodium hydroxide n/a 0.28 g (≈ 2 pellets)
Distilled water n/a 400 mL
3% Glutaraldehyde
Reagent Final concentration Amount
Sorensen’s phosphate buffer n/a 400 mL
Glutaraldehyde 25% 54 mL
Sucrose n/a 7.4 g
Adjust the pH to 7.2–7.4. Prepare the solution as stock and store at 4 °C for long-term use.
5% uranyl acetate
Reagent Final concentration Amount
Uranyl acetate 5% 50 mg
Distilled water n/a 1 mL
10× Electrophoresis running buffer
Reagent Final concentration Amount
Tris base 25 mM 30 g
Glycine 192 mM 144 g
SDS 0.1% 10 g
Make up the volume to 1,000 mL. Keep at 37 overnight to dissolve completely. Store at 4 for long-term use.
1× Electrophoresis running buffer
Reagent Final concentration Amount
10× electrophoresis running buffer n/a 100 mL
Distilled water n/a 900 mL
Transfer buffer
Reagent Final concentration Amount
Tris base 25 mM 3.0 g
Glycine 190 mM 14.2 g
Methanol n/a 200 mL
Make up the volume to 1,000 mL. Prepare the solution 3–4 h before use so that it can be completely dissolved. The solution needs to be chilled.
1.0 M Tris-HCl
Reagent Final concentration Amount
Tris base 1.0 M 6 g
Distilled water n/a 70 mL
Keep the solution at 37 overnight. Set the pH at 6.8 using concentrated HCl. Make up the final volume to 100 mL. Store at 4 .
1.5 M Tris-HCl
Reagent Final concentration Amount
Tris base 1.5 M 18.15 g
Distilled water n/a 70 mL
Keep the solution at 37 overnight. Set the pH at 8.8 using concentrated HCl. Make up the final volume to 100 mL. Store at 4 .
10% SDS
Reagent Final concentration Amount
SDS n/a 10 g
Distilled water n/a 100 mL
10% APS
Reagent Final concentration Amount
Ammonium persulphate n/a 0.1 g
Distilled water n/a 1 mL
Freshly prepare the solution every time before use. Keep the APS on ice until use.
10% polyacrylamide resolving gel
Reagent Final concentration Amount
Acrylamide-bisacrylamide solution 30% 3.3 mL
Tris-HCl 1.5 M 2.5 mL
SDS 10% 0.1 mL
Distilled water n/a 4.0 mL
APS 10% 100 μL
TEMED n/a 4 μL
12% polyacrylamide resolving gel
Reagent Final concentration Amount
Acrylamide-bisacrylamide solution 30% 4 mL
Tris-HCl 1.5 M 2.5 mL
SDS 10% 0.1 mL
Distilled water n/a 3.3 mL
APS 10% 100 μL
TEMED n/a 4 μL
5% polyacrylamide stacking gel
Reagent Final concentration Amount
Acrylamide-bisacrylamide solution 30% 330 μL
Tris-HCl (pH: 6.8) 1.0 M 250 μL
SDS 10% 20 μL
Distilled water n/a 1.4 mL
APS 10% 20 μL
TEMED n/a 2 μL
6× Loading dye (Laemmli buffer)
Reagent Final concentration Amount
SDS 9% 0.9 g
Pure glycerol 50% 5 mL
Tris-HCl 375 mM 0.591 g
Distilled water n/a 7 mL
Dissolve all three items. Set to pH 6.8 using NaOH. Add 0.03 g of bromophenol blue (0.03%). Make up the final volume to 10 mL using distilled water. Store at -20 for long term use.
Working loading dye (reducing)
Reagent Final concentration Amount
6× Loading dye n/a 91 μL
β-mercaptoethanol n/a 9 μL
Freshly prepare the dye every time before use.
CBB staining solution
Reagent Final concentration Amount
Coomassie brilliant blue 0.1% 0.05 g
Methanol n/a 20 mL
Distilled water n/a 70 mL
Glacial acetic acid n/a 10 mL
Coomassie brilliant blue is first dissolved in methanol followed by distilled water and acetic acid. Mix thoroughly and store at 4 . This solution can be used several times.
10× TBS
Reagent Final concentration Amount
Tris base n/a 24 g
NaCl n/a 88 g
Distilled water n/a 900 mL
Keep the solution at 37 overnight. Set to pH 7.6 using concentrated HCl. Make up the final volume to 1000 mL. Store at 4 for long term use.
1× TBST
Reagent Final concentration Amount
10× TBS n/a 100 mL
Distilled water n/a 900 mL
Tween 20 0.1% 1 mL
Store the solution at 4 .
5% BSA blocking buffer
Reagent Final concentration Amount
BSA 5% 2.5 g
1× TBST n/a 50 mL
Freshly prepare the solution each time.
Laboratory supplies
FinnpipetteTM F2 Good Laboratory Pipetting (GLP) Kits: 1: 1–10 μL, 10–100 μL, and 100–1000 μL (Thermo ScientificTM, catalog number: 4700870)
1.5 mL micro centrifuge tubes (Tarsons, catalog number: 500010)
96-well plate (Falcons, catalog number: 353072)
15 mL graduated centrifuge tubes, PP (Tarsons, catalog number: 546021)
50 mL graduated centrifuge tubes, PP (Tarsons, catalog number: 546041)
Disposable Petri dishes (Tarsons, catalog number: 460090-90 mm)
Costar® 6-well cell culture plate (Corning Inc, catalog number: 3516)
Sterile surgical blades
Syringe driven filters (HIMEDIA, catalog number: SF-137)
Quartz flow cell cuvette (Malvern, model number: ZEN0023)
Type 90 Ti fixed-angle titanium rotor (Beckmann Coulter, product number: 355530)
Quick-seal tube kit, 16 × 76 mm (Beckmann Coulter, product number: 348179)
Coverslips
Copper grid, carbon coated, Type B 200 mesh (TED Pella USA, model number: 01810)
Tweezer, high precision, Style 5 (Electron Microscopy Sciences, catalog number: 78325-5S)
Blotting sheets
Nitrocellulose transfer membrane 0.45 μm (HIMEDIA, catalog number: SF110B)
Polystyrene round-bottom tube 12 × 75 mm style (Falcon, catalog number: 352054)
Equipment
Airstream® Class II Type A2 biological safety cabinet (ESCO Scientific, catalog number: 2011011)
Centrifuge 5430-R with rotor F-35-6-30 (Eppendorf, catalog number: 022620659)
Ultra-sonic bath sonicator (LABWAN, model: LW-USB11)
Optima XPN 100 ultracentrifuge (Beckmann coulter, model: OPTIMA XPN-100)
Zetasizer Nano ZSP (Malvern, model: MEL0665)
JSM-IT300 InTouchScopeTM scanning electron microscope (JEOL, model: JSM-IT300)
JEOL-1400 plus Transmission electron microscope (JEOL, model: JEOL 1400 plus)
Mini-PROTEAN® TETRA CELL (Bio-Rad, catalog number: 1658004)
Mini-PROTEAN® TETRA CELL Single core (Bio-Rad, catalog number: 1658005)
PROTEAN® System casting stand (Bio-Rad, catalog number: 1658050)
Mini-PROTEAN® TETRA cell handcasting accessory kit (Bio-Rad, catalog number: 1653370)
Mini-PROTEAN® system glass plates (Bio-Rad, catalog number: 1653308)
Mini-PROTEAN® Gel releasers (Bio-Rad, catalog number: 1653320)
Mini-PROTEAN® gaskets (Bio-Rad, catalog number: 1653305)
Mini-PROTEAN® comb, 10 well (Bio-Rad, catalog number: 1653359)
Mini-TRANS-BLOT® electrophoretic transfer cell (Bio-Rad, catalog number: 1703930)
Mini-TRANS-BLOT® foam pads (Bio-Rad, catalog number: 1703933)
Mini-TRANS-BLOT® accessories (Bio-Rad, catalog number: 1703820)
Rocking shaker (Dot Scientific Inc, catalog number: SK-R1807-S)
Azure 400® imager (Azure Biosystems, model number: AZURE 400)
BD FACS Canto II flow cytometer (BD Biosciences, model: BD FACS CantoTM II)
Procedure
Procurement and culture of human visceral adipose tissue
Add approximately 5 g of visceral adipose tissue from human subjects undergoing surgery depending upon the site of adipose tissue to a sterile pre-chilled 1× DPBS vial (see Recipe 1).
Note: In this study, peri-pancreatic adipose tissue is used as visceral adipose tissue, as mentioned in biological material section. A fresh tissue sample should be taken. Frozen tissue samples should not be used, as it can decrease the quality and quantity of extracellular vesicles.
Clean the tissue thoroughly 3–4 times with 1× DBPS until the tissue is free from blood.
Note: This protocol needs to be strictly done in a biosafety cabinet class II, as the tissue has to be sterile throughout.
Transfer the tissue to a sterile Petri dish and cut the tissue in small pieces using sterile blades.
Transfer the tissue pieces to a 6-well plate and add 3 mL of exosome-free adipocyte differentiation media in the first 48 h.
Collect the conditioned media after 48 h in a sterile vial; store at -20 °C and add 3 mL of exosome-free adipocyte differentiation media again for the next 48 h.
Collect and pool the conditioned media for extracellular vesicle isolation.
Note: If not subjected to extracellular vesicle isolation directly, the conditioned media can be stored at -80 °C up to five days for the better quality and quantity of EVs.
Isolation of extracellular vesicles by ultracentrifugation
Label 15 mL sterile tubes containing conditioned media.
Centrifuge the conditioned media at 2,000× g for 20 min at 4 °C to remove any tissue debris.
Note: Isolation of the EVs should be strictly performed at 4 or on ice.
Collect the supernatant in a fresh sterile tube and place it on ice.
Filter the supernatant using a 0.45 μM syringe filter to remove large sized vesicles or apoptotic bodies.
Note: Alternatively, centrifuge the supernatant at 10,000× g for 1 h at 4 to remove large sized vesicles or apoptotic bodies.
Transfer the filtered supernatant in the ultracentrifuge polypropylene tubes on ice.
Centrifuge at 100,000× g at 4 for 2 h for EVs isolation.
Carefully discard the supernatant using a 1000 μL and 20 μL pipette. Leave approximately 200 μL of supernatant to avoid disturbing the pellet.
Fill the ultracentrifuge tube up to 14 mL (marking at the tube) with 1× DPBS.
Centrifuge at 100,000× g at 4 for 2 h to wash the EVs pellet.
Discard the supernatant carefully.
Dissolve the pellet in 200 μL of ice-cold 0.1× DPBS (Figure 1).
Note: Dilute 1× DPBS by dissolving 5 mL of 1× DPBS in 45 mL of distilled water.
Note: Sometimes, the pellet does not appear as clear and is hard to visualize; in this case, it is suggested to remove the supernatant carefully and leaving up to 50 µL.
Figure 1. Isolation of adipose tissue–derived extracellular vesicles (EVs) by ultracentrifugation. The circle shows the EVs pellet obtained after ultracentrifugation.
The EVs pellet is ready for quantification and characterization.
Quantification of total EVs protein by BCA analysis
Prepare BSA dilutions (see Table 3) in 1.5 mL microcentrifuge tubes to obtain a BCA standard curve.
Table 3. BCA standard curve preparation
Albumin standard dilution (mg/mL) Albumin stock solution (2 mg/mL) (μL) ddH2O (μL)
2.00 200 -
1.00 100 100
0.5 50 150
0.25 25 175
0.125 12.5 187.5
0.0625 6.25 193.75
Add 100 μL of 1× RIPA buffer to 100 μL of EVs (dissolved in 0.1× DPBS) and mix thoroughly (see Recipe 2).
Keep the solution for 1 h at 4 for protein enrichment. Add 2 μL of protease inhibitor cocktail.
Sonicate for 2 min with 1 min break thrice on ice.
Centrifuge at 14,000× g for 25 min.
Collect supernatant in 1.5 mL tubes and discard the pellet (if formed).
Pipette 12.5 μL of BSA standards and EVs sample in triplicates into the same 96-well plate.
Add 100 μL of BCA working solution to each well (see Recipe 3).
Incubate the plate at 37 °C for 45 min and evaluate the protein concentration at OD 562 with plate reader.
Calculate the protein concentration by plotting the BCA standard curve (Table 4 and Figure 2).
Table 4. BCA analysis. Total EVs protein calculation performed by BCA analysis. Six BSA standards in serial dilutions were used, and average absorbance was calculated at 562 nm.
Sample OD 1 OD 2 OD 3 Average OD Concentration
BSA S1 (2 mg/mL) 1.222 1.229 1.264 1.238333333
BSA S2 (1 mg/mL) 0.656 0.709 0.673 0.679333333
BSA S3 (0.5 mg/mL) 0.415 0.413 0.414
BSA S4 (0.25 mg/mL) 0.262 0.285 0.276 0.274333333
BSA S5 (0.125 mg/mL) 0.183 0.17 0.178 0.177
BSA S6 (0.0625 mg/mL) 0.134 0.131 0.133 0.132666667
Adipose tissue 1 0.899 1.005 0.984 0.962666667 1.5 mg/mL
Adipose tissue 2 0.697 0.59 0.713 0.666666667 0.975 mg/mL
Figure 2. BCA standard curve for estimation of concentration of proteins. The concentration of adipose tissue–derived extracellular vesicles is calculated using a linear equation obtained from the standard curve. EV, extracellular vesicles.
According to the Minimal Information for the Study of Extracellular Vesicles (MISEV 2023) guidelines, each preparation of EVs should be (a) defined by quantitative measures of the source of EVs; (b) characterized to the extent possible to determine abundance of EVs; (c) tested for the presence of EV components associated with subtypes, and (d) validated for the presence of other non-vesicular, co-isolated contaminants.
Size evaluation of EVs by dynamic light scattering
Dilute isolated EVs in 0.1× DPBS (1:200). Mix the solution gently and thoroughly such that a single suspension of EVs will be formed.
Transfer 200 μL of diluted EVs to the glass cuvette and perform particle size analysis.
The system takes approximately 30 s to calculate the average particle size (Figure 3).
Figure 3. Dynamic light scattering for particle size analysis of extracellular vesicles. The figure represents size evaluation of isolated extracellular vesicles from adipose tissue sample. The average size of the particle appears to be 99.11 nm, which is in the size range of extracellular vesicles, with a polydispersity index (PDI) of 0.474, which represents a heterogeneous population of extracellular vesicles in the adipose tissue.
Visualization of EVs via scanning electron microscopy
Dilute the freshly isolated EVs in 0.1× DPBS (1:500). Mix the solution gently and thoroughly such that a single suspension of EVs is formed.
Spread 50 μL of diluted EVs evenly on a coverslip.
Let the coverslip dry under sterile conditions.
To make the surface conductive, apply a coating gold-palladium by sputtering.
Visualize under the SEM at low beam energy (7 kV) (Figure 4).
Figure 4. Scanning electron microscopy for size and morphological analysis of extracellular vesicles. The figure shows a heterogeneous population of isolated extracellular vesicles in the size range of 30–1,000 nm. (A) Magnification: 50,000×; Scale: 0.5 μm (B) Magnification: 100,000×; Scale: 0.1 µm
High resolution imaging by transmission electron microscopy
Dilute the freshly isolated EVs in 0.1× DBPS (1:500). Mix the solution gently and thoroughly such that a single suspension of EVs is formed.
Filter the diluted sample using a 0.45 μM syringe filter.
Mix 5 μL of diluted EVs and 5 μL of 3% glutaraldehyde (see Recipe 5). Incubate at room temperature for 5 min.
Incubate the carbon coated copper grid on a drop of fixed EVs for 2 min.
Remove excess sample using a blotting sheet by holding the grid gently with Tweezer, Style 5 from the side.
Incubate the grid on a drop of 5% uranyl acetate (see Recipe 6) for 5 min.
Again, remove the excess sample using blotting sheet.
Air dry the grid under sterile conditions.
Transfer to gelatin capsule or microcentrifuge tubes until visualization.
For visualization, mount the grid in a TEM specimen holder and set the voltage at 120 kV.
Visualize the EVs under the TEM (Figure 5).
Figure 5. Transmission electron microscopy for the evaluation of the extracellular vesicles’ morphology. Extracellular vesicles show an exact round-shaped morphology surrounded by a membrane. The figure shows a heterogeneous extracellular vesicle population of different size ranges. (A) Scale: 200 nm (B) Scale: 500 nm
Characterization by protein composition using western blotting
Prepare 1× electrophoresis running buffer from 10× running buffer and transfer buffer (see Recipes 7–9) and keep the solutions at 4 to cool down.
For transferring of proteins, cut nitrocellulose membranes conferring to the gel size.
Prepare the polyacrylamide gel (10% for antibodies CD 63, TSG 101, Flotilin-1, and Calnexin and 12% for CD 9) (see Recipes 10–16) in PROTEAN® system casting stand. Let the gel sit for 25–30 min to get polymerized at room temperature.
Note: Different percentages of polyacrylamide are used for gel preparation depending upon the molecular weight of the proteins to be resolved: 7.5% for 50–200 kDa proteins, 10% for 15–100 kDa proteins, 12% for 10–70 kDa proteins, and 15% for 12–45 kDa proteins.
By the time gel gets polymerized, resuspend 50 µg of EVs sample (dissolved in RIPA) in working loading dye thoroughly and let it boil at 95 for 5 min in a dry bath (see Recipes 17–18).
Remove the polymerized polyacrylamide gels carefully from the casting stand and shift them into Mini-PROTEAN® TETRA CELL filled with chilled 1× electrophoresis running buffer.
Note: Chilled 1× electrophoresis running buffer is required to prevent the smile distortion effect leading to smiling or frowning bands.
Fill the wells of the gel with 1× running buffer before the loading of samples.
Inject the samples in each well using 10 μL tips.
Note: Carefully inject the samples as it may rupture the well and the sample may overflow sometimes. Join the 2–3 wells of the comb using tape when preparing polyacrylamide gel if the concentration of the sample is too low and the volume of the sample increases (>40 μL).
Run the gel at 70 V for the first 30 min for the stacking of the samples. After that, run the gel at 90 V for 150 min or until the loading buffer dye reaches the base of the gel.
Gently separate the glass plate containing gel from Mini-PROTEAN® TETRA CELL system.
Gently remove the gel from glass plates using releasers.
Note: Alternatively, before proceeding to protein transfer, it is important to visualize the separated protein bands on electrophoretic gel using CBB staining (see Recipes 19). CBB binds to the proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. For this, pour 25–30 mL of CBB stain on to the electrophoretic gel placed in the tray and incubate it at room temperature for 1 h on a rocking shaker. For clear visualization of bands, wash the gel with distilled water thrice for 10 min each (Figure 6).
Figure 6. Representative image of Coomassie brilliant blue staining. The figure represents multiple bands of isolated extracellular vesicle proteins of one sample. The concentration of the protein loaded is 10, 20, 30, 40, and 50 μg from left to right. Different concentrations were used to standardize the protocol for protein transfer. Protein ladder (extreme right) is used for the detection of molecular weight.
Note: CBB stained gel is not reusable for protein transfer. Once the electrophoresis is confirmed, run a new SDS-PAGE and continue from step 10 without CBB staining.
Place the gel into a clean tray for 5 min containing 50–100 mL of chilled freshly prepared transfer buffer such that the gel equilibrates.
Label the nitrocellulose membrane (0.45 μm) and rinse it with chilled transfer buffer.
Note: If using PVDF membrane, make sure to activate the membrane with 100% methanol for 1 min. Nitrocellulose membranes do not need to get activated.
Soak the foam pads and filter papers in chilled transfer buffer.
Assemble the filter paper, gel, and nitrocellulose membrane in the following order.
Foam pad → Filter paper → Gel nitrocellulose membrane → Filter paper → Foam pad
The arrangement should be done from the black side to the white side of the cassette.
Note: Ensure that no bubble formation occurs while transferring the gel and membrane on to the pads as it will disrupt the transfer.
Transfer the cassette in the Mini-TRANS-BLOT® electrophoretic transfer cell and fill the system with chilled transfer buffer. Arrange the cassettes in the appropriate order.
Transfer at 70 V for 2 h at 4 .
After the transfer, gently remove the membrane and transfer it to the tray with a lid containing 25–30 mL of 1× TBST (see Recipes 20–21).
Wash the membrane thrice for 10 min under gentle agitation.
Note: Before proceeding to the next step, first confirm the transfer of proteins via Ponceau S. stain, which is a reversible stain for evaluating the transfer efficiency of western blotting. Ponceau S is red colored and negatively charged, binding non-polar groups of proteins and positively charged amino groups. For this, add ready-to-use Ponceau S. stain to the membrane for 20 min and wash with 1× TBST thrice for 10 min each.
After visualization of transferred proteins, incubate the membrane in 5% BSA for 2 h at 37 under gentle agitation (see Recipes 22).
Rinse the membrane with 1× TBST three times for 5 min each under gentle agitation.
Incubate the membrane with 10 mL of primary antibody with recommended dilutions in 5% BSA (prepared in 1× TBST) overnight at 4 under gentle agitation (Table 5).
Table 5. Dilutions of primary antibody used
Antibody Dilution used
CD 63 1:1,000
CD 9 1:1,000
TSG 101 1:1,000
Calnexin 1:1,000
Note: Use the recommended concentration/dilution of the primary antibody.
Wash the membrane with 1× TBST three times for 5 min each under gentle agitation.
Incubate the membrane with 10 mL of anti-rabbit secondary antibody (1:1,000 dilution in 5% BSA) for 2 h under gentle agitation at room temperature.
Rinse membrane using 1× TBST three times for 5 min each under gentle agitation.
Prepare ECL working solution by pipetting 1 mL of ECL substrate peroxide solution and 1 mL of ECL substrate enhancer solution.
Remove the excess 1× TBST.
Add the ECL working solution to the tray containing membrane. Incubate the membrane for 30 s and place the membrane in the Azure 300.
Image the membranes (Figure 7).
Figure 7. Western blot analysis. Isolated extracellular vesicles’ (EVs) surface proteins are characterized via western blotting. (A) TSG 101, molecular weight: 50 KDa; (B) CD 9, molecular weight: 22 KDa, 35 KDa; (C) Flotilin-1, molecular weight: 50 KDa; (D) CD 63, molecular weight: 25–60 KDa. Calnexin, endoplasmic reticulum marker, was used as a negative control for extracellular vesicles characterization during identification of TSG 101, CD 9, CD 63, and Flotilin-1 proteins. Calnexin, negative control along with TSG 101, CD 63, and Flotilin-1 protein gel is from 10% resolving gel (A, C, D), whereas CD 9 protein gel (B) is from 12% resolving gel.
Characterization by flow cytometry
Dissolve freshly isolated EVs pellet in 1 mL of 1× DPBS. Divide the 200–300 μL sample in three polystyrene round-bottom tubes. Divide the sample according to the number of tubes.
Add 50 μL of CD 9 and CD63 capture beads in tube 2 and tube 3, respectively. Tube 1 is the blank/unstained tube. Resuspend the capture beads by vortex before use.
Incubate at room temperature overnight under dark conditions.
After overnight incubation, add 5 μL of conjugated primary antibody: CD 9-PE antibody in tube 2 and CD 63-FITC antibody in tube 3.
Incubate at 4 for 2 h in dark conditions.
Wash with 1 mL of 1× DPBS (500× g for 5 min at room temperature).
Carefully remove the supernatant and resuspend the pellet in 300 μL of 1× DPBS.
Mix well by vortex. Acquire the samples using BD FACS Canto II flow cytometer using the following parameters and compensation (Figure 8).
Figure 8. Parameters used for flow cytometry. A. The PMT voltages of FSC and SSC of unstained control tubes are 521 and 478, respectively. The compensation values of PE and FITC are 41.00 and 10.00. B. The PMT voltages of FSC, SSC, FITC, and PE of CD 9-PE tubes used are 521, 478, 429, and 429, respectively. The compensation values of PE and FITC are 41.00 and 10.00. (C) The PMT voltages of FSC, SSC, FITC, and PE of CD 63-PE tubes used are 521, 478, 429, and 429, respectively. The compensation values of PE and FITC are 41.00 and 10.00.
Analyze the data using BD FACS DIVA software v.6.1.3 (Figure 9 and Figure 10).
Note: The samples were acquired on the basis of unstained controls (no-antibody control) to gate the positive population of CD 9 and CD 63 extracellular vesicles. Alternatively, a corresponding isotype control specific to the host in which the antibody is raised can also be used.
Figure 9. Representative dot plot for the characterization of CD 9+ extracellular vesicles through flow cytometry. The relative percentage of CD 9+ extracellular vesicles is 97.7%, which confirmed the presence of CD 9+ surface marker, a characteristic feature of extracellular vesicles.
Figure 10. Representative dot plot for the characterization of CD 63+ extracellular vesicles through flow cytometry. The relative percentage of CD 63+ extracellular vesicles is 97.3%, which confirmed the presence of CD 63+ surface marker, a characteristic feature of extracellular vesicles.
Data analysis
All the experiments were performed in triplicates, and appropriate controls were used, as mentioned in the respective sections. The results obtained from the characterization techniques discussed above are as follows:
BCA analysis: The total protein concentration of isolated adipose tissue-derived extracellular vesicles was 1.5 mg/mL and 0.975 mg/mL, as shown in Table 4. The average protein concentration of adipose tissue–derived EVs was 1237.5 ± 131.25 µg/mL.
Dynamic light scattering: The average size of the extracellular vesicles appears to be 99.11 nm, which is in the size range of extracellular vesicles (30–1000 nm). The polydispersity index (PDI) was 0.474, representing the heterogeneous population of extracellular vesicles in the adipose tissue, as shown in Figure 3.
Scanning electron microscopy: The qualitative analysis of isolated extracellular vesicles for the morphological and size evaluation shows a heterogenous population of EVs with a round-shaped morphology and variable size ranging from 30 to 1,000 nm, as shown in Figure 4.
Transmission electron microscopy: Adipose tissue–derived extracellular vesicles showed a morphology similar to round-shaped vesicles enclosed by a membrane, as shown in Figure 5.
Western blot: Qualitative characterization of surface protein markers exclusively present on extracellular vesicles showed remarkable bands of EVs-specific markers (TSG101, CD 63, CD 9, and Flotilin-1), as shown in Figure 8. Also, the samples were negative for calnexin, which is an endoplasmic reticulum marker and is negative for extracellular vesicles. This showed that the isolated vesicles are sorted exclusively during their biogenesis.
Flow cytometry: Quantitative analysis of extracellular vesicles was performed through flow cytometry, and data was analyzed using BD FACS DIVA software v.6.1.3. The relative percentage of extracellular vesicles was determined using unstained beads incubated with EVs sample as a negative control for gating positive population of EVs. The relative percentage of CD 9+ extracellular vesicles was 97.7 % ± 0.2% (Figure 9) and CD 63+ extracellular vesicles were 97.3% ± 0.2% (Figure 10).
Validation of protocol
The experiments performed for characterizing EVs followed the latest MISEV guidelines. The procedure and data analysis section discusses all the datasets that validate this protocol. This protocol is reproducible as all the experiments were performed in triplicates, and appropriate controls were used utilizing simple molecular biology techniques. This protocol is a part of our ongoing doctoral thesis research work entitled “To study the potential of peri-pancreatic adipose tissue-derived exosomes in the progression of pancreatic adenocarcinoma.” This protocol will be duly cited in the upcoming publications from this project.
Some techniques like western blot and dynamic light scattering are already used widely by researchers to characterize EVs. For reference: Mizuta, Y., Akahoshi, T., Guo, J. et al. Exosomes from adipose tissue-derived mesenchymal stem cells ameliorate histone-induced acute lung injury by activating the PI3K/Akt pathway in endothelial cells. Stem Cell Res Ther 11, 508 (2020). https://doi.org/10.1186/s13287-020-02015-9 (Supplementary Figure 1, panel b, c).
General notes and troubleshooting
General notes
General notes for dynamic light scattering
Due to the small size of EVs, they can form aggregates after ultracentrifugation or isolation process. Always dilute the sample and mix gently before subjecting it to size evaluation.
Always clean the cuvette before use and make sure to run a blank of the solvent.
General notes for transmission electron microscopy
Always filter the EVs sample using a 0.45 μM syringe filter and dilute the sample to remove aggregates, as aggregates can provide false morphology and size evaluation.
Use the shiny/dark side of the carbon-coated copper grids for casting the sample.
Troubleshooting
Troubleshooting tips for Western blot
Problem Possible causes Solutions
No signal/faint bands
Washing for longer period/ multiple times.
Proteins are not transferred effectively.
Expired reagents due to improper handling and storage.
Not enough exposure time while imaging the blot.
Prevent the removal of detection reagents by washing the membrane gently and not aggressively. Reduce the duration of washing steps.
Reassure protein transfer via Ponceau S staining. Check if the transfer was performed in the right direction (explained in Step 14 of western blotting).
Store the solution at the prescribed temperatures. Prepare a fresh solution, if mentioned. Avoid freeze/thawing.
Increase the time of exposure from 30 s to 1 min.
Faint bands/No signal in the sample lane
Protein of interest is not present.
Primary antibody is not enough.
Not enough protein is present.
Primary and secondary antibody is not compatible.
Run a positive control.
Increase the concentration of primary antibody.
The concentration of the protein should be 20–30 μg per lane.
Use the secondary antibody generated against the primary antibody species.
High background
Non-specific binding could be the reason.
Not enough washing steps.
Choice of membrane.
Incubation temperature is high.
Primary antibody concentration is too high.
Secondary antibody is binding non-specifically.
Increase the blocking incubation period.
Increase the number of washing steps but do not wash aggressively.
Nitrocellulose membrane show less background and clear bands as compared to PVDF.
Incubate at 4 for a longer duration and RT for a shorter duration.
Optimize the primary antibody dilution.
Use the secondary antibody generated against the primary antibody species.
Multiple bands
At high molecular weight
At low molecular weight
Proteases have digested the protein.
Proteins have disulfide bonds.
Always add protease inhibitor before protein analysis.
Always use reducing agents and boil the samples in Laemmli buffer.
Unusual black dots/ hazy background Blocking reagent has clumped. Always prepare fresh blocking reagent. Mix the reagent thoroughly. It is better to filter the BSA blocking solution before use.
Smile effect/uneven bands
High voltage.
Gel has been too hot during migration.
Optimize the voltage.
Make sure to cover the gel apparatus on ice or at 4 .
Troubleshooting tips for flow cytometry
Problem Possible causes Solutions
Weak fluorescence intensity
Signal not compensated correctly.
Insufficient antibody present for detection.
Fluorochrome fluorescence has faded.
Positive single-color control should be set up on the flow cytometer correctly. Gating and compensation should be correct to capture all the events.
Increase antibody concentration.
Use fresh antibody. Always store at cold temperature or as described by the manufacturer.
Increased fluorescence intensity Too high antibody concentration. Reduce the antibody volume to each sample. Ensure adequate washing steps using wash buffers.
Two or more populations observed when there should be just one Improper binding of EVs and beads. Ensure proper binding of EVs and beads as per manufacturer instructions before staining with EVs-specific fluorochrome-labeled antibodies. Increase the blocking incubation period.
Acknowledgments
The authors are thankful to all the patients who participated in this study. Author Ankita Arora is grateful to the Department of Biotechnology, New Delhi, India, for providing a research fellowship (DBTHRDPMU/JRF/BET-21/I/2021-22/62) leading to a Ph.D.
Competing interests
The authors declare that they have no competing interests.
Ethical considerations
The study has been approved by the Institutional Ethics Committee (INT/IEC/2023/SPL-287, 21st March, 2023) of the Post Graduate Institute of Medical Education and Research, Chandigarh, India.
References
Zaborowski, M. P., Balaj, L., Breakefield, X. O. and Lai, C. P. (2015). Extracellular vesicles: composition, biological relevance, and methods of study. Bioscience. 65(8): 783–797. https://doi.org/10.1093/biosci/biv084
Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V. and Laktionov, P. P. (2018). Isolation of extracellular vesicles: general methodologies and latest trends. Biomed Res Int. 2018: 8545347. https://doi.org/10.1155/2018/8545347
Li, S. R., Man, Q. W., Gao, X., Lin, H., Wang, J., Su, F. C., Wang, H. Q., Bu, L. L., Liu, B. and Chen, G. (2021). Tissue‐derived extracellular vesicles in cancers and non‐cancer diseases: Present and future. J Extracell Vesicles. 10(14): e12175. https://doi.org/10.1002/jev2.12175
Crescitelli, R., Lässer, C. and Lötvall, J. (2021). Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat. Protoc. 16(3): 1548-1580. https://doi.org/10.1038/s41596-020-00466-1
Chen, X.-D., Zhao, J., Yan, Z., Zhou, B. W., Huang, W.-F., Liu, W. F., Li, C. and Liu, K.-X. (2020). Isolation of extracellular vesicles from intestinal tissue in a mouse model of intestinal ischemia/reperfusion injury. Biotechniques. 68(5): 257-262. https://doi.org/10.2144/btn-2019-0159
Guo, M., Wang, J., Zhao, Y., Feng, Y., Han, S., Dong, Q., Cui, M. and Tieu, K. (2020). Microglial exosomes facilitate α-synuclein transmission in Parkinson’s disease. Brain. 143(5): 1476-1497. https://doi.org/10.1093/brain/awaa090
Leung, L. L., Riaz, M. K., Qu, X., Chan, J. and Meehan, K. (2021). Profiling of extracellular vesicles in oral cancer, from transcriptomics to proteomics. Semin Cancer Biol. 74: 3-23. https://doi.org/10.1016/j.semcancer.2021.01.002
Jingushi, K., Uemura, M., Ohnishi, N., Nakata, W., Fujita, K., Naito, T., Fujii, R., Saichi, N., Nonomura, N. and Tsujikawa, K. (2018). Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin. Int J Cancer. 142(3): 607-617. https://doi.org/10.1002/ijc.31080
Huang, Z. and Xu, A. (2021). Adipose extracellular vesicles in intercellular and inter-organ crosstalk in metabolic health and diseases. Front Immunol. 12: 608680. https://doi.org/10.3389/fimmu.2021.608680
Camino, T., Lago-Baameiro, N., Martis-Sueiro, A., Couto, I., Santos, F., Baltar, J. and Pardo, M. (2020). Deciphering adipose tissue extracellular vesicles protein cargo and its role in obesity. Int J Mol Sci. 21(24): 9366. https://doi.org/10.3390/ijms21249366
Moraes, J. A., Encarnação, C., Franco, V. A., Xavier Botelho, L. G., Rodrigues, G. P., Ramos-Andrade, I., Barja-Fidalgo, C. and Renovato-Martins, M. (2021). Adipose tissue-derived extracellular vesicles and the tumor microenvironment: revisiting the hallmarks of cancer. Cancers 13(13): 3328. https://doi.org/10.3390/cancers13133328
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CRISPR-Cas9 Protocol for Efficient Gene Knockout and Transgene-free Plant Generation
EP Enzo A. Perk
AL Ana M. Laxalt
Ignacio Cerrudo
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.5012 Views: 1050
Reviewed by: Noelia ForesiPooja Verma Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Planta May 2023
Abstract
Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.
Key features
• Two sgRNAs employed.
• Takes 6–12 months to have an edited transgene-free plant.
• Setup in tomato.
Keywords: CRISPR-Cas9 Tomato Non-transgenic Plant regeneration GoldenGate
Graphical overview
Agrobacterium-mediated transformation and plant regeneration in Solanum lycopersicum for gene editing by CRISPR-Cas9 method.
Background
Tomato (Solanum lycopersicum) is an important model organism for crop improvement studies. CRISPR-Cas9 provides an effective tool for uncovering the function complexity of tomato genes. The main objective of this protocol is to establish a robust strategy for the production of knockout lines in tomato plants. Knockouts involve targeted disruption of a specific gene, resulting in loss of function. This specific variation allows researchers to examine the role of individual genes in a variety of biological processes, from development to environmental responses. It could also be used for plant genetic improvement. To achieve disruption of target genes, the CRISPR-Cas9 system utilizes small guide RNAs (sgRNAs) that direct the Cas9 endonuclease to specific genomic regions. In this system, sgRNAs have been carefully designed to target a region preferably within the first exon downstream of the ATG codon of the gene of interest. To improve efficiency, two sgRNAs are used in this protocol. The selection of sgRNAs is based on their specificity, avoiding off-targets, and ensuring efficiency. The protocol outlines a detailed workflow, including cloning of sgRNAs, assembly of expression cassettes in a vector, and Escherichia coli and Agrobacterium transformation. Then, we provide a detailed protocol for tomato transformation and screening of edited plants in order to generate homozygous edited plants free of transgene. We rely on works previously published by Nekrasov et al. [1] and Van Eck et al. [2], along with our own personal experience [3], to create a comprehensive and effective protocol for generating knock-out plants in tomato in 6–12 months. The protocol was used to demonstrate that phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS, an increase in jasmonic acid, and a reduction in salicylic acid–response marker genes [3].
Materials and reagents
Biological materials
Escherichia coli DH5α (Thermo Fisher, catalog number: EC0112)
Tomato (Solanum lycopersicum cv. MoneyMaker Cf-0) (in-house propagated)
Agrobacterium tumefaciens, strain: GV3101 (GoldBio, catalog number: CC-207-5x50)
pZG23C04 commercial vector (ZGene Biotech Inc, Taibei, Taiwan)
pICSL01009::AtU6p was a gift from Sophien Kamoun (Addgene plasmid #46968; http://n2t.net/addgene:46968; RRID: Addgene_46968)
pICH47751 was a gift from Sylvestre Marillonnet (Addgene plasmid #48002; http://n2t.net/addgene:48002; RRID: Addgene_48002)
pICH47761 was a gift from Sylvestre Marillonnet (Addgene plasmid #48003; http://n2t.net/addgene:48003; RRID: Addgene_48003)
pICSL11024 (pICH47732::NOSp-NPTII-OCST) was a gift from Jonathan D Jones (Addgene plasmid #51144; http://n2t.net/addgene:51144; RRID: Addgene_51144)
pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST was a gift from Sophien Kamoun (Addgene plasmid #49771; http://n2t.net/addgene:49771; RRID: Addgene_49771)
pICH41780 was a gift from Sylvestre Marillonnet (Addgene plasmid #48019; http://n2t.net/addgene:48019; RRID: Addgene_48019)
pAGM4723 was a gift from Sylvestre Marillonnet (Addgene plasmid #48015; http://n2t.net/addgene:48015; RRID: Addgene_48015)
Reagents
Agar (Britania, catalog number: B010406)
Agarose (GenBiotech, catalog number: RU1010)
Ampicillin (Amp) (GenBiotech, catalog number: A-0104-5)
Acetosyringone (Sigma-Aldrich, catalog number: D134406)
Bleach
BpiI (BbsI) (Thermo Scientific, catalog number: ER1011)
Buffer G (Thermo Scientific, catalog number: ER1011)
BsaI HF (New England BioLabs, catalog number: R3733S)
DNA Marker 100 bp (Inbio Highway, catalog number: K0177)
DNA Marker 100 bp (PBL, catalog number: MA0201)
DNA Marker Lambda/HindIII (Inbio Highway, catalog number: K0180)
Ethanol 96%
Plastic centrifuge tube, 15 and 50 mL (Henso Medical)
Gentamicin (GenBiotech, catalog number: G400-50)
HindIII (Promega, catalog number: R6041)
Buffer E (Promega, catalog number: R6041)
IPTG (GenBiotech, catalog number: I2481C5)
IAA (Merck, catalog number: 353)
Kanamycin (GenBiotech, catalog number: K0126-5)
Kinetin (Sigma, catalog number: K0753)
Kit dNTPS (Inbio Highway, catalog number: K1404)
Magenta boxes, W × L × H: 77 mm × 77 mm × 97 mm (V8505 MagentaTM vessel)
MS + Gamborg B5 vitamins (Duchefa-Biochemi, catalog number: P1278801)
NaCl (J.T. Baker, catalog number: 3624-i9)
PCR purification kit (Inbio Highway, catalog number: K1206)
Phytoagar (Sigma, catalog number: P8169)
Purification plasmid DNA kit (Qiagen, catalog number: 12123)
Rifampicin (GenBiotech, catalog number: R-120-1)
Sterile water
Sucrose (Cicarelli, catalog number: 841214)
Taq DNA polymerase (INBIO HIGHWAY, catalog number: K1007)
Taq DNA Buffer (INBIO HIGHWAY, catalog number: K1007)
MgCl2 (INBIO HIGHWAY, catalog number: K1007)
T4 ligase (Thermo Fisher, catalog number: EL0011)
T4 ligase buffer (Thermo Fisher, catalog number: EL0011)
Thiamine HCl (Sigma, catalog number: T-3902)
Timentin (GoloBio, catalog number: T-104-25)
Pipette tips (Henso Medical)
Tryptone (OXOID, catalog number: LP0042B)
X-Gal (GenBiotech, catalog number: I4281C5)
Yeast extract (OXOID, catalog number: LP0021)
2,4-D (Sigma, catalog number: D7299)
L trans-Zeatin (Golobio, catalog number: Z-105-50)
Eppendorf (Henso Medical)
Primers
Cas9_6F: 5' ACTAGCCTTGTGGCCCTACC 3'
Cas9_6R: 5' TCGATCTAGTAACATAGATGACACC 3'
RB_F1: 5' GGATAAACCTTTTCACGCCC 3'
Solutions
½ MS (see Recipes)
CIM I (see Recipes)
CIM II (see Recipes)
SIM I (see Recipes)
SIM II (see Recipes)
RIM (see Recipes)
LB (see Recipes)
Recipes
½ MS
2.15 g/L MS + Gamborg B5 vitamins
10 g/L sucrose
8 g/L agar
pH = 5.8
CIM I
4.3 g/L MS + Gamborg B5 vitamins
30 g/L sucrose
5.2 g/L Phytoagar
Sterilize and add 1 mg/L thiamine HCl, 1 mg/L 2,4-D, and 0.2 mg/L kinetin.
CIM II
4.3 g/L MS + Gamborg B5 vitamins
30 g/L sucrose
5.2 g/L Phytoagar
Sterilize and add 1 mg/L thiamine HCl, 1 mg/L 2,4-D, 0.2 mg/L Kinetin, and 200 μM acetosyringone
SIM I
4.3 g/L MS + Gamborg B5 vitamins
30 g/L sucrose
5.2 g/L Phytoagar
Sterilize and add 1 mg/L thiamine HCl, 2 mg/L trans-Zeatin, 100 mg/L kanamycin, and 250 mg/L timentin.
SIM II
4.3 g/L MS + Gamborg B5 vitamins
30 g/L sucrose
5.2 g/L Phytoagar
Sterilize and add 1 mg/L thiamine HCl, 1 mg/L trans-Zeatin, 100 mg/L kanamycin, 250 mg/L timentin, and 0.1 mg/L IAA.
RIM
4.3 g/L MS + Gamborg B5 vitamins
30 g/L sucrose
5.2 g/L Phytoagar
Sterilize and add 50 mg/L kanamycin, 250 mg/L timentin, and 1 mg/L IAA.
LB
10 g/L tryptone
5g/L NaCl
5g/L yeast extract
Laboratory supplies
Petri dishes (9 cm × 1.5 cm) (Biopetri)
Petri dishes (9 cm × 2.5 cm) (Biopetri)
Scalpel
Equipment
37 °C oven (SAN JOR, model: SE60A)
E. coli pulser transformation apparatus (Bio-Rad, model: 155103)
Heat dry bath (Benchmark, model: BSH1001-E)
Veriti 96-well thermal cycler (Applied Biosystems, model: 9902)
DNA electrophoresis apparatus (Bio-Rad, model: 55656)
Microcentrifuge Sorvall Legend Micro 17R (Thermo Fisher Scientific, model: 75002440)
30 °C oven (SAN JOR, model: SL300)
Orbital Shaker Thermo Forma (Thermo Fisher Scientific, model: 320REL)
PIPETMAN L Starter Kit, 4 Pipette Kit, P2L, P20L, P200L, P1000L (Gilson, model: F167370)
Cultivation room at 25 °C, photoperiod 16:8 h light/dark
Software and datasets
CRISPR2 (http://crispr.hzau.edu.cn/CRISPR2)
Cas-OFFinder (http://www.rgenome.net/cas-offinder/)
Solgenomics (https://solgenomics.net/)
Bioedit sequence alignment editor
Procedure
Design small guide RNAs (sgRNAs) for the target gene
Search for the DNA sequence of interest in the Sol Genomics database.
Design two sgRNAs in the first exon using the web page CRISPR-P 2.0 and Cas-OFFinder for plants (http://crispr.hzau.edu.cn/CRISPR2; http://www.rgenome.net/cas-offinder/). Choose sgRNAs based on specificity (without off-targets for chains of 20 and 12 nt), with 40%–50% GC content, without poly(T) repeats, and with an activity score greater than 0.5. The chosen sgRNA must be a good choice for both online design tools.
Design primers to amplify a region of approximately 600–700 bp where sgRNAs are included (see General note 1).
Check if the selected region shows no allelic variations in the sgRNAs regions. To determine allelic variants, extract DNA from 10 tomato plants, amplify the target region, and sequence it. It is normal for different cultivars to have nucleotide differences from reference genomes.
Level 1 vector assembly (based on Nekrasov et al. [1])
Design primers. To the guide sequence (20 bp in underlined capital letters chosen in section A), add flanking sequences as follows. BsaI site is indicated in italic letters, the transcribed sequence is represented in capital letters.
Forward sgRNA primer:
tgtggtctcaATTNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAG
And a reverse sgRNA primer:
gtcggtctcaAGCGCTCAAGAGGATAAAACCTCAC
Construct plasmid level 1 using Golden Gate cloning.
Perform sgRNAs assembly by using the primers and amplifying with the commercial CRISPR/Cas9 vector pZG23C04 as a template (Tables 1 and 2). The resulting PCR product (210 bp, Figure 1) will have the following sequence (primer sequences in bold, BsaI cutting site in italic letters, the 20 bp guide sequence in underlined capital letters, and transcribed sequence in capital letters).
tgtggtctcaATTNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTACTAGTTTTGATCTTGAAAGATCTTTTATCTTTAGAGTTAAGAACTCTTTCGTATTTTGGTGAGGTTTTATCCTCTTGAGCGCTtgagaccgac
Table 1. PCR conditions
Reagent Volume (µL)
Template DNA (CRISPR/Cas9 vector pZG23C04, 100 ng/µL) 1
MgCl2(25 mM) 4.2
10× BufferTaq 5
10 mM dNTPs 1.6
Forward primer (10 mM) 2.5
Reverse primer (10 mM) 2.5
Taq DNA Polymerase (5,000 U/mL) 0.5
H2O 32.5
Table 2. PCR program
Step Time, temperature Cycles
Initial denaturation 30 s, 98 °C 1
Denaturation 10 s, 98 °C 35
Annealing 30 s, 60 °C
Extension 30 s, 72 °C
Final extension 3 min, 72 °C 1
Final hold ∞ 4 °C 1
Figure 1. sgRNA verification. Five microliters of the PCR product was loaded onto a 2% agarose gel. MW: 100 bp marker.
Run 5 µL of PCR products on a 2% agarose gel.
Desalt 45 µL of PCR products using a PCR purification kit.
Assemble the level-1 sgRNAs expression cassette using Golden Gate Assembly. Cut-ligate the PCR products (sgRNA1 and sgRNA2) into different level-1 vectors, one into pICH47751 (CarbR) and the other into pICH47761 (CarbR) (Tables 3 and 4, Figure 2A). In the same cut-ligation reaction, the sgRNAs are placed under the control of the U6 promoter into a level-1 vector.
Figure 2. Cloning strategies. A. Insertion of two sgRNAs under the control of the U6 promoter into a level-1 vector. B. Insertion of the U6-sgRNAs, a selectable marker (NPTII), and a Cas9 cassette into a level 2 expression vector.
Table 3. Cut-ligation conditions
Reagent Volume
5× T4 ligase buffer 4 µL
BsaI-HF (10,000 U/mL) 1 µL
pICH47751 or pICH47761 300 ng
pICSL01009::AtU6p 100 ng
sgRNA PCR desalted (100 µg/µL) 5 µL
T4 ligase (5 U/μL) 3 µL
H2O to 20 µL
Table 4. Assembly reaction
Time, temperature Cycles
5 min, 37 °C 50
5 min, 20 °C
10 min, 50 °C 1
10 min, 80 °C 1
Transform 50 µL of chemically competent E. coli cells with the product from the cut-ligation reactions.
i. Place -80 °C competent E. coli cells on ice for 30 min.
ii. Incubate 50 µL of competent E. coli cells with 10 µL of each cut-ligation product on ice for 20 min.
iii. Heat shock the cells at 42 °C for 45 s.
iv. Add 700 µL of LB.
v. Incubate for 7 min on ice and then 1h at 37 °C.
vi. Plate transformation onto LB plates with 100 µg/mL Amp, 20 mg/mL X-Gal, and 100 µL of 0.1 M IPTG. Incubate at 37 °C overnight.
Pick the white colonies from each cloning reaction and plate them onto the master plate LB-Amp (100 µg/mL). Conduct a colony PCR to check for the presence of sgRNAs. Use the same primer design as in stepB1 and the PCR conditions and program mentioned in step B2 (Tables 2 and 3). Run the PCR products on a 2% agarose gel.
Select a positive colony from each cloning reaction and use it to inoculate 5 mL of LB-Amp. Incubate overnight at 37 °C on a shaker.
Purify each level-1 construct using a Qiagen plasmid DNA purification kit following manufacturer’s protocol.
Check level-1 construction by BpiI digestion at 37 °C for 1 h (Table 5). Run the reaction on a 2% agarose gel; expect to observe a 240 bp band released from the plasmid (Figure 3).
Table 5. Digest conditions
Reagent Volume (µL)
H2O 12
10× Buffer G 2
Level 1 construct 5
BpiI (10 U/ µL) 1
Figure 3. Level-1 construct. The purified level-1 construct was digested or not with BpiI at 37 °C for 1 h and the product was run on 2% agarose gel. -: without BpiI; +: with BpiI; MW: marker 100 bp.
Verify the level-1 constructs through sequencing using the RB_F1 primers.
Level 2 assembly
Combine the assembled sgRNAs with a selectable marker (NPTII) and a Cas9 cassette into a level 2 Acceptor (Tables 6 and 7, Figure 2B).
Table 6. Cut-ligation conditions
Reagent Volume
10× T4 ligase buffer 2 µL
BpiI (10 U/μL) 1.5 µL
plCH47751::U6p::sgRNA1 300 ng
plCH47761::U6p::sgRNA2 300 ng
pICSL11024 (pICH47732::NOSp-NPTII-OCST) 300 ng
pICH47742::2x35S-5’UTR-hCas9(STOP)-NOST 300 ng
plCH41780 linker 100 ng
pAGM4723 300 ng
T4 ligase (5 U/μL) 2 µL
H2O to 20 µL
Table 7. Assembly reaction
Time, temperature Cycles
5 min, 37 °C 50
5 min, 20 °C
10 min, 50 °C 1
10 min, 80 °C 1
Transform 50 µL of chemically competent E. coli cells with 10 µL of the cut-ligation product as previously described. Select on LB-Kanamycin (Kan) agar plates. Pick five white colonies and inoculate them in 5 mL of LB-Kan overnight culture at 37 °C on a shaker (see General note 2).
Purify the level-2 construct using a Qiagen plasmid DNA purification kit following the manufacturer’s protocol.
Check level-2 construction by HindIII digestion at 37 °C for 1 h (Table 8). Run the reaction on a 2% agarose gel; expect to observe bands around 4 kb, 1.8 kb, 1.4 kb, 500 bp, and 400 bp released from the plasmid (Figure 4). Additionally, verify by sequencing using the RB_F1 primer.
Table 8. Digest conditions
Reagent Volume (µL)
H2O 12
10× Buffer E 2
Level 2 construct 5
HindIII (10 U/µL) 1
Figure 4. Level-2 construct. The purified level-2 plasmid was digested with HindIII at 37 °C for 2 h and the product was run on 2% agarose gel (expected bands: around 4 kb, 1.8 kb, 1.4 kb, 500 bp, and 400 bp). MW: marker; Lambda/HindIII.
Agrobacterium transformation
Thaw the electrocompetent Agrobacterium on ice.
Incubate 2 μL of the level-2 construct with Agrobacterium for 30 min on ice.
Transfer to the electroporation cuvette (see General note 3).
Electroporate at 2.5 kV until the apparatus beeps.
Add 1 mL of LB into the cuvette, resuspend, and transfer back to an Eppendorf tube.
Incubate for 1 h at 30 °C.
Plate on LB agar plates with kanamycin (50 μg/mL), gentamicin (25 μg/mL), and rifampicin (100 μg/mL).
Grow for 48 h at 30 °C.
Plant transformation (adapted from Van Eck et al. [2])
Sterilize seeds:
Immerse 1 g of seeds in 25 mL of 70% ethanol for 2 min.
Wash the seeds with sterile water.
Add 25 mL of 20% bleach for 20 min.
Rinse the seeds three times with sterile water.
Sow the seeds in Magenta boxes with ½ MS (20–30 seeds/box). Use eight Magenta boxes per transformation.
Leave the seeds at 4 °C for two days (from Friday, day 1, to Monday).
Put the boxes at 25 °C in darkness for eight days (from Monday to Thursday, day 11).
On the morning of day 10 (Monday), release a liquid culture Agrobacterium by picking a single colony from a fresh plate (see General note 4) into 4 mL of LB with the appropriate antibiotics. Simultaneously, release a liquid culture with only LB (control) and LB + antibiotics. Put the liquid culture of Agrobacterium on a shaker at 28 °C at 12 rcf.
On Day 11, transfer the germinated plants to autoclaved glass plates containing autoclaved filter paper moistened with water in a laminar flow hood.
Cut each cotyledon at both ends with a scalpel (see General note 5). The explant is usually generated around each wound, facilitating the identification of independent transformation events in the same cotyledon.
Transfer the cotyledons to a plate with CIM I (callus induction medium I) to induce callus formation. Place them with the abaxial side facing the culture medium. Transfer the cotyledons to eight plates (30–40 cotyledons per plate).
Place the plates in dim light at 25 °C for 24 h (see General note 6).
Prepare four Erlenmeyer flasks, each with 250 mL of LB plus the appropriate antibiotics. Inoculate them with 1, 2, 5, and 10 µL of the liquid culture of Agrobacterium released previously in stepE5 (do not discard the controls). Incubate the flasks at 28 °C in a shaker at 12 rcf for 14–16 h.
On day 12, measure the OD600 of Agrobacterium cultures. The ideal OD is between 0.5 and 0.6, but any value within the range of 0.4–0.9 can be used.
Divide the Erlenmeyer flask that best fits the reference OD into two 15 mL Falcon tubes and centrifuge at 1600 rcf for 10 min.
Discard the supernatant and resuspend the pellet in a sterile solution of 10 mL of 10mM MgSO4.
Pour the Agrobacterium suspension into a sterile Petri dish (15 mL/dish).
Immerse the cotyledons in this suspension for approximately 10 min, stirring gently by hand.
Extract the cotyledons from the suspension and place them shortly on sterile filter paper (avoid drying out the explants). Immediately, transfer them to plates previously prepared with CIM II (callus induction medium II).
Leave 20 cotyledons uninfected to use as controls.
Place the plates at 25 °C with dim light for 48 h.
On day 14, transfer the explants to plates prepared with SIM I (shoot induction medium I) for the induction of shoot formation. Place 15–20 explants per plate. Transfer 10 of the cotyledons uninfected with Agrobacterium to medium without antibiotics and the other 10 to medium with antibiotics. Place the plates in a plant room at 25 °C with a light/dark cycle of 16:8 h for 14–18 days.
On day 28–32, transfer the explants to fresh agar every 15 days. When green tissue appears on the explant (Figure 5A), use a scalpel to cut and separate the remains of cotyledons and untransformed material (brown or yellow explants) and transfer them to plates prepared with SIM II (shoot induction medium II). Let the remaining explants continue in SIM I until they also generate green tissue.
Note: At this stage, individual explants cannot be identified.
Figure 5. Plant regeneration. Plant regeneration from transformed explant in different stages. A. SIM I. B. SIM II. C. RIM. Scale bar: 0.1 cm.
On day 45–60, it is expected the emergence of sprouts (Figure 5B), indicative of independent events. Cut the sprouts when they reach a sufficient height to be detached from the base without carrying remnants of explants or cutting the apex. Transfer the cut sprouts to a tall Petri dish filled with RIM (root induction medium). Simultaneously, transfer 10 positive controls and 10 negative controls. Obtain the controls from plates without incubation with Agrobacterium, without kanamycin. Transfer them to RIM without (negative) or with kanamycin (positive). Positive controls (without kanamycin) should regenerate, and negative controls should not.
Around day 55–70 (10–12 days in RIM), check for root development in the sprouts (Figure 5C). Sprouts with successful transformation should have roots growing within the medium (see General note 7). In non-transformed cases, the root either does not grow during this period or grows over the medium (escaping the medium). Identify the sprouts with successful transformation and transplant them into the ground.
Once the generated plants have developed their first pair of true leaves (approximately 20 days), conduct total gDNA extraction from leaves by CTAB method according to Capron et al. [4].
Utilize this gDNA as a template for PCR to assess the presence of the plasmid, utilizing specific primers for the vector (e.g., CAS9). In positive cases, perform another PCR with primers designed in step A3. Run 5 μL of the PCR product on a 2% agarose gel, where a double band may be observed if a large deletion occurs. Use the remaining portion for Sanger sequencing, where frameshift (as explained later) can be observed in case of editing.
Examine the plant sequences to identify those displaying a frameshift in the reading frame within the sgRNA area (indicative of heterozygotes). In Figure 6, observe how the sequencing follows a normal pattern and suddenly a frameshift occurs between base 420 and 430, indicated by an arrow.
Figure 6. Chromatogram of edited plant DNA as an example. Extraction of T0 plant gDNA, followed by PCR amplification. The resulting PCR products were sequenced, revealing a frameshift (indicated with an arrow) in the reading frame in the sgRNA2 region (note in the zoom-in that the chromatogram gets at least two peaks of different bases at the same position). Annotation includes the positions of sgRNA1 and sgRNA2, as well as the corresponding protospacer adjacent motifs (PAMs).
If a frameshift is not found, perform a sequence alignment to confirm no editing or that editing occurred on both DNA strands, although this is a rare occurrence.
Proceed with the plants exhibiting these characteristics.
Search for homozygous edited lines.
Germinate 10 plants from T1 generation and extract gDNA as in step E23. Perform PCR with primers designed in step A3. Sequence PCR product.
Note: In this step, homozygous, heterozygous, and wt lines could be obtained for the deletion.
Perform a sequence alignment (ClustalW) where it is possible to observe the deletion in homozygous lines (Figure 7).
Figure 7. Sequence alignment. Sequence comparison of wt and KO 1 and 2 for PLC2 in T1 generation plants used in Perk et al. [3].
Conduct a PCR analysis using specific primers for CAS9 (Cas9_F and Cas9_R). It is possible to observe homozygous lines without the transgene in the T1 generation (indicated by a negative PCR result for CAS9). If it is not possible to find a transgene-free line, continue with the next generation until it is found; it will lead to an edited and transgene-free plant.
Validation of protocol
This protocol was used to generate the KO lines employed in CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants more resistant to Botrytis cinerea by Perk et al. [3].
General notes and troubleshooting
General notes
Primers must hybridize at least 100 bp from the sgRNAs, so Sanger sequencing would be optimal in sgRNAs regions.
Note that orange colonies carry the empty vector.
Electroporation cuvettes should be pre-chilled on ice.
The Agrobacterium plate should not be more than 1 week old.
The leaves should not be enlarged or opened.
Plastic white bags can be used to dim the light.
If roots have not appeared by day 12, discard the plants.
Acknowledgments
This work was supported by Bayer (Grants 4traits) and ANPCyT (PICT 2017 No0601).
Competing interests
The authors declare that they have no conflict of interest.
References
Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D. and Kamoun, S. (2017). Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep7(1): 482.
Van Eck, J., Keen, P. and Tjahjadi, M. (2019). Agrobacterium tumefaciens-mediated transformation of tomato. In: Kumar, S., Barone, P. and Smith, M. (Eds.). Transgenic plants. Methods in Molecular Biology. vol 1864 (pp 225–234). Humana Press, New York.
Perk, E. A., Arruebarrena Di Palma, A., Colman, S., Mariani, O., Cerrudo, I., D’Ambrosio, J. M., Robuschi, L., Pombo, M. A., Rosli, H. G., Villareal, F., et al. (2023). CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants are more resistant to Botrytis cinerea. Planta 257(6): 117.
Capron, A., Gourgues, M., Neiva, L. S., Faure, J. E., Berger, F., Pagnussat, G., Krishnan, A., Alvarez-Mejia, C., Vielle-Calzada, J. P., Lee, Y. R., et al. (2008). Maternal Control of Male-Gamete Delivery in Arabidopsis Involves a Putative GPI-Anchored Protein Encoded by the LORELEI Gene. Plant Cell. 20(11): 3038–3049.
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© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Biological Sciences > Biological techniques > CRISPR/Cas9
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5,013 | https://bio-protocol.org/en/bpdetail?id=5013&type=0 | # Bio-Protocol Content
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Fast, Easy, and Comprehensive Techniques for Microscopic Observations of Fungal and Oomycete Organisms Inside the Roots of Herbaceous and Woody Plants
TT Tomáš Toma
JK Ján Kováč
Jaroslav Ďurkovič
Published: Vol 14, Iss 11, Jun 5, 2024
DOI: 10.21769/BioProtoc.5013 Views: 570
Reviewed by: Lucy Xie Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of Fungi Mar 2022
Abstract
The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques.
Key features
• The protocol enhances tissue-clearing techniques employing KOH or NaOH and is applicable to a broad range of roots from different plant species.
• Hydroxides are mixed with hydrogen peroxide to obtain an efficient bleaching solution, which effectively clears roots without causing significant tissue damage.
• The protocol could also be used for staining of fungi or oomycetes localized both on the root surface or inside the root tissues.
• Simple combination of non-fluorescent methyl blue and fluorescent solophenyl flavine dyes allows the observation of fungal organisms in both brightfield and fluorescence microscopy.
Keywords: Fungi staining Methyl blue staining NaOH/KOH clearing Oomycetes staining Plant root microtechniques Root clearing Roots of herbaceous plants Roots of woody plants Solophenyl flavine staining
Graphical overview
Background
Fungal and oomycete organisms, regardless of whether they are pathogenic or symbiotic, enter the roots of herbaceous or woody plants and grow inside their tissues. However, the minimal optical transparency of such roots is a key factor limiting direct microscopic observations of colonizing fungal organisms inside their root environment. Thin and relatively transparent young roots still do not allow efficient microscopic observation of their internal structures, including inside growing fungal organisms. In addition, older roots are much thicker and accumulate a lot of colorants, which deteriorates the observation at a microscopic level. Finally, when looking for fungi inside the root, they must be stained to contrast with the root tissues. To overcome these problems, diverse plant microtechniques allow different ways to visualize and effectively locate the fungal organism inside root tissues, for example, whole-mount root clearing and subsequent staining of fungal organisms [1], root hand sectioning followed by clearing and staining [2], or marking the fungi with fluorescent GFP [3]. The effectiveness of such techniques is limited by the time needed for their realization. Processing large root systems of herbs and especially woody plants requires a lot of time to find and visualize the fungal organisms in them. The main aim of this protocol is to combine the above-mentioned techniques of fluorescent marking and whole-mount clearing to offer a fast and easy way to observe the fungal and oomycete organisms inside the root tissues.
A key feature that enables staining at the root surface is the fact that fluorescent dye solophenyl flavine 7GFE 500 (syn. Direct Yellow 96) provides a fluorescent signal from the fungal structures but not from the root cells. Solophenyl flavine was introduced by Hoch et al. [4] as a new dye applicable for fungal cell-wall staining. Like calcofluor-white, a cellulose-specific fluorescent dye, solophenyl flavine also stains fungal structures. Moreover, the excitation and emission wavelengths of solophenyl flavine are shifted toward the red end of the spectrum, so the excitation is less dependent on UV or violet light. Moreover, the bright yellow–green signal from solophenyl flavine–stained fungi strongly contrasts against the background of a dark root. This makes screening effective for a quick inspection of the root surface. If deeper inspection of the root is required, clearing by removing cell protoplasm makes root tissues transparent. This is normally performed by using chemicals like NaOH or KOH [5]. If dark colorants still persist inside the root tissues, they need to be bleached before staining [6]. Hydrogen peroxide can bleach the roots and make them white. In this protocol, we use a mixture of NaOH (or KOH) with hydrogen peroxide as an efficient clearing/bleaching solution [7]. Metals that could be released from the samples, leading to reduced activity of the hydrogen peroxide, are chelated following the addition of sodium citrate. As hydrogen peroxide is extremely reactive, which results in bubble release, magnesium sulfate is added to prevent tissue damage caused by the air bubbles [8]. A key feature that allows staining inside the root is a mild clearing procedure that protects the root tissue structures and the fungal structures inside the root. Clearing also protects cell-wall binding sites, important for both cell wall–dye interaction and effective staining. Roots cleared this way can be stained again using solophenyl flavine. In some cases, however, there is a need for brightfield staining of fungal organisms. Dyes commonly used for non-fluorescent fungal staining, such as trypan blue [9], chlorazol black [10], or lactophenol cotton blue [11], are effective but harmful substances. In this protocol we use modified lactophenol cotton blue solution without phenol, which is harmful. Instead, methyl blue is mixed with glycerol and lactic acid to obtain blue staining of fungal organisms. If required, the protocol also offers a possibility of mixing methyl blue with solophenyl flavine and staining for both brightfield and fluorescence microscopy in one step. The proposed protocol improves the existing root-clearing techniques by using a shorter time and a lower clearing temperature. This improvement is achieved by combining clearing and bleaching into a single step and delicate sample handling.
Materials and reagents
Biological materials
Alnus glutinosa, Oryza sativa, or Arabidopsis thaliana roots or any other root material
Reagents
Potassium hydroxide (KOH) (Centralchem, CAS: 1310-58-3)
Sodium hydroxide (NaOH) (Centralchem, CAS: 1310-73-2)
Hydrogen peroxide, 50% (H2O2) (Centralchem, CAS: 7722-84-1)
Sodium citrate dihydrate [HOC(COONa)(CH2COONa)2·2H2O] (Sklochem-Agroekolab, CAS: 6132-04-3)
Magnesium sulphate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, CAS: 10034-99-8)
Methyl blue (C37H27N3Na2O9S3) (Fisher Chemical, CAS: 28983-56-4) (please do not confuse with methylene blue)
Glycerol [HOCH2CH(OH)CH2OH] (Duchefa Biochemie, CAS: 56-81-5)
Lactic acid, 80% [CH3CH(OH)COOH] (Mikrochem, CAS: 50-21-5)
Solophenyl flavine 7GFE 500 (syn. Direct Yellow 96) (C39H34N10O13S4) (abcr Germany, CAS: 61725-08-4)
1% HCl
Solutions
1% NaOH bleaching solution—sample clearing and bleaching (see Recipes)
10% KOH bleaching solution—sample clearing and bleaching (see Recipes)
0.06% methyl blue—staining solution (stock) (see Recipes)
0.006% methyl blue—non-fluorescent fungi staining (working solution)
0.1% solophenyl flavine—fluorescent fungi staining (see Recipes)
Methyl blue + solophenyl flavine mixture—combined fluorescent and non-fluorescent fungi staining (see Recipes)
Recipes
1% NaOH bleaching solution (BS)
To prepare 100 mL of 1% NaOH BS, first dissolve 0.9 g of NaOH in 90 mL of distilled water. Add 1 g of sodium citrate dihydrate and 0.03 g of magnesium sulphate heptahydrate into 90 mL of 1% NaOH. Using a stirrer to dissolve all components. A large volume of prepared solution may be stored for months in a dark place. Mix thoroughly before using.
Prior to clearing, add 10 mL of 50% H2O2 into 90 mL of the prepared NaOH + sodium citrate dihydrate + magnesium sulphate heptahydrate solution. Stir shortly to minimize hydrogen peroxide reaction but be sure to mix the prepared solution well.
Reagent Final concentration Quantity or Volume
NaOH 1% (w/v) 0.9 g
Sodium citrate dihydrate 1% (w/v) 1 g
Magnesium sulphate heptahydrate 0.03% (w/v) 0.03 g
Hydrogen peroxide 5% (v/v) 10 mL
H2O 90 mL
Total 100 mL
10% KOH bleaching solution (BS)
Alternatively, for 10% potassium hydroxide bleaching solution (KOH BS), dissolve 9 g of KOH in 90 mL of distilled water. Other chemicals and steps are applied in the same order as for Recipe 1.
Reagent Final concentration Quantity or Volume
KOH 10% (w/v) 9 g
Sodium citrate dihydrate 1% (w/v) 1 g
Magnesium sulphate heptahydrate 0.03% (w/v) 0.03 g
Hydrogen peroxide 5% (v/v) 10 mL
H2O 90 mL
Total 100 mL
Although hydrogen peroxide alone can bleach, mixing NaOH or KOH with hydrogen peroxide shortens the time required for clearing. Individual clearing with NaOH or KOH followed by hydrogen peroxide will work too, but mixed compounds work faster.
0.06% methyl blue stock solution
To make 0.06% methyl blue stock solution, dissolve methyl blue in water using a stirrer. Then, mix glycerol with lactic acid. Finally, add methyl blue solution into glycerol/lactic acid mixture and stir well. This solution can be stored in a cool, dark place for months. Before staining, prepare 0.006% working solution by diluting 5 mL of methyl blue stock solution in 50 mL of distilled water.
Reagent Final concentration Quantity or Volume
Methyl blue 0.06% (w/v) 0.03 g
Glycerol 50% (v/v) 25 mL
Lactic acid 80% 25% (v/v) 12.5 mL
H2O 12.5 mL
Total 50 mL
0.1% solophenyl flavine staining solution
To prepare a 0.1% solophenyl flavine staining solution, dissolve 0.025 g of solophenyl flavine powder in 25 mL of distilled water and mix thoroughly. This solution can be stored in a cool, dark place for one month.
Reagent Final concentration Quantity or Volume
Solophenyl flavine 0.1% (w/v) 0.025 g
H2O 25 mL
Total 25 mL
Methyl blue + solophenyl flavine mixture
To obtain 50 mL of methyl blue + solophenyl flavine mixture, add 1 mL of 0.1% solophenyl flavine into 49 mL of 0.006% methyl blue working solution. Stir well.
Laboratory supplies
Petri dishes of various size (it depends on the size of the examined root system) for root processing and staining:
Petri dish Ø150 mm (Fisher Slovakia, catalog number: 1215.1525)
Petri dish Ø120 mm (Fisher Slovakia, catalog number: 1215.1220)
Petri dish Ø50 mm (Fisher Slovakia, catalog number: 1215.0512)
Petri dish Ø40 mm (Fisher Slovakia, catalog number: 1215.0412)
Crystallizing dish 500 mL (Fisher Slovakia, catalog number: 1212.0115)
Beaker 250 mL (Fisher Slovakia, catalog number: 1112.0250)
Beaker 150 mL (Fisher Slovakia, catalog number: 1112.0150)
Beaker 50 mL (Fisher Slovakia, catalog number: 1112.0050)
Glass slide 76 mm × 26 mm (Fisher Slovakia, catalog number: 1820.1100)
Cover glass 24 mm × 50 mm (Fisher Slovakia, catalog number: 1820.1250)
Parafilm M 50 mm × 50 mm (Fisher Slovakia, catalog number: 2105.6001)
Razor blade (Agar Scientific, catalog number: AGT5115)
Laboratory scissors (Fisher Slovakia, catalog number: 2305.7711)
Laboratory needle (Fisher Slovakia, catalog number: 2305.4822)
Laboratory tweezer (Fisher Slovakia, catalog number: 2305.4143)
Scalpel (Fisher Slovakia, catalog number: 2305.9004)
Disposable plastic Pasteur pipette (Fisher Slovakia, catalog number: 2101.6460)
Pasteur glass pipette (Fisher Slovakia, catalog number: 1780.0150)
Magnetic stir bar (Fisher Slovakia, catalog number: 6115.4008)
Micropipette 1,000 µL (Fisher Slovakia, catalog number: 4052.0050)
Flat paint brush
Equipment
Heating plate with regulated temperature (30–70 °C)
Light (brightfield) and fluorescent microscope (Leica, model: DM4000) equipped with a Leica fluorescence filter A, D, I3 or any other filter with the following characteristics: excitation 400 nm and emission 470 nm
Freeze dryer (Christ Alpha 1-2 LD plus) (or any other freeze-drying machine), if freeze-drying is considered to be carried out
Procedure
Fast root-surface screening using solophenyl flavine staining
Gently rinse the root system in water to wash out the fixatives (if used) and remove soil particles or debris by using brush, needles, or forceps. Be careful as some fungal organisms are only slightly attached to the root surface; also, soil particles or debris may contain some fungal organisms. Sometimes, it is not possible to remove all soil particles; however, that can be advantageous and provide additional information about the interaction between the fungus, root, and soil.
Submerge the roots into a minimum of 5 mL of 0.1% solophenyl flavine solution for 5–7 min at laboratory temperature (20–25 °C). If necessary (if roots are mature or thick), keep in warm solution (30–40 °C) on a heating plate without stirring (stirring can damage fragile roots) for 10–30 min. According to the root system size, use enough solophenyl flavine dye and be sure that all roots are submerged.
Wash samples briefly in distilled water, using an approximately 50–100 times larger volume of water than the volume of used dye.
Transfer roots into a drop of water on a glass slide. If the roots are too large to be observed as a one piece, use scissors, scalpel, or tweezers and divide the root into appropriately smaller pieces.
Observe samples under the fluorescence light using the blue filter set (e.g., Leica A or D filter set) with excitation around 400 nm and emission around 470 nm. For more information, please see solophenyl flavine fluorescence characteristics.
Stained roots are suitable for whole-mount clearing, sectioning (e.g., cryosectioning), or freeze-drying.
Root whole-mount clearing and staining
For whole-mount clearing, we recommend using surface-stained roots from previously described steps. Nevertheless, non-stained roots are also good (Figure 1A).
Figure 1. Laboratory steps for root clearing and staining. A. Overview of roots in a Petri dish before clearing. B. Clearing and bleaching of roots in bleaching solutions on a heating plate. C. Washing roots after clearing. D. Cleared roots in a Petri dish before staining. E. Staining roots with solophenyl flavine (yellow color) and methyl blue (blue color) in small Petri dishes. F. Completed root specimens prepared for microscopic observations.
Transfer the roots to a bleaching solution (NaOH BS or KOH BS). Keep solution warm (60 °C) on a heating plate without stirring (stirring can damage fragile roots) for 1 h. A volume of 50–100 mL of solution should be enough, but it strongly depends on the root tissue mass and discoloration. If the roots are too dark, use more bleaching solution or prolong time (Figure 1B).
Put the cleared roots into the preheated dH2O (60 °C, at least 500 mL) and let them cool down at laboratory temperature for an appropriate time (approximately 30–60 min) (Figure 1C).
Roots are now prepared for staining (Figure 1D).
Gently transfer cleared roots into the staining solution (e.g., on the tip of a laboratory needle or tweezer, avoiding pressurizing roots with a tweezer jaw).
Depending on the desired outcome, stain roots with methyl blue for brightfield observations, with solophenyl flavine for fluorescence observations, or with methyl blue + solophenyl flavine mixture for observations in both types of microscopies in parallel (Figure 1E).
Methyl blue staining: Add a minimum of 5 mL of methyl blue working solution into a small Petri dish and insert the cleared roots. Stain at laboratory temperature (20–25 °C) for 10–15 min (enough for most of the roots) (Figure 1E). Shortly wash in distilled water (0.5 min, as prolonged washing may remove dye from roots). Methyl blue–stained samples should be acidified. Put a drop of 1% HCl on a glass slide, submerge the stained samples into it, and observe covered with a cover glass (Figure 1F). Use brightfield microscopy. Fungal and oomycetes structures are stained blue.
Solophenyl flavine staining: Add a minimum of 5 mL of solophenyl flavine into a small Petri dish and insert cleared roots. Stain at laboratory temperature (20–25 °C) for 5 min (Figure 1E). Wash in distilled water until all free dye is released from the stained roots (5–7 min; prolonged washing does not decrease staining intensity of roots), put on a glass slide in a drop of distilled water, and observe it under a cover glass (Figure 1F). Use fluorescence microscopy. Fungal and oomycetes structures are stained blue, green, or yellow.
Methyl blue + solophenyl flavine mixture staining: Add a minimum of 5 mL of solophenyl flavine + methyl blue mixture into a small Petri dish and insert cleared roots. Timing and temperature depend on the root origin and characteristics such as thickness and age. We suggest submerging for 5 min at laboratory temperature (20–25 °C) for soft and thin roots (in vitro–cultivated plants, small herbaceous species). On the other hand, we suggest submerging for 30 min at elevated temperature (50 °C) for thicker and older roots (woody plants). After staining, wash samples in distilled water for 5 min, put a drop of 1% HCl on a glass slide, and cover with cover glass. Use brightfield and/or fluorescence microscopy for observation (Figure 2A–F).
Figure 2. Microscopy images of fungi and oomycetes inside the roots of selected plant species (Alnus glutinosa, Arabidopsis thaliana, Oryza sativa) after the clearing and staining steps described in the protocol. A. Oomycetes (arrows) inside Alnus glutinosa roots, KOH BS clearing, methyl blue + solophenyl flavine mixture staining, fluorescence microscopy. B. Oomycetes (arrows) inside Alnus glutinosa roots, NaOH BS clearing, solophenyl flavine staining, fluorescence microscopy. C. Fungal hyphae inside Oryza sativa roots, KOH BS clearing, methyl blue + solophenyl flavine mixture staining, fluorescence microscopy. D. Fungal hyphae (arrow) inside Oryza sativa roots, NaOH BS clearing, methyl blue staining, brightfield microscopy. E. Endophytic fungus (arrows) on the Arabidopsis thaliana root surface, non-cleared, methyl blue staining, brightfield microscopy. F. Oomycete hypha (arrows) inside Alnus glutinosa root, non-cleared, solophenyl flavine staining, scanning electron microscopy. The inset shows the identical hypha (arrows) in fluorescence microscopy before freeze-drying and SEM observation. Scale bars: A–E = 20 µm, F = 10 µm.
Freeze-drying of stained samples
The following steps are optional and may follow after step A6. The majority of surface-stained samples (without clearing and bleaching) are suitable for the subsequent assessment using scanning electron microscopy (SEM).
Insert two parafilm strips between the glass slide and the border of the cover glass to prevent root squeezing (Figure 3).
Figure 3. Example of inserting the parafilm between the glass slide and cover glass
Find the desired object (fungal structure) on a root surface using fluorescence microscopy.
Put samples on the glass slide into a freeze-drying machine and freeze it. Start vacuuming and drying the samples.
When the samples are dry (time of drying strongly depends on both the size of sample and the volume of water inside the sample; approximately 2 h), gently remove the cover glass and transfer samples onto the adhesive carbon tape mounted on the SEM specimen tub.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Corcobado et al. [13]. Metabolomic and Physiological Changes in Fagus sylvatica Seedlings Infected with Phytophthora plurivora and the A1 and A2 Mating Types of P. ×cambivora. Journal of Fungi 8(3): 298, Figure 2.
Moreover, our procedure is validated by Figure 2 shown in this protocol.
General notes and troubleshooting
General notes
It is not easy to determine the exact volume needed for individual solutions. Examined roots can have various sizes. The volume of clearing/bleaching solution should be 10 times higher than the volume of the examined root system. To wash in distilled water, we recommend using a 4–5 times larger volume than the volume of the clearing/bleaching solution. If solophenyl flavine or methyl blue are used as staining dye, the stained roots should be fully submerged into the staining solution. It is not necessary to add the staining solution in excess. A drop (approximately 50 µL) of mounting medium (water or 1% HCl) is used for microscopic observations.
If better-quality staining of surface or sub-surface fungal organisms is required, it is recommended to use solophenyl flavine dissolved in ClearSee solution. The examined samples are incubated at laboratory temperature for days or weeks. This procedure completely protects root tissues against the damage that is caused by clearing and bleaching. The same procedure is also suitable for subsequent sectioning or freeze-drying. However, ClearSee staining is a time-consuming process. We recommend using a 0.1% solution of solophenyl flavine in ClearSee. For more details about ClearSee solution, see Ursache et al. [12].
Be sure that all baths for sample washing or bleaching are preheated and samples are transferred to the next step without any rapid change in temperature (e.g., during the clearing/bleaching step at 60 °C in the bleaching solution, distilled water prepared for washing should be heated to the same temperature as the bleaching solution).
Except for staining with methyl blue + solophenyl flavine mixture, there is also a possibility to stain with solophenyl flavine at first, then wash the samples in distilled water, and subsequently stain with methyl blue. This double-staining helps to quench undesirable fluorescence of cell walls that is caused by solophenyl flavine. However, as this staining procedure is complementary, some fungal structures become stained blue with methyl blue and may lose their fluorescence from solophenyl flavine staining.
Troubleshooting
Clearing and bleaching methods described above are suitable for a broad range of roots that are sampled from soil. Bleaching time and temperature for agar- or hydroponics-cultivated fine roots should be adequately adjusted. For example, bleaching for agar-cultivated A. thaliana seedlings should be 30 min at 40–50 °C. Fine roots that are sampled from soil can also be cleared in a shorter time than 30 min. If the roots remain white-brown or white-yellow after 1 h of clearing and bleaching, we recommend extending the clearing/bleaching step for another 10 or 15 min. However, if the root system consists of roots with a large diameter variation, some thin roots can be already white, whereas thick roots still remain partially discolored.
After clearing and bleaching, fungal structures show some weak fluorescence if excited with violet/blue light. However, this signal is much more stable, and excitation is shifted toward the red (blue-green) spectrum, after staining with methyl blue + solophenyl flavine mixture.
Methyl blue is prone to nonspecific staining that has to be carefully recognized. If the roots are not cleared properly, cell nuclei or cytoplasm may be stained. Cell nucleuses are evenly organized inside root tissues: one nucleus in one cell. Fungal structures are distributed randomly. If stained non-specifically, other structures in roots can be recognized and distinguished from fungal structures according to their shape and morphology.
For certain unknown reasons, some samples cannot be stained with methyl blue. There may be a problem due to the clearing or the nature of the sample. In some samples, both fungal structures and root tissues are intensively stained with solophenyl flavine, resulting in a weak contrast. In both cases, double-staining with solophenyl flavine and methyl blue dyes or staining with methyl blue + solophenyl flavine mixture helps.
Stopping point
A stopping point is relevant mainly for section B. At step B4, if the roots are prepared for staining, they can be stored before staining in a refrigerator (1–2 days, 4 °C). At step B8, there is also a possible stopping point. If the SF-stained samples are placed on a glass slide, not in a drop of water but in a drop of water:glycerol mixture (1:1), they can be stored for several days in the refrigerator. However, ensure that the mounting medium does not evaporate from the glass slide and the specimen is still mounted. If not, add a little of the water/glycerol mixture again until the sample is completely mounted.
Extra caution
Caution is needed when mixing NaOH or KOH bleaching solutions. These reactions are exothermic, and heat is released. One should also be careful with a volume of hydrogen peroxide. If too much hydrogen peroxide is added, extreme bubbling during clearing/bleaching may damage root samples. Always protect the heating device (heating place) against small drops coming from the bleaching solution or bleached samples during a transfer. They may contaminate or damage the surface coating of your device. If using HCl for methyl blue staining, be careful as it may also etch the mechanical stage of your microscope.
Acknowledgments
This work was supported by the Slovak scientific grant agency VEGA (1/0108/23) and the TUZVO internal grant agency IPA (1/2023). A part of this protocol was published by Corcobado et al. [13].
Competing interests
The authors declare no conflicts of interest.
References
Kowal, J., Arrigoni, E. and Lane, S. (2020). Acidified Blue Ink-staining Procedure for the Observation of Fungal Structures Inside Roots of Two Disparate Plant Lineages. Bio Protoc. 10(20): e3786.
Brundrett, M. C. and Tedersoo, L. (2020). Resolving the mycorrhizal status of important northern hemisphere trees. Plant Soil. 454: 3–34.
Nonomura, T., Tajima, H., Kitagawa, Y., Sekiya, N., Shitomi, K., Tanaka, M., Maeda, K., Matsuda, Y. and Toyoda, H. (2003). Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked Fusarium oxysporum strains simultaneously colonizing root surfaces. J Gen Plant Pathol. 69(1): 45–48.
Hoch, H., Galvani, C., Szarowski, D. and Turner, J. (2005). Two new fluorescent dyes applicable for visualization of fungal cell walls. Mycologia. 97(3): 580–588.
Lux, A., Vaculík, M. and Kováč, J. (2015). Improved methods for clearing and staining of plant samples. In: Yeung, E. C. T., Stasolla, C., Sumner, M. J. and Huang, B. Q. (Eds.). Plant Microtechniques and Protocols. Springer International Publishing, Cham, Switzerland, pp. 167–178.
Ruzin, S. E. (1999). Plant Microtechnique and Microscopy. Oxford University Press, New York.
Li, Y., Fu, Q., Rojas, R., Yan, M., Lawoko, M. and Berglund, L. (2017). Lignin‐Retaining Transparent Wood. ChemSusChem. 10(17): 3445–3451.
Gladyshev, N. F., Dvoretskii, S. I., Zhdanov, D. V., Ul'yanova, M. A. and Ferapontov, Y. A. (2003). Choice of a Stabilizer for the Reaction of KOH with Hydrogen Peroxide to Produce Potassium Superoxide. Russ J Appl Chem. 76(11): 1858–1859.
Wilkes, T. I., Warner, D. J., Edmonds-Brown, V., Davies, K. G. and Denholm, I. (2020). A comparison of methodologies for the staining and quantification of intracellular components of arbuscular mycorrhizal fungi in the root cortex of two varieties of winter wheat. Access Microbiol. 2(2): e000083.
Chenchouni, H., Mekahlia, M. N. and Beddiar, A. (2020). Effect of inoculation with native and commercial arbuscular mycorrhizal fungi on growth and mycorrhizal colonization of olive (Olea europaea L.). Sci Hortic. 261: 108969.
Marques, J. P. R. and Nuevo, L. G. (2022). Double-Staining Method to Detect Pectin in Plant-Fungus Interaction. J Visualized Exp.: e63432.
Ursache, R., Andersen, T. G., Marhavý, P. and Geldner, N. (2018). A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J. 93(2): 399–412.
Corcobado, T., Milenković, I., Saiz-Fernández, I., Kudláček, T., Plichta, R., Májek, T., Bačová, A., Ďatková, H., Dálya, L. B., Trifković, M., et al. (2022). Metabolomic and Physiological Changes in Fagus sylvatica Seedlings Infected with Phytophthora plurivora and the A1 and A2 Mating Types of P. ×cambivora. J Fungi. 8(3): 298.
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5,014 | https://bio-protocol.org/en/bpdetail?id=5014&type=0 | # Bio-Protocol Content
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Measuring Sleep and Activity Patterns in Adult Zebrafish
FD Fusun Doldur-Balli
AZ Amber J. Zimmerman
CS Christoph Seiler
OV Olivia Veatch
AP Allan I. Pack
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5014 Views: 729
Reviewed by: Alberto RissoneErica BrescianiSalim Gasmi
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Abstract
Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (Danio rerio) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration.
Key features
• Allows an isolated and controllable environment to carry out activity and sleep assays in juvenile and adult zebrafish.
• Measures activity of zebrafish in life stages later than early development, which requires feeding animals during the assay.
• Requires use of a commercially available equipment system and six tanks.
• The activity of zebrafish can be tracked for five days including an acclimation step.
Keywords: Adult zebrafish Sleep Activity Melatonin Juvenile
Background
Sleep is a conserved behavior despite introducing some disadvantages to the organism, such as limiting responsiveness to the environment and preventing food foraging, which are critical activities for animals [1]. We spend nearly one-third of our lifetime sleeping. Although negative consequences of chronic sleep deprivation have been well-documented [1], the function of sleep is not fully known [2]. Major topics in sleep research include the investigation of why we sleep, what biological processes occur during sleep, and how sleep is regulated. Model organisms including zebrafish have been instrumental in addressing these fundamental questions in sleep research. Zebrafish meet all the behavioral criteria for defining sleep [3]. Because electrophysiological recordings of brain activity are not accessible in zebrafish, sleep is defined based on activity; one minute of inactivity in larvae [3,4] and six seconds of inactivity in adults [5] are defined as sleep. Sleep depth and arousability are possible to measure in larvae as well [6,7]. We and others have successfully identified sleep phenotypes in larval zebrafish when drug compounds were administered to their environment [8,9] and when particular genes were targeted via CRISPR/Cas9 mutation [6,10–12]. Nevertheless, there is a need to measure activity patterns and sleep in adult stages to seek answers to the following questions [13]:
1) Do sleep disturbances persist in juvenile mutant fish and adult mutant fish?
2) Do the genes of interest or the administered drug have a developmental (only at larval stage) role or a regulatory role (also at adult stages) in sleep?
For example, overexpression of the gene of interest via heat shock induction in a knockout model can be tested to see if sleep phenotypes at different stages of life are rescued. If the phenotype is rescued in larvae but not in adult fish, it indicates that the mutation irreversibly affects developmental circuits controlling sleep. Heat shock induction to mediate conditional gene expression has been applicable in juvenile [14] and adult zebrafish [15]. Sleep/wake patterns in adult zebrafish have been reported in previous studies that employed homemade solutions [5,7,16]. The methodology we propose here includes the use of a commercially available system, which ensures standardized and reproducible measurements. Sleep and activity phenotyping in larval zebrafish is high throughput; it is possible to include 96 animals in one assay [4]. Such assays in older age groups require the use of much larger tanks to allow normal swimming and exploration; therefore, one can phenotype behaviors of only 6–8 animals at once. We employed this automated video tracking in adult zebrafish (5 months old) and validated our protocol by administering melatonin, a known sleep-promoting compound. We demonstrated a sharp decrease in activity and wakefulness in adult zebrafish compared to the DMSO controls. Further studies are needed to carry out more detailed analyses of sleep parameters such as sleep bout length, sleep latency, and sleep bout numbers in age groups older than larval stages.
Materials and reagents
Biological materials
Adult wild type zebrafish (AB line). Adult zebrafish are housed under a 13:11 h light/dark cycle at the zebrafish core facility of the Children Hospital of Philadelphia.
Note: If possible, fish should be of equal size and pigment. Although the measure is meant to detect the center of the body, discrepancies can be found when comparing fish of drastically different sizes/pigments.
Reagents
Water from tank system (Aquaneering, San Diego, USA)
Gemma micro zebrafish food 150ZF (Skretting, Gemma micro-150)
Tap water to warm up and circulate around the tanks
Laboratory supplies
Six zebrafish breeding tanks, 1 L capacity [Aquaneering, ZHC100T (tank) and ZHC100L (lid)]
Equipment
Zebracube and regulator (ViewPoint Life Sciences Inc)
Zebracube is the chamber that houses juvenile or adult zebrafish during the experiment. It is connected to a computer and software that enable automated video tracking under cycling lights. A camera inside the Zebracube captures data based on the settings defined in the software by the researcher. The regulator facilitates circulation of warm water (28.5 °C) in and out of the water bath where the tanks are placed.
Temperature control unit (Julabo GmbH, model: 200F)
The temperature control unit heats tap water to 28.5 °C in order to keep zebrafish at the optimal growth temperature during the experiment.
Software and datasets
ZebraCube v5.18.0.0 (Viewpoint Life Sciences, Inc., access date, 09/26/2021)
Microsoft Excel (Excel 2016, access date, 02/16/2024)
MATLAB v2021b (Mathworks, https://mathworks.com, access date, 02/16/2024)
GraphPad Prism V10 (access date, 02/16/2024)
Procedure
We monitored activity patterns of 16 (5-month-old) adult zebrafish across 72 continuous hours using the commercially available automated system Zebracube to determine the optimal acclimation period for circadian cycle stabilization. We concluded that the acclimation period (first two days), as previously reported by Yokogawa et al. (2007) [5], shows robust sleep/wake patterns in line with 13:11 h light/dark cycle. We then captured activity data following the acclimation step, and we were able to replicate the activity patterns demonstrated previously [7] using the same parameters. Previous literature reported the use of homemade setups to record activity patterns of adult zebrafish [5,7,16]. We hereby introduce the use of a standardized protocol by employing a commercially available video tracking system to monitor juvenile and adult zebrafish sleep and activity patterns.
Animal handling
Start the regulator and temperature control unit to warm up the water surrounding the tanks (Figure 1). Set the water temperature to 28.5 °C.
Measure the temperature of water inside the water bath to ensure it is 28.5 °C. One may need to set the temperature on the control unit to a higher temperature due to the long tubing between equipment.
Add 500 mL of system water to each tank. Transfer one adult fish per tank. Use six tanks (Figure 2). We recommend alternating locations by genotype and/or treatment to avoid potential detection differences in the camera. When multiple replicate rounds are run, the group order should be alternated.
Note: Eight tanks will fit in this setup, but we found more reliable readings with six. We used the outer tank and the lid of a breeding tank. Lids were open during data capture to avoid condensation. The tanks are portable. We placed them in the water bath and then defined the virtual areas to collect data. Warm water at 28.5 °C circulates around the tanks.
Figure 1. Video monitoring system to measure locomotor activity patterns in juvenile and adult zebrafish. The experimental system is composed of the Zebracube system (ViewPoint Life Sciences, Inc.), a camera located at the top of the equipment to capture locomotor activity data of zebrafish, a water bath for circulating warm water during experiments, a regulator, and a temperature control unit. In and out pipes for water circulation are marked on the picture.
Adult zebrafish monitoring
Acclimation
Day 1 (acclimation): Draw rectangles by outlining the rectangle of the tanks so that the virtual area for data collection overlaps with the physical boundaries of the tanks that house adult fish (Figures 2 and 3). Apply distance calibration (Figure 3). Define light/dark cycle under the stimulus driving feature (Figure 4). Start the software using the following settings in tracking mode:
[background threshold: 40, inactive/small movement cutoff: 1.3 cm/s, small/large movement cutoff: 8 cm/s] on the main page (see General notes 1).
Figure 2. Adult zebrafish activity monitoring assay. The swimming behavior of six adult zebrafish is monitored in separate tanks. Large movement, small movement, and inactivity can be tracked.
Figure 3. Tools in the software to specify the virtual areas (rectangular icon marked with purple circle) for collecting activity data of adult zebrafish and to apply distance calibration (ruler icon marked with black circle). Location name needs to be changed with each box to provide a unique identifier in the output file. Make sure to keep a note of which genotype/treatment is in each location. Each location is identified as Loc1–6 to differentiate each animal.
Figure 4. Stimulus driving tab to define light and dark cycle. We specified 13 h of light cycle starting at 07:45 am and 11 h of dark cycle starting at 8:45 pm to be consistent with the light settings at the core facility. Time of lights on and duration of light and dark cycle may be redefined in the software. Make sure to test the light source with a lux meter at the level of the tanks to ensure consistent lux readings across experiments. Alterations in brightness and hue can alter behavior. Select the option loop until the software is closed to keep the same light/dark cycle until the end of the experiment.
Day 2 (acclimation): Refresh 20% of system water in the tanks at 9 am, feed the fish, and remove debris using a transfer pipette. Day 3 can also be included as acclimation, but we use it as a baseline reference day if drug is to be added.
The zebrafish were transferred from their original tanks to the tanks used in the experiment. Handling may cause stress in fish. We looked for stabilization of the sleep-wake behavior of the adult zebrafish at the acclimation step. Zebrafish are very responsive to the light. A stabilized sleep/wake cycle aligned to the timing of the light/dark cycle without large swings in activity throughout the day/night displays that acclimation has been achieved and the setup is working.
Data collection
Day 3 (assay): Refresh 20% of system water in the tanks, feed the fish, and remove debris using a transfer pipette at 9 am. Collect data to analyze sleep and activity patterns of adult zebrafish using the same settings from acclimation step during day and night. Data were collected every 6 s, as 6-s bouts of inactivity have previously been shown to be indicative of a sleep-like state by an increase in arousal threshold in adult fish [5].
Note: If adding drugs such as melatonin, care should be taken to segregate drug administration and feeding so that the effect of the drug is not masked by the innate drive to feed. Please see the validation section for more details. We avoided drug and food overlap by feeding the fish at 9 am and administering melatonin and DMSO at 11:45 am.
Day 4: Stop the assay after the 72-h period is completed or after the lights turn on.
Data analysis
Saving the data: After the assay is completed, data files will automatically populate in the selected output folder. The Excel file contains the data for analysis, but all files in the output folder should be saved to a drive, in case recreation of the Excel file is necessary.
Open the Excel file (Figure 5) and create a column called “sumdist” to the right of “lardist.” In Excel, use the sum function to add the “smldist” and “lardist” columns into the new “sumdist” column. This can be done in R or MATLAB, but for ease of use by others, we show how to do this in Excel.
Figure 5. A representative output Excel spreadsheet showing locomotor activity record of adult zebrafish. To analyze the data, we created a new variable called “sumdist,” which is the sum of the activity in the “smldist” and “lardist” columns. Small movement distance (smldist) and large movement distance (lardist) are calculated by the software using the determined thresholds.
Transform the data using MATLAB. We adapted the code provided by Lee et al. (https://bio-protocol.org/en/bpdetail?id=4313&type=0) [4] to transform the files for easier analysis. This transformation creates an output file ordered by clock time (zeitgeber time) with individual fish ordered at the top by box location. It collects the data from the “sumdist” column and places it in the corresponding “FISH” column. The value in the “CLOCK” column is zeitgeber time with 0 representing the lights-on and 13 representing the lights-off. Each row is a 6-s bin and represents the total distance traveled during that 6 s.
Note: This was adapted in the MATLAB script, as the script provided by Lee et al. uses a 14:10 light cycle. The Lee et al. code is also made for larvae, using 1 min bins instead of 6 s bins. We therefore changed the code to account for this. Use code: Viewpoint_to_Matlab_forAdults. – %comments are added where code was adapted from code provided by https://bio-protocol.org/en/bpdetail?id=4313&type=0
Analyzing the data. This can be done in a number of ways. To create the graphs displayed in Figure 6, we summed the data across hourly windows by summing the values every 600 rows in the Excel file beginning at CLOCK = 0 for each individual fish.
Note: It is important to start at CLOCK = 0 AFTER the acclimation period.
The hourly sums are then transferred to GraphPad Prism V10 for statistical analyses and plotting. We recommend including a 24-h period for acclimation. The acclimation data are not analyzed in the finalized dataset but provide a window for behavioral stabilization in the novel environment.
Depending on the experimental timeline, the hour following the acclimation period will be used for data analysis. For example: if a 48-h period is used for analysis, you will measure activity from ZT0 of the second day to the final bin before ZT0 or the 4th day (i.e., 23.99...). We recommend at least one full 24-h window for data analysis, but extra hours may be included if needed.
The data are loaded into a “grouped” table with sub-columns representing each fish per group. Hourly data are placed in each row of the table and data are plotted using the XY “points and connecting lines” graph format to create a plot as in Figure 6 with mean and SEM. We recommend plotting values for individual fish first to inspect data for outliers or spurious values. If a fish is a very different size from the others, the detection can be skewed. Individual boxes may also lose detection or fish may be extremely inactive skewing the results. Video review should be performed for suspicious results to justify removal.
To detect the prolonged response to melatonin, we plotted the hourly sums for the 9 h following drug administration (to lights-off) (Figure 6B). The immediate response to melatonin was calculated as the total distance traveled in the 1 h after drug administration (12–1 pm) (Figure 6C). We also compared this to the same time window on the previous baseline day to show the activity did not differ between groups prior to melatonin administration.
Validation of protocol
We tested the effects of melatonin on activity patterns of adult zebrafish to validate the protocol. We tracked distance traveled before and after melatonin administration to capture activity changes in light and dark cycles. Melatonin dissolved in DMSO (1.5 mL 50 µM) was administered to 500 mL final volume of water in which fish were housed during experiments. Therefore, the final concentration of melatonin was 150 µM. In control groups, 1.5 mL of DMSO was administered to 500 mL of tank water. The final concentration of DMSO was 0.3%. Melatonin and DMSO were added on day 4 at 11:45 am as endogenous melatonin levels are low at this time of day [17]. Data were captured until the next morning. Total distance traveled for the subsequent 9 h of the light cycle following drug administration was compared between experiment and control groups using RM-ANOVA (Figure 6B) [F(1,14) = 2.36, P = 0.15)]. Distance traveled after melatonin administration was compared between the experimental group and control group for the baseline day (12–1 pm) and the post-melatonin period (12–1 pm) by two-way repeated measures ANOVA with Bonferroni’s multiple comparisons correction applied [main effect of time X treatment; F(1,14) = 5.63, P = 0.03] [Bonferroni’s adjusted p-value; post-melatonin (P = 0.03)]. (Figure 6C). Exogenous melatonin administration at noon promoted inactivity peaking 1 h after administration as shown in larvae [8] with a modest effect persisting through the remainder of the light cycle (Figure 6A–B). Thus, we validated this methodology by demonstrating a decrease in activity and wakefulness in adult zebrafish compared to the control group.
Figure 6. Exogenous melatonin decreases daytime wakefulness. A. Distance traveled (mean and SEM) captured across three continuous days. Melatonin (150 µM final concentration in 0.3% DMSO) or 0.3% DMSO (control) was administered at 11:45 am (right arrow). Activity is captured on a 13:11 h light/dark cycle and shown as zeitgeber time. B. Total distance traveled each hour of light cycle following drug administration. C. Total distance traveled in the hour following drug administration (12 pm–1 pm). Two-way RM-ANOVA with Bonferroni’s multiple comparisons test was performed in B–C. *p < 0.05 adjusted. Data are presented as mean and SEM. n = 8 fish/group from three biological replicates.
General notes and troubleshooting
General notes
Background thresholds might vary based on the body size of juvenile zebrafish and adult zebrafish. One needs to observe if the colored tracks are generated for each fish (i.e., the line should track with the center of the body).
Using larvae in sleep assays is higher throughput when compared to using juveniles and adults; however, there are some specific questions that require the use of older age groups as indicated under the background section. Therefore, performing these assays in a standard and replicable environment is critical.
We continued to follow the 13:11 h light/dark cycle, which was applied in the zebrafish core facility at the time. We preferred this schedule in our experiment to not introduce a sudden change in the circadian rhythm of the adult zebrafish; however, we recommend setting the light/dark cycle to the usual 14:10 h cycle if possible. Duration of light/dark cycle and the time to start the cycle are possible to redefine in the software (see Figure 4).
System water used in the beginning of the experiment setup was at room temperature. We first started the temperature control unit, warmed up the system water inside the tanks by taking advantage of the circulating water, and finally placed the fish inside each tank individually. Circulating water was at 28.5 °C. We made sure of this temperature by measuring the water inside the water bath. The water used to refresh the tank water was at room temperature. Since it is 20% of the tank water, it was quickly warmed by the circulating warm water.
Troubleshooting
Problem: Software does not track the fish.
Possible cause: The virtual area that overlaps with the physical boundaries of the tank was defined in the previous assay.
Solution: Define the virtual areas at the beginning of each assay.
Problem: The lines are not centered on the body of the fish or are delayed.
Solution: Redefine the threshold for each box individually to ensure each fish is being detected over the background.
Acknowledgments
We thank Adele Donahue for technical assistance. Data in this work were supported by funding from Fulbright Visiting Scholar Program–Postdoctoral Grant, grant number FY-2017-TR-PD-07 (FDB), and National Institutes of Health grant, The Program Project Grant (PPG) P01 HL160471-01A1 (AIP).
Competing interests
The authors declare no competing interests.
Ethical considerations
All experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines of the University of Pennsylvania and the Children Hospital of Philadelphia.
References
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Lee, D. A., Oikonomou, G. and Prober, D. A. (2022). Large-scale Analysis of Sleep in Zebrafish. Bio Protoc. 12(3): e4313. https://doi.org/10.21769/BioProtoc.4313.
Yokogawa, T., Marin, W., Faraco, J., Pezeron, G., Appelbaum, L., Zhang, J., Rosa, F., Mourrain, P. and Mignot, E. (2007). Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants. PLoS Biol .5(10): e277. https://doi.org/10.1371/journal.pbio.0050277.
Doldur-Balli, F., Zimmerman, A. J., Keenan, B. T., Shetty, Z. Y., Grant, S. F. A., Seiler, C., Veatch, O. J. and Pack, A. I. (2023). Pleiotropic effects of a high confidence Autism Spectrum Disorder gene, arid1b, on zebrafish sleep. Neurobiol Sleep Circadian Rhythms. 14: 100096. https://doi.org/10.1016/j.nbscr.2023.100096.
Chen, S., Reichert, S., Singh, C., Oikonomou, G., Rihel, J. and Prober, D. A. (2017). Light-Dependent Regulation of Sleep and Wake States by Prokineticin 2 in Zebrafish. Neuron. 95(1): 153–168 e156. https://doi.org/10.1016/j.neuron.2017.06.001.
Doldur-Balli, F., Smieszek, S., Keenan, B. T., Zimmerman, A. J., Veatch, O. J., Polymeropoulos, C. M., Birznieks, G. and Polymeropoulos, M. H. (2024). Screening effects of HCN channel blockers on sleep/wake behavior in zebrafish. Front Neurosci. 18. https://doi.org/10.3389/fnins.2024.1375484.
Rihel, J., Prober, D. A., Arvanites, A., Lam, K., Zimmerman, S., Jang, S., Haggarty, S. J., Kokel, D., Rubin, L. L., Peterson, R. T., et al. (2010). Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science. 327(5963): 348–351. https://doi.org/10.1126/science.1183090.
Zimmerman, A. J., Doldur-Balli, F., Keenan, B. T., Shetty, Z. Y., Palermo, J., Chesi, A., Sonti, S., Pahl, M. C., Brown, E. B., Pippin, J. A., et al. (Preprint). Zebrafish screen of high-confidence effector genes at insomnia GWAS loci implicates conserved regulators of sleep-wake behaviors. Preprint. 2022.2010.2005.511011. https://doi.org/10.1101/2022.10.05.511011.
Ruzzo, E. K., Perez-Cano, L., Jung, J. Y., Wang, L. K., Kashef-Haghighi, D., Hartl, C., Singh, C., Xu, J., Hoekstra, J. N., Leventhal, O., et al. (2019). Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks. Cell. 178(4): 850–866 e826. https://doi.org/10.1016/j.cell.2019.07.015.
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Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants
GB Gabriele Bradamante
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5015 Views: 787
Reviewed by: Sarah C. PlechaJohn P Phelan Anonymous reviewer(s)
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Abstract
All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli.
Key features
• Novel dissection and imaging method of the shoot apical meristem of Arabidopsis.
• Procedure performed with intact, soil-grown, flowering plants.
• Possibility of long-term live imaging of the shoot apical meristem.
• Protocol can be adapted to different plant species.
Keywords: Shoot apical meristem Plant stem cells In vivo imaging Confocal microscopy Arabidopsis thaliana
Background
In plants, above-ground organs are continuously generated from stem cells in the shoot apical meristem (SAM). This process is best described in the dicot plant Arabidopsis thaliana (reviewed in Fuchs and Lohmann [1]; Holt et al. [2]; Hong and Fletcher [3]; Janocha and Lohmann [4]). The initial vegetative SAM produces only leaves but changes into a reproductive SAM after transition to flowering. A subset of cells within the SAM will eventually lead to the formation of gametes. Mature flowers and siliques are formed after bolting when the main stem emerges from the basal rosette and the SAM changes into an inflorescence meristem (IM). Following the fate of stem cells during these important transitions of the meristem is therefore of utmost importance to understand this developmental program.
Different approaches have delivered detailed information about meristem development [5–7]. However, most techniques require dissection, staining, or cell separation and are not compatible with the observation of dynamic processes. Live imaging of the SAM has overcome this limitation to some extent but is usually performed by detaching the shoot tip of a bolting plant, placing it in a microscopy-suited vessel filled with growth medium and submerging it in water, followed by removing flowers and buds before microscopy observation [8–11].
In an alternative setup, Grandjean et al. [12] recorded SAM development in intact plants using an inverted microscope, with the drawback of working against gravity. This was later overcome by Heisler et al. [13] and Tobin and Meyerowitz [14]. These authors proposed observing the SAM under physiological conditions, after removing surrounding flower buds from a plant prior to bolting without separating it from the plant body, keeping it completely underwater. However, this limited the observations to an early stage, and the complete submergence of the plant can cause hypoxia and corresponding side effects on the development [15]. These side effects could be reduced by placing only a water drop between the plant tip and the front lens of a water immersion objective [14], however, at the price of low stability and restricted time for the acquisition.
The protocol described here provides an innovative method to observe the SAM of bolting Arabidopsis plants without detaching it from the stem. An “easy-to-built” custom-made device allows the preparation of the main inflorescence of a soil-grown plant for subsequent observation with an upright confocal microscope. This system limits submergence to the very top and permits precise positioning of the exposed SAM in all dimensions. In addition, the observation of the living SAM can be extended over several days, while the plant grows in regular substrate, light, and oxygen supply. The setup allows further control and variation of environmental conditions like drought, salinity, light stress, hormone or drug application, and pathogen infections. The procedure can likely be modified to also study meristem development in species with similar size and growth architecture as Arabidopsis.
Materials and reagents
Biological material
Plants (Arabidopsis thaliana) in a suitable stage of inflorescence meristem development, e.g., three weeks grown at 8:16 h light/dark cycles followed by two weeks at 16:8 h light/dark cycles (short- and long-day regimes, respectively) at 21 °C with 60% relative humidity and 150 µmol m-2·s-1 light intensity), grown in soil in round 5 cm diameter pots
For proof of concept used in this protocol: a transgenic Arabidopsis plant carrying a stem cell–specific nuclear marker (ProCLV3:H2B-mCherry, Gutzat et al. [16]) in addition to the ubiquitously expressed nuclear marker ProHTR5:H2A-mNeonGreen (unpublished construct, assembled with modules described in Donà et al. [17])
Reagents
Low melting agarose (LMA) (Roth, catalog number: 6351)
Distilled water
Solutions
1.5% LMA in water. Aliquots can be stored in Eppendorf tubes at 4 °C
Laboratory Supplies
Sterilin Petri dishes 50 mm (Thermo Fisher Scientific, catalog number: 124-17)
FEP tubing (inner diameter 2.8 mm, outer diameter 3.2 mm, Wolf-Technik)
Sample tubes 0.6 mL, microcentrifuge tubes low retention (Thermo Fisher Scientific, catalog number: 3446) for LMA aliquots
Glass capillary Transferpettor, caps cert. 100 µL (Brand, catalog number: 701910)
Piston rod Transferpettor (Brand, catalog number: 701938)
Patafix glue pads (Uhu, catalog number: 64797)
Round plant pots with 5 cm diameter (e.g., singularized from trays, HerkuPlast HP D 60/5.5 R)
Standard soil (e.g., Profi Substrat, Gramoflor)
Equipment
Dissection desk (table with opening on the top, with appropriate height range for the lifting station (width 58 cm, depth 40 cm, height 38 cm; hole diameter 11 cm)
Stereomicroscope with opening on the stage (Leica MZ6)
Lifting station (e.g., Swiss Boy Lab jack)
Petri dish prepared with a central opening into which a 1.5 cm plastic tube is fixed (prepared in a workshop by drilling a central opening and gluing the plastic tube, e.g., with AL-FIX Füllstoff Noviqua glue)
Adjustable stem holder fitting the opening of the stereomicroscope stage and the modified Petri dish with a central tube [prepared in a workshop from laminated wood (light blue), plastic material (black disc), metal parts, and a screw for movement]
Thermoblock (Eppendorf Themomixer comfort) matching LMA sample tubes
Fine forceps (Ideal-tek 3C.SA or Dumont No. 5)
Needles (Sterican Gr.1: 0.90 mm × 40 mm, yellow)
Syringes (Tuberculin Luer. Chirana 1 mL CHTUB01) with attached plastic tube or pipette
Upright confocal microscope without condenser (Zeiss LSM700 Axio Imager 2)
Water dipping lenses (Zeiss W N-Achroplan 40×/0.75)
Plexiglass microscope stage insert with central opening (prepared in a workshop by drilling a central opening; width 15 cm, depth, 15 cm, height 0.5 cm; hole diameter 3 cm)
LED light illumination (Model TXD-6, GRENEBO)
Plant growth chamber (e.g., Photon System Instruments)
Software and datasets
Microscope software: ZEN 2010 Version 6.0
Visualization software: Imaris 10
Procedure
Preparation of the meristem under the stereomicroscope
Melt an LMA aliquot at 80 °C in the thermoblock, then keep it at 37 °C and use the aliquot within 1 h.
Place the stereomicroscope onto the dissection desk (Figure 1A, B).
Figure 1. Setting up the dissection desk. A. Dissection desk with stereomicroscope on top. B. Stereomicroscope stage without lid on top of the dissection desk opening. C, D. Positioning of the lifting station under the dissection desk. E. Alignment of microscope stage opening, dissection desk opening, and lifting station.
Place the lifting station under the dissection desk (Figure 1C–1E).
Remove siliques, flowers, and big flower buds from the main shoot of a plant with fine forceps (Figure 2A–2C).
Figure 2. Manual dissection of the shoot apical meristem (SAM). A. Soil-grown, flowering Arabidopsis plant. B. Close-up of the shoot tip. C. Dissected apical stem. Scale bar = 4 mm.
Place the plant pot onto the lifting station and adjust the height so that the plant tip comes out (approximately 1 cm) from the base of the stereomicroscope (Figure 3C, D).
Figure 3. Positioning of the dissected plant and adjustable stem holder onto the dissection desk. A. Top view of the adjustable stem holder. B. Bottom view of the adjustable stem holder. C. Positioning of the dissected plant onto the lifting station. D. Lowering of the lifting station. E. Positioning of the adjustable stem holder onto the stereomicroscope stage. F. Close-up of the adjustable stem holder with the plant apex visible in the center.
Open the adjustable stem holder to approximately 1 cm and insert it into the dissection desk opening of the stereomicroscope base so that the plant tip can go through it (Video 1; Figure 3A, B, E, F; Figure S1).
Video 1. Adjusting the stem holder.This video shows how to operate the adjustable stem holder to stabilize the Petri dish before fine dissection of the shoot tip.
Insert a plunger into a glass capillary and then insert this capillary into the plastic tube of the modified Petri dish. Place the lower end of the plunger between the endpoints of the capillary and the plastic tube (Figure 4A, B; Video 2).
Figure 4. Fine dissection of the shoot apical meristem (SAM). A. Modified Petri dish with a tube connected at its center, plunger, and glass capillary. B. Plunger inserted into the capillary, further connected to the tube of the modified Petri dish. C. Removal of capillary and plunger after filling the tube with LMA. D. Syringe with needle for fine dissection of the apical meristem. E. Exposed SAM as seen from the stereomicroscope. The meristem dome is indicated by the black arrow. Scale bar = 100 µm. F. Disassembly of all the tools and plant release from the dissection desk.
Video 2. Assembly of the modified Petri dish.This video shows how to assemble the plunger and capillary and how to connect them to the modified Petri dish. The plunger is inserted into the capillary, which is then inserted into the tube of the modified Petri dish.
By moving the plunger upwards, fill the plastic tube with LMA (approximately 90 µL) from the Eppendorf tube, then remove the capillary and plunger simultaneously (Video 3; Figure 4C).
Video 3. Preparation of the modified Petri dish with LMA.This video shows how to fill the tube of the modified Petri dish with LMA by moving the plunger upwards.
Immediately after removing the capillary and plunger and as long as the agarose plug is liquid, gently push the tip of the plant through the agarose to expose the apex in the air. If necessary, use forceps to perform this procedure (Video 4).
Video 4. Positioning of the modified Petri dish with LMA on the plant tip.This video shows how to handle the Petri dish filled with LMA and how to position it on the plant tip in the adjustable stem holder.
Wait 5 min until the agarose is solidified.
Stabilize the Petri dish on the dissection desk by tightening the screw on the stem holder (Video 5).
Video 5. Securing the Petri dish on the adjustable stem holder.This video shows how to stabilize the Petri dish on the adjustable stem holder by rotating the outer screw.
Fill the Petri dish with distilled water to cover the meristem (approximately 20 mL) (Video 6).
Video 6. Filling the Petri dish with water.This video shows how to fill the Petri dish with water once the LMA is solidified.
Expose the meristem optimally under the stereomicroscope using needle and forceps. Stabilize the stem with forceps and use the needle to detach the flower buds from the inflorescence. Start by removing larger flower buds and then dissect the smaller ones, making sure to excise them at their connection with the main stem (Figure 4D, E).
Remove the water from the Petri dish with a tube connected to a syringe or with a pipette.
Release the Petri dish from the dissection desk insert and remove it from the plant stem (Figure 4F).
Remove residual agarose from the stem with forceps.
Imaging of the meristem under the confocal microscope
Remove the condenser from an upright confocal microscope (Figure 5A, B)
Insert the plant into the condenser holder (Figure 5C–5E).
Figure 5. Positioning of the prepared plant under the microscope. A. Upright confocal microscope. B. Removal of the condenser. C, D, F. Positioning of the plant pot onto the condenser holder (top, bottom, and front view).
Place the plexiglass insert onto the microscope stage and, by rotating the condenser knobs, bring the plant up so that the tip goes through the opening in the plexiglass insert (Figure 6A).
Figure 6. Plant preparation for imaging. A. Positioning of transparent plexiglass insert with central opening onto the microscope stage. B. Plant tip exposed in the Petri dish with the stem stabilized by solidified LMA C. Petri dish filled with water. D. Positioning of water immersion lenses on top of the plant tip. E. Petri dish stabilization with patafix at opposite sides. F. Illumination of the plant with LED. G. Close-up of meristem tip under LED illumination.
Repeat steps A7–A10 as described above, this time on the microscope stage (Figure 6B).
Fill the Petri dish with distilled water (Figure 6C).
Align the plant tip with the objective (Figure 6D).
Stabilize the Petri dish on the plexiglass insert with small pieces of patafix at opposite sides (Figure 6E).
Acquire 3D acquisitions (Figure 7A, B) with water dipping lenses.
Figure 7. Confocal acquisition of an Arabidopsis shoot apical meristem (SAM). A. Maximum intensity projection of confocal image stacks viewed from the top. B. 3D rendering of the same stack. Two imaging tracks were used with 488 nm and 555 nm excitation lasers for mNeonGreen and mCherry, respectively. Z-stack of 1.12 µm step for a total of 97 slices. Magenta: stem cell nuclei (ProCLV3:H2B-mCherry). Light blue: ubiquitous nuclear marker (ProHTR5:H2A-mNeonGreen). Scale bar = 50 µm.
Validation of protocol
This protocol is validated by the figures and movies provided to show the functionality of the experimental setup. The images acquired by confocal acquisition of the dissected meristem, as presented in Figure 7, show that the plant apex with an intact SAM can be prepared in the described way and allow anybody to reproduce it step-by-step for studies of the dynamic processes in this organ.
General notes and troubleshooting
The adjustable stem holder does not necessarily need to be manufactured as presented in the equipment section (Figure 3A, B; Video 1; Figure S1). A simpler device keeping the Petri dish stable is sufficient.
The ideal length of the plant stem depends on plant growth conditions, the dimensions of the pot, and the space of the condenser holder. In the setup presented here, the plant stem has to be at least 3.5 cm long.
Aliquots are best stored in 0.6 mL tubes; refer to the laboratory supply section.
LMA in the tube of the modified Petri dish takes approximately 4 min to solidify at room temperature. If the agarose solidifies before the stem has gone through, it can be removed from the tube with the plunger.
Exposure to the air of the dissected meristem (between steps A4 and A11, and between steps A14 and B4) should be minimized. Once the step is completed, move directly to the next one.
Optional for long-term studies: place LED lights above the stage, on the side of the revolving nosepiece (Figure 6F, G).
XYZ drift correction tools (e.g., Huygens Object Stabilizer) might be applied to the 3D acquisition to correct minor plant movements occurring during acquisition.
Acknowledgments
The author is grateful to Ortrun Mittelsten Scheid for assistance and comments on the manuscript. He also appreciates the excellent support from Pawel Pasierbek and Alberto Moreno Cencerrado (BioOptics Core Facility). Many thanks go also to Martin Colombini from VBC workshop and Roberto Bradamante for manufacturing the necessary equipment.
Competing interests
The author declares not to have any conflict of interest.
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Gutzat, R., Rembart, K., Nussbaumer, T., Hofmann, F., Pisupati, R., Bradamante, G., Daubel, N., Gaidora, A., Lettner, N., Donà, M., et al. (2020). Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns. EMBO J. 39(20): e103667.
Donà, M., Bradamante, G., Bogojevic, Z., Gutzat, R., Streubel, S., Mosiolek, M., Dolan, L. and Scheid, O. M. (2023). A versatile CRISPR-based system for lineage tracing in living plants. Plant J. 115(5): 1169–1184.
Supplementary information
The following supporting information can be downloaded here:
Figure S1. Schematics of the adjustable stem holder.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Plant Science > Plant developmental biology > General
Cell Biology > Cell imaging > Live-cell imaging
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5,016 | https://bio-protocol.org/en/bpdetail?id=5016&type=0 | # Bio-Protocol Content
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Peer-reviewed
Transfection of Babesia duncani: A Genetic Toolbox of This Pathogen to Advance Babesia Biology
SW Sen Wang
JW Jianyu Wang
DL Dongfang Li
FC Fangwei Chen
WL Wanxin Luo
JZ Junlong Zhao
LH Lan He
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5016 Views: 611
Reviewed by: Alba BlesaRITU SOM Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Frontiers in Cellular Infection Microbiology Apr 2022
Abstract
Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis.
Keywords: Babesiosis Babesia duncani Stable transfection Gene manipulation Homologous recombination
Graphical overview
Background
Babesia are parasitic protozoa of the Apicomplexa phylum, which encompasses other important human pathogens such as the agents of malaria and toxoplasmosis. Babesia parasites are transmitted primarily by ticks and can infect various mammalian species [2]. In humans, babesiosis is caused by several Babesia species including B. microti, B. duncani, B. divergens, and B. venatorum [3]. Clinical manifestations of the disease include fever, hemolytic anemia, and in severe cases, complications such as cardiac failure, respiratory distress, and pulmonary issues, which can ultimately lead to death. Patients with underlying immunological disorders, undergoing immunosuppressive treatment, or having undergone splenectomy are particularly susceptible to experiencing severe symptoms and increased mortality if infected by Babesia [3–5]. B. duncani is found primarily in western United States and exhibits higher virulence in animal models compared to B. microti, leading to acute mortality in mice and in hamsters [6–9]. Morphologically, there are no significant distinguishing features between B. duncani and B. microti [10].
Gene editing has become an invaluable tool for the investigation of gene function and validation of potential drug targets [11]. Advancing our understanding of the fundamental biology of Babesia parasites requires the development of genetic manipulation techniques [12]. Previous studies have demonstrated successful genetic manipulation methods in various protozoan pathogens, including Babesia bovis, B. gibsoni, B. ovis, B. ovata, Theileria annulata, and Theileria parva [13–18]. Successful transfection of piroplasmids was achieved in B. bovis [1] using the elongation factor 1-aB (ef-1aB) promoter to drive expression of a reporter GFP-BSD consisting of a fusion between the green fluorescent protein (GFP) and the blasticidin S deaminase (BSD) [19], which was integrated into the ef-1αA region through homologous recombination. Subsequent studies employed a similar approach for stable transfection in B. gibsoni, B. ovata, and Babesia sp. Xinjiang [17,18,20]. While a genetic modification method has been reported for B. microti, it mainly relies on fluorescent tags due to the lack of effective in vivo drug screening markers, challenging the generation of gene-edited strains in B. microti [21]. Genetic manipulation of human Babesia parasites has also been facilitated by the availability of a continuous in vitro culture system in human red blood cells and the sequencing, assembly, and annotation of their genomes [5,9,22–24]. Unlike B. microti, for which only a short-term culture could be achieved, long-term and continuous in vitro culture of B. duncani has been established using hamster and human erythrocytes [24–27]. Furthermore, in B. duncani, an animal model of lethal infection has been optimized, thus paving the path for evaluating the importance of specific genes in vivo [8,27]. The first genetic modification of B. duncani [28] was achieved using a transient transfection technique to express the mCherry reporter in the parasite under the regulatory control of the ef-1αB promoter. Subsequently, a stable expression of eGFP was achieved in B. duncani using the human dihydrofolate reductase (hDHFR) gene selectable marker, which confers resistance to the antifolate WR99210. The development of genetic tools for the manipulation of B. duncani provides a unique opportunity to study gene function in B. duncani and to gain further insights into its biology and pathogenesis.
Development of the protocol
The electroporation method has been widely utilized for various apicomplexan parasites, including Toxoplasma gondii (the causative agent of toxoplasmosis) and Plasmodium spp. (the agents of human malaria). Electroporation is an effective means of introducing foreign DNA into these parasites for genetic manipulation and functional studies. Electroporation methods are indeed categorized into two main types based on the electrical parameters. The first is low voltage–long pulse, which employs a lower electrical voltage for a relatively longer pulse duration. This approach is typically considered gentler and is often used when cell viability is a primary concern. The high voltage–short pulse method involves using a high electrical voltage for a very short pulse duration. It is often referred to as the burst or high-field electroporation. This approach is more efficient in terms of introducing foreign material into cells but may have a greater impact on cell viability. The choice between these methods depends on the specific requirements of the experiment, such as the type of cells or organisms being electroporated and the desired level of transfection efficiency vs. cell survival. In B. duncani, as in previously reported cases in B. bovis [1], the high voltage–short pulse electroporation method results in higher transfection efficiency. To enhance transfection efficiency in B. duncani, our optimization efforts focused on changing the parameters of electroporation, including voltage, volume, and frequency, with the aim of achieving higher levels of transfection efficacy.
When comparing different electroporation instruments, we usually find variations in their effectiveness for transfection. We found that a higher transfection efficiency could be achieved using the Gemini SC Electroporation System from BTX, whereas transfection using the Bio-Rad Gene Pulser Xcell was not successful.
Materials and reagents
Biological materials
Chemically competent E. coli DH5α [F− supE44 ΔlacU169 (φ80 lacZDM15) hsdR17 (rk−mk) recA1 endA1 thi1 gyrA relA] is used for routine cloning and plasmid maintenance. The competent cells are available via ThermoFisher (catalog number: 18265017)
Hamster RBCs are used for B. duncani culture. The hamster RBCs are obtained from Syrian golden hamsters [Crl: LVG (SYR)] and can be prepared following the protocol.
B. duncani WA1 strain (ATCC PRA-302™) is expanded through in vitro cultivation and the protocol.
Reagents
MilliQ water
Bacto tryptone (Becton Dickinson, catalog number: 211705)
Bacto yeast extract (Becton Dickinson, catalog number: 212750)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886)
Bacto agar (Becton Dickinson, catalog number: 214010)
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518)
Phanta Max Super-Fidelity DNA Polymerase (Vazyme, catalog number: P505)
2× Rapid Taq Master Mix (Vazyme, catalog number: P222)
ClonExpress MultiS One Step Cloning Kit (Vazyme, catalog number: C113)
100 bp DNA ladder (Vazyme, catalog number: MD104-01)
EasyPure® Quick Gel Extraction kit (Trans, catalog number: EG-101)
EasyPure® Plasmid MiniPrep kit (Trans, catalog number: EM-101)
EndoFree Maxi plasmid kit (TIANGEN, catalog number: DP117)
TIANamp Geneomin DNA kit (TIANGEN, catalog number: DP304)
VP-SFM ATGTM (Thermo Fisher Scientific, Gibco, catalog number: 12559019)
Ala-Gln (MACKLIN, catalog number: A800584)
AlbuMax II (Thermo Fisher Scientific, Gibco, catalog number: 11021037)
Antimycotic (antibiotic) (Thermo Fisher Scientific, Gibco, catalog number: 15240-062)
WR99210 (MCE, catalog number: HY-116387)
MBP146-78 (TargetMol, catalog number: T7321)
Giemsa’s stain (Sigma-Aldrich, catalog number: G4507)
Immersion oil (Sigma-Aldrich, catalog number: 1046990100)
Trisodium citrate (Sigma-Aldrich, catalog number: V900443)
Citric acid (Sigma-Aldrich, catalog number: C0759)
Glucose (Sigma-Aldrich, catalog number: G8270)
Sodium dihydrogen phosphate (Sigma-Aldrich, catalog number: 5438400100)
Adenine (Sigma-Aldrich, catalog number: A2786)
Mannitol (Sigma-Aldrich, catalog number: M4125)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: S5886)
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670)
Dipotassium hydrogen phosphate (K2HPO4) (Sigma-Aldrich, catalog number: P8281)
Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P5655)
4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number:H4034)
Ethylene glycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889)
Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2393)
Saponin (Sigma-Aldrich, catalog number: S7900)
Absolute ethanol (Sigma-Aldrich, catalog number: 1009831011)
Alsever’s solution (Sigma-Aldrich, catalog number: A3551)
DMSO (Sigma-Aldrich, catalog number: D8418)
Glycerol (Sigma-Aldrich, catalog number: G5516)
Tris (MACKLIN, catalog number: T819512)
Ethylenediaminetetraacetic acid (EDTA) (MACKLIN, catalog number: E809068)
Acetic acid (MACKLIN, catalog number: A801295)
Potassium hydroxide (KOH, MACKLIN, catalog number: P816399)
Agarose (MACKLIN, catalog number: A800342)
GelRed (MACKLIN, catalog number: G917739)
Ethidium bromide (EB, MACKLIN, catalog number: E808961)
Sodium acetate buffer, 3 M, pH 5.2 (MACKLIN, catalog number: S885174)
Hoechst (Beyotime, catalog number: CH1024)
Solutions
LB medium (see Recipes)
LB plates (see Recipes)
Ampicillin stock solutions (100 mg/mL) (see Recipes)
TAE buffer (see Recipes)
VP-SFM complete media (see Recipes)
Red blood cell preservation solution (see Recipes)
Cytomix buffer (see Recipes)
20% Saponin stock solution (see Recipes)
5 mM WR99210 storage solution (see Recipes)
Freezing solution (see Recipes)
Recipes
LB medium
Prepare LB medium by dissolving the following ingredients (with the help of a magnetic stirrer) in MilliQ water: 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl.
Aliquot the broth in clean bottles, autoclave at 121 °C for 15 min, and let it cool down before use.
LB medium can be stored at room temperature for up to six months.
LB plates
Prepare the LB medium as described above, aliquot in clean bottles, and add 15 g/L Bacto agar. Autoclave at 121 °C for 15 min and let cool down to ~50 °C at room temperature before the addition of antibiotics or other supplements (when needed). Mix well and pour ~20 mL into Petri dishes. Plates can be stored at 4 °C for up to one month.
Ampicillin stock solutions (100 mg/mL)
Weigh 1,000 mg of ampicillin sodium salt using an electronic scale and dissolve in 10 mL of MilliQ water. Filter the solution through a 0.2 μm filter in the laminar flow hood and prepare 1 mL aliquots in sterilized 1.5 mL microcentrifuge tubes. Antibiotic stock solutions can be stored at -20 °C for up to one year.
TAE buffer
The 50× TAE stock solution contains 2 mol/L Tris (242 g/L), 0.1 mol/L EDTA disodium salt (37.2 g/L), and 57.1 mL/L acetic acid. Add double-distilled water (ddH2O) to a final volume of 1 L, place the solution on the magnetic stirrer, and stir until it is completely dissolved. Once dissolved, adjust the pH to 8.0. The 50× TAE stock solution can be stored at room temperature for several years; dilute it with ddH2O to 1× TAE buffer before use.
VP-SFM complete media
Prepare VP-SFM complete medium by dissolving the following ingredients (with the help of a magnetic stirrer) in MilliQ water: 17.6 g/L VP-SFM ATGTM, 0.86 g/L Ala-Gln, 2 g/L AlbuMax II, and 10 mL/L 100× antibiotic/antimycotic. Filter the solution through a 0.2 μm filter in the laminar flow hood and prepare 50 mL aliquots in a sterilized centrifuge tube. Medium can be stored at 4 for up to six months.
Red blood cell preservation solution
Prepare red blood cell preservation solution by dissolving the following ingredients (with the help of a magnetic stirrer) in MilliQ water: 1.5 g/L trisodium citrate, 0.2 g/L citric acid, 7.93 g/L glucose, 0.94 g/L sodium dihydrogen phosphate, 0.14 g/L adenine, 4.97 g/L sodium chloride, and 14.57 g/L mannitol. Aliquot the solution in clean bottles, autoclave at 121 °C for 15 min, and let it cool down before use. Red blood cell preservation solution can be stored at 4 for up to six months.
Cytomix buffer
In a clean and sterile container or bottle, add approximately 800 mL of MilliQ water. Carefully add the dry ingredients in the following order while gently stirring to ensure proper dissolution: 6.7 g KCl, 0.012 g CaCl2, 1.30 g K2HPO4, 1.02 g KH2PO4, 4.46 g HEPES, 0.76 g EGTA, and 0.762 g MgCl2·6H2O; adjust the pH 7.5 with potassium hydroxide (KOH). After all the components are dissolved, carefully adjust the volume to 1 L using MilliQ water and mix thoroughly to ensure the solution is homogenous. Filter the solution through a 0.2 μm filter in the laminar flow hood and prepare 50 mL aliquots in a sterilized centrifuge tube. Medium can be stored at 4 for up to six months.
20% Saponin stock solution
Weigh 20 g of saponin using an electronic scale and dissolve in 100 mL of 1× PBS (8.0 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, PH 7.4). Filter the solution through a 0.2 μm filter in the laminar flow hood and prepare 1 mL aliquots in sterilized 1.5 mL microcentrifuge tubes. Saponin stock solutions can be stored at −20 °C for up to one year. Dilute the saponin solution to 0.1% with PBS when lysing red blood cells.
5 mM WR99210 storage solution
Add 0.5067 mL of DMSO to 1 mg of WR99210 powder. Gently swirl to dissolve. Prepare small 10 μL aliquots and store at -20 °C for up to 12 months. Prepare a 5 μM working concentration by diluting it in DMSO at a 1:1,000 ratio before use.
Freezing solution
In a 50 mL centrifuge tube, add 15 mL of glycerol, followed by 35 mL of Alsever's solution. Mix the solution by vortex thoroughly and then filter it using a 0.2 μm filter. Freezing solution can be stored at 4 °C for up to six months.
Laboratory supplies
Micropipette refill tips (10, 200, and 1,000 µL)
1.5 mL Eppendorf tube (Pullen, catalog number: PL03001)
PCR tubes, 8 tubes per strip, 125 strips per unit (Crystalgen (GY), catalog number: L-2081)
PCR tubes, domed 8 caps per strip, 125 strips per unit (Crystalgen (GY), catalog number: L-2082)
24-well plates (Corning, catalog number: 3524)
96-well plates (Corning, catalog number: 3917)
15 mL centrifuge tube (PULLEN, catalog number: PL01005)
50 mL centrifuge tube (PULLEN, catalog number: PL01006)
1 mL syringe (Beyotime, catalog number: FS802)
Equipment
Micropipette set, Eppendorf Research plus (0.1–2.5, 2–20, 20–200, and 100–1,000 µL) (Eppendorf, catalog number: 3123000012, 3123000098, 3123000055, and 3123000063)
Laboratory glass bottles (250, 500, and 1,000 mL)
T100 thermal cycler (Bio-Rad, catalog number: 1861096)
Petri dishes (Ø × H: 92 × 16 mm, with ventilation cams)
Benchtop centrifuge (Eppendorf, catalog number: 5810R)
Electric thermostatic incubator (Shanghai Jinghong Instrument, catalog number: DNP-9272)
Floored large refrigerated shaking incubator (Shanghai Zhichu Instrument, catalog number: ZQLY-300S)
Medical icebox (Panasonic, catalog number: MPR-710)
Low temperature freezer (-20 °C) (Panasonic, catalog number: MDF-539)
Gemini SC electroporation system (BTX, catalog number: 45-2042)
pH meter (Sartorius, model: PB-11)
Gene Pulser/MicroPulser electroporation cuvettes, 0.2 cm gap (Bio-Rad, catalog number: 1652082)
Modular incubator chamber (Billups-Rothenberg, Inc, model: MIC-101)
Millipore 0.2 μM filters (Sigma-Aldrich, catalog number: SLGNDZ5)
Blue-light gel cutter (Sangon, model: G600312-000)
NanoDrop 2000 (Thermo Fisher, model: pedestal mode)
Water bath (Beyotime, model: E0530)
Gel imaging system (Bio-Rad, model: XXX)
OLYMPUS FRAME_BX63 scanning confocal microscope (OLYMPUS FRAME, model: BX63)
Cell counter (Watson, model: 177-112C)
Nalgene Mr. Frosty Freezing Container (Thermo Scientific, catalog number: 5100-0036)
Software
SnapGene (RRID: SCR_015052, https://www.snapgene.com, SnapGene 7.0.2)
Procedure
Plasmid design
Promoter region: For the promoter region, select the 250 base pairs located upstream of the transcription start site as shown in Figure 1A. Commonly used promoters for drug screening tags include ef-1aB promoter and enolase promoter.
Marker selection: Choose from commonly used fluorescent reporter genes such as eGFP, mCherry, or luciferase, as indicated in Figure 1A. For drug selection markers, commonly used options are human dihydrofolate-reductase (hDHFR) and puromycin-N-acetyltransferase (PAC). The addition of the drug WR99210 disables the Babesia DHFR enzyme and halts nucleic acid synthesis. By expressing hDHFR, which is resistant to the drug, the function of the babesia DHFR is replaced. PAC confers resistance to puromycin.
Terminators region: Downstream of the gene ORF (open reading frame), commonly used terminators include ef-1a, rap-1, and DHFR-TS (dihydrofolate reductase-thymidylate synthase). Most termination sites are between 200 and 600 bp in length.
Homologous arms: When performing gene insertion and replacement, it is essential to have a segment of homologous arms to target the gene of interest. Typically, the homologous arms consist of 750 bp located both upstream and downstream of the modification site, as illustrated in Figure 1B.
Figure 1. Vector maps common for B. duncani. A. Plasmid map for transient transfection. B. Plasmid for genetic modification.
Design the oligonucleotides to construct the plasmid. Firstly, identify the gene of interest in the genome and extract 1,500 bp of flanking upstream and downstream of this gene. Select 750 bp of sequence upstream and downstream of the gene locus for the homologous arms. The genome of B. duncani has been uploaded to NCBI with the accession number GCA_028658345.1.
Design primers according to the ClonExpress MultiS One Step Cloning kit instructions for amplifying the homologous arms, drug selection marker, and linearized vector. Dilute the ordered primer (5 nmol) with nuclease-free H2O (500 μL) to a final concentration of 10 μM.
Amplify the homologous arms, drug selection marker, and linearized plasmid by PCR. The template for PCR of homologous arms is genomic DNA (20 ng) isolated using the TIANamp Geneomin DNA kit, while the templates for the drug selection marker and the linearized plasmid are plasmid DNA (1 ng) isolated with the EasyPure® Plasmid MiniPrep kit. Extract the DNA using the Tissue DNA kit following the manufacturer’s instructions. Prepare the PCR mixture according to Table 1.
Table 1. Phanta Max Super-Fidelity DNA Polymerase reaction system
Component Volume Final concentration
Template 1 μL
Primer-F 10 μM 1 μL 0.2 μM
Primer-R 10 μM 1 μL 0.2 μM
2 × Phanta Max buffer 25 μL
dNTP mix (10 mM each) 1 μL 0.2 mM
Phanta Max Super-Fidelity DNA Polymerase 1 μL
Nuclease-free H2O 20 μL
Total 50 μL
Perform the PCR reaction under the following cycling conditions (Table 2):
Table 2. Phanta Max Super-Fidelity DNA Polymerase reaction protocol settings
Cycle number Denature Anneal Extend
1 95 °C, 5 min
2–36 95 °C, 15 s 52 °C, 15 s 72 °C, 2 min
37 72 °C, 5 min
PCR product purification: Purify the PCR products by using agarose gel electrophoresis [1.5% (wt/vol) in TAE buffer, supplemented with 1:10,000 (vol/vol) GelRed or ethidium bromide]. Add 6 μL of 10× DNA loading buffer to the 50 μL of PCR product and then load the mixture into a well of the agarose gel. Load 5 μL of 100 bp DNA ladder in a flanking well of the same gel. Run the gel in 1× TAE buffer at 120 V for 30 min. Excise the target DNA band with a blue-light gel cutter and extract the DNA fragment by using a EasyPure® Quick Gel Extraction kit, following the manufacturer’s instructions.
After extracting the DNA fragment, measure the concentration of extracted DNA fragment on a NanoDrop 2000.
Use the ClonExpress MultiS One Step Cloning kit to ligation the fragments obtained in step A5. According to the manufacturer's instructions, mix the DNA fragments. Prepare the ligation mixture according to the following table (Table 3):
Table 3. Ligation mixture system
Component Quantity Volume
pBluescript fragment 60 ng
Marker Selection fragment 72 ng
5HR fragment 30 ng
3HR fragment 30 ng
5× CE MultiS buffer 2 μL
Exnase MultiS 1 μL
ddH2O To 10 μL
Total 10 μL
Incubate the ligate mixture from step A7 at 37 °C for 45 min in a T100 thermal cycler.
Add the entire ligate mixture from Step A8 to 100 μL of DH5α chemically competent cells and incubate the Eppendorf tube containing the competent cells on ice for 30 min.
Heat-shock the competent cells in a 42 °C water bath for 90 s and then immediately keep them on ice for 1 min.
Add 500 μL of LB medium (without any antibiotics) into the tube, put the tube in a thermostatic shaker, and shake the tube at 580× g for 45 min at 37 °C.
Centrifuge the recovered bacteria at 1,500× g for 5 min at room temperature and then remove most of the supernatant by pipetting, leaving ~50 μL of LB medium to resuspend the bacteria. Evenly apply the bacterial solution to an LB agar plate with ampicillin (100 μg/mL) using a glass rod or beads, invert the plate, and incubate at 37 °C overnight.
After overnight incubation, single clones are visible on the LB agar plate. Pick up five individual clones and culture each in 1 mL of LB medium with ampicillin (100 μg/mL) individually. Shake them in a 37 °C shaker incubator at 580× g for 12–14 h.
Verifying spacer cloning by PCR (cPCR): To perform a cPCR in a 10 µL reaction volume, to check for transformants harboring the correctly cloned spacer inside the pBluescript, prepare the following reaction mix (Table 4):
Table 4. Rapid Taq Master Mix reaction system
Component Quantity (μL) Final concentration
M13F 10 μM 0.25 0.25 μM
M13R 10 μM 0.25 0.25 μM
2× Rapid Taq Master Mix 5 1×
Bacterial from step A13 1
ddH2O 3.5
Total 10
Perform the PCR reaction under the following cycling conditions (Table 5):
Table 5. Rapid Taq Master Mix reaction protocol settings
Cycle number Denature Anneal Extend
1 95 °C, 5 min
2–36 95 °C, 15 s 52 °C, 15 s 72 °C, 2 min
37 72 °C, 5 min
Pipette 5 μL of each PCR reaction along with the GeneRuler 100 bp DNA ladder on a 1.5% (wt/vol) agarose gel prepared in 1× TAE buffer and stained with ethidium bromide. Run the gel at 120 V for 30 min and visualize the bands using the Gel Jet Documentation System. A 3,500 nt band is expected.
Use EasyPure® Plasmid MiniPrep kit to extract the plasmids from the clones in Step A13, following the manufacturer’s instructions, and then send these plasmids for Sanger sequencing to confirm that the sequence of the constructed Pbs-TPX-1 KO is correct.
Use EndoFree Maxi Plasmid kit to extract the plasmids from the clones in Step A14, following the manufacturer’s instructions.
After extraction, perform ethanol precipitation to obtain a pellet of plasmid DNA. Ethanol precipitation is a DNA purification technique. Here are the specific steps:
Transfer the extracted DNA solution to a new centrifuge tube.
Add 0.1 volume of NaOAc buffer (3 M, pH 5.2) and 2.5 volumes of cold ethanol (either absolute ethanol or isopropanol). Mix the solution thoroughly.
Place the tube in a -20 freezer for 15 min or freeze it at -80 for at least 30 min to allow DNA precipitation.
Centrifuge the tube at 12,000–16,000× g for 10–15 min at 4 . This will cause the DNA to form a pellet at the bottom of the tube.
Carefully pour off the supernatant without disturbing the DNA pellet. Take care not to disrupt the pellet.
Wash the DNA pellet by adding 70% ethanol. Gently swirl the tube to ensure thorough washing.
Centrifuge again at 12,000–16,000× g for 5 min at 4 to remove the ethanol wash.
Carefully pour off the ethanol, ensuring that the DNA pellet is left to air-dry as much as possible.
Rinse the walls of the tube gently with wash solution to remove any residual ethanol.
Air-dry the DNA pellet at room temperature for 5–10 min or use a low-speed centrifuge to remove any remaining liquid.
Add an appropriate volume of solvent, such as Tris-EDTA buffer, to completely dissolve the DNA pellet.
Please note that during the ethanol precipitation method, it is important to use sterile reagents and maintain a sterile working environment to avoid DNA contamination. Additionally, handle the samples with care to prevent DNA damage or loss.
(Optional) Linearization: Plasmid linearization is achieved by using restriction enzymes to cleave the plasmid at specific recognition sites. Following linearization, the DNA fragments can be concentrated using the ethanol precipitation method.
Note: Linearizing the plasmid is an optional step; using circular plasmids can also yield the desired results. Linearized plasmids are more prone to recombine with the chromosome compared to non-linearized plasmids. Circular plasmids may exist in the parasite as episomal vector.
Syrian golden hamster erythrocyte collection
Anesthetize hamsters using a respiratory anesthesia device and administer isoflurane at a concentration of 2.5% (refer to Figure 2A).
Collect blood from the orbital vein of each hamster using a 1-mL syringe (refer to Figure 2B). Each hamster can provide 150 μL of blood with a 2-week interval between collections. After collecting the blood, immediately add it to an equal volume of red blood cell preservation solution and gently shake it until thoroughly mixed.
Figure 2. Representative image depicting Syrian golden hamster erythrocyte collection.
To the collected blood, add twice the volume of red blood cell preservation solution. Place the mixture in a centrifuge at 500× g for 10 min at 4 . Carefully remove the upper layer of liquid after each centrifugation, repeating the process three times.
After completing the washing process, add an equal volume of red blood cell preservation solution to the washed red blood cells. Store the solution at 4 °C.
Note: The washed RBCs (50% hematocrit) can be immediately used for in vitro culture or stored at 4 °C for further use. The washed RBCs can be stored at 4 °C and used for up to two weeks in cell culture.
B. duncani culture
To perform a culture medium change for B. duncani, follow the steps outlined below:
Prewarm the culture medium: Before use, warm the culture medium to 37 . This ensures that the medium is at the optimal temperature for the growth of B. duncani.
Remove the culture supernatant: Carefully discard the culture supernatant from the culture vessel, ensuring not to disturb the settled red blood cells at the bottom.
Add fresh culture medium: Add an appropriate amount of prewarmed culture medium to the culture vessel to achieve the desired total volume. The exact volume required will depend on your specific culture system’s requirements.
Mix the red blood cells: Gently agitate the culture vessel to mix the red blood cells and the new culture medium by aspirator, ensuring that they are evenly distributed. It is important to avoid vigorous agitation that may potentially damage the red blood cells.
Medium change frequency: The frequency of medium changes depends on the parasitemia level, which refers to the percentage of infected red blood cells. If the parasitemia is below 5%, you can change the medium every 48 h. However, if parasitemia exceeds 5%, more frequent medium changes are necessary, typically every 24 h.
Note:
Culture system for B. duncani: B. duncani parasites can be cultured in different sizes of culture dishes or cell culture bottles. For instance, in a 24-well plate, each well contains 950 μL of culture medium and 50 μL of red blood cells. Refer to Table 6 below for the recommended amounts of culture medium and red blood cells for different culture containers.
Table 6. Culture system for B. duncani
Culture vessel Culture medium volume Red blood cell volume Culture medium replacement volume
96-well plate 95 μL 5 μL 70 μL
48-well plate 475 μL 25 μL 400 μL
24-well plate 950 μL 50 μL 800 μL
12-well plate 1,900 μL 100 μL 1,600 μL
6-well plate 4.75 mL 250 μL 4 mL
T25-cell culture bottle 4.5 mL 500 μL 4 mL
T75-cell culture bottle 27 mL 3 mL 24 mL
Culturing environment: To achieve optimal growth, B. duncani requires 37 °C, saturated humidity, and a specific gas composition in the culturing environment. The recommended composition is 2% O2, 5% CO2, and 93% N2. There are two common methods to establish this environment:
Gas-infused culture flask: A culture flask designed for gas infusion can be utilized. The pre-mixed gas with the desired composition of 2% O2, 5% CO2, and 93% N2 is introduced into the flask, creating the appropriate culturing environment. The flask is typically equipped with a gas exchange system to regulate and maintain the desired gas levels.
Tri-gas incubator: Alternatively, a tri-gas incubator can be employed. This incubation system offers the capability to adjust gas proportions. It is equipped with controls to regulate the flow of gases and maintain the desired composition of 2% O2, 5% CO2, and 93% N2 within the enclosed chamber where the culture vessels are placed. The incubator provides a stable and controlled environment for the optimal growth of B. duncani.
Both methods ensure the availability of the required gas composition for the culturing process, enabling the optimal growth of B. duncani.
Continuous cell culture
Prewarm the medium at 37 °C.
Utilize a 24-well plate and maintain 1 mL cultures. Monitor the parasitemia, aiming to keep it between 4% and 8% (though it can reach as high as 30%).
Adjust the culture parasitemia to 1% and maintain the hematocrit at 5% by splitting the culture. For example, if the parasitemia is at 1 mL culture at 5% hematocrit, mix culture well and transfer 250 μL into a new well, diluting it down to 1%.
Add 750 μL of VP-SFM and add 37.5 μL of RBC to the new well.
To prepare blood smears from cultured B. duncani samples, please follow the steps below:
During medium change, tilt the culture dish and carefully remove the red blood cells from the bottom using a pipette.
Place two glass slides side by side, slightly overlapping each other.
Using a pipette, transfer a small drop of the collected red blood cells onto the center of the slide.
Quickly and smoothly spread the drop of blood across the slides using the edge of another slide at a 45-degree angle.
Allow the smears to air dry completely.
Once dried, fix the smears by immersing them in methanol for approximately 1–2 min.
Prepare the Giemsa stain according to the instructions provided by the manufacturer.
Submerge the dried smears in the Giemsa stain for the recommended staining duration, typically around 10–20 min.
Gently rinse the smears with distilled water to remove excess stain.
Allow the smears to air dry completely before examining them under a microscope.
By following these steps, you can create blood smears from the cultured B. duncani samples. These smears can then be further examined and analyzed using a microscope for diagnostic or research purposes.
Transfection of B. duncani
Obtaining infected red blood cells: Three days prior to transfection, prepare 100 μL of B. duncani with an initial parasitemia of 1%. It is advisable to change the culture medium daily. On the day of transfection, the parasitemia should be between 15% and 20%.
(Optional) On the third day of culture, when the parasitemia is at 15%–20%, change the medium to one containing 1 μM of MBP146–78 and continue culturing at 37 for 12–24 h. This step is intended to synchronize the B. duncani parasites to the tetrad stage, which increases their survival rate after electroporation.
(CRITICAL STEP) Carefully remove the supernatant medium and resuspend 100 μL of infected red blood cells in 1 mL of cytomix buffer. Centrifuge the mixture at 1,500× g for 2 min at 4 and discard the supernatant buffer. Repeat this washing step twice with cytomix buffer, discarding the supernatant after each wash.
Prepare a 24-well plate and add 50 μL of red blood cells and 950 μL of culture medium. Place the 24-well plate in a 37 °C incubator and keep it aside.
Dissolve the DNA precipitate obtained in step A18 with 200 μL of cytomix buffer and take up 10 μL of the DNA mixture. Measure the DNA concentration by NanoDrop2000.
Combine the harvested parasite-infected red blood cells, DNA from step D5, and cytomix buffer in a sterile microcentrifuge tube. Prepare the electroporation mixture according to Table 7.
Table 7. Electroporation transfection for B. duncani
Component Amount (μL) Final concentration
Parasite-infected red blood cells 100
DNA 50 μg 0.25 μg/μL
Cytomix buffer Add to 200
total 200
Gently mix the contents and transfer the mixture to a 0.2 cm electroporation cuvette, ensuring there are no air bubbles. Keep the cuvette on ice.
Set the BTX electroporator to the following settings: 1,200 V, 25 μF, 200 Ω, and prepare for electroporation.
Place the electroporation cuvette in the electroporation apparatus. After clicking on Ω, the screen will display the set transfection conditions (refer to Figure 3A). Then, click on GO to initiate the electroporation. The screen will show the actual electroporation conditions during the process (refer to Figure 3B). The resulting time constant should range between 0.4 and 0.7 ms.
Figure 3. Representative image for electroporation. (A) Electroporation protocol setup. (B) Electroporation results.
Repeat the operation described in step D9 for a second round of electroporation.
Note: Before each pulse, gently tap the electroporation cuvette to remove any generated air bubbles. During the second electroporation, splashing of the mixture might occur.
After completing the electroporation, transfer the whole mixture to the wells prepared in step D4 by transfer pipette (10–200 μL), gently mix, and place it in a cell culture incubator.
After 3 h following transfection, replace the prewarmed medium.
Genetic modification strain screening
Twenty-four hours after transfection, replace the culture medium with a medium containing 5 nM WR99210. Prepare blood smears as described in step C3 of B. duncani culture to calculate the transfection efficiency.
Subsequently, change the medium containing 5 nM WR99210 daily and add 25 μL of fresh red blood cells on the seventh day post-transfection.
After 7–12 days of drug selection, resuspend the culture and transfer 100 μL to a new well. Then, add 45 μL of uninfected red blood cells and 855 μL of medium.
(Optional) Withdraw 2 μL of the parasitized red blood cells for live cell fluorescence microscopy to confirm the expression of the fluorescent protein that co-expresses with the drug selection marker. For live-cell imaging, the parasite-infected blood was first washed twice with PBS using a centrifuge set at 1500× g for 2 min each time. The cells were then stained with 1 μg/mL Hoechst 33342 in PBS for a duration of 5 min. All images were captured and processed using identical settings in the OLYMPUS FRAME_BX63 scanning confocal microscope with a 100× numerical-aperture (NA) oil objective.
Note: Live cell fluorescence is optional. The expression of the fluorescent protein does not necessarily confirm the successful construction of the genetically modified parasite strain. Further confirmation through PCR is required.
Extract the remaining DNA from the parasitized red blood cells using the TIANamp Genomic DNA kit following the manufacturer's instructions.
Design primers that anneal to the flanking regions of interest. Confirm the presence of the desired genetic modification using PCR identification.
Note: To prevent false positives caused by plasmid DNA, set the forward primer approximately 200 bp upstream of 5H and the reverse primer approximately 200 bp downstream of 3H.
Analyze 5–10 μL of each PCR reaction along with an appropriate ladder (GeneRuler 100 bp DNA ladder) on a 1%–2.5% (wt/vol) agarose gel prepared in 1× TAE buffer and stained with ethidium bromide. Run the gel at 120 V for at least 30 min and visualize the size of the PCR product using the Gel Jet Documentation System.
Note: PCR1 is used to confirm the correct recombination of the 5’ end of the insert fragment with the genome, PCR2 is used to confirm the correct insertion of the 3’ end of the insert fragment, and PCR3 is used to confirm the deletion of the target gene. Refer to Figure 5B to see the presence of bands of varying sizes in the modified parasite strains, including those of the same size as the WT.
Monoclonal screening
Due to the variability in drug selection efficiency, achieving stable genetically modified parasite strains solely through drug selection is challenging. Therefore, clonal selection is necessary to obtain stable genetically modified parasite strains.
Cultivate the strains with the correct insertion (as confirmed by PCR in steps E5–E7 above) until the parasitemia reaches 5%.
Centrifuge the culture at 1,500× g for 5 min.
Take 1 μL of the red blood cell pellet (approximately 1 × 107 cells) and resuspend it in 1 mL of PBS. Mix well and use a cell counter to determine the cell count.
Dilute the parasitized red blood cells with PBS to achieve a concentration of 100 red blood cells per microliter (with approximately five parasites per microliter).
Add 6 mL of culture medium without WR99210 and 300 μL of fresh uninfected red blood cells to a 15 mL centrifuge tube and mix well.
Add 6 μL of the diluted solution (containing the parasite-infected red blood cells) to the mixture of culture medium and red blood cells, to achieve a total of 30 parasites in the mixture.
After mixing the solution, add 100 μL of the mixture to each well of a 96-well cell culture plate to achieve a concentration of 0.5 parasites per well.
Change the medium every three days, replacing 70 μL of the medium.
On the second medium change, use a culture medium containing 5% red blood cells.
On the fourth medium change, perform blood smears to observe the Babesia parasites.
CRITICAL STEP: Typically, around 20 wells out of the 60 should contain parasites. If more than 30 wells yield parasites, it is likely that the obtained parasites are not monoclonal.
Upon observing that the monoclonal parasite strains have grown to a parasitemia of 5%, mix the culture well and then transfer 10 μL to a new 96-well plate. Add 90 µL of culture medium containing 5% red blood cells to each well.
Place the clonal parasite strains in a cell culture incubator for further cultivation.
Transfer the remaining 90 μL of monoclonal culture medium to a clean PCR tube. Centrifuge for 2 min using a microcentrifuge.
Discard the supernatant and retain the red blood cells.
Add 100 μL of PBS containing 0.1% saponin to lyse the red blood cells for 5 min.
Centrifuge the Babesia parasites using a microcentrifuge for 5 min to pellet them at the bottom of the tube.
Discard the supernatant and add 20 μL of nuclease-free water. Mix well, then boil the mixture in a water bath for 10 min.
Centrifuge the boiled sample in a microcentrifuge for 5 min. The resulting supernatant contains the DNA of the clonal parasite strain.
To confirm that the clonal parasite strain has the correct insertion, perform three PCR reactions. Use the setup and thermal cycler program for the expected size of the mutant or the WT PCR fragment. Include a colony of the WT strain as a nonedited control.
Analyze 5–10 μL of each PCR reaction along with an appropriate ladder (GeneRuler 100 bp DNA ladder) on a 1%–2.5% (wt/vol) agarose gel prepared in 1× TAE buffer and stained with EB. Run the gel at 120 V for at least 30 min and visualize the size of the PCR product using the Gel Jet Documentation System.
Sequencing of PCR products of the expected size is conducted to confirm the correct insertion.
Transfer the parasite strain with the desired genetic modification from the 96-well plate to a new 24-well cell culture plate for amplification.
Freeze single clone strains when the parasitemia is at 20%. The method for freezing B. duncani is as follows:
Take the mixture of culture medium and red blood cells and transfer it to a new 1.5ml Eppendorf tube.
Use a centrifuge at 1,500× g for 2 min at 4 to separate the supernatant.
Carefully remove the supernatant to avoid losing the cell pellet. Add 100 µL of freezing solution to the pellet and mix to ensure an even distribution.
Transfer the mixture to a new cryogenic storage tube.
Place the storage tube in a Mr. Frosty™ Freezing container set at -80 °C to gradually decrease the temperature over two days. This helps prevent cell damage.
After completion of the freezing process, immediately transfer the storage tube to a liquid nitrogen container for long-term preservation of B. duncani parasites.
This method can be used to freeze B. duncani parasites for future experiments. In liquid nitrogen, the parasites can be stored for an extended period. However, it is essential to follow the appropriate biosafety and laboratory protocols to ensure the safe storage and handling of the samples.
General notes and troubleshooting
Expanding B. duncani for transfection
Because the transfection method for B. duncani results in a high mortality rate, increasing the parasite's infection rate can enhance the number of parasites post-transfection. By using VP-SFM medium and hamster red blood cells, the parasitemia of B. duncani can be raised to over 30%. Increasing the initial parasitemia to 2%–4% during passages ensures that sufficient parasitemia can be obtained for transfection by the third day.
Improve the post-electroporation survival rate of Babesia parasites
Electric transfection of B. duncani employs high voltage and low pulse times, which results in a substantial loss of parasites after electroporation. The reasons for parasite death include both the stimulus from high voltage and, significantly, the rupture of red blood cells caused by electroporation. As a result, only fully developed tetrad-stage parasites can continue to grow. Thus, increasing the proportion of tetrad-stage Babesia parasites is essential to enhance their survival rate following electroporation. Given that Babesia replication is asynchronous, obtaining synchronized tetrad-stage parasites through regular culturing is challenging. While increasing parasitemia does raise the proportion of tetrads to some extent, the impact on post-electroporation survival is not significant. Babesia parasites egress via the cGMP pathway, and the use of a cGMP-dependent protein kinase (PKG) inhibitor can block egress, allowing for the synchronization of Babesia parasites to the tetrad stage. Compound C1 (MBP146-78) is a known PKG inhibitor, and its effect is reversible; removing C1 permits the normal growth of the parasites. The use of compound C1 to treat B. duncani synchronizes them to the tetrad stage, increasing post-electroporation survival rates from 2.5%–5% to 10%–15%, significantly improving subsequent screening efficiency.
Data analysis
Live-cell imaging for B. duncani
Fusing the drug selection marker with GFP allows for the observation of drug selection marker expression in B. duncani parasites using fluorescence microscopy (refer to Figure 4). Through fluorescence microscopy, the expression of the green fluorescent protein in the cytoplasm of B. duncani parasites can be detected. It is important to note that observing the expression of the fluorescent protein does not guarantee that the drug selection marker has recombined correctly as expected; there may still be cases of single-side homologous recombination.
Figure 4. Live-cell imaging for B. duncani. Green fluorescence corresponds to the transfected parasite expressing eGFP, Hoechst staining represents the nucleus of the parasite, and DIC (differential interference contrast microscope) image shows a parasitized red blood cell (RBC). Merged image represents the overlap of all images. Scale bar = 5 μm.
Confirming gene modifications through PCR
PCR is particularly useful for detecting the presence of homologous integration events using a combination of a plasmid-specific oligonucleotide (not specific to the gene-targeting sequence) and one directed to the genomic sequence located immediately outside of the gene-targeting fragment found in the plasmid. To confirm the correct insertion of the drug selection marker into the target gene locus, amplify the upstream and downstream sequences connected to the drug selection marker. The presence of such a product (which should be sequenced for confirmation) demonstrates that homologous integration has indeed occurred.
Screen the selected monoclonal parasite strains through PCR1 and PCR2 to confirm the precise insertion of the drug selection marker into the TPX-1 gene locus in B. duncani. Additionally, PCR3 is used to verify the deletion of the TPX-1 gene (refer to Figure 5).
Figure 5. Targeted disruption of the B. duncani TPX-1 gene. A. Plasmid construct for disruption of the B. duncani TPX-1 gene. B. PCR confirmation of the disruption of the B. duncani TPX-1 gene. Monoclonal strains T1, T2, and T3 were identified by PCR1, PCR2, and PCR3, with WT strain used as the control.
Limitations
Optimizing the transfection efficiency in intracellular parasites like B. duncani can be challenging due to the need for external DNA to traverse both the red blood cell and the parasite's cell membrane. Achieving a transfection efficiency of 0.5%–1% through optimization is a notable accomplishment. However, it is important to consider that the use of higher voltage can reduce the post-transfection survival rate of the parasites, typically to approximately 2.5%–5%. This lower survival rate can prolong the time needed to select edited parasite strains, often requiring approximately one month. These challenges highlight the need for continued refinement and optimization of transfection methods for this parasite.
In addition, this protocol specifically outlines a method for gene editing through homologous recombination. To further advance research on genes that are crucial for the growth and development of B. duncani, it would be beneficial to develop conditional knockout systems such as Di-Cre. These conditional knockout systems can allow for the precise control and study of genes that are vital to the life cycle of the parasite.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Wang, S. et al. (2022). Establishment of a Transient and Stable Transfection System for Babesia duncani Using a Homologous Recombination Strategy. Front Cell Infect Microbiol.
Acknowledgments
Funding was provided by grant 2022YFD1801700 from the National Key Research and Development Program of China, grants 32172879 and 31930108 from the National Natural Science Foundation of China, funds from the Top-notch Young Talent Supporting Program (L.H.), and grant 2262022DKYJ001 from the Fundamental Research Funds for the Central Universities. We also sincerely appreciate Choukri Ben Mamoun from Department of Infectious Diseases, School of Medicine, Yale University, for his help in editing the manuscript.
Ethical considerations
This study was approved by the Scientific Ethic Committee of Huazhong Agricultural University (permit number: HZAUMO-2017-040). All mice were handled in accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
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Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5017 Views: 621
Reviewed by: Anthony SignoreMarcelo S. da Silva Anonymous reviewer(s)
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Abstract
Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites.
Key features
• Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables.
• Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost.
• Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes.
Keywords: Foot-and-mouth disease virus FMDV Nanopore sequencing Direct RNA Sequencing NGS Point of care
Graphical overview
Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).
Background
Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease that causes vesicular disease in cloven-hoofed domestic and wild animals. Outbreaks can lead to serious economic losses due to decreased livestock productivity and restrictions on movement and trade of animals and their products [1]. The causative agent of the disease is foot-and-mouth disease virus (FMDV), an Aphthovirus from the family Picornaviridae, with seven antigenically distinct serotypes including the EuroAsiatic serotypes A, O, C, and Asia 1, and the Southern African territories (SAT) serotypes 1, 2, and 3. Cross-protection is not conferred between serotypes following infection or vaccination and may not be conferred between different subtypes or variants of the same serotype [2]. The key factor to prevent the disease and control its spread is early detection and identification of the serotype(s) and subtype(s) of outbreak strains so that directed vaccination may be initiated. The antigenic determinants of FMDV reside within the structural proteins of the viral capsid (VP1-VP4), encoded by the genes 1A (VP4), 1B (VP2), 1C (VP3), and 1D (VP1). Historically, FMDV molecular epidemiology of outbreaks has focused on analyses of partial or full-length sequences of the 1D gene that encodes the structural protein VP1 [3]. Currently, over 60% (9571/14952 in December 2023) of FMDV records in the GenBank database have a total nucleic acid length of 400–700 bp and cover the partial or full 1D gene. Unfortunately, relying solely on the VP1 coding sequence does not provide all of the information required to fully characterize important phenotypic traits of FMDV strains, since several antigenic determinants are located on other viral capsid proteins, such as VP2 and VP3 [4,5]. Moreover, during shorter epidemic time scales, sequencing of 1D cannot provide enough resolution to discriminate the viruses collected from adjacent premises within the same outbreak clusters due to the viral populations not having diverged substantially [6]. In this regard, numerous reports have been published using the whole P1 region, encoding all structural proteins (i.e., VP1 to VP4) for strain genotyping and phylogenetic analysis [7,8]. The characterization of the whole P1 region (1A to 1D) is important for tracking the emergence or spread of FMD and for selecting vaccines in case of an outbreak. However, restricting analyses to P1 sequences may not detect recombination events that can occur between multiple serotypes or topotypes in the event of co-circulating FMDV within infected animals [5,9,10]. Next-generation sequencing (NGS) techniques offer much promise to perform viral whole genome sequencing (WGS) without prior knowledge about the target sequence and provide a huge amount of data from a limited quantity of starting material in a rapid, cost-effective manner. Valdazo-González and colleagues (2012)[11] have demonstrated that WGS of FMDV is a powerful tool for the reconstruction of transmission trees.
WGS of FMDV has been previously conducted on different platforms including Oxford Nanopore Technology (ONT) and Illumina [6,10,12–14]. These methods use reverse transcriptase to convert RNA to cDNA and/or go through one or two FMDV-specific or non-specific PCR amplifications. Some are labor-intensive with cumbersome procedures; others may have relatively longer turnaround times. Moreover, viral RNA manipulation can introduce biases into the data [15], which may not represent the actual diversity within samples [6]. Here, we introduce for the first time an FMDV direct RNA sequencing method using Nanopore sequencing on Flongle flow cells with MinION Mk1C (or GridION). Compared to the original ONT SQK-RNA002 protocol, we omitted the optional reverse transcription step to make the process even shorter by saving ~35 minutes as the ONT device only sequences the RNA molecule, not the cDNA. Our method is low-cost, fast, and reliable and can be deployed to low-resource settings when using an Mk1C device, such as a remote or field laboratory with alternative RNA extraction methods. This POC method requires less than 50 min of manipulation time from RNA to NGS run (Graphical Abstract), followed by a rapid identification of the whole FMDV genome within minutes.
Materials and reagents
Biological materials
Seven FMDV isolates were used to develop the RNA direct sequencing method, one of each serotype (see Table 4 in the Evaluation of Protocol section below). The isolates were obtained from the Biorepository in the Reagents and Vaccine Services Section of FADDL as viral stocks propagated from cell lines identified in Table 4.
Reagents
MagMAXTM CORE Nucleic Acid Purification kit (Thermo Fisher, catalog number: A32702 or A32700)
Qubit RNA High Sensitivity (HS) Assay kit (Thermo Fisher, catalog number: Q32852 or Q32855)
Ethyl alcohol, molecular grade (e.g., Thermo Fisher, catalog number: T032021000)
RNAClean XP beads (Beckman Coulter, catalog number: A63987)
Direct RNA Sequencing kit (ONT, catalog number: SQK-RNA002)
Flow Cell Priming kit (ONT, catalog number: EXP-FLP001)
Flongle sequencing expansion (ONT, catalog number: EXP-FSE001)
NEBNext® quick ligation reaction buffer (New England Biolabs, catalog number: B6058)
T4 DNA ligase 2,000,000 units/mL (New England Biolabs, catalog number: M0202)
Flongle flow cell (ONT, catalog number: FLO-FLG001)
Laboratory supplies
Calibrated pipettes single (P1000, P200, P100, P20, P10, P2) (e.g., Rainin)
Pipette tips, aerosol resistant, RNase free (P1000, P1000X, P200, P200X, P20) (Rainin, catalog numbers: 30389212, 30389323, 0389239, 30389242, 30389225)
1.5 mL DNA LoBind® tubes (Eppendorf, catalog number: 022431021)
Qubit tubes (Thermo Fisher, catalog number: Q32856)
Nuclease-free water (Thermo Fisher, catalog number: AM9937)
Racks for 1.5 mL tubes
Magnetic stands (e.g., Thermo Fisher, catalog number: 12321D)
Timers (e.g., Cole-Parmer, catalog number: EW-90225-35)
Ice or ice packs (e.g., Fisher Scientific, Corning Ice Pan Mini 1L, catalog number: Corning 432116)
PPE: Disposable lab coats, nitrile gloves, eye goggles
Equipment
KingFisherTM Duo Prime purification system (Thermo Fisher, catalog number: 5400110)
Qubit 4 fluorometer (Thermo Fisher, catalog number: Q33238)
Scilogex SCI-M analog microplate mixer (Scilogex, catalog number: 822000049999)
Scilogex Mixer accessories: universal circular adapter (Scilogex, catalog number: 18900067)
Scilogex Mixer accessories: foam test tube insert for 12 tubes (Scilogex, catalog number: 18900022)
Microcentrifuges to hold 1.5–2 mL tubes and 0.2 mL PCR tubes
MinION Mk1C (Oxford Nanopore Technologies)
GridION (Oxford Nanopore Technologies)
Flongle adapter (ONT, catalog number: ADP-FLG001)
Cold storage devices: Refrigerator (+4 °C) for flow cells and some reagents, freezer (-20 °C) for enzymes and other reagents, ultra-low freezer (-70 °C) for storage of RNA samples
Software and datasets
MinION Mk1C (or GridION) devices with up to date MinKNOW software
The host genome FASTA files were downloaded from GenBank (Table 1), uploaded to MinION Mk1C (or GridION), and then used as references on the adaptive sampling step of the MinKNOW runtime setting to deplete the host sequence reads
Table 1. Host genome used as references on the adaptive sampling step of MinKNOW
Species Genome version used
Bovine Bovine UMD3.1_chromosomes
Swine GCF_000003025.6_Sscrofa11.1_genomic
Guinea pig GCF_000151735.1_Cavpor3.0_guineaPig
Sheep Ovis aries (sheep) GCA_016772045.1_ARS-UI_Ramb_v2.0
Procedure
This protocol was developed with the capacity to rapidly detect and resolve the full genome sequence from an RNA extraction containing full-length FMDV genomes. The whole process takes less than 50 min on the bench, and usually within 25 min to detect the full genome after the run starting on the MinION Mk1C or GridION.
Reagent preparation
Remove the following reagents (Table 2) from the refrigerator and keep at room temperature (RT) for at least 30 min before use.
Table 2. Required reagents stored in a refrigerator
Reagents Step to use Note
Qubit RNA HS Assay kit B.2, F.12
RNAClean XP beads C.3, F.1
NEBNext quick ligation buffer C.1, D.1, E.1
Flongle flow cell G.1 Take one or two extras in case of finding low pores flow cell(s)
Remove the following reagents (Table 3) from the frozen ONT kits and completely thaw before use.
Table 3. Reagents stored in a freezer
Reagents Step to use Note
RT adapter (RTA) C.1 From SQK-RNA002, thaw on ice
RNA adapter mix (RMX) E.1 From SQK-RNA002, thaw on ice
Wash buffer (WSB) F.4 From SQK-RNA002, thaw at RT
Elution buffer (ELB) F.8 From SQK-RNA002, thaw at RT
RNA running buffer (RRB) G.4 From SQK-RNA002, thaw at RT
Flush tether (FLT) G.2 From EXP-FLP002, thaw at RT
Flush buffer (FB) G.2 From EXP-FSE001, thaw at RT
Quantification and quality of extracted RNA
Extract total genomic DNA and RNA from 200 μL of frozen FMDV viral stock for each isolate used (Table 4) using MagMax Core kits following the manufacturer’s instructions on an automated extraction instrument (KingFisher Duo Prime). A final elution of 70 μL of elution buffer provided by the kit is used as the input material for preparation of the libraries.
Table 4. FMDV isolates used in this study
Serotype Isolate name Cell line Cell line origin
A A 8 Parma GPVF Guinea pig
Asia 1 Asia 1, Shamir BTTP Bovine
O O 9 Nueve De Julio IBRS2 Swine
C C1 Noville MVPK Swine
SAT1 SAT1 Ken 4/98 LK Sheep
SAT2 Egypt MVPK Swine
SAT3 SAT 3/4 Bech 1/65 IBRS2 Swine
Quantification of the extracted total genomic RNA is performed using the Qubit 4 fluorometer and the Qubit RNA high-sensitivity assay kit, following the manufacturer’s guidelines. The quantity of RNA used in the reaction has been modified from the original ONT protocol; see Note 1 in the General notes and Troubleshooting section below.
Primer annealing and ligation
In a 1.5 mL low-binding tube, add the following reagents and mix by pipetting several times after adding each component.
NEBNext quick ligation reaction buffer (5×) 3 μL
RNA 9.5 μL
RTA 1 μL
T4 DNA ligase (2M U/mL) 1.5 μL
Total 15 μL
Put the tube into a microcentrifuge and run the centrifuge for approximately 1 s (briefly spin) to bring down all the components.
Keep the tube at RT for 10 min.
Purification with Agencourt RNAClean XP beads
During the above 10 min incubation time (step C3), prepare the following:
Use the 5× NEBNext quick ligation reaction buffer to make 30 μL of 1× ligation buffer by mixing 6 μL of 5× buffer with 24 μL of nuclease-free water.
Prepare 70% ethanol from ethyl alcohol by mixing 140 μL of ethanol with 60 μL of nuclease-free water. The wash step requires 150 μL of 70% ethanol per sample.
After the incubation, transfer 25 μL of 1× ligation buffer prepared at step D1a into the ligation tube (step C1) to make a final volume of 40 μL.
Pipette 72 μL of RNAClean XP beads to the above tube for a ratio of 1:1.8; then, tap the tube several times to mix the beads with the sample.
Insert the tube in the test tube foam on a Scilogex mixer and shake at ~300 rpm for 5 min at RT.
Spin down the sample and pellet on a magnetic stand for ~1.5 min. Keep the tube on the magnet and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 150 μL of 70% ethanol (prepared at step D1b) without disturbing the pellet as described below:
Keeping the magnetic stand on the benchtop, rotate the bead-containing tube at 180°. Wait for the beads to migrate toward the magnet and form a pellet.
Rotate the tube 180° again (back to the starting position) and wait for the beads to pellet.
Keeping the tube on the magnetic stand, remove the 70% ethanol with a pipette and discard.
Centrifuge briefly, place the tube back on the magnetic stand, and pipette off any residual liquid.
Remove the tube from the magnetic stand and add 23.5 μL of nuclease-free water.
Insert the tube in the test tube foam on a Scilogex mixer and shake it at ~300 rpm for 2 min at RT.
Centrifuge the sample briefly and pellet on a magnetic stand for ~1.5 min.
Transfer 23 μL of the eluate to a new 1.5 mL LoBind tube.
Ligation of sequencing adapters
In the tube with purified RNA (step D12), add the following reagents and mix by pipetting several times after adding each component.
NEBNext quick ligation reaction buffer (5×) 8 μL
RMX 6 μL
T4 DNA ligase (2M U/mL) 3 μL
Total 40 μL
Put the tube into a microcentrifuge and briefly spin to bring down all the components.
Incubate at RT for 10 min.
Purification with Agencourt RNAClean XP beads
After the incubation (step E3), add 40 μL of RNAClean XP beads to the above tube for a ratio of 1:1; then, tap the tube several times to mix the beads with the sample.
Insert the tube in the test tube foam on a Scilogex mixer and shake at ~300 rpm for 5 min at RT.
Centrifuge the sample briefly and pellet on a magnetic stand for ~1.5 min. Keep the tube on the stand and pipette off the supernatant.
Take the tube from the magnetic stand and add 150 μL of WSB solution. Resuspend the beads completely by flicking the tube, then spin down briefly and put the tube back on the magnetic stand to pellet the beads.
Without disturbing the pellet, discard the supernatant with a pipette.
Repeat steps F4 and F5 once more for a second wash with the WSB solution.
Centrifuge briefly and place the tube back on a magnetic stand; then, remove the residual WSB by pipetting without touching the pellet.
Remove the tube from the magnetic stand and add 17 μL of ELB solution to resuspend the beads.
Insert the tube in the test tube foam on a Scilogex mixer and shake at ~300 rpm for 2 min at RT.
Centrifuge the sample and pellet on a magnetic stand for ~1.5 min.
Transfer the 17 μL of library to a new LoBind tube.
It is optional to measure 1 μL of library in Qubit with the HS RNA kit.
Flongle loading
We changed the MinION/GridION flow cell in the original ONT SQK-RNA002 to Flongle flow cell; see Note 2 in the General Notes and Troubleshooting section below.
Following the ONT manufacturer protocol, put the Flongle flow cell on the MinION Mk1C (or GridION) with a Flongle adapter. Using the MinKNOW software, ensure the flow cell has 50 or more pores and open the sample port.
Prepare the flush buffer by combining 117 μL of FB with 3 μL of FLT. Vortex to mix, then centrifuge briefly.
Use a 200 μL pipette to load 120 μL of mixed flush buffer into the Flongle sample port without introducing any air into the port.
Add an equal volume of RRB buffer to the tube containing the purified library from step F11. Mix well by pipetting and then centrifuge briefly.
Use a 200 μL pipette to load all the library with running buffer into the sample port of the Flongle. Seal the port and start the run.
The runs were performed with the default setting of the MinKNOW software (version: 21.05.12), except the adaptive sampling being enabled.
Once the run generates a read file, data analysis can be performed during the runtime. The run can proceed for up to 24 h.
Data analysis
The base calls were generated using the default setting of the MinKNOW program on the MinION Mk1C or GridION. The data analysis was conducted using the ASFV fast pipeline previously developed by O’Donnell VK et al. with slight modifications and changing the ASFV database to an FMDV isolate database [15]. In short, Minimap2 was used to align reads to sequences of publicly available, complete FMDV genomes obtained from the National Center for Biotechnology Information (NCBI) GenBank as of November 22, 2023. Average alignment score from the resulting alignments was assessed to determine the best reference genome, and data were re-aligned to the corresponding reference genome that had the highest alignment score averaged across the genome length. Average depth of coverage and percent of the genome covered by greater than one read over time were calculated using the default output files from the MinKNOW software and visualized in R.
Validation of protocol
The seven FMDV isolates used in this study are listed in Table 5 with the condition of the seven Flongle runs performed with the protocol above on fresh RNA extractions from seven FMDV serotypes. The analyzed run results are displayed in Figures 1 and 2. All serotype genomes were resolved within 25 min of runtime, and the data generated were enough to cover the full genome of the reference strain used, except the SAT 1 sample that had over 90% genome coverage after 25 min and reached 99.8 % coverage after 50 min (Figure 1). The reads coverage depth of the reference genomes are displayed in Figure 2: one graph for each serotype. There was increased coverage on the 3′ end of the genome vs. the 5′ end, since ONT RNA Direct Sequencing kit requires the RNA molecule to have a poly-A tail at the 3′ end, and any FMDV RNA splitting forms without poly-A could not be sequenced. To better display the depth of coverage, the y-axis of Figure 2 is displayed on the natural log scale, with unit 0 representing 1 read coverage and unit 8 representing ~2981 read coverage. Although only FMDV isolates were used in the study, this method may be used to sequence any viral RNA with a poly-A tail.
Table 5. Summary of the seven FMDV samples direct RNA sequencing runs.
The adaptive sampling option was used in the MinKNOW software of MinION or GridION to deplete the reads that align to the host genome—the cell line used to generate the viral stocks. The last two columns display the analysis results of the fastq files of each sample after running for 24 h and passing the ONT default quality controls, aligned to the given reference sequence.
FMDV serotype Input RNA (ng) Library loaded (ng) Adaptive sampling Reference genome Average depth Percentage of genome coverage to reference
A 176 86.4 Guinea pig AY593792 1553 99.9
Asia 1 458 186 Bovine JF739177 3499 99.9
O 500 150 Swine AY593819 1573 99.9
C 293 93.3 Swine AY593804 463 99.8
SAT1 327 122 Sheep KM268899 975 99.8
SAT2 274 165 Swine JX014255 1453 99.8
SAT3 125 63 Swine AY593853 953 99.8
Figure 1.Accumulation of sequencing reads that align to the given foot-and-mouth disease virus (FMDV) reference sequence of the seven serotypes, showing the percentage coverage of the reference genomes.
Figure 2. Coverage depth on the natural log scale for the reference genome sequence of the seven Flongle runs. Due to the different size of the reference isolates used (see Table 5), the x-axis is normalized to the percentage of the given isolate genome: starting from 0% and ending at 100% of the genome sequence length.
General notes and troubleshooting
The ONT SQK-RNA002 original protocol recommends 500 ng of poly-A+ tailed RNA in 9 μL for library preparation. We used 9.5 μL of RNA by omitting the control RNA provided in the kit, and as low as 125 ng total RNA for the library preparation.
We ran the RNA library (Step G in the Procedure above) on a Flongle flow cell instead of a MinION/GridION flow cell. As a result, the cost of flow cell alone for our protocol was reduced dramatically, as a Flongle costs ~1/10 of a MinION/GridION flow cell. In addition, during our tests, as low as 63 ng of library was loaded into the Flongle (see Table 5), compared to 200 ng in the original protocol, which generated enough sequencing data to cover the full length of the FMDV genome (Table 5, Figures 1 and 2).
General notes
Initially, the adaptive sampling option was enabled on MinKNOW to deplete the reads aligned to the host (cell line origin) genome after uploading the host fasta files to the MinION Mk1C or GridION, but this step was determined to be optional since the adaptive sampling has no significant impact on the final result (data not shown).
The quality and quantity of FMDV genomic RNA are critical for RNA direct sequencing. Real-time reverse transcription PCR (RT-qPCR) [16] is commonly used to detect FMDV and assess RNA quality and quantity. However, there is no direct correlation between NGS (including RNA direct sequencing) results and Ct values. RT-qPCR relies on specific primers/probes to amplify a short sequence (usually around 100 bp), while RNA direct sequencing is independent of the target sequence. Mutations in the primer/probe site can cause RT-qPCR failure or abnormally high Ct values, but NGS can still achieve overall genome sequencing. In our experience, samples with low Ct values sometimes yield poor NGS data due to viral genome degradation, even when the short region targeted by RT-qPCR primers/probe remains amplifiable. Ultimately, RNA direct sequencing data quality depends on factors beyond Ct values.
Troubleshooting
When encountering low bead recovery during the XP beads purification in the Primer annealing step (step D), consider the following two factors to ensure optimal results:
1) Homogenization of stock beads solution before adding it to the reaction. When the AMPure beads-to-sample ratio falls below 0.4:1, there will be no nucleic acid recovered by the beads at all.
2) At least 70% ethanol concentration in the washing solution. If the ethanol concentration is lower, nucleic acid may be eluted from the beads during washing. The reduced ethanol concentration in washing buffer could be caused by several factors, such as an older solution not freshly made during the same day of the experiment; the stock of ethanol used to prepare the washing solution has a lower concentration than indicated on the label due to prolonged storage or inadequate sealing after previous usages.
Acknowledgments
We thank the Reagents and Vaccine Services Section and Diagnostic Services Section, Foreign Animal Disease Diagnostic Laboratory, National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, New York, USA, for providing the FMDV viral isolates. We thank David Vierra for initiating the direct RNA sequencing project.
This research was funded by USDA Animal and Plant Health Inspection Service, through the Foreign Animal Disease Diagnostic Laboratory. It was supported in part by an appointment to the Plum Island Animal Disease Center (PIADC) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664.
Competing interests
The authors declare no conflicts of interest.
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© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Microbiology > Pathogen detection
Microbiology > Microbe-host interactions > Virus
Molecular Biology > RNA > RNA detection
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5,018 | https://bio-protocol.org/en/bpdetail?id=5018&type=0 | # Bio-Protocol Content
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This protocol has been corrected. See the correction notice.
Peer-reviewed
Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease
JC Jillian E. J. Cox
KP Kevin D. Pham
AK Alex W. Keck
ZW Zsabre Wright
MT Manu A. Thomas
WF Willard M. Freeman
SO Sarah R. Ocañas
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5018 Views: 1052
Reviewed by: Geoffrey C. Y. LauSuresh Kumar Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of Neuroinflammation Aug 2023
Abstract
Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq.
Key features
• Analysis of microglial phenotypes from murine brain without the need for cell sorting, imaging, or scRNA-seq.
• This protocol can distinguish between homeostatic, disease-associated (DAM), lipid-droplet accumulating (LDAM), and interferon response (IRM) microglia from any murine brain region and/or disease model of interest.
• This protocol can be modified to incorporate additional markers of interest or dyes when using a cell analyzer capable of multiple color detections.
Keywords: Microglia Microglial Phenotypes Flow Cytometry Autofluorescence Neuroinflammation Disease-associated microglia Lipid-droplet microglia Interferon-response microglia
Graphical overview
Background
Microglia are brain-resident immune cells that contribute to the neuroenvironment through diverse functions, including debris phagocytosis, neuronal support, and synaptic pruning. Their multifaceted roles align with distinct phenotypic states, largely influenced by environmental stimuli and intrinsic signaling cascades [1]. These specialized cells are involved in many neurological diseases, such as Alzheimer’s disease (AD). For instance, the disease-associated microglia (DAM) phenotype was first identified in an AD mouse model and has since been further validated to play a role in response to neurodegeneration [2]. Differentiating and quantifying microglial phenotypic states offers valuable insights into pathways associated with development and disease processes, presenting promising avenues for therapeutic exploration. Although fluorescent immunolabeling and microscopy can be used to distinguish microglial phenotypes, this approach is time-consuming, and quantification requires multiple tissue sections. Alternatively, sequencing-based approaches, though providing high-throughput and high-resolution data, are costly and demand specialized analytical training. Here, we present a fluorescence-based flow cytometry method to distinguish microglial phenotypic states, allowing for high-throughput, multiparametric analysis of single-cell suspensions with straightforward quantification tools.
In this protocol, we describe a simple and time-effective preparation of single-cell suspensions from murine brain tissue for flow cytometric analysis to distinguish multiple microglial phenotypes. Microglia have been previously described as either “M1” (pro-inflammatory) or “M2” (anti-inflammatory) to indicate “active” or “resting” states, respectively. However, the field has since moved to more descriptive terms such as “homeostatic” or specific “reactive” phenotypes, given that microglia are in a constant state of activity to respond to stimuli [3]. Therefore, this panel can distinguish between non-reactive/surveying (homeostatic) or reactive [DAM, interferon-response microglia (IRM), and lipid-droplet accumulating microglia (LDAM)] microglial phenotypes [3]. This protocol was previously used to examine sex differences in microglial phenotypic states during aging [4]. The markers for each of the examined phenotypes (homeostatic, DAM, IRM, and LDAM) were selected from several publications [2,5–10], ensuring no overlap between states, and are listed within the Materials and Reagents section.
A notable advantage to this protocol is the adaptability of this microglial phenotype panel, allowing for additional fluorescent antibodies or dyes depending on the laser and filter capabilities of the chosen cell analyzer. The use of fluorescent dyes offers in vivo examination of microglial phenotypes, including Methoxy-X04 (amyloid-β plaques) [11] and BODIPY (lipid accumulations) [10], where colocalization with these markers is useful in identifying DAM and LDAM phenotypes, respectively. This flexibility facilitates versatile applications in studying microglia. While initially utilized to examine sex differences in hippocampal microglial aging, this protocol can be applied to address various biological questions relevant to multiple murine disease models and other brain regions.
Materials and reagents
Biological materials
Mice: C57BL/6N (or any mouse line of interest)
Note: The data presented in the primary research article was generated in adult mice between 3 and 25 months of age. This protocol has not been tested on microglia isolated from neonatal or juvenile mice.
Reagents
Adult Brain Dissociation kit (Miltenyi Biotec, catalog number: 130-107-677)
D-PBS, calcium, magnesium, glucose, pyruvate (Gibco, catalog number: 14-287-072)
Cell staining buffer (BioLegend, catalog number: 420201)
TruStain FcX (BioLegend, catalog number: 101319)
Actinomycin D (Streptomyces species) (Sigma-Aldrich, catalog number: A1410-5MG)
Anisomycin (Streptomyces griseolus) (Sigma-Aldrich, catalog number: A9789-25MG)
Triptolide (Sigma-Aldrich, catalog number: T3652-1MG)
Dimethyl Sulfoxide (DMSO) (Fisher Scientific, catalog number: D1281)
Antibodies used for each microglia phenotype (Table 1)
Table 1. Antibody list for microglial phenotypes
Phenotype Antibody Conc. Fluorophore Clone Host Vendor Catalog # Dilution
Canonical CD11b 0.2 mg/mL Brilliant Violet 421TM M1/70 Rat BioLegend 101235 1:50
Canonical (hematopoietic) CD45 0.2 mg/mL Brilliant Violet 785TM 30-F11 Rat BioLegend 103149 1:40
Homeostatic P2RY12 0.2 mg/mL APC/FireTM 810 S16007D Rat BioLegend 848013 1:50
Disease-associated microglia (DAM) CD11c 0.2 mg/mL Brilliant Violet 605TM N418 Arm. Hamster BioLegend 117334 1:50
Disease-associated microglia (DAM) CD282 0.2 mg/mL PE/Cyanine7 QA16A01 Mouse BioLegend 153011 1:50
Disease-associated microglia (DAM) CLEC7A 9 μg/300 μL APC REA154 Human Miltenyi 130102985 1:50
Interferon response Microglia (IRM) CD317 0.2 mg/mL Brilliant Violet 650TM 927 Rat BioLegend 127019 1:50
Lipid-droplet accumulating microglia (LDAM) CD63 0.2 mg/mL PE NVG-2 Rat BioLegend 143903 1:40
Solutions
Actinomycin D (5 mg/mL stock solution) (see Recipes)
Triptolide (10 mM stock solution) (see Recipes)
Anisomycin (10 mg/mL stock solution) (see Recipes)
Recipes
Actinomycin D (5 mg/mL stock solution)
Reconstitute actinomycin D in DMSO to a concentration of 5 mg/mL.
Aliquot and store at -20 °C protected from light.
Triptolide (10 mM stock solution)
Reconstitute triptolide in DMSO to a concentration of 10 mM.
Aliquot and store at -20 °C protected from light.
Anisomycin (10 mg/mL stock solution)
Reconstitute anisomycin in DMSO to a concentration of 10 mg/mL.
Aliquot and store at 4 °C protected from light.
Laboratory supplies
GentleMACS C tubes (Miltenyi Biotec, catalog number: 130-096-334)
MACS SmartStrainer (70 μm) (Miltenyi Biotec, catalog number: 130-110-916)
15 mL centrifuge tubes (Corning, catalog number: 0553859A)
5 mL round-bottom Polystyrene test tube (Falcon, catalog number: 352054)
Strainer caps for FACS tubes (Olympus Plastics, catalog number: 28-155)
Equipment
GentleMACS Octo dissociator with heaters (Miltenyi Biotec, catalog number: 130-096-427)
Allegra X-30R centrifuge with swinging bucket (Beckman Coulter, catalog number: B08540)
Cytek Aurora 5 laser spectral flow cytometer (Cytek Biosciences, model: U0488, U1188)
Software and datasets
FlowJo v10.9.0
GraphPad Prism v9.5
Procedure
Below, we describe the step-by-step procedure for performing a flow cytometry experiment to identify microglial phenotypic states from murine brain tissue. This procedure has been used to identify sex differences in microglial phenotypic states from young and aged murine hippocampal tissue. The biological markers used in this study were selected by cross-referencing markers in common from multiple publicly available microglia data sets [2,5–10] and the commercial availability of non-overlapping fluorescent antibodies. This protocol can be optimized for additional murine disease models and brain regions.
*For all steps: Buffers, solutions, and suspensions must be kept at 4 unless otherwise noted (i.e., gentleMACS Octo dissociator with heaters).
** It is recommended to label all necessary tubes for the appropriate steps (i.e., C tubes, 15 mL, 5 mL, etc.) before starting the protocol.
Cell preparation
Note: Cell preparation and debris removal will follow the manufacturer’s instructions for the Adult Brain Dissociation kit with minor modifications, as detailed below.
Set the swinging bucket centrifuge to 4 .
Prepare the solutions from the Miltenyi Adult Brain Dissociation kit according to the manufacturer’s instructions.
Prepare Enzyme Mix 1: Buffer Z (1,900 μL/sample) + Enzyme P (50 μL/sample).
Prepare Enzyme Mix 2: Buffer Y (20 μL/sample) + Enzyme A (10 μL/sample).
Add 1,950 μL of Enzyme Mix 1 to a gentleMACS C tube.
Supplement with transcription and translation inhibitors (2 μL/sample of each) to prevent microglial ex vivo activation [12].
Actinomycin D (5 mg/mL stock solution)
Anisomycin (10 mg/mL stock solution)
Triptolide (10 mM stock solution)
Carefully dissect the hippocampus on surfaces chilled by ice.
Note: Although hippocampal tissue is used in this procedure, this protocol can be adapted for any brain region.
Euthanize mice according to the approved euthanasia method.
Note: Cardiac perfusion or intravenous labeling of circulating immune cells is recommended to exclude circulating immune cells from analysis.
Using small scissors, cut the skin carefully down the midline of the head, ending at the nasal bridge, and separate the skin from the skull laterally.
Break the nasal bridge using large scissors.
Using small scissors, cut the skull along the longitudinal fissure, beginning caudal and moving rostral.
Two distinct sides of the skull are formed. Using forceps, carefully separate the skull from the brain laterally. Avoid puncturing the brain tissue.
Remove the brain from the skull cavity and place it into a dish containing ~10 mL of ice-cold D-PBS to rinse the tissue.
Extract the brain from the dish and gently dry it with a dry wipe on an appropriate dissection surface (i.e., an aluminum plate on ice).
Using a small dissection spatula, cut into the cortex along the midline of the brain, roughly mid-brain deep.
To dissect the hippocampus, carefully peel away the cortex from one hemisphere, exposing the hippocampus. Once identified, scoop the hippocampus away from the cortex. Repeat on the opposite side.
Once isolated, cut the hippocampus into approximately four pieces and place into the appropriately labeled C tube.
Add 30 μL of Enzyme Mix 2 (from step A2b above) to each C tube containing brain tissue.
Attach the C tube to the gentleMACS Octo dissociator with heaters and use program 37C_ABDK_02 (20-100 mg of sample) or 37C_ABDK_01 (>100 mg of sample)(Miltenyi Biotech preset program).
Note: Ensure all tissue pieces are within the buffer solution to obtain the highest yield.
After completion of the program, remove the C tubes and centrifuge briefly at 300× g (immediately stop once it reaches 300× g) to collect the dissociated sample at the bottom of the tube.
Using 1 mL of D-PBS, pre-wet a 70 μm MACS SmartStrainer on a labeled 15 mL conical tube for each sample.
Resuspend each cell pellet and transfer it onto the appropriate 70 μm MACS SmartStrainer.
To retain the remaining tissue, add 10 mL of cold D-PBS to the C tube, close the tube, shake gently, and filter the complete volume to the appropriate 70 μm MACS SmartStrainer and 15 mL conical tube.
Using a swinging bucket centrifuge, centrifuge the 15 mL tube containing the cell suspensions at 300× g for 10 min at 4 .
Completely aspirate supernatant and proceed immediately to section B.
Debris removal
Resuspend (do not vortex) the pellet with 1,550 μL of D-PBS and transfer suspension to a labeled 5 mL round-bottom FACS tube.
Add 450 μL of debris removal solution.
Mix well by setting pipette volume ≥ 1,000 μL and gently mix ≥ 20 times.
Note: Proper mixing of the debris removal solution is necessary to ensure separation of the layers.
Gently overlay the cell suspension with 2 mL of D-PBS, avoiding the mixture of layers (see General Note 1).
Note: Gently tilt the 5 mL tube to a near-horizontal plane to allow for more controlled pipetting. Within the tube, place the pipette tip slightly above the top of the tilted solution and very slowly eject D-PBS, ensuring that the layersdo not mix. Pipette 1,000 µL twice for a smaller, more controllable volume.
Centrifuge the layered suspension at 3,000× g for 10 min at 4 .
Centrifugation will form three layers. Completely aspirate layers 1 & 2 (Figure 1).
Figure 1. Representative image of debris removal step post-centrifugation. Layer 1: buffer; layer 2: debris; layer 3: remaining buffer with cell pellet.
Bring volume up to 5 mL with D-PBS, cap the tube, and gently invert three times to mix.
Centrifuge the cell suspension at 1,000× g for 10 min at 4 with full acceleration and brake.
Completely aspirate the supernatant and proceed to section C.
Cell staining
Resuspend the cell pellet carefully in 1 mL of cell staining buffer.
Using 100 μL of cell staining buffer, pre-wet the filter cap of a 5 mL round-bottom FACS flow tube and filter the cell suspension through the cap, collecting the filtered suspension into the appropriately labeled flow tubes.
Note: Snap the filter cap on and ensure the ability to move the cap up and down to avoid a seal that prevents the flow of the solution. Slowly add the suspension to the cap and avoid overfilling the filter. The initial volume may need assistance passing through the filter by tapping the tube on a hard surface.
Centrifuge the cell suspension at 300× g for 10 min at 4 .
While the cells are spinning, prepare the Fc blocking solution at a 1:200 dilution (i.e., 1 μL of TruStain FcX + 199 μL of cell staining buffer). You will need 50 μL of Fc blocking solution per sample.
Once centrifugation is finished, decant the supernatant (leaves ~30 μL of solution).
Note: Decant by quickly inverting the tube over an appropriate waste container without shaking the tube. Allow most of the liquid to drain and blot the remainder on the rim using a disposable absorbent material placed flat on a benchtop. Quickly orient the tube without shaking or disturbing the pellet.
Resuspend the cells in 50 μL of Fc blocking solution and incubate at room temperature for 5 min protected from light.
Label tubes for each sample and controls [i.e., single-antibody, fluorescence minus one (FMO), and isotype controls].
Resuspend control samples in a volume that results in 100 μL for each tube.
Note: This step is for the distribution of the control samples into a number of tubes that correlates to the total number of antibodies used plus one unstained sample (i.e., 1,100 μL for 10 antibodies that leaves 100 μL remaining for the unstained control).
Transfer 100 μL of each control sample into its corresponding tube.
For the remaining tubes, add 20 µL of cell staining buffer to bring volume to ~100 μL for staining.
Add each antibody to their corresponding tube at the appropriate concentrations for a 100 μL volume.
Incubate the antibody cell mixture at 4 for 30 min, protected from light.
Wash the cells using 1 mL of cell staining buffer and spin at 300× g for 10 min at 4 ℃.
Aspirate or decant the supernatant completely and resuspend in 250 μL of cell staining buffer for flow analysis.
Data analysis
Data collection, data analysis, gating strategy, and identification of microglial phenotypes
Data collection
Make the appropriate adjustments (i.e., laser power, forward/side scatter, etc.) during data collection to position the data points of the unstained controls in the lower left quadrant. Each single-stained control should display a distinct positive population within the desired channel (fluorophore overlap should be corrected before collecting experimental data).
Note: It is recommended to collect data using a cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) with autofluorescence exclusion capabilities due to the high autofluorescence found in murine brain tissue that can negatively affect downstream analysis.
Determining the cell population
On a density plot, select forward scatter vs. side scatter (FSC vs. SSC) on the X and Y axes, respectively. FSC indicates cell size, while SSC indicates cell granularity. The cell population is determined by excluding the leftmost population (debris) (Figure 2A).
Figure 2. Gating strategy for distinguishing microglia. A. Recommended parameters to establish live cell population. B. Single-cell selection to exclude doublets. C. Selection of an autofluorescent negative population using the Cytek Aurora 5 laser spectral flow cytometer. D. Identification of microglial population (CD11b+CD45Mid).
Single-cell selection
To exclude doublets, select forward scatter area vs. forward scatter height (FSC-A vs. FSC-H) on the X and Y axes, respectively. Gate the densest population located along the diagonal (Figure 2B).
Autofluorescence correction
To correct for autofluorescence, select FSC-A vs. autofluorescence area (FSC-A vs. AF-A) on the X and Y axes, respectively. Place a gate on the negative population, extending the gate to negative values located off the displayed plot (Figure 2C). This will ensure that all cell populations are included in the data set.
Note: Cells positive for autofluorescence should be excluded due to the possibility of a false positive signal.
Identifying microglial population
To select the microglia population for downstream phenotyping, select the canonical microglial marker CD11b vs. the canonical hematopoietic marker CD45 on the X and Y axes, respectively. Microglia populations will display a CD11b+CD45Mid expression as described in previous reports [1] (Figure 2D). Use this population to identify microglia phenotypes.
Microglial phenotypes
The antibodies from this panel can be used to identify four distinct microglial phenotypes, as shown in Figure 3 (Note: More variations can be used to visualize these phenotypes). Homeostatic microglia are defined as P2RY12+CLEC7A- (Figure 3A). Disease-associated microglia (DAM) are determined by CD11chighCLEC7Ahigh and/or CD282+CLEC7Ahigh (Figure 3B). Interferon-response microglia (IRM) (Figure 3C) and lipid-droplet accumulating microglia (LDAM) (Figure 3D) are measured by the mean fluorescent intensity (MFI) of CD317+ and CD63+, respectively. When using the MFI, comparisons should be made between sample groups to determine differences in the IRM and LDAM populations for biological relevance.
Figure 3. Distinguishing microglial phenotypes. A. Homeostatic microglia are defined as P2RY12+CLEC7a-. B. Disease-associated microglia determined by CD11chighCLEC7Ahigh and CD282+CLEC7Ahigh. C. Interferon-response microglia determined by CD317 histogram. D. Lipid-droplet accumulating microglia determined by CD63 histogram.
Statistical analysis
GraphPad Prism v9.5 was used to conduct statistical analyses using a mixed-effects model, matching by age and collection date, and Sidak’s multiple comparisons test. Outliers due to technical variation were removed using the ROUT outlier test (Q+1%). The number of biological replicates to reach statistical power for this type of experiment depends on the expected proportion of each phenotypic state for the given murine model and brain region.
Validation of protocol
This protocol or parts of it have been used and validated in the following research article(s):
Ocañas et al. (2023). Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer’s disease. Journal of Neuroinflammation. (Figure 6, panels A-I).
General notes and troubleshooting
General notes
Debris removal—step B4: Proper overlay with D-PBS is crucial for clean data output, which is dependent on the separation of the layers. Mixing of the layers will result in excessive debris that can pollute the quality of the sample. Ensure careful adherence to the note listed in this step.
Fluorescent dyes, such as Methoxy-X04 [11] and BODIPY [10,11], may be used to examine in vivo microglial activity of DAM and LDAM phenotypes, respectively.
Appropriate controls, including single antibody, FMO, and isotype controls, should be included to ensure the observed signals are specific to the markers of interest.
This protocol has only been tested on murine hippocampal and cortical regions. However, other brain regions and/or disease models may be suitable with minimal optimization.
Cardiac perfusion or in vivo labeling of circulating immune cells is recommended to reduce 1) the amount of infiltrating peripheral macrophages that may confound distinct microglial populations, 2) autofluorescence from peripheral blood, and 3) overall debris that would result in cleaner data output.
Murine brain tissue has significant autofluorescence, with increased signals in aged tissue. The Cytek Aurora 5 laser spectral flow cytometer was used in this protocol, which allows gated removal of autofluorescent signal, but this feature may not be available in other flow cytometers. If using a cell analyzer without this autofluorescent detection, consider adapting this protocol with Burns et al. 2021 [13,14].
Previous reports have indicated that a subset of microglia may exist in AF+ populations due to the accumulation of lipofuscin during phagocytosis of cellular debris, with increased incidence in aged brains [15,16]. However, this protocol does not address AF correction during the cell preparation steps that would be necessary to include this lipofuscin+ population in phenotypic differentiation. Therefore, all AF+ populations are excluded during analysis to avoid false positive results.
This protocol used control samples from the same tissue due to variability in fluorescence intensity. Compensation beads may be used instead to provide more consistent and reusable control samples; however, this will require optimization to select the appropriate fluorescence intensity according to the tissue sample of interest.
The original intent of this protocol is to distinguish changes in microglial populations during aging, as described in the referenced primary research article [17]. However, the same cell preparation and staining steps can be adapted and optimized for cell sorting of these microglial phenotypes for additional downstream experiments.
Acknowledgments
This work was supported by grants from the National Institute of Health (NIH): DP5OD033443, R01AG059430, T32AG052363, 1S10OD028479-01. This work was also supported in part by awards I01BX003906, IK6BX006033, and ISIBX004797 from the United States (U.S.) Department of Veterans Affairs, Biomedical Laboratory Research and Development Service. This work was supported by a grant from the Alzheimer's Association (SAGA23-1072406).
Competing interests
The authors declare no competing interests.
Ethical considerations
All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oklahoma Medical Research Foundation (OMRF).
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Ocañas, S. R., Pham, K. D., Cox, J. E., Keck, A. W., Ko, S., Ampadu, F. A., Porter, H. L., Ansere, V. A., Kulpa, A. and Kellogg, C. M. (2023). Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer’s disease. bioRxiv: 2023.2003. 2007.531562. https://doi.org/10.1101/2023.03.07.531562
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Biochemistry > Protein > Labeling
Cell Biology > Cell-based analysis > Flow cytometry
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A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast
YO Yoshitaka Ogawa
TU Taisei P. Ueda
KO Keisuke Obara
KN Kohei Nishimura
TK Takumi Kamura
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5019 Views: 677
Reviewed by: Lucy XieOlga SinJoyce Chiu
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Original Research Article:
The authors used this protocol in PLOS Genetics Jun 2023
Abstract
The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems—the super-sensitive AID and AID 2—were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker–based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)–dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts.
Key features
• AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA–depending manner.
• Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.
Keywords: Auxin-inducible degron (AID) Super-sensitive AID (ssAID) Affinity linker–based super-sensitive AID (AlissAID) Nanobody (Nb) Budding yeast
Background
Targeted protein degradation (TPD) is a powerful tool for investigating protein function by rapidly depleting the target proteins in cells. Auxin-inducible degron (AID) is a TPD system that enables auxin-dependent AID-tagged target protein degradation [1]. This technique has been widely used in various eukaryotic species, including yeast, Drosophila, Caenorhabditis elegans, and vertebrate cells [2–5]. In this system, expression of the AID-tagged target protein and the auxin receptor Oriza sative TIR1 (OsTIR1) are necessary for target degradation. The OsTIR1 interacts with the AID degron, an interaction that is stabilized by auxin. It further recruits the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF), which leads to the ubiquitination and degradation of target proteins.
The conventional AID system requires a concentration of 100 µM or higher of auxin for the target protein degradation. However, such a high dose of auxin may lead to cytotoxicity [6]. To address this issue, two alternative systems, super-sensitive AID (ssAID) [7] and AID2 [8] have been developed using the bump and hole technique. The ssAID system uses a high-affinity pair of synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA), and a modified auxin receptor, OsTIR1F74A. This advanced system enables the degradation of AID-tagged target proteins at nanomolar concentrations of 5-Ad-IAA.
Recently, we developed an affinity-linker–based supersensitive AID (AlissAID) system using a single polypeptide antibody known as a nanobody (Figure 1) [9]. In this system, the addition of 5-Ad-IAA induces the degradation of GFP- or mCherry-tagged proteins by stabilizing the interaction between OsTIR1F74A and the minimized AID-tagged nanobody (mAID-Nb). [9]. The binding of OsTIR1F74A and mAID-tag depends on the concentration of 5-Ad-IAA. This advancement enables the use of GFP or mCherry proteins as degradation tags instead of the conventional AID tags in the AlissAID system.
GFP and mCherry are well-known fluorescent tags used in various eukaryotic cells. In budding yeast, GFP fusion with endogenous proteins is easily achievable through genetic manipulation. Furthermore, the GFP tag can be employed in a Yeast GFP Clone collection [10], with a GFP-tagged Open Reading Frame (ORF) at its chromosomal locus, containing 75% of the yeast proteome. Here, we describe a protocol for easy AlissAID strain generation from GFP- or mCherry-tagged strains in budding yeast.
Figure 1. Schematic illustration of generating AlissAID strains from GFP-tagged strains. (A) To generate the AlissAID strain, the pAlissAID anti-GFP plasmid is transformed into a GFP-tagged strain. (B) OsTIR1F74A and mAID-Nb (GFP) are constitutively expressed from the pAlissAID anti-GFP plasmid. The expressed OsTIR1F74A forms the SCF-OsTIR1F74A complex and then polyubiquitinates the GFP fusion protein in a 5-Ad-IAA dependent manner.
Materials and reagents
Biological materials
AlissAID plasmids targeting GFP fusion proteins
Note: These plasmids are available from National BioResource Project (NBRP)– Yeast
https://yeast.nig.ac.jp/yeast/top.xhtml
pAlissAID anti-GFP_313 (HIS3 marker) (NBRP, catalog number: BYP10393)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10393.pdf
pAlissAID anti-GFP_314 (TRP1 marker) (NBRP, catalog number: BYP10394)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10394.pdf
pAlissAID anti-GFP_315 (LEU2 marker) (NBRP, catalog number: BYP10395)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10395.pdf
pAlissAID anti-GFP_316 (URA3 marker) (NBRP, catalog number: BYP10392)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10392.pdf
AlissAID plasmids targeting for mCherry fusion proteins
pAlissAID anti-mCherry_313 (HIS3 marker) (NBRP, catalog number: BYP10396)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10396.pdf
pAlissAID anti-mCherry_314 (TRP1 marker) (NBRP, catalog number: BYP10397)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10397.pdf
pAlissAID anti-mCherry_315 (LEU2 marker) (NBRP, catalog number: BYP10398)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10398.pdf
pAlissAID anti-mCherry_316 (URA3 marker) (NBRP, catalog number: BYP10399)
Vector map: https://yeast.nig.ac.jp/yeast/pdf/byp/BYP10399.pdf
Budding yeast strains (commonly used strains are useable, BY4741 W303-1a, etc…)
Antibodies (anti-OsTIR1 (MBL, catalog number: PD048), anti-Pgk1 (our Laboratory), anti-AIDtag (gifted from Prof. Karim Labib)
Reagents
Lithium acetate (LioAc) (Wako, catalog number: 127-01545)
Salmon sperm DNA (ssDNA) (Wako; catalog number: 043-31381)
Poly-ethylene glycol 4,000 (PEG) (Wako, catalog number: 162-09115)
Dimethyl sulfoxide (DMSO) (Wako, catalog number: 043-07216)
5-Adamantyl-IAA (5-Ad-IAA) (Tokyo Chemical Industry (catalog number: A3390).
D-Glucose (Wako, catalog number: 045-31167)
Extract yeast dried (Nacalai Tesque, catalog number: 15838-45)
Polypeptone (Wako, catalog number: 398-02117)
Adenine hydrochloride (Biosynth, catalog number: FA02944)
Yeast nitrogen base without amino acids (ForMedium, catalog number: CYN0410)
SC Quadruple Drop Out: -His, -Leu, -Trp, -Ura (ForMedium, catalog number: DSCK1027)
Agar (Nacalai Tesque, catalog number: 01028-85)
Solutions
YPD medium (see Recipes)
SD medium (see Recipes)
Amino acids and Uracil solution (see Recipes)
LiOAc solution (0.1 M, 1 M) (see Recipes)
Single-strand DNA (ssDNA; 2.0 mg/mL) (see Recipes)
PEG (50% w/v) (see Recipes)
5-Ad-IAA (see Recipes)
Recipes
YPD medium
Reagent Final concentration Amount
D-glucose 2% 20 g
Extract yeast dried 1% 10 g
peptone 2% 20 g
Adenine hydrochloride 0.01% 100 mg
MILLI-Q
Total 1,000 mL
SD medium
Reagent Final concentration Amount
D-glucose 2% 20 g
Adenine hydrochloride 0.01% 100 mg
Yeast nitrogen base without amino acids 0.69% 6.9 g
SC Quadruple Drop Out: -His, -Leu, -Trp, -Ura 0.06% 0.6 g
5 M NaOH 5 mM 1 mL
agar (for plate) 2% 20 g
Amino acids and Uracil solution 10 mL
MILLI-Q
Total 1,000 mL
For auxotrophic selection, supplement appropriate amino acids and Uracil.
Amino acids and Uracil solution
Histidine solution 10 g/L
Leucine solution 12 g/L
Tryptophan solution 10 g/L
Uracil solution 2 g/L
LiOAc solution (0.1 M, 1 M)
Dissolve in MILLI-Q at the prescribed concentration and autoclave.
Single-strand DNA (ssDNA; 2.0 mg/mL)
Dissolve in MILLI-Q at a concentration of 2.0 mg/mL and autoclave.
PEG (50% w/v)
Dissolve in MILLI-Q water at 50% (w/v) and autoclave. The transformation efficiency will decrease if used for a long time; therefore, approximately 10 samples should be prepared at a time.
5-Ad-IAA
Dissolve in DMSO at a concentration of 5 mM and store at -30 °C. When it is used for liquid medium, add 1/1,000 of the amount of 5 mM 5-Ad-IAA to the medium. When it is used for plate media, autoclaved media should be cooled to approximately 50 °C before adding 5-Ad-IAA. 5-Ad-IAA containing plates can be stocked at 4°C, hidden from direct light, for at least one month.
Laboratory supplies
Laboratory disposables:
1.5 mL tubes (Watson, catalog number: 131-7155C)
15 mL tubes (Greiner, catalog number: 188 271- 013)
50 mL tubes (Greiner, catalog number: 227 261)
1000 μL tips (Watson, catalog number: 110-706C)
200 μL tips (Watson, catalog number: 110-705C)
Petri dishes (STAR, catalog number: RSU-SD9015-2)
Equipment
Cool incubator (As one, catalog number: A1201)
Shaking incubator (N-BIOTEK, catalog number: NB-205L)
Heat block (WAKENYAKU, catalog number: WKN-9626)
Vortex (Scientific Industries, Inc., catalog number: SI-0286)
Centrifuge (Eppendorf, catalog number: 5420000237
Microscope (AxioObserver Z1 (Carl Zeiss, Oberkochen, Germany) equipped with a CCD camera (AxioCam MRm; Carl Zeiss))
Procedure
Preparation of GFP- or mCherry-tagged strains
Prepare strains in which target proteins are tagged with GFP or mCherry. It is easy to add fluorescent tags to an ORF in its chromosomal location through homologous recombination in budding yeast [10]. Alternatively, the appropriate strains were selected from the GFP Clone Collection [10]. In this paper, we used W303-1a strains that were tagged at the C-terminus of ASK1 on the genome and selected with HIS3 using the method previously reported [9].
Note: Although we have not tested the protein degradation of N-terminal–tagged proteins, in principle, the AlissAID system would work on N-terminal tagged proteins.
Transformation of the pAlissAID plasmid
Note: Pay attention to the selection marker; in the case of the GFP Clone Collection, the GFP sequence is inserted at the C-terminus of the target gene using HIS3 as the selection marker. Therefore, we used the pAlissAID plasmids encoding TRP1, LEU2, and URA3 (Figure 2). All these pAlissAID plasmids (HIS3, TRP1, LEU2, and URA3 encoding) are available from NBRP.
Figure 2. Overview of pAlissAID plasmids. (A, D) Schematic illustrations of pAlissAID (anti-GFP or anti-mCherry) plasmids. pAlissAID plasmids contain an expression cassette of OsTIR1F74A-T2A-mAID-Nb (GFP or mCherry) under the control of constitutive ADH1 promoter. The expressed protein is cleaved at the self-cleavage site of T2A to produce OsTIR1F74A and mAID-Nb, separately. (B, E) Lists of pAlissAID plasmids. Each plasmid has a different selection marker HIS3, TRP1, LEU2, or URA3. (C, F) Immunoblot analysis of OsTIR1F74A and mAID-Nb. Each pAlissAID plasmid with a different selection marker was transformed into the W303-1a cells to express both OsTIR1F74A and mAID-Nb. Pgk1 was used as a loading control.
Day 1
Grow yeast cells overnight in 5 mL of YPD medium at 30 °C with shaking at 230 rpm.
Day 2
Dilute the cells to OD600 = 0.3 and regrow the cells in 5 mL of YPD medium at 30 °C with shaking at 230 rpm for 3–5 h.
When cells have grown to approximately OD600 = 1.0, centrifuge them (1,000× g, 5 min, approximately 25 °C) and discard the supernatant.
Suspend cells in 1 mL of sterilized water and transfer to a sterilized 1.5 mL tube.
Centrifuge (20,000× g, 30 s, 25 °C) and discard the supernatant.
Resuspend in 1 mL of 0.1 M LiOAc.
After centrifugation (20,000× g, 30 s, 25 °C), discard the supernatant and centrifuge again to completely remove the supernatant.
Denature the ssDNA at 90 °C for approximately 5 min and cool on ice.
Add the following solutions and mix using a vortex.
2.0 mg/mL ssDNA, 25 μL
pAlissAID plasmid, 1 μg
sterilized water, up to 50 μL
Add 240 μL of 50% w/v PEG and 36 μL of 1 M LiOAc. Vortex the mixture and incubate in a heat block at 30 °C for 30 min.
Add 40 μL of DMSO. Vortex the mixture and incubate in a heat block at 42 °C for 20 min.
Centrifuge (20,000× g, 30 sec, room temperature) and discard the supernatant.
Resuspend the pellet in the remaining supernatant (at least 50 μL is needed) and plate the cells onto an SD plate medium (lacking histidine, leucine, tryptophan, or uracil).
Incubate at 30 °C for 2–3 days.
Day 4 or 5
Pick up the colonies that contain pAlissAID plasmids (Figure 3A and 4A).
Figure 3.Degradation of GFP fusion proteins in AlissAID strains. (A) AlissAID strains were generated by the transformation of pAlissAID plasmids with a different selection marker (TRP1, LEU2, or URA3) into the Ask1-GFP strain. (B) Microscopic observations of fluorescent signals of Ask1-GFP in AlissAID strains. Cells were treated with or without 5.0 µM 5-Ad-IAA for 3 h at 30 . Scale bar, 10 µm. (C) Serial dilution spotting of control and AlissAID strains on SD medium plate with or without 5.0 µM 5-Ad-IAA. 10-fold serial dilutions were made from the cells with OD600 = 0.3 and spotted into the plate. The strain that expresses Ask1 instead of Ask1-GFP and contains pAlissAID plasmid is used as a control. Cells were grown for 48 h at 30 °C.
Figure 4. Degradation of mCherry fusion proteins in AlissAID strains. (A) AlissAID strains were generated by the transformation of pAlissAID plasmids with a different selection marker (TRP1, LEU2, or URA3) into the Ask1-mCherry strain. (B) Microscopic observations of fluorescent Ask1-mCherry signals in AlissAID strains. Cells were treated with or without 5.0 µM of 5-Ad-IAA for 3 h at 30 . Scale bar, 10 µm. (C) Serial dilution spotting of control and AlissAID strains on SD medium plate with or without 5.0 µM 5-Ad-IAA. 10-fold serial dilutions were made from the cells with OD600 = 0.3 and spotted into the plate. The strain that expresses Ask1 instead of Ask1-mCherry and contains pAlissAID plasmid is used as a control. Cells were grown for 48 h at 30 °C.
After picking up the colonies and growing them in SD medium, confirm successful plasmid transformation into the yeast by any method (Immunoblot, microscope observation, phenotyping, etc.) (Figure 3B and C, 4B and C).
Note: Expressed nanobodies bind to GFP or mCherry instead of target protein in the AlissAID strains. It is unlikely that these nanobody interfere with the target protein’s function.
5-Ad-IAA treatment
When it is used for liquid medium, add 1/1,000 of the amount of 5 mM 5-Ad-IAA to the medium (final concentration 5 μM of 5-Ad-IAA). When it is used for plate media, autoclaved media should be cooled to approximately 50 °C before adding 5-Ad-IAA.
Check the protein degradation by fluorescence microscopy
Degradation of target proteins can be confirmed by immunoblot or fluorescence microscopy. If the target is an essential protein, it can also be verified by a serial dilution assay in 5-Ad-IAA–containing plate medium. These detailed methods are described in the original paper [9]. Here, we briefly introduce an example of verification using fluorescence microscopy.
Day 1
Grow yeast cells overnight in 5 mL of SD medium at 30 °C with shaking at 230 rpm.
Day 2
Dilute the cells to OD600 = 0.3 and regrow the cells in 5 ml of SD medium at 30 °C with shaking at 230 rpm for 3–5 h.
When the cells have grown to approximately OD600 =1.0, add 5 μL of 5 mM 5-Ad-IAA and incubate at 30 shaking at 230 rpm for 1–3 h.
Collect the cells and observe fluorescent signals of live cells by using a fluorescence microscope.
Note: PFA fixation by using 4% paraformaldehyde is also available for microscopic observation.
Note: The efficiency of protein degradation depends on the expression level and localization of the target protein, so in some cases immunoblots would be more suitable for confirming protein reduction.
Data analysis
Representative data
AlissAID strains induce target protein depletion in the presence of 5 µM of 5-Ad-IAA (Figures 3B and 4B). When targeting essential genes, the AlissAID strains exhibited severe growth defects in plates containing 5-Ad-IAA (Figures 3C and 4C). Immunoblot, microscopic observation, and serial dilution assay methods followed those detailed in the original paper [9].
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Ogawa et al. (2023). Development of AlissAID system targeting GFP or mCherry fusion protein. PLOS Genetics (Figure 5).
Acknowledgments
This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers, JP19K06611, JP20K21423 and JP22K05558 to K.N.; JP22K06141 to K.O.; JP20H03208 to T.K. This work was also supported by the Institute for Fermentation, Osaka (IFO), and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. Y. O. was funded by the Japan Society for the Promotion of Science fellowship (DC2).
Original research paper—Ogawa et al. (2023): Development of AlissAID system targeting GFP or mCherry fusion protein. https://doi.org/10.1371/journal.pgen.1010731
Competing interests
The authors declare no financial or non-financial competing interests.
References
Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. and Kanemaki, M. (2009). An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods. 6(12): 917–922. https://doi.org/10.1038/nmeth.1401.
Morawska, M. and Ulrich, H. D. (2013). An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30(9): 341–351. https://doi.org/10.1002/yea.2967.
Trost, M., Blattner, A. C. and Lehner, C. F. (2016). Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster. Fly (Austin) 10(1): 35–46. https://doi.org/10.1080/19336934.2016.1168552.
Zhang, L., Ward, J. D., Cheng, Z. and Dernburg, A. F. (2015). The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142(24): 4374–4384. https://doi.org/10.1242/dev.129635.
Holland, A. J., Fachinetti, D., Han, J. S. and Cleveland, D. W. (2012). Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc Natl Acad Sci USA. 109(49): E3350–3357. https://doi.org/10.1073/pnas.1216880109.
Hac-Wydro, K. and Flasinski, M. (2015). The studies on the toxicity mechanism of environmentally hazardous natural (IAA) and synthetic (NAA) auxin--The experiments on model Arabidopsis thaliana and rat liver plasma membranes. Colloids Surf B Biointerfaces. 130: 53–60. https://doi.org/10.1016/j.colsurfb.2015.03.064.
Nishimura, K., Yamada, R., Hagihara, S., Iwasaki, R., Uchida, N., Kamura, T., Takahashi, K., Torii, K. U. and Fukagawa, T. (2020). A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair. Nucleic Acids Res. 48(18): e108. https://doi.org/10.1093/nar/gkaa748.
Yesbolatova, A., Saito, Y., Kitamoto, N., Makino-Itou, H., Ajima, R., Nakano, R., Nakaoka, H., Fukui, K., Gamo, K., Tominari, Y., et al. (2020). The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun. 11(1): 5701. https://doi.org/10.1038/s41467-020-19532-z.
Ogawa, Y., Nishimura, K., Obara, K. and Kamura, T. (2023). Development of AlissAID system targeting GFP or mCherry fusion protein. PLoS Genet. 19(6): e1010731. https://doi.org/10.1371/journal.pgen.1010731.
Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S. and O'Shea, E. K. (2003). Global analysis of protein localization in budding yeast. Nature 425(6959): 686–691. https://doi.org/10.1038/nature02026.
Article Information
Copyright
© 2024 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
Microbiology > Microbial genetics > Plasmid
Cell Biology > Cell engineering
Biochemistry > Protein > Degradation
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Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing
HL Hongzhe Li
SB Sandro Bräunig
SS Stefan Scheding
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5020 Views: 646
Reviewed by: Vivien J. Coulson-ThomasMaria Walker Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in eLIFE Mar 2023
Abstract
The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME’s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche.
Key features
• Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq
• Targeted strategy for enriching low-frequency stromal cells
Keywords: Human bone marrow microenvironment Non-hematopoietic cells Bone marrow stromal cells Flow cytometry CD271 CD45 CD235a
Graphical overview
Background
In human bone marrow, hematopoietic stem cells (HSCs) and their progenies are contained in a specialized microenvironment that regulates HSC maintenance and differentiation. Despite the important role of this hematopoietic environment (HME), its cellular composition, potential heterogeneity, and cellular hierarchy remain poorly defined. This is mainly due to the extremely low frequency of the HME-forming cells, the so-called bone marrow stromal (stem) cells, BMSCs [1,2].
The conventional approach to studying the human bone marrow microenvironment is mainly based on the analysis of different cell types defined by the expression of a limited number of known surface markers, which results in an underestimation of cellular complexity. Novel single-cell–based omics approaches, on the other hand, have the potential to provide detailed insights into complex cellular organization and function. However, whereas bulk preparations of bone marrow cells allow for analysis of the majority of cells, important low-frequency cell populations such as BMSCs will escape detailed analysis. Therefore, we developed a strategy to combine single-cell RNA sequencing of sorted non-hematopoietic BM cells with highly enriched BMSCs to resolve the cellular heterogeneity of the human bone marrow microenvironment at the highest possible resolution based on transcriptomic profiling [3].
Our approach is based on the expression of CD45, CD235a, and CD271. CD45 is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene (protein tyrosine phosphatase receptor type C). CD45 is considered a pan-hematopoietic marker and is widely used to select all hematopoietic cells and precursors except erythroid cells [4]. CD235a, also known as glycophorin A (GYPA), is a major intrinsic membrane protein of erythrocytes and a distinct marker of erythroblasts [5]. Therefore, we chose to use the combination of CD45 and CD235a to enrich non-hematopoietic human bone marrow microenvironment cells based on their low or absent expression of both CD45 and CD235a. Finally, BMSCs were highly enriched by sorting CD45low/-CD235a-/CD271+ cells, which is based on data by us and others demonstrating that the CD271 positive BM cell population contains all assayable stromal cells [5–7].
This paper describes a step-by-step protocol to isolate cells from the human bone marrow microenvironment for single-cell RNA sequencing [3] that can certainly be applied to other state-of-the-art omics approaches. Thus, this protocol contributes valuable information that, when combined with future research efforts, will contribute to a deeper understanding of the intricate cellular interactions within the bone marrow niche.
Materials and reagents
The following materials and equipment are recommended for this protocol, but alternative reagents and equipment from other sources than those recommended herein can be used when shown equivalent.
Biological materials
Human iliac crest bone marrow aspirates from a healthy donor (ca. 50–60 mL), collected in 20 mL syringes prefilled with 1.6 mL of heparin (5,000 IU/mL) (Skåne University Hospital, Lund, Sweden)
Reagents
Phosphate-buffered saline (PBS) without Ca2+ & Mg2+ (HyClone, catalog number: SH30256.01)
Bovine Serum Albumin (BSA) (Merck, catalog number: A7906)
Fetal Bovine Serum (FBS) (Gibco, catalog number: 10270-106)
Gammanorm human normal immunoglobulins (Octapharma, catalog number: 096178)
Ficoll-Paque Premium (Cytiva, catalog number: 17544203)
Mouse anti-human CD45-FITC antibody (BD, clone: 2D1, catalog number: 345808)
Mouse anti-human CD235a-PE-Cy5 antibody (BD, clone: GA-R2 (HIR2), catalog number: 561776)
Mouse anti-human CD271-APC antibody (Miltenyi, clone: REA844, catalog number: 130-112-602)
DAPI stock solution (1 mg/mL) (Sigma, catalog number: D9564)
Mouse IgG1-FITC (BD, catalog number: 345815)
Mouse IgG2b-PE-Cy5 (BD, catalog number: 555744)
Mouse IgG1-APC (BD, catalog number: 345818)
BD Pharm LyseTM Lysing Buffer (BD, catalog number: 555899)
Anticoagulant Citrate Dextrose Solution USP (ACD) Solution A (ACDA) (Terumo BCT, catalog number: 77960-010)
Solutions
Ficoll buffer (see Recipes)
Sorting buffer (see Recipes)
Blocking buffer (see Recipes)
Collection buffer (see Recipes)
Recipes
Ficoll buffer
PBS with 0.6% ACDA and 2% FBS
Sorting buffer
PBS with 1% BSA
Blocking buffer
PBS 1:50 Gammanorm, 1% FBS (sterile-filtered)
Collection buffer
PBS with 0.04% BSA
Laboratory supplies
Falcon conical tubes 50 mL (Fisher Scientific, catalog number: 11819650)
T-75 culture flask (Merck, catalog number: CLS3290)
Filcon 30 μm, sterile, cup-type (BD, catalog number: 340626)
Equipment
Easypet (Eppendorf, catalog number: 4430000018)
Centrifuge (Hettich, model: ROTANTA 460R)
Cell counter (chemometec, model: NucleoCounter NC-250)
Cell sorter (BD, model: Aria II)
Software and datasets
FACSDiva (BD, version 9.0)
ChemoMetec NucleoView NC-250 (ChemoMetec, version 1.2.0.0)
Procedure
MNC isolation
Prepare five 50 mL Falcon tubes with 15 mL of Ficoll-Paque Premium.
Transfer the bone marrow aspirate to a sterile T-75 culture flask.
Add 100 mL of Ficoll buffer to the flask and mix by pipetting up and down.
Carefully layer 30 mL of bone marrow-Ficoll buffer mix over Ficoll-Paque Premium in each 50 mL Falcon tube.
Centrifuge at 300× g for 30 min at room temperature without breaks [acceleration rate: 6 (maximum 10); deceleration rate: 0] utilizing a centrifuge equipped with a swinging bucket rotor.
Collect interphases containing mononuclear cells (Figure 1) into five new 50 mL Falcon tubes and fill up with Ficoll buffer to 50 mL.
Figure 1. Ficoll-Paque separation demonstration and expected layers after density gradient centrifugation. Each layer represents components with different densities, allowing for the separation and isolation of specific cell populations from the bone marrow aspirates. Cell separation layers starting from the top: plasma; interphase with mononuclear cells; Ficoll-Paque; and bottom fraction with leucocytes, granulocytes, and erythrocytes.
Centrifuge for 15 min at 400× g at 4 °C.
Prepare 35 mL of 1× Pharm Lyse by mixing 3.5 mL of BD Pharm LyseTM Lysing Buffer with 31.5 mL of sterile distilled water.
When centrifugation (step 7) is complete, aspirate the supernatants and re-suspend the pellets by adding 7 mL of 1× Pharm Lyse into each tube and gently pipetting up and down.
Combine the resultant resuspended cell mixtures from the five tubes into one 50 mL Falcon tube.
Gently vortex the sample and keep it for 15 min at room temperature.
Centrifuge at 200× g for 15 min at 4 °C.
Carefully aspirate the supernatant.
Resuspend the pellet in 250 μL of Blocking buffer.
Take 5 μL of cells in blocking buffer and mix with 995 μL of Blocking buffer to prepare a 2 to 100 dilution for cell counting.
Count cell numbers using NucleoCounter NC-250 and calculate the original cell number by multiplying with the dilution factor 200.
Adjust the cell concentration to 2 × 108–2 × 109/mL by adding appropriate volume of Blocking buffer.
FACS staining
Incubate cells in Blocking buffer for 20 min at room temperature.
Aliquot the cells and antibodies according to Table 1 to isolate CD45low/-CD235a- cells. Aliquot the cells and antibodies according to Table 2 to isolate CD45low/-CD235a-CD271+ cells.
Table 1. FACS staining panel for CD45low/-CD235a- cell isolation
Tube ID Tube name CD45 FITC (µL) CD235a PE-Cy5 (µL) IgG1 FITC (µL) IgG2b PE-Cy5 (µL) Cells (µL) Blocking buffer (µL)
a Unstained cells - - - - 5 45
b Compensation FITC 5 - - - 5 40
c Compensation PE-Cy5 - 5 - - 5 40
d *FMO-FITC - 5 5 - 5 35
e *FMO-PE-Cy5 5 - - 5 5 35
f Sample 25 25 - - 175 25
*FMO: fluorescence minus one control
Table 2. FACS staining panel for CD45low/-CD235a-CD271+ cell isolation
Tube ID Tube name CD45 FITC (µL) CD235a PE-Cy5 (µL) CD271 APC (µL) IgG1 FITC (µL) IgG2b PE-Cy5 (µL) IgG1 APC (µL) Cells (µL) Blocking buffer (µL)
a’ Unstained cells - - - - - - 5 45
b’ Compensation FITC 5 - - - - - 5 40
c’ Compensation PE-Cy5 - 5 - - - - 5 40
d’ Compensation APC - - 5 - - - 5 40
e’ FMO-FITC - 5 5 5 - - 5 30
f’ FMO-PE-Cy5 5 - 5 - 5 - 5 30
g’ FMO-APC 5 5 - - - 5 5 30
h’ Sample 25 25 25 - - - 175 -
Incubate the staining tubes for 30 min at 4 °C in the dark.
Wash the stained cells by adding 1 mL of Sorting buffer to the tubes and centrifuge tubes for 5 min at 800× g at 4 °C.
Resuspend Tubes a-e (a’–g’ for panel 2) with 500 μL of Sorting buffer and add 2.5 μL of sterile DAPI stock.
Resuspend Tube f (h’ for panel 2) in 3 mL of Sorting buffer and add 15 μL of DAPI stock.
Pass the cells through a 30 μm Filcon or any equivalent strainer.
Proceed quickly with FACS sorting.
FACS sorting
Open the BD FACSDiva Software in the software interface and locate the workspace where you can create plots. Create a plot and choose the parameters to make a Forward Scatter-Area (FSC-A) versus Side Scatter-Area (SSC-A) plot. Adjust the individual FSC and SSC photomultiplier tube settings to visualize the expected cell populations (Figure 2A).
Figure 2. Gating strategies for isolation of human bone marrow CD45low/-CD235a- and CD45low/-CD235a-CD271+ cells. Representative FACS plots illustrate the sequential gating strategy to eliminate cell debris (A), doublets (B), and dead cells (C). Sorting gates for isolation of CD45low/-CD235a- (D) and CD45low/-CD235a-CD271+ (E) cells according to the appropriate FMO controls.
Set up a gate to remove FSC-low populations by drawing a polygonal gate around the regions containing FSC-medium and FSC-high particles, as the FSC-low population consists of cell debris, air bubbles, and laser noise (see Figure 2A).
Create a Forward Scatter-Height (FSC-H) versus FSC-A plot and exclude doublets and multiplets by gating out cells with higher area signal values (FSC-A) (Figure 2B).
Create a FSC-A versus DAPI plot to exclude non-viable cells by gating out the DAPI-high cells (Figure 2C).
Create a FITC versus PE-Cy5 plot to exclude CD45-high and CD235-expressing cells (Figure 2D).
Create a FSC-A versus APC plot to exclude CD271 negative cells (Figure 2E).
Sort 100 events from the CD45low/-CD235a- or CD45low/-CD235a-CD271+ cell fractions into a tube containing 100 µL of ice-cold Collection buffer.
Perform reanalysis with the sorted sample to evaluate the sorting purity (> 85%).
Collect the target cell fraction into a tube containing 700 µL of the ice-cold Collection buffer.
After sorting is complete, count the cell number and re-suspend 20,000 cells with 47 µL of ice-cold Collection buffer.
Proceed immediately to perform single-cell RNA sequencing using Chromium Controller (10× Genomics) and Chromium Single Cell Gene Expression 3’ v3 Reagent Kit.
Data analysis
To evaluate the effectiveness of cell isolation, various parameters were assessed, including (1) the efficiency of Ficoll-Paque gradient separation, (2) the viability of bone marrow mononuclear cells, (3) the real-time gating strategy utilizing BD FACS Diva, and (4) the efficiency of cell sorting.
Ficoll-Paque gradient separation
After centrifugation, observe the tube to identify different layers. Typically, layers include plasma, a mononuclear cell layer (buffy coat), a Ficoll-Paque layer, and a red blood cell layer. A well-performed Ficoll-Paque gradient separation should result in four distinct layers with the mononuclear cell layer containing a high concentration of nucleated cells. The efficiency of separation can be evaluated by the presence of a clear interface between layers and minimal contamination between different cell populations.
Cell viability
The quality of isolated mononuclear cells can be evaluated using various viability assays. A cell viability of over 90% is recommended, particularly for cell sorting procedures. Cell viability is calculated using the following formula:
Viability (%) = (1 - Total number of stained cells / Total number of cells) × 100
Gating strategy
An effective real-time gating strategy is paramount for the precise collection of high-quality, viable non-hematopoietic cells for single-cell RNA sequencing. As illustrated in Figure 2, the gating strategy employed ensured the targeted isolation of the desired cell population while meticulously excluding debris (Figure 2A), doublets (Figure 2B), non-viable or damaged cells (Figure 2C), and unwanted hematopoietic cells (Figure 2D). The sequential application of these gates progressively refined the cell population, culminating in the final selection of CD271-positive cells (Figure 2E) for subsequent downstream analyses.
Sorting efficiency
After the initial sorting process, a critical step in ensuring data integrity is the reanalysis of sorted cells. This involves a reassessment of the sorted cell population using the same flow cytometry gating strategy. By performing sorting reanalysis, we could evaluate the sorting purity and efficiency, identify contaminants and refine the gating strategy if necessary. Through meticulous verification and validation, researchers can ensure the consistency and accuracy of sorted cell samples, facilitating trustworthy results in subsequent experiments and analyses. It is advisable to aim for a sorting purity exceeding 85% for optimal suitability in subsequent analyses.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Li et al. [3]. Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing. eLife (Figure 1, panel A).
General notes and troubleshooting
General notes
High-quality BM aspirates should be used fresh and contain sufficient numbers of cells. We routinely collect 50–60 mL of bone marrow from 2–3 aspirations from the same donor. However, lower volumes might also be sufficient. In case BM cells are obtained from biopsies, MNC isolation has to be performed by methods such as “crushing and/or flushing” either with or without the use of enzymes such as collagenase [8,9].
CD45 is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene (protein tyrosine phosphatase, receptor type C). Conventionally, CD45 is considered a pan-hematopoietic marker and is widely used to select for hematopoietic cells. In this protocol, we chose to explore the human bone marrow microenvironment by using bone marrow mononuclear cells that showed low or absent expression of CD45. Our gating strategy aimed to enrich all non-hematopoietic cells but not to exclude potential stromal cells by too rigorous gating. Therefore, several hematopoietic cell types including B cells, NK cells, CD235a- late-stage erythroid progenitors, megakaryocytes, monocytes, dendritic cells, granulocytes, and CD34-expressing putative hematopoietic stem and progenitor cell (HSPC) populations could be identified within this gate. This is consistent with previous murine studies that used comparable gating to enrich non-hematopoietic bone marrow cells, which included multiple CD45- hematopoietic cell populations [10,11]. The hematopoietic cells could be easily identified based on their gene expression profiles.
It has been widely known that primary bone marrow stromal cells are difficult to isolate due to the extremely low frequency of this cell type. We therefore recommend including CD271 in the staining panel (Table 2) to further enrich the stromal cells if this is the target population for detailed analysis, as bone marrow stromal stem/progenitor cells are highly and exclusively enriched in CD271-expressing cells [12].
Having an effective Ficoll-Paque density gradient separation is one of the crucial steps in obtaining high-quality human bone marrow mononuclear cells for FACS-based cell isolation.
Bone marrow samples should be processed for mononuclear cell isolation immediately after aspiration (within 30 min) to achieve the best sample quality with the highest cell viability and cell yield.
It is important to use a swing-out rotor (also known as a bucket rotor) instead of a fixed-angle rotor for Ficoll-Paque gradient centrifugation to ensure a better separation of cell layers during centrifugation and a higher cell recovery rate. This is especially crucial when working with limited cell numbers or rare cell populations.
In this protocol, we used BD Pharm LyseTM lysing solution, which is an ammonium chloride (NH4Cl)-based lysing reagent, to lyse red blood cells. Incubating cells treated with NH4Cl at room temperature for 15 min allows for the complete lysis of red blood cells through osmotic shock. The duration of 15 min for incubating cells has been empirically determined to be sufficient for effective lysis while minimizing damage to other cell types when processing 60 mL human bone marrow samples. The duration of the red blood cell removal step (Procedure A11) can be adjusted depending on the individual sample. Pharm Lyse treatment can be reduced to as short as 2 min if red blood cell contamination is minimal.
Although we used CD45-FITC, CD235a-PE-Cy5, and CD271-APC antibodies in this protocol to distinguish CD45/CD235a double-negative cells from the cells that are positive for one or both surface markers and to enrich CD271-expressing cells, any fluorochromes conjugated to these three antibodies could be used to isolate cells from the human bone marrow microenvironment. When selecting antibodies with distinct conjugates, it is advisable to opt for conjugates that have minimal overlap in their emission spectra. As FACS-based cell isolation is highly dependent on the quality of antibody staining, it is recommended to titrate each antibody using bone marrow samples to avoid the antibody saturation effect, optimize signal-to-noise ratio, minimize antibody non-specific binding, enhance consistency and reproducibility, and achieve the best sorting outcome.
As the flow cytometric cell sorter plays an essential role in obtaining accurate and reliable results in this protocol, it is extremely important to ensure that the cell sorter is operated under optimal conditions.
Machine-specific preparatory steps should be performed according to the manual.
The cell sorter should be cleaned properly before the experiment to minimize any potential contamination.
Photomultiplier tube (PMT) voltage and detector sensitivity settings need to be adjusted for each fluorochrome.
Perform compensation calculation using single-stained compensation controls. Double-confirm the compensation calculation by using FMO controls need to be included.
A gating strategy to distinguish positive and negative events based on FMO samples has to be established.
The sample flow rate has to be kept within the recommended range to avoid overlapping events (< 3,000 events/second for a 70 µm nozzle, BD Aria II).
Ice-cold collection buffer should be used to increase the viability of sorted cells.
While our strategy focuses on isolating and characterizing non-hematopoietic cells from the human bone marrow microenvironment using a specific marker panel, it is important to acknowledge a limitation in the underrepresentation of bone-associated cells such as osteoblasts and chondrocytes from the analysis.
Lastly, as human samples generally demonstrate high person-to-person diversity, it is recommended to collect as many samples as possible to ensure reproducible results.
Acknowledgments
This work was supported by funds from the StemTherapy Program, the Swedish Cancer Foundation, the Swedish Childhood Cancer Foundation, the Swedish Bloodcancer Association (Blodcancerförbundet), Foundation Siv-Inger and Per-Erik Anderssons minnesfond, John Persson Foundation, ALF (Government Public Health Grant), and the Skåne County Council Research Foundation. The authors would like to thank Helene Larsson and Maria Nilsson for their help to collect bone marrow samples and the Lund Stem Cell Center FACS facility personnel for technical assistance. The protocol described in this paper is published in eLife (Li et al. [3]. Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing. doi: 10.7554/eLife.81656).
Competing interests
The authors declare no conflicts of interest.
Ethical considerations
Human bone marrow was aspirated from the iliac crest bone of consenting healthy donors. The use of human samples was approved by the Regional Ethics Review Board in Lund, Sweden.
References
Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., Marshak, D. R., et al. (1999). Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science. 284(5411): 143–147.
Li, H., Ghazanfari, R., Zacharaki, D., Ditzel, N., Isern, J., Ekblom, M., Méndez-Ferrer, S., Kassem, M. and Scheding, S. (2014). Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow. Stem Cell Rep. 3(6): 965–974.
Li, H., Bräunig, S., Dhapolar, P., Karlsson, G., Lang, S. and Scheding, S. (2023). Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing. eLife. 12: e81656.
Penninger, J. M., Irie-Sasaki, J., Sasaki, T. and Oliveira-dos-Santos, A. J. (2001). CD45: new jobs for an old acquaintance. Nat Immunol. 2(5): 389–396.
Mao, B., Huang, S., Lu, X., Sun, W., Zhou, Y., Pan, X., Yu, J., Lai, M., Chen, B., Zhou, Q., et al. (2016). Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a, CD34, and CD36. Stem Cell Rep. 7(5): 869–883.
Jones, E. A., Kinsey, S. E., English, A., Jones, R. A., Straszynski, L., Meredith, D. M., Markham, A. F., Jack, A., Emery, P., McGonagle, D., et al. (2002). Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum. 46(12): 3349–3360.
Quirici, N., Soligo, D., Bossolasco, P., Servida, F., Lumini, C. and Deliliers, G. L. (2002). Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol. 30(7): 783–791.
Ahrens, N., Tormin, A., Paulus, M., Roosterman, D., Salama, A., Krenn, V., Neumann, U. and Scheding, S. (2004). Mesenchymal Stem Cell Content of Human Vertebral Bone Marrow. Transplantation. 78(6): 925–929.
Gleitz, H. F., Snoeren, I. A., Fuchs, S. N., Leimkühler, N. B. and Schneider, R. K. (2021). Isolation of human bone marrow stromal cells from bone marrow biopsies for single-cell RNA sequencing. STAR Protoc. 2(2): 100538.
Baryawno, N., Przybylski, D., Kowalczyk, M. S., Kfoury, Y., Severe, N., Gustafsson, K., Kokkaliaris, K. D., Mercier, F., Tabaka, M., Hofree, M., et al. (2019). A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia. Cell. 177(7): 1915–1932.e16.
Boulais, P. E., Mizoguchi, T., Zimmerman, S., Nakahara, F., Vivié, J., Mar, J. C., van Oudenaarden, A. and Frenette, P. S. (2018). The Majority of CD45– Ter119– CD31– Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors. Immunity. 49(4): 627–639.e6.
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Stem Cell > Adult stem cell > Hematopoietic stem cell
Cell Biology > Cell isolation and culture > Cell isolation
Cell Biology > Single cell analysis > Flow cytometry
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A Flow Cytometry–Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells
AS Alex Shepherd
BB Bigitha Bennychen
ZA Zafer Ahmed
RW Risini D. Weeratna
Scott McComb
Published: Vol 14, Iss 12, Jun 20, 2024
DOI: 10.21769/BioProtoc.5021 Views: 1039
Reviewed by: Alka MehraMartin V KolevNavnita Dutta
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Original Research Article:
The authors used this protocol in Frontiers in Immunology Jul 2022
Abstract
Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector–target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality.
Key features
• Determines CAR receptor cellular interaction kinetics using a Jurkat cell model.
• Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets.
• Experiments can be performed in under two hours with no staining using a standard flow cytometer.
• Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.
Keywords: CAR-T Jurkat Screening T cell Cellular avidity High throughput Flow cytometry Cell-to-cell interaction Cellular kinetics
Graphical overview
Background
Chimeric antigen receptor (CAR) T-cell therapy is an engineered cellular cancer therapy that reprograms a patient’s T cells to target a specific protein, mimicking natural T-cell receptor (TCR) function but redirected toward antigens found on the surface of cancer cells. CAR-T cells have emerged as a highly successful tool for providing robust therapeutic responses against refractory or relapsed hematological cancers, with ongoing research across many domains of immunology to extend the range and efficacy of CAR-T treatments for solid tumor malignancies [1–3].
Many advancements have been made in understanding the novel biology of CAR-modified T cells; however, CAR-T-cell screening models still struggle to choose candidate binding elements based on in vitro assessments of CAR-T activation and cytotoxicity assays. Whilst activation markers, such as CD69, are well-documented using CAR-Jurkat and primary CAR-T-cell screening methods, it is unclear how well these markers can predict in vivo efficacy of the CAR-T cells [4,5]. Recently, cellular avidity was recognized as a property of CAR-expressing cells that varies between different CAR constructs and correlates with important functional properties such as CAR expansion, CAR-activated trogocytosis, and CAR-T exhaustion [6–9].
Cellular avidity is defined by the total sum of cell-to-cell interactions, encompassing the whole immunological synapse and many cellular adhesion processes [10]. Given that cell binding is a near-immediate consequence of CAR signaling and proximal T-cell response processes, understanding the nature of this interaction and how it differs between standard TCR interaction vs. CARs is an important area of research. Investigating how cellular avidity changes with different CAR binding domains or structural designs is also vital for improving CAR discovery and development. It is important to note that while the specific properties of an antibody used to create a CAR impact the overall cellular avidity, the affinity and avidity of an antibody are entirely distinct from the complete cell-to-cell interaction that defines “cellular avidity”.
For soluble proteins such as antibodies, avidity is defined as the overall accumulated strength of interaction through multiple interactions, but it can vary widely depending on the nature of the target antigen and the specific conditions of the assay. The measured avidity of an antibody is usually reported as the half-maximal binding concentration observed over a titrated binding assay, this property being referred to as the apparent affinity (KD). In contrast to antibodies, using the assay described herein, we have observed that the half-maximal binding of CAR-expressing cells over a titrated range of target cell concentration does not vary between CAR proteins; rather, we find that CAR cells consistently reach a point of saturated maximal binding, wherein a varying proportion of CAR-expressing cells will form a strong bond with target cells (we refer to this as a CAR-target doublet). The strength of this interaction, while not encompassing the entirety of the kinetics, approximates what is commonly called cellular avidity. This observation of a varying binding rate between CAR-expressing and target cells has similarly been reported using an assay based on a resonance force ramp microscopy using the Lumicks zMovi device [6], which directly tests the strength of interaction by attempting to measure the force at which CAR-target pairs are dissociated. We find that the rate of CAR-target doublet formation increases with higher target cell density, eventually reaching a saturation point. Furthermore, we find the rate of doublet formation varies between different CARs [11] or CAR designs [5]. While this assay does not directly test the strength of interaction and therefore is only an approximation of overall cellular avidity, we believe the unique target binding properties for each CAR serve as valuable metrics for CAR screening and assessment. As such, this assay could be used as a replacement to, or in conjunction with, direct avidity assays for optimal CAR selection.
Here, we provide a complete description of our cellular binding assay that serves as a quick, high-throughput addition to CAR-T screening workflows using a standard flow cytometer. Through the quantification of cell/target doublets formed within CAR Jurkat target cell co-cultures over a wide range of effector-to-target ratios, the unique cell binding properties associated with each CAR can be determined. This assay provides consistent results and can be employed with both solid tumors (see validation of protocol) and hematological tumor models.
Materials and reagents
Biological materials
Jurkat e6.1 (ATCC, catalog number: TIB 152) expressing a variety of CAR proteins
Ramos (ATCC, catalog number: CRL-1596) or other relevant target tumor cell line(s)
Target cells have been engineered with Nuclight Lenti-Red (Sartorius, Germany; 4476). It is also possible to use non-fluorescent cells for this assay, but antibody prestaining of targets or effectors is recommended as detailed in section B.
Reagents
Fetal Bovine Serum (FBS) (Sigma Life Sciences, catalog number: F2442-500ML)
L-Glutamate (Gibco, catalog number: 25030-081)
Penicillin/Streptomycin (Pen/Strep) (Gibco, catalog number: 15140-122)
RPMI 1640 (Gibco, catalog number: 21870-076)
Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, catalog number: 14190-144)
VHH or scFv-specific fluorescent antibody (optional) (generated in-house)
An analogous anti-VHH polyclonal product (Jackson Laboratories, catalog number: 128-605-232)
For other CARs, various anti-scFv or anti-linker antibodies are available
CD45 or other Jurkat/T-cell specific antibody (optional) (BD Pharmingen, catalog number: 560178)
CD19 or other target-specific antibody (optional) (BD Horizon, catalog number: 612938)
Solutions
R10 complete (see Recipes)
Recipes
R10 complete
Reagent Final concentration Quantity or Volume
RPMI 1640 500 mL
FBS 100 mL/L 50 mL
L-Glutamate 10 mL/L 5 mL
Pen/strep 10 mL/L 5 mL
Laboratory supplies
96-well U-bottom plate (Falcon, catalog number: 353077)
Equipment
BD LSRFortessa flow cytometer
BD LSRFortessa flow cytometer HTS plate reader
Software and datasets
FACS Diva
FlowJo
GraphPad Prism 10
Procedure
Stable CAR Jurkat preparation
Notes:
1) This assay has been optimized to use both target and effector cells to sort for 100% CAR or reporter-positive cells. Doing so will significantly improve data analysis and data quality.
2) We also recommend testing any cells being used for mycoplasma before use in this assay.
3) CAR Jurkat cells and target cells should be in healthy log-phase growth conditions prior to initiating the avidity assay, as described below. This can be accomplished by splitting cells 1–2 days before use.
4) If using adherent cells, aim to have cells with a confluency of 70%–80%.
5) Jurkat cells can be visually assessed for cell health (healthy Jurkat cells will be round and form small clumps) alongside a viability stain when counting. Do not use cells that are less than 80% viable. We have found that cell health can have a major impact on the consistency of results for this assay.
Cells used to make stable CAR Jurkat or Nuclight target cells should be healthy, log-phase cells prior to lentivirus exposure. Lentiviral transduction should only be performed by trained professionals and handled with all safety protocols in mind. A full lentivirus creation and transduction protocol can be found in Tandon et al. [12]. Please note that all work using active lentivirus requires BSL2+ safety and training.
While details are not provided here, a general workflow for generating stable CAR Jurkat cells involves the following steps: CAR-lentiviral particle production as per linked protocol; (optional) lentiviral concentration using high-speed centrifugation; transduction of Jurkat cells with CAR-lentiviral particles; cell sorting to isolate a pure population of CAR Jurkat cells; and (optional) cryopreservation of the CAR Jurkat cell line for various downstream analyses, such as the one described here.
Once transduced cells are viral vector–free (typically after three media changes and at least 7 days at 37 °C), cells can be safely sorted for 100% CAR or fluorescent marker expression for best results. To generate target cells with stable Nuclight-Lenti (Sartorius, USA) expression, puromycin selection can be used. For stable cells with no resistance genes, the best option is the derivation of single-cell clone populations using cell sorting or limiting dilutions.
CAR Jurkat binding assay plate setup
Remove your target and stable CAR Jurkat cells from the incubator and count them, ensuring cells have at least 80% viability and are in the log phase of growth. We recommend not using cells that have been in culture for more than three months.
Spin down and bring CAR Jurkat and target cells to 1 million cells/mL in separate suspensions in R10 complete media. To enhance discrimination between effector cells, target cells, or doublets, you may use prestaining with antibodies that are specific to effector cells (e.g., CD45 or anti-VHH/scFv) or target cells (e.g., CD19). This is especially helpful if you are not using cells with stable fluorescent protein expression, as shown in Figure 1. If you are using an antibody stain for your CAR or target cells, spin down and stain your cells now.
For staining, resuspend the cell pellet in 100 µL of R10 complete medium, stain, and leave in the dark at room temperate for 15 min before washing off the excess stain. To wash, add 5 mL of 1× PBS and spin down the cells at 500× g for 3 min before removing the PBS and resuspending the cells in R10 complete medium, bringing the cell concentration back up to 1 M/mL.
Exemplary data provided here uses the Ramos human lymphoma cell line as the target cell line, although this assay can be successfully performed with a wide variety of adherent and non-adherent target cells.
Add 50 µL of R10 complete medium to each well of your 96-well plate.
Add target cells to each well at the ratio described in Table 1 below. An example plate map is also given in Table 2.
Table 1. Effector-to-target dilution chart
Ratio # of cells (effector/target) Vol. needed at 1 M cell/mL effector (µL) Vol. needed at 1 M cell/mL target (µL) Row letter
1:10 5,000/45,000 5 45 A
1:5 10,000/40,000 10 40 B
1:2 15,000/30,000 15 30 C
1:1 25,000/25,000 25 25 D
2:1 30,000/15,000 30 15 E
5:1 40,000/10,000 40 10 F
10:1 45,000/5,000 45 5 G
Table 2. Example plate map
E:T
CAR Samples
CAR1 CAR2 CAR3 CAR4 CAR5 Control (irrelevant CAR)
1:10
1:5
1:2
1:1
2:1
5:1
10:1
No target
Plate Jurkat cells at the appropriate concentration as shown above. If done correctly, all wells should contain 100 µL of sample. Manually and gently shake the plate from side to side.
Place the 96-well plate in an incubator at 37 °C and 5% CO2 for 30 min.
If desired, this assay can be run at 4 °C as a control. Doublets should not form at this temperature.
This assay has been performed in as little as 10 min and as long as 4 h. Thirty minutes is our recommended minimum time for best results. It should be noted that, if this assay is being performed with an adherent target cell line, the incubation should not last long enough for cells to adhere.
CAR Jurkat avidity flow cytometry
During the plate incubation, start up the flow cytometer and perform Cytometer Setup and Tracking (CS&T) and a system prime of the high-throughput sampler (HTS) to ensure the flow cytometer is functioning properly.
Once the incubation period has elapsed, run the plate on the flow.
This assay is optimized for an HTS setup for the BD LSRFortessa. Depending on your equipment, some modifications may be necessary.
While some settings may vary, those used for our setup are as follows:
1) Samples were run on a 96-well U-bottom plate, collecting 75 µL of sample or 50,000 events per well.
2) Cells were mixed by the cytometer and collected at a rate of 3 µL/s.
The relevant voltage settings are as follows:
a) FSC 180 V
b) SSC 250 V
c) FITC 480 V
d) PerCP-Cy5-5 730 V
e) APC 600 V
Data analysis
Data analysis of the raw flow data should be performed in the latest version of FlowJo but could be similarly performed with alternative flow cytometry analysis software. To start, gate out dead cells or cellular debris using forward and side scatter (Figure 1, top left). From here, analysis can be performed in two separate ways to obtain cellular avidity reading. If you are using stained cells or cell lines with a reporter, set your laser reading to display your cellular stains for the CAR and your target cell line with acceptable signal strength. The example shown below uses stable fluorescent markers: NeonGreen (FITC) for the CAR and Nuclight Red (or mKate2; PerCP Cy5.5) for the target cell lines. Should the cells bind, this should show three or four separate populations: unbound CAR Jurkat cells, unbound target cells, and bound doublets that express both the CAR and target colors (Figure 1, top middle). If your stable population of CAR Jurkat is unsorted, you may see an unstained population of non-CAR expressing Jurkats as well.
Figure 1. Gating method for fluorescently labeled and sorted cells. Dead cells and debris are gated out (top left), then unbound targets are removed (top middle). Finally, doublets are separated from unbound chimeric antigen receptor (CAR) Jurkat cells and displayed as a percentage of parent (top right). CAR confirmation of doublets can be confirmed using a CAR-specific surface stain (APC channel here), removing auto-fluorescent false positives from doublets that are dead cells or cellular debris (bottom, left). An alternative method of analyzing doublets using their increased size on forward scatter (bottom, middle). This method will require some additional calculations to find the number of living Jurkat cells.
If you do not have stains in your CAR or target cells, instead display the forward scatter height by the forward scatter width. Cell doublets present in the well should be wider than the rest of the cells and can be selected alongside the other unbound Jurkats to give a similar result (see Figure 1, bottom middle). It should be noted that if the target cells are significantly bigger than the Jurkat cells, this method may not work as well, so we recommend using a staining method if possible.
To generate a CAR:target curve, use flow cytometry analysis software to exclude unbound target cells and then record your doublet population as a percentage of parents of the entire Jurkat population in the well (Figure 1, right). This will give the percentage of the CAR Jurkat population bound at that time and ratio. The ratio of bound doublet cells to free CAR Jurkat can be calculated manually as follows:
This data can be graphed using appropriate data visualization software such as GraphPad PRISM 10, resulting in a curve with maximum binding occurring in an excess of target cells (>1:10–1:25 effector:target ratio) (see Figure 2).
Figure 2. Exemplary data of CD22 chimeric antigen receptor (CAR) Jurkat binding kinetics against Ramos WT cells. The data displayed was performed in triplicate and displays data plus standard error of mean (SEM). Note that the “Irrelevant CAR” data set (green) has been nudged up two data points for visibility. Exported data was imported into PRISM 10 and graphed in the “grouped” format.
Validation of protocol
This protocol or parts of it have been used and validated in the following research articles:
McComb et al. [5]. Programmable attenuation of antigenic sensitivity for a nanobody-based EGFR chimeric antigen receptor through hinge domain truncation. Frontiers in Immunology. 13. (Figure 4, Supplementary Figure 4)
McComb et al. [11]. Discovery and Pre-Clinical Development of a Therapeutically Active Nanobody-based Chimeric Antigen Receptor targeting human CD22. Molecular Therapy Oncology. 32, 1, 200775. (Figure 2F)
General notes and troubleshooting
General notes
As mentioned above, this assay is biological in nature and requires active cellular signaling to induce cell-to-cell interaction. No cell-to-cell binding will occur at 4 °C, as signaling processes are inactive at this temperature. This can be used to test for non-specific binding and establish a background (Figure 3). Additionally, we recommend running the assay in parallel with a negative cell line and a non-specific/irrelevant specificity CAR to establish non-specific binding control.
Figure 3. Chimeric antigen receptor (CAR)-Jurkat binding assessment using a temperature curve. (A) CD22 CAR-J cells were run first at 1:25 effector:target starting at 4 °C and incrementing the temperature gradually until 37 °C. Then, the binding assay was repeated in full at 4 °C. CAR-J cells cannot bind targets at 4 °C and only begin to show binding at approximately 21 °C. (B) The standard assay described above performed with target antigen-negative cells. Experimental data was performed in triplicate. Error bars show standard error of mean (SEM).
This assay is not CAR exclusive. This can work with any biological that causes T cells to bind, such as bispecific T cell engagers (BiTEs), that cause cell binding and T-cell synapse formation.
We are currently working on a version of this assay using lentiviral-transduced T cells. The primary cell version can be successful, but the nature of expanded donor T cells requires additional attention to experimental details to yield consistent results. Specifically, the proliferation state, transduction rate, and donor source are all key variables that can interfere with the measurement of CAR cellular avidity. Thus, we feel that this assay is best measured using Jurkat T cells for several reasons: (1) Jurkat cells lack a cytotoxic response and have limited cytokine activity, with no risk of the bound cell dying before passing through the flow cytometer; (2) Jurkat CAR cells can be sorted to 100% CAR cells without issues due to their steady proliferation; (3) Jurkat cells maintain a consistent differentiation profile, while primary T cells can vary widely throughout manipulations. For these reasons, we recommend using Jurkat cells for performing this assay.
Troubleshooting
Problem 1: Cells do not form doublets in co-culture within 30 min.
Possible cause: Wrong culture plate used.
Solution: Change the assay plate to a 96-well treated U-bottom plate. This assay is optimized for U-bottom 96-well plates. Attempts in larger wells or wells with flat bottoms generally result in sub-optimal results.
Possible cause: Cells are unhealthy.
Solution: Re-attempt assay with cells in log-phase of growth. Cell health has been shown to have a direct impact on the speed and count of doublets in solution.
Problem 2: Cells do not form doublets ever.
Possible cause: Incubation is performed in untreated plastic or the wrong plastic type.
Solution: Ensure the assay is performed in a 96-well plate made of non-pyrogenic vacuum gas plasma-treated polystyrene. Incubations done in polypropylene tubes such as PCR tubes or 1.5 mL Eppendorf or in untreated polystyrene have been unsuccessful, as it appears the cells adhere to the plastic and have difficulty binding.
Acknowledgments
This project was funded and supported by the Cancer Immunology team, of the Human Health Therapeutics branch of the Canadian National Research Council. Graphical overview made in BioRender.com.
Competing interests
There are no conflicts of interest or competing interests.
References
Lu, J. and Jiang, G. (2022). The journey of CAR-T therapy in hematological malignancies. Mol Cancer. 21(1): 194.
Marofi, F., Motavalli, R., Safonov, V. A., Thangavelu, L., Yumashev, A. V., Alexander, M., Shomali, N., Chartrand, M. S., Pathak, Y., Jarahian, M., et al. (2021). CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res Ther. 12(1): 81.
Zhang, X., Zhu, L., Zhang, H., Chen, S. and Xiao, Y. (2022). CAR-T Cell Therapy in Hematological Malignancies: Current Opportunities and Challenges. Front Immunol. 13: e927153.
Lee, Y. G., Chu, H., Lu, Y., Leamon, C. P., Srinivasarao, M., Putt, K. S. and Low, P. S. (2019). Regulation of CAR T cell-mediated cytokine release syndrome-like toxicity using low molecular weight adapters. Nat Commun. 10(1): 2681.
McComb, S., Nguyen, T., Shepherd, A., Henry, K. A., Bloemberg, D., Marcil, A., Maclean, S., Zafer, A., Gilbert, R., Gadoury, C., et al. (2022). Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation. Front Immunol. 13: e864868.
Halim, L., Das, K. K., Larcombe-Young, D., Ajina, A., Candelli, A., Benjamin, R., Dillon, R., Davies, D. M. and Maher, J. (2022). Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation. Front Immunol. 13: e836549.
Leick, M. B., Silva, H., Scarfò, I., Larson, R., Choi, B. D., Bouffard, A. A., Gallagher, K., Schmidts, A., Bailey, S. R., Kann, M. C., et al. (2022). Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia. Cancer Cell. 40(5): 494–508.e5.
Olson, M. L., Mause, E. R. V., Radhakrishnan, S. V., Brody, J. D., Rapoport, A. P., Welm, A. L., Atanackovic, D. and Luetkens, T. (2022). Low-affinity CAR T cells exhibit reduced trogocytosis, preventing rapid antigen loss, and increasing CAR T cell expansion. Leukemia. 36(7): 1943–1946.
Zhang, Y., Patel, R. P., Kim, K. H., Cho, H., Jo, J. C., Jeong, S. H., Oh, S. Y., Choi, Y. S., Kim, S. H., Lee, J. H., et al. (2023). Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study. Mol Cancer. 22(1): 200.
Erlendsson, S. and Teilum, K. (2021). Binding Revisited—Avidity in Cellular Function and Signaling. Front Mol Biosci. 7: e615565.
McComb, S., Arbabi-Ghahroudi, M., Hay, K. A., Keller, B. A., Faulkes, S., Rutherford, M., Nguyen, T., Shepherd, A., Wu, C., Marcil, A., et al. (2024). Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22. Mol Ther Oncol. 32(1): 200775.
Tandon, N., Thakkar, K., LaGory, E., Liu, Y. and Giaccia, A. (2018). Generation of Stable Expression Mammalian Cell Lines Using Lentivirus. Bio Protoc. 8(21): e3073.
Supplementary information
The following supporting information can be downloaded here:
Figure S1. Cytometer and HTS setup.
Article Information
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Category
Immunology > Immunotherapy > CAR-T
Cell Biology > Cell-based analysis > Cell adhesion
Cancer Biology > Tumor immunology > Cancer therapy
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5,022 | https://bio-protocol.org/en/bpdetail?id=5022&type=0 | # Bio-Protocol Content
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Peer-reviewed
Bilateral Common Carotid Artery Stenosis in Mice: A Model of Chronic Cerebral Hypoperfusion-Induced Vascular Cognitive Impairment
MK Masashi Kakae
AK Ayaka Kawashita
HO Haruya Onogi
TN Takayuki Nakagawa
Hisashi Shirakawa
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5022 Views: 835
Reviewed by: Xiaoyi Zheng Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Advances Jul 2023
Abstract
Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease and is associated with various types of dementia. Chronic cerebral hypoperfusion (CCH) is one of the major contributors to VCI. Among the various rodent models used to study CCH-induced VCI, we have found the mouse bilateral common carotid artery stenosis (BCAS) model to be highly suitable. Here, we introduce the BCAS model of C57BL/6J mice generated using microcoils with an internal diameter of 0.18 mm. To produce the mouse BCAS model, the bilateral common carotid arteries are isolated from the adhering tissues and vagus nerves and twined around the microcoils. This model shows cognitive impairment and white matter lesions preceding neuronal dysfunction around postoperative day 28, which is similar to the human clinical picture. Overall, the mouse BCAS model will continue to be useful in studying CCH-induced VCI.
Key features
• This mouse BCAS model requires approximately 4 weeks to show phenotypes such as cognitive impairment and white matter injury.
Keywords: Bilateral common carotid artery stenosis (BCAS) Vascular cognitive impairment (VCI) Chronic cerebral hypoperfusion (CCH) White matter injury Microcoil Vagus nerve
Graphical overview
Overview of the mouse BCAS model. The BCAS-operated mouse develops CCH and white matter injury, leading to cognitive impairment. This BCAS model is considered to be a suitable and useful CCH-induced VCI model. BCAS, bilateral common carotid artery stenosis; CCA, common carotid artery; CCH, chronic cerebral hypoperfusion; ECA, external carotid artery; ICA, internal carotid artery; VCI, vascular cognitive impairment.
Background
Vascular cognitive impairment (VCI) refers to cognitive alterations related to vascular disease and is associated with various types of dementia [1]. Chronic cerebral hypoperfusion (CCH)-associated small vessel disease is one of the major contributors to VCI [2,3]. CCH is elicited by aging and various lifestyle diseases, such as metabolic syndromes, atherosclerosis, hypertension, obesity [4], and hypotension [5]. It has also been suggested that CCH induces white matter lesions, which are key characteristics of VCI. In addition, many patients with various types of dementia including VCI have white matter lesions [6]. To understand the precise pathological mechanism underlying CCH-induced VCI including white matter lesions, multiple rodent models have been used in many studies.
Some widely used rodent models of CCH-induced VCI are the rat bilateral common carotid artery occlusion (BCCAO), mouse unilateral common carotid artery occlusion (UCCAO), rat and mouse 2-vessel gradual occlusion (2-VGO), mouse asymmetric common carotid artery surgery (ACAS), and mouse bilateral common carotid artery stenosis (BCAS) models. However, rodent models have several limitations. First, the visual pathway is damaged and behavioral tests to assess cognitive function are affected in the rat BCCAO and mouse UCCAO models [7,8]. Second, the device used in the rat and mouse 2-VGO models and mouse ACAS model is expensive [9]. The mouse BCAS model was reported as a CCH model in 2004 by Shibata et al. [10] and is a more suitable and useful tool for research of CCH-induced VCI. This model shows cognitive impairment and a decrease in myelin sheaths without severe damage to the visual pathway [9]. In addition, BCAS-induced cognitive impairment and white matter lesions precede neuronal death [11,12]. Therefore, the mouse BCAS model reproduces the clinical picture in human VCI patients [13]. However, surgery is harder to perform in this model than in the rat BCCAO and mouse UCCAO models, and expertise is needed to create a stable mouse model. Therefore, we present a detailed and robust method that can stably create the mouse BCAS model used in our previous studies [11,12,14] for further development of VCI research.
Materials and reagents
Mice: Male C57BL/6J, 8–12 weeks old, 20–30 g
Isoflurane (Viatris, catalog number: 901036504)
Equipment
Animal anesthetizer (Biomachinery, catalog number: TK-7)
Surgical microscope (Carl Zeiss, catalog number: OPMI11/S21)
Surgical equipment and materials (Figure 1)
Curved suture needle (Natsume Seisakusho, catalog number: C-24-500-1)
5-0, white braided silk sutures (Akiyama Medical, catalog number: EWB0514)
Needle holder (SHIN-EI, catalog number: MNH-1)
Micro spring scissors (Natsume Seisakusyo, catalog number: MB-56)
Two forceps, straight sharp (Fine Science Tools, catalog number: 11293-00)
Graefe forceps (Fine Science Tools, catalog number: 11051-10)
Microcoils with a wire diameter of 0.08 mm, an internal diameter of 0.18 mm, a pitch of 0.50 mm, and a total length of 2.5 mm (Name abbreviation: microcoil 0.08 × 0.18 × 0.50 × 2.5), which are custom-made (Sawane Spring or Komatsu Spring). To obtain microcoils, contact the manufacturers in English via e-mail or website form as follows:
Sawane Spring Co., Ltd.
Website: https://www.sawane-spring.com
Contact form: https://www.sawane-spring.com/cgi-bin/contact/form.cgi
E-mail address: [email protected]
Komatsu Spring Industrial Co., Ltd.
Website: https://www.komatsubane.com/english/
Contact form: https://www.komatsubane.com/english/contact_us/
Handmade wire hook [kite string-tied bent 27 G needle with the tips cut off (TERUMO, catalog number: NN-2719)]
Paper towel (NIPPON PAPER CRECIA, catalog number: 61001)
Figure 1. Surgical equipment and materials. A. Curved suture needle and silk suture. B. Needle holder. C. Micro spring scissors. D. Two straight sharp forceps. E. Curved forceps. F. Handmade wire hook. G. Microcoil with an internal diameter of 0.18 mm.
Procedure
Anesthesia (presumably, any would be possible)
Anesthetize the mice with 3% isoflurane in 30% O2 and 70% N2O for ~3 min in the incubation box.
Maintain anesthesia throughout surgery with 1.5% isoflurane in 30% O2 and 70% N2O using a face mask.
Isolation of the common carotid artery (CCA)
Place an anesthetized mouse in the supine position with the four limbs spread out.
Make a ~1–1.5 cm ventral cervical skin incision in the midline (Figure 2A).
Separate the submandibular glands laterally to make the trachea visible (Figure 2B).
Move the sternocleidomastoid muscle laterally to make the CCA sheath visible by a handmade wire hook with a weight to open the surgical area (Figure 2C).
Carefully isolate the CCA from the adhering tissues and vagus nerve using forceps. Be careful not to damage the CCA and vagus nerve (Figure 2D, Video 1).
Note: The CCA and vagus nerve are wrapped in a transparent thin membrane, and the vagus nerve is often seen at a deep depth (dorsal) and on the lateral side of its sheath (Figure 2E).
Figure 2. Generation of a ventral cervical skin incision and isolation of the CCA (section B). A. A cervical skin incision was made, and the submandibular glands were visualized. B. The submandibular glands were separated, and the trachea was exposed. C. The surgical area was opened by moving the sternocleidomastoid muscle laterally and the CCA was visible. D. The CCA was isolated from the adhering tissues and vagus nerve with forceps. E. Before isolation of the CCA, the vagus nerve was wrapped in a transparent membrane with the CCA. The dotted line shows the vagus nerve. CCA, common carotid artery.
Putting microcoils on the CCAs
Gently lift the isolated CCA and put a microcoil under it vertically. Put the CCA in the center of the microcoil (Figure 3A, Video 1).
Grab one side of the microcoil and twine the CCA around the other side of the microcoil (Figure 3B, Video 1).
Twine the CCA around the other side of the microcoil in the same way as described in step C2 (Figure 3C, Video 1).
Remove the wire hook and place the sternocleidomastoid muscle and submandibular glands back.
Repeat the procedure described in steps C3 and C4 with the other CCA. The other CCA is on the other side of the trachea at the symmetrical position. As described in step B4, the other CCA sheath is also visible after moving the other sternocleidomastoid muscle laterally.
Figure 3. Placement of a microcoil on the CCA and completion of stenosis (section C). A. A microcoil was put under the isolated CCA. B. The CCA was twined around half of the microcoil. C. The CCA was twined around the other side of the microcoil, and stenosis was completed. CCA, common carotid artery.
Video 1. Surgical procedure for the isolation of the CCA and stenosis by a microcoil (steps B5–C3)
Completion of surgery
Suture the surgical cervical skin incision with a 5-0 silk suture in three interrupted sutures.
Put the mouse back into a clean cage and house the mice at a constant ambient temperature of 22 ± 2 °C under a 12/12 h light/dark cycle. Allow them food and water ad libitum.
Procedure of the sham operation
Conduct the procedure described in steps A1–B5.
Place the sternocleidomastoid muscle and submandibular glands back in the same way as described in step C4 without putting a microcoil on the CCA and repeat for the other CCA.
Suture the surgical incision and put the mouse back into the cage as described in steps D1 and D2.
Data analysis
We measured regional cerebral blood flow (rCBF) at 60 min after surgery with laser Doppler flowmetry [12]. BCAS surgery successfully reduced rCBF to ~65% of the baseline, which is similar to the data reported by Shibata et al. [10]. In our previous studies, this mouse BCAS model generated using wildtype mice (8–12 weeks old, 20–30 g) showed a decrease in the myelin density by myelin staining and cognitive impairment in a novel object recognition test on postoperative day 28, whereas there were no changes in the number of neuronal cells detected by immunostaining or spatial memory in a novel location recognition test [11,12,14].
Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:
Shibata et al. [10]. White Matter Lesions and Glial Activation in a Novel Mouse Model of Chronic Cerebral Hypoperfusion. Stroke (Figures 1A–C, 2B, and Table 1).
Miyanohara et al. [11]. TRPM2 Channel Aggravates CNS Inflammation and Cognitive Impairment via Activation of Microglia in Chronic Cerebral Hypoperfusion. J Neurosci (Figures 1F and 2A, B, D, E).
Kakae et al. [12]. The astrocytic TRPA1 channel mediates an intrinsic protective response to vascular cognitive impairment via LIF production. Sci Adv (Figure 1P–R and supplementary figure 1D).
General notes and troubleshooting
Do not damage CCAs and the vagus nerves. In particular, carefully conduct the procedures during isolation and twining of CCAs.
Fully twine CCAs on microcoils. If CCAs are positioned off the end of microcoils, re-twine them or rotate microcoils.
Conduct the procedures as quickly as possible. If possible, complete this surgery within ~10–20 min and take at most 30 min.
Put microcoils on the lower branch of the carotid artery, not the internal carotid artery or external carotid artery.
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from MEXT/JSPS (to H.S., JP19H03377, JP23H02639), Grant-in-Aid for Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan (to M.K., N-194402), Grants-in-Aid for JSPS Fellows (to M.K., JP20J20232), and also by the Takeda Science Foundation (to H.S.) and the Uehara Memorial Foundation (to H.S.). This protocol was adapted from the publication Kakae et al. [12].
Competing interests
The authors declare that they have no competing interests.
Ethical considerations
All animal experiments were conducted in accordance with the ethical guidelines of the Kyoto University animal experimentation committee and the Japanese Pharmacological Society. All animal use and study protocols were approved by the Kyoto University animal experimentation committee (approval number: 20-42, 20-42-2, 20-42-3, 20-42-4).
References
Rosenberg, G. A., Wallin, A., Wardlaw, J. M., Markus, H. S., Montaner, J., Wolfson, L., Iadecola, C., Zlokovic, B. V., Joutel, A., Dichgans, M., et al. (2015). Consensus statement for diagnosis of subcortical small vessel disease. J Cereb Blood Flow Metab. 36(1): 6–25.
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Article Information
Publication history
Received: Mar 25, 2024
Accepted: Jun 4, 2024
Available online: Jun 18, 2024
Published: Jul 5, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Phylogenomics of Plant NLR Immune Receptors to Identify Functionally Conserved Sequence Motifs
TS Toshiyuki Sakai
AT AmirAli Toghani
HA Hiroaki Adachi
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5023 Views: 1111
Reviewed by: Wenrong HeDemosthenis ChronisLiyuan Wang
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Original Research Article:
The authors used this protocol in eLIFE Nov 2019
Abstract
In recent years, the increase in genome sequencing across diverse plant species has provided a significant advantage for phylogenomics studies, allowing the analysis of one of the most diverse gene families in plants: nucleotide-binding leucine-rich repeat receptors (NLRs). However, due to the sequence diversity of the NLR gene family, identifying key molecular features and functionally conserved sequence patterns is challenging through multiple sequence alignment. Here, we present a step-by-step protocol for a computational pipeline designed to identify evolutionarily conserved motifs in plant NLR proteins. In this protocol, we use a large-scale NLR dataset, including 1,862 NLR genes annotated from monocot and dicot species, to predict conserved sequence motifs, such as the MADA and EDVID motifs, within the coiled-coil (CC)-NLR subfamily. Our pipeline can be applied to identify molecular signatures that have remained conserved in the gene family over evolutionary time across plant species.
Key features
• Phylogenomics analysis of plant NLR immune receptor family.
• Identification of functionally conserved sequence patterns among plant NLRs.
Keywords: Gene annotation Phylogenetic analysis Motif prediction NLR immune receptors Plant immunity
Background
Nucleotide-binding leucine-rich repeat receptors (NLRs) play a pivotal role in plants' innate immune system by recognizing pathogen-secreted molecules, ultimately triggering robust defense responses known as the hypersensitive response [1]. Plant NLRs are characterized by three principal domains: an N-terminal coiled coil (CC) or toll/Interleukin-1 receptor (TIR) domain, a central nucleotide-binding domain with APAF-1, various R proteins, and a CED-4 (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR) domain [2]. Although plant NLR proteins share the common domain architecture, plant NLRs often undergo rapid evolution and remarkable sequence diversification, reflecting a continuous arms race between plants and pathogens [3–5]. This sequence diversity in the NLR immune receptor family enables plants to recognize a wide range of pathogen-derived molecules, which are typically fast-evolving, to modulate the multi-layered plant immune system for successful pathogen infection. Therefore, understanding the evolutionary dynamics of the NLR gene family across diverse plant species provides valuable insights into the molecular mechanisms underlying the plant immune system and also for the breeding program of disease-resistant crops.
Previous studies have elucidated the molecular function of NLR proteins by site-directed mutations targeting conserved regions and motifs. For instance, mutations in the P-loop and MHD motifs within the central NB-ARC domain can render plant NLRs nonfunctional and autoactive, respectively [6,7]. Therefore, identifying evolutionally conserved motifs is a key strategy to pinpoint and understand yet unknown plant NLR functions. For smaller sets of sequences, multiple sequence alignment is an effective tool to uncover specific conserved regions and amino acid residues [8]. However, the multiple sequence alignment method encounters technical challenges with larger datasets, where identifying conserved regions becomes complicated due to the presence of gaps and deletions, especially in the diverse gene family of plant NLRs. To address this, we have developed a computational pipeline tailored for identifying conserved sequence patterns in the plant NLR family [9]. This approach integrates NLR gene annotation, phylogenetic analysis, clustering protein families, and prediction of conserved sequence motifs. Here, using a test NLR dataset from six representative plant species (previously analyzed in Adachi et al. [9]), we provide a step-by-step procedure to identify functionally conserved motifs in CC-type NLR (CC-NLR) subfamily (Figure 1). Given the development of genome sequencing technologies and rising access to genome databases of diverse plant species, our pipeline has the potential to be applied for further phylogenomics studies of plant NLR and other gene families.
Figure 1. Workflow to identify conserved sequence patterns in nucleotide-binding leucine-rich repeat receptors (NLR) family proteins by phylogenomics
Equipment
64-bit Linux operating system; alternatively, Mac OS X operating system
Software and datasets
Protein or nucleotide sequences from reference genome databases
InterProScan 5.53-87.0 [10]
NLRtracker v1.0.3 [2]
NLR-Annotator v2.1 [11]
MAFFT v7 [12]
RAxML v8.2.12 [13]
iTOL (Interactive Tree Of Life) [14]
BLAST+ v2.12.0 [15]
MCL v14-137 [16]
MEME Suite v5.5.5 [17]; same analysis can be performed through MEME website (https://meme-suite.org/meme/tools/meme)
HMMER v3.4 [18]
Test data are included as supplemental datasets in this manuscript and have been deposited in GitHub: https://github.com/slt666666/MADA_motif_protocol. Supplemental Python scripts can be used through Google Collaboratory: https://colab.research.google.com/github/slt666666/MADA_motif_protocol/blob/master/Supplemental_scripts.ipynb (Access date, 22/01/24). The instructions for using Supplemental Scripts are provided in Supplemental video file (Video S1).
Procedure
Software installation
InterProScan 5.53-87.0
InterProScan is a software that characterizes protein function. This program can be downloaded and installed by following the instructions provided at https://www.ebi.ac.uk/interpro/download/. It is compatible with 64-bit Linux operating systems. In this protocol, the InterProScan is utilized in the NLRtracker pipeline.
NLRtracker v1.0.3
NLRtracker is an NLR annotation tool that utilizes a protein sequence file as the input dataset. This program can be downloaded and installed by following the instructions provided at https://github.com/slt666666/NLRtracker. NLRtracker is more sensitive and accurate than other available tools for extracting NLRs from a given plant proteome [2]. Alternatively, NLR-Annotator v2.1 can be downloaded and installed by following the instructions available at https://github.com/steuernb/NLR-Annotator. NLR-Annotator is an NLR annotation tool designed to work with nucleotide sequence files as input datasets. This program is suitable for users who do not have access to a Linux system.
MAFFT v7
MAFFT is a program for multiple sequence alignment. This program can be downloaded and installed by following the instructions provided at https://mafft.cbrc.jp/alignment/software/.
RAxML v8.2.12
RAxML is a program for maximum likelihood-based inference of large phylogenetic trees. To download and install this program, please refer to PART and in the manual available at https://cme.h-its.org/exelixis/resource/download/NewManual.pdf. Alternatively, RAxML-NG v1.2.1 can be downloaded and installed on Unix/Linux and macOS systems by following the instructions provided at https://github.com/amkozlov/raxml-ng?tab=readme-ov-file.
BLAST+ v2.12.0
BLAST+ is a command-line tool to run BLAST in your own local environment. This program can be downloaded and installed by following the instructions provided at https://www.ncbi.nlm.nih.gov/books/NBK569861/.
MCL v14-137
MCL software is a program for clustering weighted or simple networks. To install blast packages in the MCL software, “--enable-blast” option is required. The installation commands are as follows:
wget http://www.micans.org/mcl/src/mcl-14-137.tar.gz
tar -zxvf mcl-14-137.tar.gz
cd mcl-14-137
./configure --enable-blast
Make
make install
MEME Suite v5.5.5
MEME Suite is a motif-based sequence analysis tool. This tool can be downloaded and installed by following the instructions provided at https://meme-suite.org/meme/doc/install.html. Alternatively, the same analysis can be performed through the MEME website at https://meme-suite.org/meme/tools/meme.
HMMER v3.4
HMMER is utilized for searching sequence homologs from sequence databases and for making sequence alignments. This program can be installed by following the instructions provided at https://github.com/EddyRivasLab/hmmer.
Annotate NLR gene family from proteome datasets
Download protein sequence files from reference genome databases.
As a test dataset, we used proteomes from six representative plant species: Arabidopsis thaliana, Beta vulgaris (sugar beet), Solanum lycopersicum (tomato), Nicotiana benthamiana, Oryza sativa (rice), and Hordeum vulgare (barley). The protein sequences were downloaded from the reference genome databases of Arabidopsis (https://www.araport.org/, Araport11), sugar beet (http://bvseq.molgen.mpg.de/index.shtml, RefBeet-1.2), tomato (https://solgenomics.net/, tomato ITAG release 2.4), N. benthamiana (https://solgenomics.net/, N. benthamiana genome v0.4.4), rice (http://rice.plantbiology.msu.edu/, rice gene models in Release 7) and barley (https://www.barleygenome.org.uk/, IBSC_v2) as used in Adachi et al. [9]. The protein sequences were compiled into a single fasta file named “NLRtracker_input_protein.fasta” (Dataset S1).
Annotate NLRs from protein sequences.
NLRs were annotated from the input protein sequence file “NLRtracker_input_protein.fasta” by running NLRtracker using the following command:
./NLRtracker -s NLRtracker_input_protein.fasta -o NLRtracker_output
The output NLR protein sequences were saved as “NLR.fasta” in the “NLRtracker_output” folder. In total, we identified 1,862 NLRs from six representative plant species.
Note: In a previous study, we used a tool, NLR-Annotator, to annotate NLR genes [11]. However, since NLR-Annotator may not detect a few functionally validated NLRs (e.g., ADR1), we employed NLRtracker [2] in this protocol. Therefore, test datasets in the following analyses slightly differ from the data reported in Adachi et al. [9].
Extract specific NLR subfamily sequences based on phylogenetic analysis.
In a previous study [9], we characterized a conserved sequence pattern (MADA motif) crucial for CC-NLRs to trigger immune responses. To identify conserved sequence patterns in each NLR subfamily, we initially classified NLRs through phylogenetic analysis. Here, the NLR sequences obtained in step B2 were combined with 31 functionally characterized CC-NLRs and saved as “NLR_set.fasta” (Dataset S2). Protein sequences in the input file “NLR_set.fasta” were aligned using MAFFT:
mafft NLR_set.fasta > NLR_set_alignment_output.fasta
For the phylogenetic analysis of the NLR family, NB-ARC domain sequences were extracted from the output alignment file “NLR_set_alignment_output.fasta” based on the NB-ARC domain sequence of Arabidopsis ZAR1 (Dataset S3). Extraction of NB-ARC domain sequences can be performed manually using alignment software or using our script (Supplemental script 1). In this script, protein sequences lacking the intact p-loop motif (G/AxxxxGKT/S) required for NLR protein function are automatically discarded from the dataset. Sequence gaps in the aligned NB-ARC domain sequences are also automatically deleted in this script. The sequences were saved as “NLR_set_alignment_NBARC_RemGap.fasta” (Dataset S4), which can be used as the input file for further phylogenetic analysis.
Note: We use conserved NB-ARC domain sequences for phylogenetic analyses of the NLR gene family because other domains, such as N-terminal domains and C-terminal LRR domain, are often too diversified and not suitable for inferring phylogenetic relations in NLRs.
The maximum likelihood phylogenetic tree was inferred by RAxML using the following command:
raxmlHPC-PTHREADS-AVX2 -s NLR_set_alignment_NBARC_RemGap.fasta -n NLR_MLtree -m PROTGAMMAAUTO -f a -# 100 -x 1024 -p 121
Note: The ‘-f’ and ‘-#’ options were set for 100 iterations of bootstrap. The ‘-x’ and ‘-p’ options were random seeds.
NLRs that belong to the CC-NLR phylogenetic subclade were classified with functionally characterized CC-NLRs on the NLR phylogenetic tree output file “RAxML_bipartitions.NLR_MLtree” (Figure 2; Dataset S5). We extracted 1,305 protein IDs (Dataset S6) of the CC-NLR clade in the NLR phylogenetic tree using iTOL [14]. For further sequence analysis, we extracted protein sequences of CC-NLRs from the input file “NLR_set.fasta” (Dataset S2) and “CCNLR_IDs.txt” (Dataset S6) using Supplemental script 2 and saved them as the output file “CCNLR_set.fasta” (Dataset S7).
Figure 2. Maximum likelihood phylogenetic tree for the classification of nucleotide-binding leucine-rich repeat receptors (NLR) subfamilies. Functionally characterized CC-NLRs are labeled in the phylogenetic tree using iTOL (Interactive Tree Of Life) [14]. The orange branches represent coiled-coil (CC)-NLRs used for further sequence analyses.
Classify N-terminal domain sequences of CC-NLRs using Markov cluster (MCL) algorithm
Extract N-terminal sequences from CC-NLRs.
Sequences prior to the NB-ARC domain were defined as N-terminal domain sequences. To extract N-terminal domain sequences, protein sequences in the input file “CCNLR_set.fasta” were aligned using MAFFT as described in section B3. N-terminal domain sequences of CC-NLRs were extracted from the alignment result, based on the start position of the NB-ARC domain of Arabidopsis ZAR1 (Dataset S3). Extraction of the N-terminal domain sequences can be done manually using alignment software or using our script (Supplemental script 3). The extracted N-terminal domain sequences were saved as the output file “CCNLR_Ndomain_set.fasta” (Dataset S8).
Cluster N-terminal domain sequences into protein families.
N-terminal domain sequences of CC-NLRs were clustered into protein families based on sequence similarity using the MCL algorithm. The method is based on a graph that contains similarity information obtained from BLAST searches. The similarity information was obtained using the following commands with the input file “CCNLR_Ndomain_set.fasta” (Dataset S8):
makeblastdb -in CCNLR_Ndomain_set.fasta -dbtype prot
blastp -query CCNLR_Ndomain_set.fasta -db CCNLR_Ndomain_set.fasta -out blast_results.txt -evalue 1e-8
Note: The outcome of this BLAST search is dependent on the e-value cutoff set by investigators. We set the BLASTP e-value cutoff to <10−8 by checking the sequence similarity of raw BLAST hits.
Clustering N-terminal domain sequences from the BLAST output file was performed using the following mclblastline command:
mclblastline --mcl-I=1.4 blast_results.txt
Note: The mcl-I option, which affects cluster granularity, was set to 1.4.
Provided N-terminal domain sequences were classified into several tribes in the output file “dump.out.blast_results.txt.l14” (Dataset S9). Among the output tribes, we focused on a tribe including ZAR1, RPP13, R2, and Rpi-vnt1.3 (tribe 3) for further sequence analyses, as described in Adachi et al. [9]. We then extracted IDs of N-terminal domain sequences from CC-NLRs grouped into tribe 3 using Supplemental script 4. For the analysis of conserved sequences, we extracted N-terminal domain sequences of tribe 3 from the input file “CCNLR_Ndomain_set.fasta” (Dataset S8) using Supplemental script 2 and saved as fasta file “Nseq_Tribe3.fasta” (Dataset S10).
Identify conserved sequence motifs in the N-terminal domain of NLRs
Identify conserved sequence patterns in the N-terminal domain tribes.
Conserved sequence patterns in tribe 3 were identified using MEME (Multiple EM for Motif Elicitation) with the following command with the input file “Nseq_Tribe3.fasta” (Dataset S10):
meme Nseq_Tribe3.fasta -protein -oc. -nostatus -time 14400 -mod zoops -nmotifs 5 -minw 6 -maxw 50 -objfun classic -minsites 62 -markov_order 0
Note: The ‘-minsites’ cutoff was set to 70% of the number of sequences to identify high occurrent sequence motifs. Other options are default settings in the MEME website (https://meme-suite.org/meme/tools/meme).
From our test data, we identified five conserved sequence patterns in the N-terminal domain of tribe 3 CC-NLRs (Figure 3). Among the identified motifs in the output “meme.html”, a motif located at the very N terminus was defined as the MADA motif based on the deduced 21 amino acid consensus sequence “MADAxVSFxVxKLxxLLxxEx” [9], conserved in approximately 78% of tribe 3 CC-NLRs. The EDVID motif, which functions in stabilizing the structure of CC-NLR proteins [19], is conserved in approximately 85% of tribe 3 CC-NLRs (Figure 3).
Figure 3. Consensus sequence patterns detected in the N-terminal domain of tribe 3 coiled-coil nucleotide-binding leucine-rich repeat receptors (CC-NLRs). Conserved motifs were identified by MEME from 88 tribe 3 CC-NLR members. The motif logos describe the N-terminal consensus patterns, as reported in Adachi et al. [9]. Figure is modified from Adachi et al. [9].
Search the MADA motif using the hidden Markov model (HMM)
Build the HMM of the conserved sequence pattern corresponding to the MADA motif.
Protein sequences corresponding to the MADA motif were extracted from a MEME output file and saved as the input file “MEME_alignment.txt” (Dataset S11). The HMM for the MADA motif was built using HMMER with the following command:
hmmbuild MADA.hmm MEME_alignment.txt
Perform HMMER search to query NLR proteomes using the MADA motif HMM.
HMMER detects sequence homologs and calculates HMM scores based on the degree of match. We performed a HMMER search to query NLR proteomes (Dataset S2) using the MADA motif HMM with the following command:
hmmsearch --max --tblout MADA.hmmsearch.tsv -o MADA.hmmsearch.txt MADA.hmm NLR_set.fasta
We set an HMM score cutoff at 10.0, which is most optimal for high-confidence searches of MADA containing CC-NLR proteins (MADA-CC-NLRs) [9]. We also defined NLR proteins with HMM scores from 0 to 10.0 as MADA-like CC-NLRs. From our test data, we identified 108 MADA-CC-NLRs and 161 MADA-like CC-NLRs. Based on conserved sequence patterns of NLRs, we can predict evolutionally conserved molecular functions of NLRs and can apply this for mutant analyses in molecular biology, biochemistry, and cell biology experiments as described in recent studies [1,9,20,21].
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Adachi et al. [9]. An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species. eLife (Figures 3–6).
Chia et al. [22]. The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages. Plant Cell (Figures 3 and 5).
Acknowledgments
This protocol is based on and modified from our previous publication [9]. We are thankful to Sophien Kamoun, Mauricio P. Contreras, Adeline Harant, Chih-hang Wu, Lida Derevnina, Cian Duggan, Eleonora Moratto, Tolga O. Bozkurt, Abbas Maqbool, Joe Win, and our colleagues in the Sophien Kamoun’s group at The Sainsbury Laboratory (UK) for their contributions to the original research paper. H.A. was funded by Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (JPMJPR21D1).
Competing interests
There are no conflicts of interest or competing interests.
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Supplementary information
The following supporting information can be downloaded here:
Dataset S1. Proteome fasta file used as input for NLRtracker.
Dataset S2. Test dataset of full-length NLR proteins used for phylogenetic analysis.
Dataset S3. NB-ARC domain sequence of Arabidopsis ZAR1.
Dataset S4. Test dataset of aligned NB-ARC domain sequences used for phylogenetic analysis.
Dataset S5. Test dataset of maximum likelihood tree.
Dataset S6. Test dataset of protein IDs of CC-NLR subfamily.
Dataset S7. Test dataset of CC-NLR subfamily sequences.
Dataset S8. Test dataset of N-terminal domain sequences extracted from CC-NLRs.
Dataset S9. Test dataset of protein IDs for each tribe.
Dataset S10. Test dataset of N-terminal domain sequences extracted from tribe 3.
Dataset S11. Test dataset of the MADA motif sequences extracted in MEME.
Video S1. Simple instructions for using Supplemental Scripts.
Article Information
Publication history
Received: Feb 19, 2024
Accepted: May 30, 2024
Available online: Jun 18, 2024
Published: Jul 5, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Plant Science > Plant molecular biology > Genetic analysis
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Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry
HV Hope K. Vanzo-Sparks
SW Sarah E. Webster
MW Mark K. Webster
Cindy L. Linn
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5024 Views: 511
Reviewed by: Joel Jovanovic Anonymous reviewer(s)
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Abstract
Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia–derived progenitor cells that arise after treatment with PNU-282987.
Key features
• Neurogenesis in the adult mammalian retina.
• Flow cytometry of retinal cells.
• PNU-282987-induced mitotic activity in the retina.
• Dissociation of the retina for flow cytometry analysis.
Keywords: Neurogenesis Flow cytometry Retina PNU-282987 Alpha7 nAChR agonist Müller glia Retinal ganglion cells Photoreceptors Proliferation Mitosis
Graphical overview
Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3–6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.
Background
Adult mammals cannot typically regenerate retinal neurons after injury [1–3]. However, previous research from this lab using adult mice has shown that the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, can induce neurogenesis in adult mammals when applied as eyedrops in the presence or absence of any retinal injury [4–10]. PNU-282987 is believed to act on alpha7 nAChRs in the retinal pigment epithelium to release signaling molecules onto the end feet of Müller glia to introduce cell cycle re-entry. From there, Müller glia de-differentiate and form retinal progenitor cells that eventually develop into mature retinal neurons [6,9]. However, these studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus restricting the number of antibodies that can be multiplexed and limiting the examination of cells undergoing mitosis and proliferation. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with mitotically active and proliferating cells that arise after treatment with PNU-282987.
Materials and reagents
Papain dissociation system, which includes papain, DNase I, ovomucoid inhibitor, and EBSS (Worthington Biochemical Corporation, catalog number: LK003150)
Phosphate buffered saline (PBS) 10× concentrate (Sigma-Aldrich, catalog number: P5493)
PNU-282987 hydrate (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride) (Sigma-Aldrich, catalog number: P6499)
BrdU (5-Bromo-2’-deoxyuridine) (Sigma-Aldrich, catalog number: 19-160)
Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: A5670701)
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418)
BD HorizonTM brilliant stain buffer (BD Biosciences, catalog number: 563794)
Fc receptor binding inhibitor polyclonal antibody, eBioscienceTM (Thermo Fisher Scientific, catalog number: 14-9161-73)
Fluorescent antibodies and unconjugated blocking antibodies (see Table 1)
UltraComp eBeadsTM compensation beads (Invitrogen, catalog number: 01-2222-42)
Table 1. Antibody panel used in this protocol
Antibody Fluorophore Clone Host Company Catalog number Dilution (per 1 × 106 cells)
Rhodopsin Unconjugated RET-P1 Mouse Invitrogen MAS-11741 3 μL
Zenon kit Alexa-405 - - Invitrogen Z25013 -
Thy 1.2 APC-eFluor-780 53-2.1 Rat eBioscience 47-0902-82 1 μL
Vimentin Biotinylated 280618 Rat R&D Systems BAM2105 2 μL
Streptavidin BUV-395 - - BD Biosciences BD 564176 2 μL
Ki67 BUV-737 20Raj1 Mouse eBioscience 367-5699-42 5 μL
BrdU PerCP-eFluor-710 BU20A Mouse eBioscience 46-5071-42 1 μL
Solutions
DMSO/PNU-282987/BrdU eyedrop solution (see Recipes)
DMSO/BrdU eyedrop solution (see Recipes)
4% PFA (see Recipes)
FACS buffer with 4% FBS (see Recipes)
0.4% Triton X-100 (see Recipes)
0.1 M HCl (see Recipes)
Recipes
DMSO/PNU-282987/BrdU eyedrop solution
Caution: Appropriate PPE must be worn when handling chemicals.
Reconstitute 50 mg of PNU-282987 in 1.66 mL of DMSO to make a 100 mM stock that can be stored at 4 for several months. Dissolve 5 mg of BrdU in 5 mL of DMSO to make a stock solution of 100 mg/mL BrdU that can be stored at -20 for several months. To make PNU-282987/BrdU eye drop solution, mix 9.8 mL of 1× PBS, 100 μL of 100 mM PNU-282987, and 100 μL of BrdU to achieve a final solution of 1 mg/mL BrdU and 1 mM PNU-282987. Store at 4 for one month.
DMSO/BrdU eyedrop solution
Mix 9.8 mL of 1× PBS, 100 μL of DMSO, and 100 μL of BrdU to achieve a final solution of 1 mg/mL BrdU. Store at 4 for one month.
4% PFA
Caution: Preparation of PFA is hazardous. Avoid skin contact and inhalation. Use appropriate PPE and a chemical fume hood.
Mix 4 g of PFA and 100 mL of 1× PBS in a media storage bottle containing a stir bar. Add drops of 2 M NaOH while the solution is stirring until a pH of approximately 7.4 has been reached or the PFA has completely dissolved. Please note the solution may become more acidic as PFA dissolves and the addition of more NaOH may be necessary. Place the bottle on a hot plate heated to 85 for approximately 20 min or until there are no visible particles. Allow the solution to cool at 4 . Add 1 M HCl drops until the solution reaches a pH of 6.9. Store at 4 for one week.
FACS buffer with 4% FBS
Add 4 mL of FBS to 96 mL of 1× PBS. Store at 4 for one month.
0.4% Triton X-100
Add 4 mL of Triton X-100 to 96 mL of 1× PBS. Store at 4 for several months.
0.1 M HCl
Add 1.5 mL of 1 M HCl to 13.5 mL of MilliQ water for a final concentration of 0.1 M HCl. Store at room temperature for several months.
Laboratory supplies
10 mm Petri dish (VWR, catalog number: 10799-192)
90 mm Petri dish (Sigma-Aldrich, catalog number: P10903)
General lab supplies (pipettes, tips, tubes, etc.)
5 mL Falcon round-bottom Polystyrene test tubes with 70 μm cell strainer (Fisher, catalog number: 08-771-23)
Equipment
Dumont #7 curved forceps (Fine Science Tools, catalog number: 11274-20)
Dumont #5 forceps (Fine Science Tools, catalog number: 11251-10)
Spring scissors (Fine Science Tools, catalog number: 15000-00)
Scalpel blades #11 (Fine Science Tools, catalog number: 10011-00)
Scalpel handle (Fine Science Tools, catalog number: 10003-12)
Corning® LSETM digital water bath, 6 L, 120 V (Corning, catalog number: 6783)
FisherbrandTM analog hotplate stirrer (Fisher, catalog number: FB30786160)
pH indicator strips (Sigma-Aldrich, catalog number: 1095350001)
VWR® water jacketed CO incubator (VWR, catalog number: 10810-744)
Pipet-Aid (Corning, catalog number: 07-202-350)
Centrifuge (Thermo Fisher Scientific, model: Heraeus Multifuge X3)
Eppendorf benchtop centrifuge (for microcentrifuge tubes at room temperature)
Benchtop vortex
Bright-Line counting chamber, which includes slides (VWR, catalog number: 100503-092)
Note: Alternatively, an automated cell counter can be used for this protocol.
BD LSRFortessaTM cell analyzer (BD Biosciences)
Software and datasets
FlowJoTM software v10.10 (BD Life Sciences) was used for flow cytometry data analysis
Procedure
Eyedrop application
Treat adult mice (3–6 month old) with eyedrops containing either 1% DMSO and 1 mg/mL BrdU or 1 mM PNU-282987 and 1 mg/mL BrdU for 28 days. Detailed instructions on how to apply eyedrops can be found in Linn et al. [11]. Briefly, the mouse is immobilized by scuffing the skin over their neck. One drop of DMSO/BrdU or DMSO/PNU-282987/BrdU eyedrop solution is placed on the bulbar conjunctiva of the right eye using a transfer pipette positioned upward by twisting the restrainer’s hand. The drop should cover the entire eye and should sit on the top of the eye for 1–2 s. Next, the restrainer twists their hand so that the left eye is positioned upward. A drop of DMSO/BrdU or DMSO/PNU-282987/BrdU eyedrop solution is placed on the bulbar conjunctiva of the left eye and allowed to sit for 1–2 s.
Retina harvesting
After euthanasia by carbon dioxide asphyxiation, place Dumont #7 curved forceps around the posterior aspect of the eyeball near the optic nerve. Apply gentle pressure and lift the eyeball out of the cavity. Place in a 10 mm Petri dish filled with 3 mL of ice-cold 1× PBS (Figure 1A).
Grasping the sclera with Dumont #5 forceps, move the eye out of the Petri dish. Create a puncture hole using a scalpel along the corneal equator (Figure 1B and 1C).
Hold the edge of the puncture hole with Dumont #5 forceps and place the eye back into the Petri dish. Insert Spring scissors into the puncture hole to create a circumferential incision along the corneal equator (Figure 1D).
Using a second pair of Dumont #5 forceps, begin to peel away the cornea and the iris from the posterior eye cup. Grasping the optic nerve with Dumont #5 forceps to generate traction may be necessary at this step (Figure 1E and 1F).
Once the cornea and iris are peeled away, the lens will be fully revealed. Remove the lens from the posterior eye cup with Dumont #5 forceps (Figure 1G).
Evert the posterior eyecup with Dumont #5 forceps to prolapse the retina (Figure 1H).
Holding the sclera with Dumont #5 forceps, remove the retina from the posterior eye cup with Spring scissors (Figure 1I). Remove any remaining, black-pigmented retinal epithelium with Spring scissors.
Rinse the isolated retina by submerging it in a new 10 mm Petri dish filled with 3 mL of ice-cold 1× PBS (Figure 1J).
Figure 1. Illustration demonstrating retina dissection. (A) Submerge the enucleated mouse eye in ice-cold 1× PBS in a 10 mm Petri dish. (B) Using Dumont #5 forceps, grasp the sclera and move the eye outside of Petri dish. While securing the eye in place with Dumont #5 forceps, create a scleral puncture using a scalpel along the corneal equator. (C) The black rectangle surrounded by the dotted circle depicts where the puncture wound should be placed. (D) Using Dumont #5 forceps, grasp the edge of the puncture wound to place the eye back into the dish. Insert Spring scissors into the puncture wound and create a circumferential incision along the corneal equator. (E) Using a second pair of Dumont #5 forceps, start peeling away the cornea and the iris from the posterior eye cup. (F) Grasp the optic nerve to generate traction. (G) Remove the lens while grasping the posterior eyecup with Dumont #5 forceps. (H) Prolapse the retina by everting the posterior eyecup with Dumont #5 forceps. (I) Secure the posterior eyecup in place with Dumont #5 forceps. Detach the retina from the posterior eyecup with Spring scissors. If any black pigmented retinal pigment epithelium remains, gently remove it with Spring scissors. (J) View depicting the isolated retina after dissection. After dissection, the isolated retina should be rinsed by submerging it in a new Petri dish filled with ice-cold 1× PBS.
Retina dissociation
Follow the manufacturer’s instructions for preparing papain, DNase I, and ovomucoid inhibitor to yield the proper concentrations.
Transfer the four retinae using Dumont #5 forceps into a 1.5 mL microcentrifuge tube filled with 500 μL of papain that has been warmed to 37 for at least 10 min in a water bath.
Mince the retinae into eight pieces using Spring scissors.
Add 25 μL of 1 mg/mL DNase I to papain.
Close the microcentrifuge tube lid and place it in an incubator heated to 37 on a rocker for constant agitation for 60 min.
Note: Optimal papain incubation time was determined in a separate experiment to investigate which incubation time yielded the highest number of living cells (Figure S1).
Pour the solution into a 90 mm Petri dish. Use warm EBSS to rinse out any remaining retinal tissue.
Triturate the tissue with a 10 mL pipette at least five times or until the solution appears cloudy and there are no large pieces of retinal tissue remaining. Transfer contents to a 15 mL conical tube.
Caution: The introduction of bubbles during trituration should be avoided. Bubbles have a high surface tension that can cause lysis.
Centrifuge at 300× g for 5 min.
While contents are centrifuging, create a resuspension solution in a 15 mL conical tube containing 2.7 mL of EBSS, 300 μL of ovomucoid inhibitor solution, and 150 μL of DNase I.
Remove the supernatant. Using a 10 mL pipette, transfer the entire resuspension solution created in step C9 to the conical tube containing the cell pellet and resuspend the cell pellet by triturating it 1–2 times.
Prepare discontinuous density gradient. In a new 15 mL conical tube, transfer 5 mL of ovomucoid inhibitor solution prepared in step C1 using a 10 mL pipette. Next, using a 10 mL pipette, add the entirety of the cell suspension created in step C10 on top of the 5 mL of albumin-ovomucoid solution to create a two-phase solution.
Caution: It is important to avoid mixing the two-phase solution. We have found that the best way to avoid mixing is to tilt the Pipet-Aid containing the cell suspension horizontally and release the liquid on the “slow” setting.
Centrifuge at 70× g for 6 min.
Note: A flow cytometry plot depicting a freshly dissociated retina is represented in Figure 2.
Figure 2. Unfixed and unpermeabilized dissociated retinae. (A) Plot depicting unfixed and unpermeabilized dissociated retinae with no fluorescent antibodies added. (B) Parameters include FSC (forward scatter; size) and SSC (side scatter; granularity). The corresponding voltages were implemented to detect an optimal signal above the noise and resolve positive and negative populations.
Fixation and permeabilization
Discard supernatant and resuspend the cell pellet by triturating it 3–5 times in 2 mL of 4% PFA using a 10 mL pipette. Let this sit for 15 min at room temperature for fixation.
Centrifuge at 500× g for 5 min.
Discard supernatant in hazardous waste and wash cell pellet by resuspension, triturating it 1–2 times in 2 mL of FACS buffer.
Centrifuge at 500× g for 5 min.
Discard the supernatant and resuspend the cell pellet by triturating it 3–5 times in 2 mL of 0.1 M HCl that has been prewarmed to 37 using a 10 mL pipette. Incubate at 37 in a warmer for 15 min.
Centrifuge at 500× g for 5 min.
Discard the supernatant and resuspend the cell pellet by triturating it 3–5 times in 2 mL of 0.4% Triton-X 100 using a 10 mL pipette. Let this sit for 15 min at room temperature for permeabilization.
Note: A flow cytometry plot depicting a fixed and permeabilized retina is represented in Figure 3.
Figure 3. Fixed and permeabilized dissociated retinae. (A) Plot depicting fixed and permeabilized dissociated retinae with no fluorescent antibodies added. The light-scatter properties of cells that have been fixed and permeabilized are typically altered, as evidenced by comparing Figure 2 and Figure 3. However, after excluding debris with gating, the two plots are comparable. (B) Parameters include FSC (forward scatter; identifies size), SSC (side scatter; identifies granularity), APC-eFluor-780 (Thy1.2), Alexa-405 (rhodopsin), BUV 395 (vimentin), PerCP-eFluor-710 (BrdU), and BUV 737 (Ki67). The corresponding voltages were implemented to detect an optimal signal above the noise and resolve positive and negative populations.
While incubating, collect 20 μL of the suspension and count cells using an automated cell counter or manually using a hemocytometer.
Note: Cell counts are approximately 1 × 106 per two retinas.
Centrifuge at 500× g for 5 min.
Remove the supernatant. Add 20 μL of Fc block and 50 μL of BD Horizon Brillant Stain Buffer to all experimental tubes.
Incubate for 15 min at room temperature.
Add 2 mL of FACS buffer using a 10 mL pipette and centrifuge at 500× g for 5 min.
Discard the supernatant and resuspend cells in 1 mL of FACS buffer by triturating it 1–2 times using a 1 mL glass pipette. Remove suspension from 15 mL conical using a 1 mL glass pipette and add it to a 1.5 mL microcentrifuge tube. The following groups should be created: A) single-stain compensation beads for each color in separate 1.5 mL microcentrifuge tubes (Table 2); B) untreated/unstained cells (Table 3); C) DMSO control (Table 3); and D) PNU-282987 treated (Table 3). Our panel included the following controls: A) compensation beads + BUV-395 only, compensation beads + APC-Efluor-780 only, compensation beads + Alexa-405 only, compensation beads + PerCP-Efluor-710 only, compensation beads + BUV-737 only; B) untreated/unstained cells; and the following experimental groups: C) DMSO control cells with Vimentin_Strep-BUV-395; Thy1.2_APC-eFluor-780; Rho_Alexa-405; BrdU_PerCP-eFluor-710; Ki67_BUV-737; and D) PNU-282987-treated cells with Vimentin_Strep-BUV-395; Thy1.2_APC-eFluor-780; Rho_Alexa-405; BrdU_PerCP-eFluor-710; Ki67_BUV-737.
Notes:
Compensation controls are necessary to set instrument voltages and compensate samples. Compensation controls must be repeated for every experiment. In the case of the retina, we found beads are the most appropriate material for compensation as compared to retina cells.
Please note that FMOs are utilized to determine positive vs. negative gates for the fluorophores included in the panel. FMOs need to be run during panel setup. An example of the FMO panels used in this experiment can be found in Figure S2.
Table 2. Compensation controls used in this protocol
Amount of compensation beads (per tube) Cell type Stain Amount of antibody added
1 drop Unstained - -
1 drop Müller glia Vimentin_Strep-BUV-395 1 μL
1 drop Retinal ganglion cells Thy1.2_APC-eFluor-780 1 μL
1 drop Rod photoreceptors Rho_Alexa-405 1 μL
1 drop Proliferating cells Ki67_BUV-737 1 μL
1 drop Mitotically active cells BrdU_PerCP-eFluor-710 1 μL
Table 3. Experimental groups used in this protocol
Experimental groups # retinae (per tube) Approximate cell count Cell type Stain
Untreated/unstained 4 ~2 M - -
DMSO control 4 ~2 M Müller glia, retinal ganglion cells, rod photoreceptors, proliferating cells, mitotically active cells Vimentin_Strep-BUV-395, Thy1.2_APC-eFluor-780, Rho_Alexa-405, Ki67_BUV-737, BrdU_PerCP-eFluor-710
PNU-282987 treated 4 ~2 M Müller glia, retinal ganglion cells, rod photoreceptors, proliferating cells, mitotically active cells Vimentin_Strep-BUV-395, Thy1.2_APC-eFluor-780, Rho_Alexa-405, Ki67_BUV-737, BrdU_PerCP-eFluor-710
Staining for flow cytometry
Compensation beads
Vortex compensation beads.
Add 1 drop of compensation beads into a 1.5 mL microcentrifuge tube.
Add 1 μL of antibody/stain to each tube.
Incubate on ice for 30 min.
Add 1 mL of FACS buffer to each tube.
Centrifuge at 150× g for 5 min.
Remove supernatant and resuspend the pellet by triturating it 1–2 times in 500 μL of FACS buffer using a p1000 pipette.
Transfer the sample into a 5 mL Falcon round-bottom polystyrene test tube by passing it through the 70 μm cell strainer.
Vimentin
Add the appropriate amount of antibody (see Table 1) that is used to label Müller glia using vimentin to the 1.5 mL microcentrifuge tube containing the cell pellet resuspended in 1 mL of FACS buffer. Briefly vortex the sample.
Allow this to sit for 30 min at room temperature.
Using a p1000 pipette, add 500 μL of FACS buffer.
Centrifuge at 150× g for 5 min.
Remove supernatant and resuspend cells by triturating it 1–2 times in 1 mL of FACS buffer using a p1000 pipette.
After briefly vortexing the streptavidin BUV-395, add it to the cell suspension at a concentration of 2 µL per 1 × 106 cells.
Allow this to sit for 30 min at room temperature.
Centrifuge at 150× g for 5 min.
Remove supernatant and triturate the cell pellet 1–2 times in 500 μL of FACS buffer using a p1000 pipette
Transfer the sample into a 5 mL Falcon round-bottom polystyrene test tube by passing it through the 70 μm cell strainer.
Rhodopsin
Prepare the rhodopsin antibody solution by combining 5 μL of mouse α Rhodopsin IgG, 5 μL of ZenonTM mouse IgG labeling reagent (Component A), and ≤ 20 μL of 1× PBS in a separate 1.5 mL microcentrifuge tube.
Incubate in the dark for 5 min at room temperature.
Add 5 μL of ZenonTM blocking reagent (Component B) to the antibody solution.
Incubate in the dark for 5 min at room temperature.
Add the appropriate amount of antibody (see Table 1) that is used to label Müller glia using vimentin to the 1.5 mL microcentrifuge tube containing the cell pellet resuspended in 1 mL of FACS buffer. Briefly vortex the sample.
Incubate in the dark at room temperature for 30 min.
Add 500 μL of FACS buffer.
Centrifuge at 150× g for 5 min.
Remove the supernatant and resuspend the cell pellet by triturating it 1–2 times in 500 μL of FACS buffer using a p1000 pipette.
Transfer the sample into a 5 mL Falcon round-bottom polystyrene test tube by passing it through the 70 μm cell strainer.
All other antibodies used
Add the appropriate volume of each antibody (see Table 1) to the 1.5 mL microcentrifuge tube containing the cell pellet resuspended in 1 mL of FACS buffer. Briefly vortex the sample.
Note: Optimal antibody concentrations were determined using a stain index.
Incubate in the dark for 30 min.
Add 500 μL of FACS buffer.
Centrifuge at 150× g for 5 min.
Remove supernatant and resuspend the cell pellet by triturating it 1–2 times in 500 μL of FACS buffer using a p1000 pipette.
Transfer the sample into a 5 mL Falcon round-bottom polystyrene test tube by passing it through the 70 μm cell strainer.
Data analysis
Selecting cells and excluding debris:
Use the Forward Scatter Area vs. Side Scatter Area (FSC-A vs. SSC-A) density plot to gate for the cell population while excluding cellular debris. Cellular debris is eliminated from the analysis by using a gate that excludes signals with a low level of forward scatter, usually aggregated at the bottom left corner (Figure 4A and Figure 4F).
Selecting singlets:
Use the Forward Scatter Area vs. Forward Scatter Height (FSC-A vs. FSC-H) density plot to gate for singlets (single cells) usually aggregated on the 45° diagonal line (Figure 4B and Figure 4G). Next, use the Side Scatter Area vs. Side Scatter Height (SSC-A vs. SSC-H) density plot to further refine the single-cell population aggregated on the 45° diagonal line (Figure 4C and Figure 4H).
Identifying populations with specific phenotype markers:
For each color of interest, select the positive population as identified in FMO (Figure 4D and Figure 4I). Then, gate on the proliferating population as identified in FMO (Figure 4E and Figure 4J).
Validation of protocol
Representative results from 28 days of DMSO and PNU-282987 treatment are presented in Figure 4. Quantitative results are shown in Figure 5.
Figure 4. Representative gating strategy used for gating vimentin+ and Ki67+ cells after 28 days of DMSO and PNU-282987 treatment. (A) and (F) A gate is made around the main cell population to exclude debris (SSC-A vs. FSC-A). (B–C) and (G–H) Singlet gates are drawn using FSC-H vs. FSC-A and SSC-H vs. SSC-A. (D) and (I) Vimentin+ cells are gated using FMOs to determine the cell population positive for vimentin (FSC-A vs. BUV-395-A). Back-gating figure for vimentin, displaying vimentin+ cells in the parent populations, is depicted in Figure S3. (E) and (J) Vimentin+ cells that are also labeled for the proliferation marker Ki67 are gated around. FMOs were utilized to determine positive cell populations (BUV-737-A vs. BUV-395-A).
Figure 5. Müller glia and proliferating Müller glia are increased in PNU-282987-treated samples. Adult (3–6 month old) mice were treated with 1× PBS eyedrops containing 1% DMSO and 1 mg/mL BrdU or 1 mM PNU-282987 and 1 mg/mL BrdU for 28 days. Subsequently, retinae were dissociated and processed for analysis by flow cytometry. (A) Graph showing the percent increase of vimentin+ Müller glia cells in DMSO vs. PNU-282987-treated samples. (B) Graph showing the percent increase of vimentin+Ki67+ Müller glia in DMSO vs. PNU-282987-treated samples. Total retinae: DMSO n = 12; PNU-282987 n = 12. Results are based on three independent experiments with retinae from mice pooled in each of the three experiments. Statistics: unpaired t-test with Welch’s correction; *p < 0.05.
General notes and troubleshooting
The discontinuous density gradient where the two layers of the gradient are visible, created in the dissociation protocol, is essential for a high cell yield. We have found that the best way to create a proper gradient is to turn the Pipet-Aid speed setting to slow, hold the Pipet-Aid horizontally so that the opening of the serological pipette is touching the inside of the tube, and slowly release the suspension so that it delicately layers.
Gentle or overly vigorous trituration of retinal tissue after incubation with papain during the dissociation process will result in a low yield of cells. Additionally, using a pipette that has a small opening could shear the cells. Therefore, we have found the tissue must be triturated at least five times with a 10 mL serological pipette so that the resulting solution is cloudy. However, if the resulting cell numbers are low with five triturations, lowering the number of triturations could result in a higher yield.
Acknowledgments
This study was funded by an NEI NIH award (#EY035803) and a WMU FRACAA award (#2040) issued to CL Linn. A special thanks to Michael Clemente and the Flow Cytometry and Imaging Core at Western Michigan University Homer Stryker M.D. School of Medicine for assistance and training in flow cytometry experiments. Graphical overview and Figure 1 created with BioRender.com.
Competing interests
There are no conflicts of interest or competing interests.
References
Dhomen, N. S., Balaggan, K. S., Pearson, R. A., Bainbridge, J. W., Levine, E. M., Ali, R. R. and Sowden, J. C. (2006). Absence ofChx10Causes Neural Progenitors to Persist in the Adult Retina. Invest Ophthalmol Vis Sci. 47(1): 386.
Lamba, D. A., Karl, M. O., Ware, C. B. and Reh, T. A. (2006). Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci USA. 103(34): 12769–12774.
Salman, A., McClements, M. and MacLaren, R. (2021). Insights on the Regeneration Potential of Müller Glia in the Mammalian Retina. Cells. 10(8): 1957.
Webster, M. K., Cooley-Themm, C. A., Barnett, J. D., Bach, H. B., Vainner, J. M., Webster, S. E. and Linn, C. L. (2017). Evidence of BrdU-positive retinal neurons after application of an Alpha7 nicotinic acetylcholine receptor agonist. Neuroscience. 346: 437–446.
Webster, M. K., Barnett, B. J., Stanchfield, M. L., Paris, J. R., Webster, S. E., Cooley-Themm, C. A., Levine, E. M., Otteson, D. C. and Linn, C. L. (2019). Stimulation of Retinal Pigment Epithelium With an α7 nAChR Agonist Leads to Müller Glia Dependent Neurogenesis in the Adult Mammalian Retina. Invest Ophthalmol Vis Sci. 60(2): 570.
Stanchfield, M. L., Webster, S. E., Webster, M. K. and Linn, C. L. (2020). Involvement of HB-EGF/Ascl1/Lin28a Genes in Dedifferentiation of Adult Mammalian Müller Glia. Front Mol Biosci. 7: e00200.
Paris, J. R., Sklar, N. C. and Linn, C. L. (2021). BrdU Positive Cells Induced in a Genetic Mouse Model of Glaucoma.J Ophthalmol Vis Sci. 6(1):1046.
Webster, S. E., Sklar, N. C., Spitsbergen, J. B., Stanchfield, M. L., Webster, M. K., Linn, D. M., Otteson, D. C. and Linn, C. L. (2021). Stimulation of α7 nAChR leads to regeneration of damaged neurons in adult mammalian retinal disease models. Exp Eye Res. 210: 108717.
Webster, S. E., Spitsbergen, J. B., Linn, D. M., Webster, M. K., Otteson, D., Cooley-Themm, C. and Linn, C. L. (2022). Transcriptome Changes in Retinal Pigment Epithelium Post-PNU-282987 Treatment Associated with Adult Retinal Neurogenesis in Mice. J Mol Neurosci. 72(9): 1990–2010.
Spitsbergen, J. B., Webster, S. E. and Linn, C. L. (2023). Functional Changes in the Adult Mouse Retina using an Alpha7 Nicotinic Acetylcholine Receptor Agonist after Blast Exposure. Neuroscience. 512: 1–15.
Linn, C., Webster, S. and Webster, M. (2018). Eye Drops for Delivery of Bioactive Compounds and BrdU to Stimulate Proliferation and Label Mitotically Active Cells in the Adult Rodent Retina. Bio Protoc. 8(21): e3076.
Supplementary information
The following supporting information can be downloaded here:
Figure S1. Sixty minutes is the optimal time for retinae papain digestion.
Figure S2. Fluorescence minus one(FMO) for photoreceptors, retinal ganglion cells, and mitotically active cells.
Figure S3. Back-gating for vimentin+ Müller glia.
Article Information
Publication history
Received: Feb 26, 2024
Accepted: May 23, 2024
Available online: Jun 18, 2024
Published: Jul 5, 2024
Copyright
© 2024 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 > Sensory and motor systems > Retina
Cell Biology > Cell-based analysis > Flow cytometry
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Quantification of Autophagosomes in Human Fibroblasts Using Cyto-ID® Staining and Cytation Imaging
BH Barbara Hochecker
KM Katja C. Matt
AM Alica L. Meßmer
MS Melanie M. Scherer
JB Jörg Bergemann
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5025 Views: 520
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Original Research Article:
The authors used this protocol in Journal of Thermal Biology Feb 2024
Abstract
As an essential process for the maintenance of cellular homeostasis and function, autophagy is responsible for the lysosome-mediated degradation of damaged proteins and organelles; therefore, dysregulation of autophagy in humans can lead to a variety of diseases. The link between impaired autophagy and disease highlights the need to investigate possible interventions to address dysregulations. One possible intervention is hyperthermia, which is described in this protocol. To investigate these interventions, a method for absolute quantification of autophagosomal compartments is required that allows comparison of autophagosomal activity under different conditions. Existing methods such as western blotting and immunohistochemistry for analysing the location and relative abundance of intracellular proteins associated with autophagy, or transmission electron microscopy (TEM), which are either very time-consuming, expensive, or both, are less suitable for this purpose. The method described in this protocol allows the absolute quantification of autophagosomes per cell in human fibroblasts using the CYTO-ID® Autophagy Detection Kit after heat therapy compared to a control. The Cyto-ID® assay is based on the use of a specific dye that selectively stains autophagic compartments, combined with an additional Hoechst 33342 dye for nuclear staining. The subsequent recognition of these stained compartments by the Cytation Imager enables the software to determine the number of autophagosomes per nucleus in living cells. Additionally, this absolute quantification uses an image-based method, and the protocol is easy to use and not time-consuming. Furthermore, the method is not only suitable for heat therapy but can also be adapted to any other desired therapy or substance.
Key features
• Absolute quantification of autophagic compartments in living cells.
• Optimised protocol for the determination of autophagy in primary human skin fibroblasts.
• Allows the testing of active substances and treatments concerning autophagy.
• Imaging-based method for the determination of autophagy.
Keywords: Autophagy Hyperthermia Primary human skin fibroblasts Absolute quantification Imaging-based method
Graphical overview
Background
As part of normal housekeeping, mammalian cells regularly replace damaged organelles or misfolded proteins to prevent potentially dangerous components from accumulating and overwhelming the cell. Autophagy removes and recycles this waste by isolating the targeted materials within a double-membrane vesicle called an autophagosome, which fuses with the lysosome. The fusion is facilitated by tethering factors that bind to proteins on the autophagosome and the lysosome [1]. After fusion with the lysosomes, the cellular waste is degraded by acidic lysosomal hydrolases (Figure 1). Disruption of the autophagy process is implicated in numerous human diseases and pathophysiological conditions, including neurodegenerative, infectious, autoimmune, cardiovascular, rheumatic, metabolic, pulmonary, and malignant diseases and ageing [2,3]. For this reason, it is important to find interventions such as treatment methods and/or substances that counteract impaired autophagy. Autophagy is known to be triggered under stress conditions such as starvation, hypoxia, DNA damage, ER stress, pathogen infection [4], and, in the case of this protocol, hyperthermia. The hyperthermia or heat treatment was carried out using an IRAcubator to induce autophagy. This device is a portable incubator that uses infrared-A radiation in the wavelength range from 780 to 1,400 nm, which heats the human fibroblasts to 39 °C. This is just one example of a possible treatment method for influencing autophagy. Regardless of the investigated intervention, however, a reliable and reproducible method is required for such measurements. Common methods for detecting autophagy are transmission electron microscopy (TEM) or western blot. However, TEM is very expensive and more suitable for analysing the location rather than the number of autophagosomes. Western blot requires a large amount of sample material, is very time-consuming, and expensive. Other methods such as the LC3 HiBiT reporter assay or other reporter systems are more suitable for cell lines than for primary cells. Another possible method, which was also used in our paper [5], could be the quantification by flow cytometry after staining with anti-LC3 FITC conjugated antibodies. However, this method is currently only established in our laboratory for peripheral blood mononuclear cells (PBMCs) and not for human fibroblasts.
The CYTO-ID® Autophagy Detection Kit used in this protocol contains a green dye that has been optimised by identifying titratable functional components that allow minimal staining of lysosomes while showing bright fluorescence when incorporated into pre-autophagosomes, autophagosomes, and autolysosomes. The kit also contains Hoechst dye for nuclear staining to enable the detection of autophagosomes per nucleus. In addition, the lysosomal inhibitor chloroquine can be added as a positive control. By inhibiting lysosomal degradation, the autophagosomal process is interrupted and autophagosomes accumulate (Figure 1). This means that in the wells that also contain chloroquine, a higher number of autophagosomes per nucleus should be seen if the staining has worked properly. For this reason, additional wells with added chloroquine were prepared for both conditions as a positive control for the assay.
Figure 1. Autophagy pathway under different conditions. After maturation of the autophagosomes, the vesicles are either degraded by fusion with lysosomes or accumulate due to the inhibition of lysosomal fusion by chloroquine.
The advantages of this method are that it is easy to use and not time-consuming. Furthermore, it is not only suitable for heat therapy but can also be adapted to any other desired therapy or substance. It can also be adapted to other adherent cells such as HeLa cells. The method is not suitable for small, round cells such as peripheral blood mononuclear cells (PBMCs).
Materials and reagents
Biological materials
Primary human skin fibroblasts, prepuce of a healthy eight-year-old male donor, passage 08–09
Reagents
Fetal bovine serum (FBS) (Gibco, catalog number: 11573397)
Trypsin-EDTA (0.5%) (Gibco, catalog number: 10779413)
Chloroquine diphosphate (TOCRIS, catalog number 4109)
Dimethyl sulphoxide (DMSO) ≥ 99.5%, BioScience grade (Carl Roth, catalog number: A994.2)
Dulbecco’s modified Eagle’s medium, high glucose (DMEM) (Gibco, catalog number: 41966052)
Gentamycin (10 mg/mL) (Gibco, catalog number: 11500506)
Sodium chloride (NaCl) (Carl Roth, catalog number: 3957)
Di-sodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) (Carl Roth, catalog number: N350)
Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: 3904)
Potassium chloride (KCl) (Carl Roth, catalog number: 6781)
CYTO-ID® Autophagy Detection Kit 2.0 (Enzo Life Sciences GmbH, catalog number: ENZ-KIT175-0200)
Solutions
1× Phosphate buffered saline (PBS) (see Recipes)
DMEM for fibroblasts (see Recipes)
Stop solution (see Recipes)
Cryomedium (see Recipes)
1× assay buffer (see Recipes)
Staining solution (see Recipes)
Recipes
1× Phosphate buffered saline (PBS)
Note: Adjust pH value to 7.4. Autoclave 10× PBS; for 1× PBS, dilute 10× stock 1:10 with deionized water (50 mL of 10× PBS + 450 mL of deionized water), autoclave, and store at room temperature.
Reagent Final concentration (10×) Quantity or volume (10×)
NaCl 8.0% (w/v) 80.0 g
KCl 0.2% (w/v) 2.0 g
Na2HPO4·12H2O 1.78% (w/v) 17.8 g
KH2PO4 0.24% (w/v) 2.4 g
Deionized water n/a ad 1,000 mL
Total (optional) n/a 1,000 mL
DMEM for fibroblasts (medium)
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
DMEM 90.5% (v/v) 500 mL
FBS 9.05% (v/v) 50 mL
Gentamycin 0.45% (v/v) 2.5 mL
Total (optional) 100% 552.5 mL
Stop solution
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
1× PBS 90.91% (v/v) 500 mL
FBS 9.09% (v/v) 50 mL
Total (optional) 100% 550 mL
Cryomedium
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
FBS 90% (v/v) 45 mL
DMSO 10% (v/v) 5 mL
Total (optional) 100% 50 mL
1× assay buffer
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
10× assay buffer (from CYTO-ID®) 1× 1 mL
Deionized water 9 mL
Total (optional) n/a 10 mL
Staining solution (CYTO-ID®)
Note: Prepare immediately before use, do not store. All listed ingredients are part of the CYTO-ID® Autophagy Detection Kit 2.0.
Reagent Final concentration Quantity or Volume
1× assay buffer n/a 2.4 mL
CYTO-ID® green detection reagent n/a 4.8 µL
Hoechst 33342 Nuclear Stain n/a 2.4 µL
Total (optional) n/a 2407.2 µL
Laboratory supplies
Centrifuge tubes, 15 and 50 mL (SARSTEDT, catalog numbers: 62.554.502, 62.547.254)
Reaction tube, 1.5 and 2 mL (SARSTEDT, catalog numbers: 72.706, 72.695.500)
CryoPure vial, 1.6 mL (SARSTEDT, catalog number: 72.380.992)
Pipette tips, 20, 200, and 1,000 μL (SARSTEDT, catalog numbers: 701.114.210, 70.3030, and 70.3050)
Serological pipettes, 5, 10, and 25 mL (SARSTEDT, catalog numbers: 86.1253.001, 86.1254.001, and 86.1685.001)
Cell culture plate, 96-well microplate (Greiner, catalog number: 655090)
Cell culture flask, T175, Cell+ (SARSTEDT, catalog number: 83.3912.302)
Equipment
Pipettes: Eppendorf Research® Plus 10 μL, 20 μL, 200 μL, 1,000 μL (Eppendorf, catalog numbers: 3123000020, 3123000039, 3123000055, 3123000063)
Multichannel microliter pipette Transferpette®, 20–200 µL (BRAND GMBH + CO.KG, catalog number: 9280173)
Pipetting aid: PipetBoy acu 2 (Integra Biosciences, catalog number: 155 000)
Neubauer counting chamber improved (Carl Roth, catalog number: PC72.1)
Inverted microscope: Primovert, Objektiv Plan-Achromat 4×/0.10, 10×/0.25 Ph1 (Carl Zeiss, catalog numbers: 415510-1100-000, 415500-1600-001, 415500-1605-001)
Water bath (GFL, catalog number: 1003)
Microcentrifuge Eppendorf 5424 (Eppendorf, catalog number: 05-400-005)
Swing out rotor centrifuge Eppendorf 5804 R (Eppendorf, catalog numbers: 5805000010, 5804709004)
Water purification system ELGA® PURELAB flex 3 (Veolia, catalog number: PF3XXXXM1)
pH meter FE20 FiveEasyTM (Mettler Toledo, catalog number: 30266626)
Heat treatment IRAcubator (Von Ardenne Institut für Angewandte Medizinische Forschung GmbH)
Incubator at 37 °C with 5% CO2, 90% humidity (HERA Cell 240, catalog number: 2510-413-01)
BioTek Cytation 1 cell imaging multimode reader, DAPI 377, 447; GFP 469, 525 (Agilent, model: CYT1AGV)
Software and datasets
Gen 5 Image Prime (3.09.07, 01/10/2020), license needed
Procedure
Cell expansion
Thaw 5 × 106 primary human skin fibroblasts (passage 08) in 30 mL of stop solution.
Centrifuge at 500× g for 3 min at room temperature.
Discard the stop solution.
Seed fibroblasts in T175 bottles. Distribute the 5 × 106 cells into five T175 cell culture flasks (approximately 1 × 106 cells per T175 flask) with 20 mL of DMEM for fibroblasts medium per flask.
Check cells by microscopy and cultivate them under standard tissue culture conditions at 37 °C and 5% CO2 in a humidified incubator.
If necessary, change the medium and divide the cells into new T175 bottles when they have reached 70% confluence.
Expand the cells until the number of cells required for all planned experiences is reached.
Note: All experiments should be carried out with the same cell passage in order to achieve better reproducibility and comparability.
Freeze all cells in 0.5 × 106 cells (passage 09) per portion in 1 mL of cryomedium in CryoPure vials.
Cell thawing for experiment
Thaw 0.5 × 106 primary human skin fibroblasts (passage 09) of the previous expanded cells in 10 mL of stop solution.
Centrifuge at 500× g for 3 min at room temperature.
Discard the stop solution.
Seed all cells in a T175 cell flask with 20 mL of DMEM for fibroblasts medium
Cultivate human fibroblasts under standard tissue culture conditions at 37 °C and 5% CO2 in a humidified incubator.
Caution: The human fibroblasts should be expanded for at least two days after thawing to allow the cells to regenerate after the freeze/thaw process. Cells should not exceed a confluence of 70%.
Cell seeding and treatment
After an appropriate incubation period, remove the cells with 0.05% Trypsin-EDTA.
Note: Incubate at 37 °C for approximately 2 min and observe the detachment process under the microscope.
Prepare 12 wells per plate (control plate, treatment plate) with 5,000 cells each in 100 µL of DMEM for fibroblasts medium.
Note: In order to achieve an even cell seeding, leave the plate for 1 h at room temperature without moving.
Incubate cells for approximately 24 h at 37 °C and 5% CO2.
The next day, proceed in the same way for both plates (Figure 2):
Add 100 µL of DMEM for fibroblasts per well to six wells.
Add 100 µL of 0.5 µM chloroquine (diluted in DMEM for fibroblasts) per well to the other six wells.
Caution: The duration of the chloroquine treatment should be approximately 1 h, so steps C4–C7 must be carried out quickly.
Figure 2. Plate layouts. Both plates, the control plate and the treatment plate, contain six wells with medium (a.) for the measurement and six wells with 0.5 µM chloroquine (b.) as a positive control.
Mix all prepared wells by pipetting.
Caution: Mix the solutions gently, as harsh pipetting could detach and stress the cells.
The IRAcubator should be preheated to 39 °C.
Place the control plate in a humidified incubator at 37 °C and 5% CO2 for 1 h.
Place the treatment plate in the IRAcubator at 39 °C for 1 h (Figure 3).
Note: Ensure that the 96-well plate is centred over the temperature sensor.
Figure 3. Placement of the treatment plate in the IRAcubator heat treatment device
Cyto-ID® staining
Discard the supernatant (DMEM for fibroblasts, 0.5 µM chloroquine) by turning the plate over on laboratory tissue paper.
Note: The Cytation Imager can only read one plate at a time; therefore, a delay is required between the two plates so that they can be measured one after the other. Start staining with the treatment plate until step D4 and then continue with the control plate after approximately 10 min.
Wash all wells with 200 µL of 1× assay buffer and discard the supernatant by turning the plate over on laboratory tissue paper.
Note: Allow the assay buffer to warm to room temperature.
Add 100 µL of staining solution (see Recipes).
Incubate the plate in the dark in a humidified incubator at 37 °C and 5% CO2 for 30 min.
Wash all wells twice with 200 µL of 1× assay buffer and discard supernatant by turning the plate over on laboratory tissue paper.
Add 100 µL of 1× assay buffer to each well and image the cells immediately.
Cell imaging
Define the imaging settings (paper button) as shown in Figure 4. Capture two images in the middle of each well.
Note: The button in the right-hand corner (e.g., 30/96) allows you to specify which wells of the plate are to be measured. These settings should match the layout of the panel.
Figure 4. Imaging settings
Define the plate layout (plate button) as shown in Figure 5.
Figure 5. Plate layout definition
Define the parameters for image processing (calculator button) and cell analysis as specified in Table 1.
Table 1. Settings of the Gen 5 image analysis software
Imaging preprocessing Image deconvolution
Image set DAPI Image set
Tsf [GFP 469,525]
Tsf [DAPI 377,447]
Background Dark Point spread function Auto, based on objective
Rolling bar diameter 66 µm Iterations 5
Image smoothing strength 0 Data out prefix Deconvolved
Image set GFP
Background Dark
Rolling bar diameter 1.5 µm
Priority Fine results
Image smoothing strength 0
Cellular analysis
Primary mask
and count
Deconvolved
[Tsf [DAPI 377,447]] Deconvolved
[Tsf [GFP 469,525]]
Secondary Mask
Deconvolved
[Tsf [GFP 469,525]]
Threshold Auto Background Dark
Background Dark
Measure within a
primary mask
Checked
Use Primary Mask
Object selection
Min. object size
Max. object size
5 µm
40 µm
Measure within a secondary mask Checked
Expand primary mask 50 µm
Threshold Unchecked
Count spots Checked
Size 0.5–5 µm
Advanced options Count spots options
Rolling ball size Default
Threshold 1200
Curve Analysis: Autophagosomes per nucleus
Start the process by pressing the Start button (play button).
Data analysis
As indicated, six technical replicates are recommended per condition. Two images are taken in the middle of the well for each technical replicate. Figure 6 shows an example of successful cell imaging with subsequent image processing and cell analysis.
Figure 6. Example of successful cell imaging (A), image processing and cell analysis (B1, B2). After image processing, the predefined masks are placed around the cell nuclei (B1). All autophagosomes in this area are counted and assigned to the respective cell nucleus (B2).
Unfortunately, the imager is not always able to find the right focus to capture a sharp image (Figure 7). For this reason, it is necessary to go through all the images, check their sharpness, and sort them out by masking them, so that they are not included in the calculation.
Figure 7. Example of a sharp image (A) and an image out of focus (B). Once the sharpness of the images has been checked, the calculated data is exported to Excel and analysed using statistical software of your choice.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article:
Hochecker et al. [5]. Heat treatment in health and disease: How water-filtered infrared-A (wIRA) irradiation affects key cellular mechanisms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients compared to healthy donors. Journal of Thermal Biology (Figure 1).
General notes and troubleshooting
General notes
In order to maintain ideal growth conditions for the cells, they should not exceed a confluency of 70%. In the case of mortal cell lines, the cell lines change as a result of cultivation, and the phenotype becomes increasingly different from the original cells. Therefore, no cells above passage 12 should be used for the experiments.
The software is not capable to distinguish the individual autophagosomes in small and round cells like PBMCs (Figure 8). This can be explained by the difficulty in finding the correct focus in round and non-planar objects, as well as the overlapping of autophagosomes, which is also due to the round cell morphology and the limited space in the small cells.
Figure 8. Example of unsuccessful cell imaging of human peripheral blood mononuclear cells (PBMCs) (A), image processing and cell analysis (B1, B2)
Troubleshooting
Problem 1: No or too few cells in the cell imaging.
Possible causes: Too few cells sown or careless washing of the cells.
Solutions: Plate more cells or wash more carefully.
Problem 2: The imager cannot find the right focus.
Possible causes: Too few cells in the image, insufficient staining, or technical aspects.
Solutions: Adjust the cell count, wash more carefully, or adjust staining.
Problem 3: Inadequate staining or background fluorescence.
Possible causes: Staining solution is not suitable or too long incubation with the staining solution.
Solutions: Optimise the concentrations of the staining solution compartments or adjust the incubation time.
Problem 4: The chloroquine control does not show a higher amount of autophagosomes compared to the samples without chloroquine (established cells and chloroquine concentration).
Possible causes: Something went wrong during the assay (treatment or staining process).
Solutions: As the results are not reliable, the test should be repeated.
Problem 5: The chloroquine control does not show a higher amount of autophagosomes compared to the samples without chloroquine (new cells and/or treatment).
Possible causes: Chloroquine concentration too low to achieve an effect, very strong effect of the investigated treatment on autophagy → saturation.
Solutions: Optimise the chloroquine concentration, adapt the treatment conditions if possible.
Problem 6: Cells are not, too little, or too strongly stained.
Possible causes: The colouring solution was forgotten, prepared incorrectly, or the concentrations of the components are not suitable.
Solutions: Repeat the experiment or optimise the staining solution.
Problem 7: Only nuclei or only autophagosomes are visible and not both.
Possible causes: Hoechst or CYTO-ID® green detection reagents were forgotten.
Solution: Repeat the experiment.
Further troubleshooting instructions for the CTYO-ID® Autophagy Detection Kit can be found in the product manual.
Further troubleshooting instructions for the Gen 5 software and the Cytation 1 Imager can be found in the respective product manuals.
Acknowledgments
The authors thank Dr. Astfalk for kindly providing skin samples. They also thank Dr. Alexander von Ardenne and Noah Molinski for the construction of the IRAcubator and the good cooperation. This work was funded by the “Professor Manfred von Ardenne Forschungsförderungsgesellschaft e.V.” This study was supported by the Baden-Württemberg Ministry for Science, Research and Art (MWK Baden-Württemberg) through the “Kooperatives Promotionskolleg (KPK) InViTe2 Sigmaringen / Konstanz,” in which Barbara Hochecker is an associated PhD student. This protocol was used in Hochecker et al. [5].
Competing interests
The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Barbara Hochecker reports financial support was provided by “Professor Manfred von Ardenne Forschungsförderungsgesellschaft e.V.”
Ethical considerations
All experiments were performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Medical Association of Baden-Württemberg, Germany. Patients were informed in advance and gave their written consent to the use of their samples.
References
Yim, W. Y. and Mizushima, N. (2020). Lysosome biology in autophagy. Cell Discov. 6(1): 6.
Rabinowitz, J. D. and White, E. (2010). Autophagy and metabolism. Science. 330(6009): 1344–1348.
Galluzzi, L., Baehrecke, E. H., Ballabio, A., Boya, P., Bravo‐San Pedro, J. M., Cecconi, F., Choi, A. M., Chu, C. T., Codogno, P., Colombo, M. I., et al. (2017). Molecular definitions of autophagy and related processes. EMBO J. 36(13): 1811–1836.
King, J. S., Veltman, D. M. and Insall, R. H. (2011). The induction of autophagy by mechanical stress. Autophagy. 7(12): 1490–1499.
Hochecker, B., Molinski, N., Matt, K., Meßmer, A., Scherer, M., von Ardenne, A. and Bergemann, J. (2024). Heat treatment in health and disease: How water-filtered infrared-A (wIRA) irradiation affects key cellular mechanisms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients compared to healthy donors. J Therm Biol. 120: 103813.
Article Information
Publication history
Received: Apr 2, 2024
Accepted: May 30, 2024
Available online: Jun 23, 2024
Published: Jul 5, 2024
Copyright
© 2024 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 imaging > Live-cell imaging
Cell Biology > Cell viability > Cell death
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Imaging Assays to Detect DNA Damage in Trypanosome Parasites Using γH2A
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Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5026 Views: 477
Reviewed by: Marcelo S. da Silva Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Jun 2023
Abstract
Diseases caused by trypanosomatid parasites remain a significant unmet medical need for millions of people globally. Trypanosomatid parasites such as Trypanosoma cruzi and subspecies of Trypanosoma brucei cause Chagas disease and human African trypanosomiasis (HAT), respectively. Although efforts to find novel treatments have been successful for HAT, Chagas disease is still treated with decades-old therapies that suffer from long treatment durations and severe safety concerns. We recently described the identification and characterization of the cyanotriazole compound class that kills trypanosomes, in vitro and in vivo, by selective inhibition of the trypanosome nuclear topoisomerase II enzyme. To evaluate whether inhibition of the topoisomerase II enzyme led to parasite death due to lethal double-strand DNA breaks, we developed assays for detecting DNA damage in both intracellular amastigotes of T. cruzi and bloodstream-form T. brucei by using the canonical DNA damage marker γH2A. Herein, this article describes the protocols for detecting DNA damage using an immunofluorescence assessment of γH2A by microscopy in trypanosome parasites.
Key features
• Immunofluorescence-based assay to detect the γH2A response in T. brucei and T. cruzi parasites.
• Robust DNA damage pathway–based cellular assays to evaluate topoisomerase II poisons’ ability to cause DNA damage.
• A 384-well plate–based T. cruzi protocol allows high-resolution and high-throughput evaluation of compounds that cause DNA damage by measuring γH2A in intracellular parasites.
• This assay could be modifiable for evaluation of DNA damage responses in various intracellular and extracellular eukaryotic pathogens.
Keywords: DNA damage γH2A Double-strand DNA break Chagas HAT Trypanosomes Trypanosoma cruzi Trypanosoma brucei Parasitology Drug discovery Topoisomerase II Topo II poison Immunofluorescence assay Histones Cyanotriazoles
Background
Trypanosomatid parasites cause various medically important diseases including Chagas disease and human African trypanosomiasis, mainly affecting people living in Latin America and sub-Saharan Africa, respectively. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to humans through the triatomine bug [1]. Human African trypanosomiasis (HAT) is caused by T. brucei gambiense and T. b. rhodesiense, which are transmitted through the bite of the tsetse fly [2]. Recent efforts have led to the discovery and development of fexinidazole as a novel oral treatment for HAT [3,4], whilst Chagas disease is still treated with nitroheterocyclics that have poor tolerability [1]. New efforts are needed to develop novel therapies to treat Chagas disease that are safer, efficacious, and have the potential for shortened treatment durations.
We described the identification and characterization of a novel cyanotriazole (CT) class of compounds showing promising parasiticidal activity in murine models of Chagas and HAT diseases [5]. Extensive mode of action studies showed that CTs act as trypanosome-specific topoisomerase II (Topo II) poisons. Topoisomerase II poisons act by stabilizing the enzyme in the Topo II-DNA complex after the topoisomerase II enzyme has cleaved the double-stranded DNA. The accumulation of these double-strand DNA breaks due to Topo II poisons ultimately results in cell death (Reviewed in Vann et al. [6]).
In eukaryotes, several DNA repair pathways are known to respond to DNA damage. One of the early responses to double-strand DNA breaks is a phosphorylation in the C-terminal region of histone H2A (H2AX in humans) to generate γH2A(X) [7]. In humans and non-human primates, this phosphorylation occurs on serine-139 of the conserved SQ motif. However, in T. brucei and T. cruzi, the phosphorylation occurs on threonine-130 and threonine-131, respectively. This phosphorylation is involved in signaling cascades to recruit DNA repair machinery to the site of damage. The H2A response can be detected with phospho-antibodies that recognize the specific γH2A-associated phosphorylation of histone H2A(X). Previous work by Glover and Horn identified this response in T. brucei [8], and the antibody was reported to be weakly cross-reactive against T. cruzi epimastigotes by western blot [9]; however, we were unsuccessful in detecting γH2A in T. cruzi using the T. brucei antibody in either epimastigotes or intracellular amastigotes by western blot or immunofluorescence microscopy. Therefore, we designed a new antibody to specifically detect γH2A in T. cruzi. This new antibody, specific for the T. cruzi epitope (Figure 1B), can detect T. cruzi γH2A in both western blots and immunofluorescence assays. Herein, we describe protocols for imaging-based γH2A assays for intracellular T. cruzi and bloodstream-form T. brucei.
Figure 1. Overview of γH2A response to DNA damage. (A) Graphical representation of γH2A response to DNA damage. The graphic is not to scale. (B) Alignment of γH2A epitope between human (Homo sapiens), Trypanosoma brucei, and Trypanosoma cruzi. Sequences obtained from UniProt (P16104) [10] and TriTrypDB (Tb927.7.2820, TcCLB.508321.21) [11]. Numbering does not include initiator methionine.
Materials and reagents
Biological materials
Vero cells (ATCC CCL-81)
Trypanosoma cruzi CL Brener parasites expressing tdTomato (Tc) [12]
Bloodstream form (BSF) Trypanosoma brucei Plimmer and Bradford (ATCC PRA-382)
Reagents
RPMI 1640 (HyClone, catalog number: SH30027.02)
0.25% Trypsin-EDTA (Gibco, catalog number: 25200056)
Fibronectin powder (Corning, catalog number: 354008)
IMDM powder (Gibco, catalog number: 12200036)
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761)
Hypoxanthine (Sigma-Aldrich, catalog number: H9636)
Sodium hydroxide (Sigma-Aldrich, catalog number: S8045)
Sodium pyruvate (100 mM) (Gibco, catalog number: 11360070)
Thymidine (Sigma-Aldrich, catalog number: T1895)
L-cysteine (Sigma-Aldrich, catalog number: C7352)
Bathocuproine disulfonic acid (Sigma-Aldrich, catalog number: 146625)
Beta-mercaptoethanol (Millipore-Sigma, catalog number: M6250)
Heat-inactivated fetal bovine serum (FBS) (Gibco, catalog number: 10082147)
Penicillin-Streptomycin (Gibco, catalog number: 15070063)
Penicillin-Streptomycin-Glutamine (100×) (Gibco, catalog number: 10378016)
Phosphate buffered saline (PBS) (Gibco, catalog number: 20012027)
Paraformaldehyde 16% aqueous solution (Electron Microscopy Sciences, catalog number: 15710-S)
Triton X-100 (Sigma, catalog number: X100)
Tween-20 (Sigma, catalog number: P9416)
Bovine serum albumin (BSA) (Sigma, catalog number: A7906-500G)
Primary antibodies:
Rabbit anti-T. cruzi γH2A polyclonal antibody (custom-made by Thermo Fischer Scientific)
i. Epitope: KKARATpPSA
ii. Bleed 3, affinity purified
Rabbit anti-T. brucei γH2A polyclonal antibody (custom-made by Thermo Fischer Scientific)
i. Epitope: KHAKATpPSV
ii. Bleed 2, affinity purified
Secondary antibody: Goat anti-rabbit Alexa Fluor 488 (Abcam, catalog number: 150081)
Hoechst 33258 (Anaspec, catalog number: AS-83219)
VECTASHIELD Vibrance® antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1800-2)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650-100ML)
Phleomycin D1 (Zeocin) (Life Technologies, catalog number: R25001)
Compounds (as described in Rao et al. [5]):
GNF6702 (kinetoplastid proteasome inhibitor)
Benznidazole (Sigma-Aldrich, catalog number: 419656)
CT0
CT1
CT3
Solutions
All solutions are stored at 4 °C. Solutions that are made fresh or need to be protected from light are indicated below each recipe.
RPMI complete medium (for Vero cells and T. cruzi parasites) (see Recipes)
8% fixative solution (see Recipes)
0.5% permeabilizing solution (see Recipes)
Blocking buffer 1 (see Recipes)
IFA wash buffer (see Recipes)
T. cruzi primary antibody solution (see Recipes)
T. cruzi secondary antibody solution (see Recipes)
Hoechst dye solution (see Recipes)
HMI-9 complete medium (for T. brucei) (see Recipes)
4% fixative solution (see Recipes)
0.25% permeabilizing solution (see Recipes)
Blocking buffer 2 (see Recipes)
T. brucei primary antibody solution (see Recipes)
T. brucei secondary antibody solution (see Recipes)
Recipes
RPMI complete medium (for Vero cells and T. cruzi parasites)
Reagent Final concentration Amount
RPMI 1640 1× 1,000 mL
FBS 9% 100 mL
Penicillin-Streptomycin 0.9% 10 mL
Total n/a 1,110 mL
Filter medium through a 0.22 µm, low-protein binding filter.
8% fixative solution
Reagent Final concentration Amount
1× PBS - 15 mL
Paraformaldehyde (16%) 8% 15 mL
Total n/a 30 mL
Make fresh on the day of the experiment.
0.5% permeabilizing solution
Reagent Final concentration Amount
1× PBS 1× 500 mL
Triton X-100 0.5% 2.5 mL
Total n/a 502.5 mL
Blocking buffer 1 (for T. cruzi)
Reagent Final concentration Amount
1× PBS 1× 100 mL
BSA 4% 4 g
Tween-20 0.1% 0.1 mL
Total n/a 100 mL
IFA wash buffer
Reagent Final concentration Amount
1× PBS 1× 1,000 mL
Tween-20 0.1% 1 mL
Total n/a 1,000 mL
T. cruzi primary antibody solution
Reagent Final concentration Amount
Blocking buffer 1 1× 25 mL
Primary antibody 1:250 0.1 mL
Total n/a 25 mL
Make fresh on the day of the experiment.
T. cruzi secondary antibody solution
Reagent Final concentration Amount
Blocking buffer 1 1× 25 mL
Secondary antibody 1:1,000 0.025 mL
Total n/a 25 mL
Make fresh on the day of the experiment, protect from light.
Hoechst dye solution
Reagent Final concentration Amount
1× PBS 1× 25 mL
BSA 1% 0.25 g
Hoechst 1:250 0.1 mL
Total n/a 25 mL
Make fresh on the day of the experiment, protect from light.
HMI-9 complete medium for T. brucei
Reagent Final concentration Amount
Water n/a 730 mL
IMDM powder 1× 1 packet
Sodium bicarbonate 0.036 mM 3.02 g
Hypoxanthine* 1.0 mM 0.136 g dissolved in 10 mL 0.1 M NaOH
Sodium pyruvate 1.0 mM 10 mL
Thymidine* 0.160 mM 0.039 g dissolved in 10 mL water
L-cysteine* 1.0 mM 0.12 g dissolved in 10 mL water
β-mercaptoethanol** 0.2 mM 14 µL diluted in 10 mL water
Bathocuproine disulfonic acid* 0.050 mM 0.028 g dissolved in 10 mL water
FBS 10% 100 mL
Serum plus 10% 100 mL
Penicillin-Streptomycin-Glutamine 1% 10 mL
Total n/a 1,000 mL
*Solid salts are dissolved in 10 mL of water (or 0.1 M NaOH) before adding to medium.
**β-mercaptoethanol is diluted in 10 mL of water before adding to medium. Combine ingredients and mix well. Filter medium through a 0.22 µm, low-protein binding filter.
4% fixative solution
Reagent Final concentration Amount
1× PBS - 12 mL
Paraformaldehyde (16%) 4% 4 mL
Total n/a 16 mL
Make fresh on the day of the experiment.
0.25% permeabilizing solution
Reagent Final concentration Amount
1× PBS 1× 25 mL
Triton X-100 0.25% 0.063 mL
Total n/a 25 mL
Blocking buffer 2 (for T. brucei)
Reagent Final concentration Amount
1× PBS 1× 100 mL
BSA 3% 3 g
Total n/a 100 mL
T. brucei primary antibody solution
Reagent Final concentration Amount
Blocking buffer 2 1× 25 mL
Primary antibody 1:250 0.1 mL
Total n/a 25 mL
Make fresh on the day of the experiment.
T. brucei secondary antibody solution
Reagent Final concentration Amount
Blocking buffer 2 1× 25 mL
Secondary antibody 1:1,000 0.025 mL
Total n/a 25 mL
Make fresh on the day of the experiment, protect from light.
Laboratory supplies
384-well black, clear and flat-bottom microplates (Greiner, catalog number: 781097)
Tissue culture–treated cell culture flasks with vented caps in T-25, T-75, T-175 sizes (Corning, catalog numbers: 430639, 430641U, and 431306, respectively)
Micropipettes of various sizes and compatible tips (e.g., Rainin, catalog number 30456871)
Neubauer Improved hemocytometer (SKC, Inc. INCYTO C-ChipTM, catalog number: DHC-N015)
0.22 µm vacuum filter and storage bottle (Corning, catalog number: 431205)
15 mL and 50 mL conical tube (VWR, catalog numbers: 21008-216, 21008-242, respectively)
WypAll tissue wipes (WYPALL, catalog number: 34770)
24-well plate (Corning, catalog number: 3524)
1.7 mL microcentrifuge tube (Axygen, catalog number: MCT-175-C-S)
Poly-L-Lysine–coated coverslips, #1 thickness, 12 mm diameter (Corning, catalog number: 354085)
Parafilm (Amcor, catalog number: PM996)
Microscope slides (Propper, catalog number: 15400100)
Sharp-angled tweezers (Excelta, catalog number: 89411-808)
Blunt-end tweezers (Burkle, catalog number: 5386-0104)
KimTech Science KimWipes (Kimberley-Clark Professional, catalog number: 34120)
Immersion oil (Type F, Index = 1.518) (Nikon, catalog number: MXA22168)
Optional: Nalgene wash bottles (Thermo Fischer Scientific, catalog number: 2402-0500)
Optional: Benchtop vacuum aspirator
Equipment
Humidified incubator set at 37 °C, 5% CO2
NucleoCounter (ChemoMetec, NC-200) and counting cassettes: Via1-Cassette (ChemoMetec, model: 941-0012)
Microplate reagent dispenser (Thermo Fisher Scientific Multidrop) and cassettes:
Multidrop standard cassette (Thermo, catalog number: 24072670)
Multidrop small tube cassette (Thermo, catalog number: 24073295)
Benchtop light microscope with 20× (0.3 NA) objective (Zeiss, Primovert, catalog number: 491206-0005-000)
Microplate orbital shaker (Perkin Elmer DELFIA PlateShake, catalog number: 1296-004)
Plate sealer (Agilent PlateLoc Thermal Microplate Sealer)
Fluorescence microscope
ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices)
Ti2-E epifluorescence microscope (Nikon)
Software and datasets
Nucleoview (ChemoMetec, version 1.2.0.0)
MetaXpress (Molecular Devices, LLC., version 6.7.0.211, 8 March 2021)
Prism v9.5.1 (GraphPad, January 26, 2023)
Excel (Microsoft, v2304, 25 April 2023)
NIS Elements AR (Nikon, version 5.20.02)
Procedure
Intracellular T. cruzi
Notes:
Culture Vero cells and parasites in RPMI complete media at 37 °C and 5% CO2 in a humidified incubator.
Utilize Vero cells between passage 2 and 30 for experiments and passage between 70% and 90% confluence.
Viability of Vero cells should be 92%–100% (determined by NucleoCounter).
A1. Vero cell culture
Remove RPMI complete medium from flask.
Add 2.5 mL of trypsin-EDTA and incubate for 6 min at 37 °C and 5% CO2 in a humidified incubator.
Tap the sides of the flask to ensure cell detachment.
Gently pipette up and down to de-clump cells and add to 10 mL of RMPI complete medium in a 15 mL conical tube to collect cells.
Spin down in the conical tube at 130× g for 7 min at room temperature.
Decant off supernatant and loosen pellet by flicking the bottom of the tube or tapping on the biosafety cabinet (BSC) work surface.
Resuspend Vero cells in 5 mL of fresh RPMI complete media.
Count cells on ChemoMetec NucleoCounter and normalize count for viability.
Add cells to prewarmed media. Cell numbers for T-75 flasks (T-25 flask will require 1/3, and T-175 flask will require three times more):
1-day passage: 6 × 106 cells
2-day passage: 3 × 106 cells
3-day passage: 1 × 106 cells
4-day passage: 7 × 105 cells—avoid using these directly for experiments.
A2. T. cruzi parasite culture
Passage T. cruzi parasites every 4 or 5 days in RPMI complete medium.
Seed 3 × 106 Vero cells in a T-175 flask (5-day passage) or T-75 flask (4-day passage) in 40 mL or 20 mL of media, for 5 and 4-day passages, respectively.
Allow cells to adhere for at least 30–60 min at 37 °C and 5% CO2 in a humidified incubator before infection.
Check parasites on the benchtop light microscope; parasites should have burst with plenty of agile, moving trypomastigotes in the culture supernatant.
Holding the flask flat on the BSC work surface, shake flask vigorously 2–3 times and collect supernatant in a 50 mL conical tube by decanting.
Count parasites by hemocytometer, with a 1:10–1:50 dilution of parasites in RPMI complete medium.
For routine passage, infect the T-175 flask containing Vero cells at a multiplicity of infection (MOI) of 5 for 5-day passage or a T-75 flask at MOI of 10 for 4-day passage.
Two to three days post-infection, rinse infected flasks with 40 mL of PBS, and add 25 mL of fresh RPMI complete medium.
A3. Cell seeding, infection, and compound treatment
Standard: tissue culture–treated plastic plates.
Using the standard cassette for a MultiDrop reagent dispenser, seed 5,000 Vero CCl-81 cells in 40 µL of RPMI complete mediuma per well of a 384-well microplate.
Allow Vero cells to adhere to microplate for at least 30–60 min in the incubator.
Note: Make sure all wells have a similar number of cells and that the cells have adhered.
Collect parasites from source flask and count parasite density on hemocytometer ensuring to only count healthy-looking trypomastigotes that are actively moving and no potential amastigotes or other rounded/ unhealthy parasites in the culture.
Infect Vero cells with trypomastigotes at MOI 5 in 10 µL of RPMI complete medium per well using small tube cassette with a MultiDrop reagent dispenser.
Incubate infected microplate for 48 h at 37 °C, 5% CO2.
Add compound to desired concentration.
Appropriate vehicle control should be used. The volume of compound or vehicle added to cultures should be kept consistent.
DMSO tolerability has been tested up to 0.6%. It is recommended to keep DMSO concentrations at or below 0.5%, but 0.6% can be tolerated.
We have typically used compounds dissolved in 100% DMSO at 10 mM stock concentration.
Return microplate to incubator at 37 °C, 5% CO2 and incubate for 24 h.
Alternative: Fibronectin-coated glass plates for high-resolution imaging adapted from Jumani et al. [13].
Add 1 mL (5 mL) of deionized (or MilliQ) water to 1 mg (5 mg) of solid-form fibronectin and leave for 30 min at room temperature to obtain a final concentration of 1 mg/mL. Allow the powder to dissolve on the bench, without disturbing it. Make aliquots and store at -20 °C. Avoid freeze-thaw of the frozen aliquots.
Dilute stock of fibronectin in PBS (without calcium and magnesium) at a dilution of 1:40 to achieve 25 µg/mL. For 384-well plates, coat wells by adding at least 20 µL per well (and at least 50 µL per well for a 96-well plate), making sure that the complete surface area of the plate is covered by the liquid.
Centrifuge for 2–4 mins at 400× g, making sure the liquid covers the surface and there are no air bubbles at the bottom of the plate.
Incubate for 2 h at room temperature.
Keep at 4 °C overnight up to 1 week for longer term storage.
For cell seeding, dump out fibronectin and immediately seed cells as above without allowing wells to dry (less than 5 min); after seeding, centrifuge plates for 2 min at 130× g to ensure proper settling.
Infect, incubate, and add compound to plate as above. All subsequent steps can be carried out in the same manner for both plastic and glass plates, below.
A4. Fixation, permeabilization, and staining
Add 50 µL of 8% fixative solution to each well.
Incubate plate with fixative solution for at least 15 min at room temperature (or at 4 °C for up to 2 weeks).
Remove fixative solution by dumping microplate contents onto absorbent, disposable wipe (WypAll wipes).
Add 35 µL of 0.5% permeabilization solution.
Centrifuge microplate at 130× g for 3 min to ensure permeabilization solution is fully in contact with cell monolayer.
Incubate microplate for 20 min with permeabilization solution at 37 °C, 5% CO2.
Remove permeabilization buffer and add 40 µL of blocking buffer 1 per well.
Incubate plate for 1 h at room temperature with gentle shaking on a plate shaker (approximately 225 rpm).
Remove blocking buffer 1 and add 40 µL of T. cruzi primary antibody solution per well.
Incubate plate for 1 h at room temperature with gentle shaking on a plate shaker (approximately 225 rpm).
Wash microplate twice with 100 µL of IFA wash buffer per well and add 40 µL of T. cruzi secondary antibody solution per well.
Incubate as above but add an opaque cover to protect the microplate from light.
Wash microplate twice with 100 µL of IFA wash buffer and then add 40 µL of Hoechst dye solution per well.
Incubate plate for 15 min at room temperature with gentle shaking as above and protected from light.
Remove Hoechst dye solution and add 75 µL of 1× PBS.
Dry any potential liquid on top of microplate with a KimWipe. Optional: seal with a light protective seal for storage.
A5. Imaging and analysis
Acquire images using an ImageXpress (or similar) confocal microscope with a confocal Acquisition Mode of “Confocal: 50 µm slit.” Select the correct plate type and confirm the dimensions. This should set the initial focus of each well to approximately the center of the well. If not, adjust the plate type and dimensions until this is achieved.
Under Acquisition settings enable Autofocus by selecting Enable laser-based focusing option.
In the Autofocus section, select Configure Laser Setting and set Well to well autofocus to Focus on well bottom option.
Under the Image-based Focusing section, select Algorithm to Standard and a Binning of 1.
For the Initial well for finding sample option, select First well acquired, and set the Number of wells to attempt initial find sample to 3.
Under the Set Autofocus option, select All sites.
For wavelengths, under Number of wavelengths add 3, with TL Legacy Shading Correction Refinement Level of 2. Select wavelengths W1 as DAPI, W2 as FITC, and W3 as TexasRed; the microscope includes standard filter sets for these wavelengths. Determine exposures using positive and negative controls for each wavelength and objective with a Target max intensity set to 33000 and making sure the highest intensity is well within the intensity maximum as visualized by the intensity histogram. Adjust the focus for each wavelength using Calculate Offset function and select the Use Z-stack. For Shading Correction select FL Shading Only option.
Acquire 3 × 3 with a 20 µm spacing (total 9 images per well) around the center of the well in an automated fashion using a 20× (0.95 NA) objective with water immersion. Higher-resolution images can be acquired using a 60× (1.2 NA) objective with water immersion. The higher-resolution images are easier to visualize and quantify.
For higher resolution, glass-bottom plates can be imaged using 60× water or oil or 100× oil objectives.
Bloodstream-form T. brucei
This protocol is modified from Kumar et al. [14] and is adapted from Glover and Horn [8].
B1. Compound treatment
Culture parasites in a humidified incubator at 37 °C, 5% CO2. Maintain parasites in logarithmic growth phase between 5 × 105 and 1 × 106 /mL.
Count parasites by hemocytometer and adjust density to 1 × 106 parasites/mL in HMI-9 complete medium.
Add 1 mL of 1 × 106/mL parasite culture per well of 24-well plates.
Add compound to desired final concentration.
Appropriate vehicle control should be used. The volume of compound or vehicle added to cultures should be kept consistent. If using DMSO, final concentration of DMSO should not exceed 0.5%.
We have typically used compounds dissolved in 100% DMSO at 10 mM stock concentration.
Incubate plates in humidified incubator at 37 °C, 5% CO2 for 4 h.
B2. Fixing and staining
Note: Once parasites are adhered to the coverslips, all dispensing and aspirating steps should be done away from direct contact with the coverslip. Dispense solutions and buffers along the well wall and aspirate from gaps between the coverslip and well walls. Orbital shaker is set at approximately 225 rpm for all shaking steps.
After compound treatment, collect entire well of treated parasites in a microcentrifuge tube.
Pellet parasites at 3,000× g for 5 min at room temperature.
Aspirate supernatant and resuspend pellet in 500 µL of 4% fixative solution.
Incubate in fixative solution for at least 15 min at room temperature or at 4 °C for up to 1 week.
Pellet samples at 3,000× g for 5 min at room temperature.
Carefully aspirate all but approximately 10 µL of the supernatant.
Resuspend parasite pellet in remaining supernatant.
Dot 10 µL of sample onto parafilm fixed to benchtop.
Place poly-L-lysine coverslip on sample, ensuring full coverage of the coverslip and minimizing bubbles. We recommend marking the coverslip with a thin marker before placing it on the sample to differentiate which side has the sample.
Allow sample to adhere to the coverslip for 15 min at room temperature.
Pick up the coverslip and place it sample-side facing up in a new 24-well plate for subsequent staining steps.
Rinse coverslips briefly with PBS, two times. We recommend using a squeeze bottle to dispense PBS; the volume is not particularly important, but it should be enough to completely cover the coverslip.
Add 500 µL of 0.25% permeabilizing solution to the well, shaking gently at room temperature on an orbital shaker.
Aspirate permeabilizing solution and add 500 µL of blocking buffer 2; incubate for 1 h at room temperature with gentle shaking (or overnight at 4 °C).
Aspirate blocking buffer and add T. brucei primary antibody solution; incubate for 1 h at room temperature with gentle shaking.
Wash sample 3× with PBS with 5 min of shaking in-between washing.
Add 500 µL of secondary antibody solution; incubate for 1 h at room temperature with gentle shaking.
Wash sample 3× with PBS with 5 min of shaking in-between washing.
Remove coverslip from 24-well plate. We recommend using sharp-angled tweezers to pry up the coverslip and a second pair to grab the coverslip once it is lifted from the bottom of the plate.
Gently dab off excess liquid from the edges of the coverslip with a Kimwipe.
Dot 3 µL of hard-set DAPI in Vectashield on a microscope slide and place the coverslip sample-side facing down.
Image using the Nikon Eclipse Ti-2E epifluorescence microscope (or equivalent).
Locate parasites in transmitted light in XY and Z-focus using a 40× (0.6 NA) objective. Turn on perfect focus and adjust to bring parasites into focus. Find location of parasites in XY plane and save 3–10 locations of XY.
For the multipoint XY locations selected, set up automated imaging using standard DAPI and FITC filters. Set exposures using DMSO vehicle control and phleomycin positive control. Make sure to not saturate the camera.
Higher-resolution images can be acquired using a 60× (1.4 NA) objective with oil immersion to improve signal-to-background.
Note: Although not necessary, Z-stacks and image deconvolution can be used to further improve resolution and signal for 40× (0.6 NA) and 60× (1.4 NA) objectives.
Data analysis
Data analysis for T. cruzi and T. brucei
Quantify the total number of parasites by counting the number of nuclei for T. brucei and tdTomato-positive parasites for T. cruzi. Perform analysis for at least 200 T. brucei nuclei or T. cruzi parasites per replicate.
For T. cruzi assay, it is essential to make sure host cell monolayers are intact by analyzing nuclei staining. A reduction in host nuclei counts could suggest cytotoxicity and/or technical error (for example, cells being washed off during the staining procedure).
Using the nuclei or parasites as a mask, count the total number of parasites that are positive for γH2A signal. The threshold/background of fluorescence intensity can be set using DMSO treated parasites. Consider parasites with any signal (foci or nuclear-wide) as positive for γH2A.
The above counts can be done manually or using an image analysis software. For manual counting, it is critical that the images are blinded. For example, image acquisition can be done by one person and then image file names changed to a generic name with codes. Image counting will then be performed by another person who only has access to the generic name without information of treatment. The first person can then unblind and analyze the data.
It is critical to run DMSO vehicle (negative control) and phleomycin for T. brucei and CT for T. cruzi as positive controls for each experiment; use these to set exposures.
Anticipated results are shown in Figures 2 and 3 with chemical structure of compounds used shown in Figure 4.
Figure 2. Detection of γH2A response in intracellular T. cruzi amastigotes. (A) Graphical overview of the protocol for γH2A detection in intracellular T. cruzi. Vero host cells were infected with T. cruzi tdTomato trypomastigotes at MOI 5 for 48 h. Cells were then treated with CTs or DMSO for 24 h before fixing with PFA and staining with anti-T. cruzi γH2A antibody. Samples were then imaged on an ImageXpress confocal microscope. (B) Representative images of γH2A response (green) in T. cruzi amastigotes (red) after treatment with DMSO control or CT5 with a 60× water objective (NA = 1.2). Scale bars = 5 µm. (C) Lower resolution representative images acquired with a 20× water objective (NA = 0.95) of γH2A response (green) in T. cruzi intracellular amastigote parasites (red) following treatment with DMSO or 20 µM CT1. Scale bars = 10 µm. (D) Quantitation of γH2A response in T. cruzi amastigotes. Infected cells were treated with DMSO or 20 µM of compounds (proteasome inhibitor is GNF6702, CT0-CT5 are cyanotriazoles). Quantitation was done by counting the total number of tdTomato-positive parasites and γH2A foci per image. Averages of at least three biological replicates, error bars represent standard deviation. Statistics calculated with unpaired parametric student’s t-test (ns = not significant, ****P < 0.0001).
Figure 3. Detecting γH2A response in BSF T. brucei. (A) Graphical overview of the protocol for γH2A detection. T. brucei parasites were seeded in a 24-well plate and treated with CTs, phleomycin, or DMSO for 4 h. Parasites were then collected, fixed with PFA, stained with anti-Tb γH2A antibody, and mounted on slides with DAPI in mounting medium. Samples were imaged on a Nikon Ti2-E microscope. (B) Representative images of T. brucei parasites treated with phleomycin D1 or DMSO. Images were acquired using a 60× oil objective (NA = 1.4). Scale bars = 5 µm. (C) Representative images of T. brucei parasites after 4 h treatment with DMSO, phleomycin D1, or CT3. Images were acquired using a 40× air objective (NA = 0.6). (D) Quantitation of γH2A response in T. brucei treated with various compounds (proteasome inhibitor is GNF6702; CT0, CT1, and CT3 are cyanotriazoles) Quantitation was carried out by counting total nuclei and γH2A foci per image and calculating the percentage of nuclei positive for γH2A. Data are mean and standard deviation of at least two biological replicates (CT0, n = 1). Statistics calculated with unpaired, parametric student’s t-test (ns = not significant, ****P < 0.0001, ***P = 0.0002).
Figure 4. Chemical structures of compounds used in the study. GNF6702 is kinetoplastid proteasome inhibitor, CT0-CT5 are cyanotriazoles, Phleomycin is a known T. brucei DNA damaging agent, benznidazole is an approved drug for Chagas disease.
Validation of protocol
This protocol has been used and validated in the following research article:
Rao et al. [5]. Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections. Science (Figure 3, panels B and C).
General notes and troubleshooting
Once microplates are stained and sealed, they can be stored at 4 °C for 1–2 months. It is critical that the wells do not dry during storage.
The protocol has been developed using T. cruzi CL Brener parasites. The protocol can be adapted to other parasite strains; however, the kinetics of γH2A induction could be different. We also note differences in the speed and duration of the γH2A response to different DNA-damaging agents, so some optimization for specific treatments may need to be made. Treatment times indicated in these protocols were optimized for parasite topoisomerase poisons and cyanotriazole (CT) compounds.
For T. cruzi, in place of 384-well microplates, 35 mm dishes (MatTek Corporation, catalog number: P35G-1.5-14-C), 96-well (or any other size well) plates, or similar can be used for the protocol using fibronectin coating and cell seeding methods as described in Jumani et al. [13]. For each vessel, cell numbers and volumes should be adjusted for surface area and height, respectively, based on the manufacturer’s recommendation.
Seeding of microplates can be done manually with single or multichannel pipettes, taking care to ensure the cell suspension is uniform during seeding, as this will enable equal distribution of cells between wells.
Washing steps for microplate-based assays can be automated with a BioTek EL406 or similar plate washer. For EL406, use slow mode, optimize protocol (for example, height, depth, and angle of the washer pins) to aspirate as much liquid as possible without disrupting monolayer, and dispense at the walls of the wells and not directly on the cells. Alternatively, washing can be done with a Multidrop or multichannel (or single channel) pipette, dispensing with a slow setting and dumping out liquid manually by inverting the plate and dabbing on absorbent laboratory wipe. Make sure to dispense liquid to the walls of the vessel and not directly onto the cells.
Image analysis can be performed using software like ImageJ/Fiji (can be adapted from Jumani et al. [13] altering the thresholding and size parameters), CellProfiler, Ilastik, or in-built microscope imaging software like NIS Elements or Molecular Devices.
For T. cruzi, it is critical to add PFA directly to cells before washing. Performing washing before fixing can cause Vero cell monolayers to disassociate.
The γH2A response in T. cruzi epimastigotes was validated with classical, non-CT DNA damaging agents hydroxyurea and phleomycin by western blot.
Acknowledgments
This work is part of the Rao et al. [5] manuscript published in Science, 2023. This research work was funded by Novartis BioMedical Research, and in part by the Wellcome Trust (Project Number: 219639/Z/19/Z). The authors thank Manuel Saldivia for coordinating production of T. brucei γH2A antibody, Debjani Patra for technical support, and to Manuel Saldivia, Ujjini H. Manjunatha, Jonathan E. Gable, Yen-Liang Chen, and the rest of the Novartis team for insightful discussions. We thank Gu Feng, Thomas Krucker, Jean Claude Poilevey, Emily Tongco-Wu and team for project management, alliance management and partnering, and legal and finance support.
Competing interests
All authors are Novartis employees, and some own shares in Novartis. CT compounds have been patented by Novartis with S.P.S.R. listed as an author (US patent application no. 17/253,737,2022).
Ethical considerations
No human or animal subjects were included in this study.
References
de Sousa, A. S., Vermeij, D., Ramos, A. N. Jr, and Luquetti, A. O. (2024). Chagas disease. Lancet. 403(10422): 203–218.
Büscher, P., Cecchi, G., Jamonneau, V. and Priotto, G. (2017). Human African trypanosomiasis. Lancet. 390(10110): 2397–2409.
Mesu, V. K. B. K., Kalonji, W. M., Bardonneau, C., Mordt, O. V., Blesson, S., Simon, F., Delhomme, S., Bernhard, S., Kuziena, W., Lubaki, J. P., et al. (2018). Oral fexinidazole for late-stage African Trypanosoma brucei gambiense trypanosomiasis: a pivotal multicentre, randomised, non-inferiority trial. Lancet. 391(10116): 144–154.
Betu Kumeso, V. K., Kalonji, W. M., Rembry, S., Valverde Mordt, O., Ngolo Tete, D., Prêtre, A., Delhomme, S., Ilunga Wa Kyhi, M., Camara, M., Catusse, J., et al. (2023). Efficacy and safety of acoziborole in patients with human African trypanosomiasis caused by Trypanosoma brucei gambiense: a multicentre, open-label, single-arm, phase 2/3 trial. Lancet Infect Dis. 23(4): 463–470.
Rao, S. P. S., Gould, M. K., Noeske, J., Saldivia, M., Jumani, R. S., Ng, P. S., René, O., Chen, Y. L., Kaiser, M., Ritchie, R., et al. (2023). Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections. Science. 380(6652): 1349–1356.
Vann, K. R., Oviatt, A. A. and Osheroff, N. (2021). Topoisomerase II Poisons: Converting Essential Enzymes into Molecular Scissors. Biochemistry. 60(21): 1630–1641.
Kinner, A., Wu, W., Staudt, C. and Iliakis, G. (2008). γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res. 36(17): 5678–5694.
Glover, L. and Horn, D. (2012). Trypanosomal histone γH2A and the DNA damage response. Mol Biochem Parasitol. 183(1): 78–83.
Gomes Passos Silva, D., da Silva Santos, S., Nardelli, S. C., Mendes, I. C., Freire, A. C. G., Repolês, B. M., Resende, B. C., Costa-Silva, H. M., da Silva, V. S., Oliveira, K. A. d., et al. (2018). The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions. PLoS NeglTrop Dis. 12(11): e0006875.
UniProt, C. (2023). UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 51(D1): p. D523–D531.
Amos, B., Aurrecoechea, C., Barba, M., Barreto, A., Basenko, E. Y., Bażant, W., Belnap, R., Blevins, A. S., Böhme, U., Brestelli, J., et al. (2021). VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center. Nucleic Acids Res. 50: D898–D911.
Canavaci, A. M. C., Bustamante, J. M., Padilla, A. M., Perez Brandan, C. M., Simpson, L. J., Xu, D., Boehlke, C. L. and Tarleton, R. L. (2010). In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds. PLoS NeglTrop Dis. 4(7): e740.
Jumani, R. S., Hasan, M. M., Stebbins, E. E., Donnelly, L., Miller, P., Klopfer, C., Bessoff, K., Teixeira, J. E., Love, M. S., McNamara, C. W., et al. (2019). A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors. Nat Commun. 10(1): 1862.
Kumar, G., Thomas, B. and Mensa-Wilmot, K. (2022). Pseudokinase NRP1 facilitates endocytosis of transferrin in the African trypanosome. Sci Rep. 12(1): 18572.
Article Information
Publication history
Received: Feb 21, 2024
Accepted: May 30, 2024
Available online: Jun 24, 2024
Published: Jul 5, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Microbiology > Microbial cell biology > Cell staining
Cell Biology > Cell imaging > Confocal microscopy
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A Protocol for Preparing Mucoadhesive Emulsion Microgels and Assessing Their Mucoadhesion Properties In Vitro
MS Mariia S. Saveleva
ML Mikhail E. Lobanov
OM Oksana A. Mayorova
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5027 Views: 294
Reviewed by: Olga KopachAmira S HanafyMathilde Ullrich
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Original Research Article:
The authors used this protocol in ACS Applied Materials & Interfaces May 2023
Abstract
Intravesical instillation is an efficient therapeutic technique based on targeted administration of a drug directly into the lesion for the treatment of bladder diseases. This is an alternative to traditional systemic administration of drugs. However, this technique requires repeated procedures, which can lead to even greater inflammation and infection of the urethra. To date, novel systems that allow prolonged drug retention in the bladder cavity are actively being developed. We recently reported a targeted drug delivery system based on the mucoadhesive emulsion microgels consisting of the natural component whey protein isolate. Such micron-sized carriers possess high loading capacity, a prolonged drug release profile, and efficient mucoadhesive properties to the bladder urothelium. As a continuation of this work, we present a protocol for the synthesis of mucoadhesive emulsion microgels. Detailed procedures for preparing precursor solutions as well as studying the physico-chemical parameters of microgels (including loading capacity and drug release rate) and the mucoadhesive properties using the model of porcine bladder urothelium are discussed. Precautionary measures and nuances that are worth paying attention to during each experimental stage are given as well.
Key features
• The protocol for the synthesis of mucoadhesive emulsion microgels based on whey protein isolate is presented. The experimental conditions of emulsion microgels synthesis are discussed.
• Methods for studying the physico-chemical properties of mucoadhesive emulsion microgels (size of emulsion microgels particles, loading capacity, release kinetics) are described.
• The method for assessing mucoadhesive properties of emulsion microgels is demonstrated using the porcine bladder tissue model ex vivo.
Keywords: Emulsion microgels (EM) Whey protein isolate (WPI) Emulsion drug release Mucoadhesion Urinary bladder Urothelium
Graphical overview
Background
Currently, for the treatment of urinary system diseases, such as cystitis or bladder cancer, targeted drug delivery using a catheter directly to the site of the disease is widely used. The intravesical instillation of antibacterial or cytostatic drugs has significant advantages over systemic administration; particularly, allowing the reduction of side effects on healthy organs (namely, inducing liver function disorders, undesirable effects on the central nervous system, and blood pressure, as well as delayed mutagenic, teratogenic, and carcinogenic effects of drugs) [1,2]. However, this delivery method has a number of disadvantages as well, which are conditioned primarily by the structure and basic physiological functions of the urinary system. The inner surface of the urinary bladder wall—the so-called urothelium—is represented by umbrella cells, whose size varies depending on their stretching degree. The surface of umbrella cells is covered with glycoproteins and proteoglycans, which form the glycosaminoglycan layer. This layer acts as an efficient barrier against the penetration of substances in the urine. However, at the same time, this layer prevents the penetration of drugs instilled to the bladder. Also, the constant flow of urine inside the bladder reduces the therapeutic effect and washes the drug out of the organ. Thus, it becomes necessary to repeat this procedure several times. According to feedback from patients receiving medications through intravesical instillation, such manipulation is quite painful. At the same time, there is a high risk of infection and inflammation of the urethra if catheterization is performed incorrectly [3].
The development of drug carriers that are able to retain drugs at the urothelium surface of the bladder for a long time might overcome these limitations. In this regard, the development of micro- and nano-sized carriers for intravesical drug delivery is of great interest. The most prominent strategies described in the literature include various types of carriers including thermosensitive hydrogels capable of undergoing sol-gel transition at body temperature as well as site-specific targeted liposomes and nanoparticles. However, in the first case, the natural dilution of the gels in the bladder leads to the loss of their gel-forming properties [4,5]. In the second case, in order to obtain such micro-sized systems, synthetic hydrophobic polymers are used, which are often dissolved in organic solvents that are mostly non-biocompatible and have irritating effects on the tissues [6–8].
In our recent work, we developed biocompatible and biodegradable emulsion microgels based on whey isolate protein with sufficient mucoadhesive properties. Targeting of such micro-sized carriers into the bladder will extend the residence time of drugs, reducing the number of instillation procedures [9]. Thus, this approach will help to significantly improve the quality of life of patients suffering from diseases of the urinary system. However, it is necessary to carefully follow the protocol for the formation of emulsion microgels, since changing the ratios of precursors can lead to the formation of particles with different loading and mucoadhesive properties.
The protocol here presented describes in detail the procedure for the synthesis of fluorescently labeled emulsion microgels based on whey protein isolate. Calculations of the optimal oil-to-water ratios for obtaining the most stable emulsion systems are given. We consider the main ways to characterize the resulting particles and provide a step-by-step description of the methodologies and troubleshooting aspects involved in determining the loading capacity of emulsion microgels as well as their drug release rates. In addition, we provide precise guidance on how to perform qualitative and quantitative analysis of the mucoadhesive ability of microgels on porcine bladder tissue ex vivo.
Materials and reagents
Reagents
Linseed oil OLEOS (Russia) (unrefined cold pressed linseed oil, refractive index n20/D 1.4795, density 0.93 g/mL at 25 °C), https://oleos-info.ru/product/lnyanoe-maslo/
Whey protein isolate (WPI) California Gold Nutrition (USA), https://www.californiagoldnutrition.com/products/california-gold-nutrition-sport-whey-protein-isolate-unflavored-5-lb-2-27-kg-76479 (27 g protein, 6.2 g BCAAs, 4.7 g glutamic acid, low lactose. Lactose is used as an excipient, which is not involved in the formation of emulsion microgels. No additives or flavor enhancers)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417)
Tetramethylrhodamine isothiocyanate (TRITC) (Sigma-Aldrich, catalog number: T0820)
Fluorescein isothiocyanate isomer I (FITC) (Sigma-Aldrich, catalog number: F7250)
Rhodamine B isothiocyanate (RITC) (Sigma-Aldrich, catalog number: 283924)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911)
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C4901)
Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014)
Dimethyl sulfoxide (DMSO) (EcoChemAnalyt, Russia, CAS: 67-68-5)
Urea (CH4N2O) (ReaChem, Russia, State Standard 2081-92, CAS: 57-13-6)
Ammonium chloride (NH4Cl) (ReaChem, Russia, State Standard 3773-72, CAS: 12125-02-9)
Magnesium sulfate heptahydrate (MgSO4·7H2O) (ReaChem, Russia, State Standard 4523-77, CAS: 10034-99-8)
Sodium sulfate (Na2SO4) (ReaChem, Russia, State Standard 4166-76, CAS: 7757-82-6)
Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) (ReaChem, Russia, State Standard 245-76, CAS: 13472-35-0)
Sodium hydrogen phosphate (Na2HPO4) (ReaChem, Russia, State Standard 11773-76, CAS: 7558-79-4)
Hydrochloric acid (HCl) (EcoChemAnalyt, Russia, State Standard 14261-77, CAS: 7647-01-0)
Sodium hydroxide (NaOH) (EcoChemAnalyt, Russia, State Standard 4328-77, CAS: 1310-73-2)
Trypsin (Sigma-Aldrich, catalog number: T4799)
Milli-Q water (Merck Millipore, model: Milli-QTM Advantage A10TM, Germany)
Note: It is possible to use linseed oil from another manufacturer. However, it is worth paying attention to its properties (the refractive index n20/D of 1.4795 and the density of 0.93 g/mL at 25 °C).
Solutions
Saline 0.9% NaCl (w/v) solution (see Recipes)
2.5% WPI solution (see Recipes)
5% WPI solution (see Recipes)
7.5% WPI solution (see Recipes)
5 mg/mL FITC solution (see Recipes)
1 M NaOH solution (see Recipes)
PBS buffer (0.1 M, pH 8.3) (see Recipes)
Artificial urine solution (see Recipes)
0.5 mg/mL trypsin in Tris-HCl buffer solution (20 mL) (see Recipes)
70% ethanol (see Recipes)
Recipes
Note: In all recipes, liquid is added to the dry sample until the final specified volume is reached (“total” volume) in order to obtain an accurate solution concentration. For example, in Recipe 1, H2O is added to the dry sample of NaCl until the final volume of 1 L is reached (instead of just adding 1 L of H2O to the dry sample of NaCl). The volume of liquid is indicated in tables as a necessary recommended volume (not as a precise volume).
Saline 0.9% NaCl (w/v) solution
*w/v: weight (g) per volume (100 mL).
Reagent Final concentration Amount
NaCl 0.9 % (w/v) 9 g
H2O n/a 1 L
Total n/a 1 L
2.5% WPI (w/v) aqueous solution
Reagent Final concentration Amount
WPI 2.5 % 250 mg
Saline 0.9% NaCl solution 0.9% (w/v) 10 mL
Total n/a 10 mL
Caution: This solution should be mixed thoroughly. Before use, please wait until the foam at the surface of the solution disappears.
Note: For the FITC conjugation procedure, WPI solution is prepared as described above in Recipe 2, but instead of water the PBS (pH 8.3) is used for protein dissolution.
5% WPI (w/v) aqueous solution
Reagent Final concentration Amount
WPI 5% 500 mg
Saline 0.9% NaCl solution 0.9% (w/v) 10 mL
Total n/a 10 mL
Caution: This solution should be mixed thoroughly. Before use, please wait until the foam at the surface of the solution disappears.
Note: For the FITC conjugation procedure, WPI solution is prepared as described above in Recipe 3, but instead of water the PBS (pH 8.3) is used for protein dissolution.
7.5% WPI (w/v) aqueous solution
Reagent Final concentration Amount
WPI 7.5% 750 mg
Saline 0.9% NaCl solution 0.9% (w/v) 10 mL
Total n/a 10 mL
Caution: This solution should be mixed thoroughly. Before use, please wait until the foam at the surface of the solution disappears.
Note: For the FITC conjugation procedure, WPI solution is prepared as described above in Recipe 4, but instead of water the PBS (pH 8.3) is used for protein dissolution.
5 mg/mL FITC solution
Reagent Final concentration Amount
FITC 5 mg/mL 5 mg
DMSO n/a 1 mL
Total n/a 1 mL
Note: Since a 5 mg sample is difficult to weigh, it is recommended to prepare a larger weight of FITC, which will require a larger volume of DMSO to prepare the final solution with the given FITC concentration of 5 mg/mL. For this, you need to proportionally increase FITC mass and DMSO volume. For example, weigh out 25 mg of FITC, then add DMSO until the volume reaches 5 mL. In this way, you will obtain 5 mL of a final FITC solution of 5 mg/mL.
1 M NaOH solution (100 mL)
Reagent Final concentration Amount
NaOH 1 M 4 g
H2O n/a 100 mL
Total n/a 100 mL
PBS solution (0.1 M, pH = 8.3)
Reagent Final concentration Amount
PBS 1× 1 tablet
H2O n/a 200 mL
Total n/a 200 mL
Note: Adjust pH of the resulting solution to 8.3 using 1 M NaOH and 1 M HCl solutions.
Artificial urine solution (pH 6.2)
Reagent Final concentration Amount
Urea n/a 24.27 g
NaCl n/a 6.34 g
KCl n/a 4.50 g
NH4Cl n/a 1.61 g
CaCl2 n/a 0.67 g
MgSO4·7H2O n/a 1.0 g
NaHCO3 n/a 0.34 g
Na2SO4 n/a 0.26 g
NaH2PO4·H2O n/a 1.0 g
Na2HPO4 n/a 0.11 g
H2O n/a 2 L
Total n/a 2 L
Note: The dry weights and the liquid volume can be changed proportionally to each other depending on needs (for example, for 1 L, the masses of dry samples are correspondingly reduced by two times relative to the masses given in the table). It is recommended to add weights in the order of priority shown in the table. The solution should be stirred for 3 h at 22 °C. The prepared artificial urine solution should be stored at 2–8 .
0.5 mg/mL trypsin in Tris-HCl buffer solution (20 mL)
Reagent Final concentration Amount
Trypsin 0.5 mg/mL 10 mg
Tris base 1 M 2.42 g
H2O n/a 20 mL
Total n/a 20 mL
Note: Adjust pH of the Tris solution to 7.5 using HCl 1 M. The solution should be stored at 2–8 °C.
Ethanol 70%
Reagent Final concentration Amount
Ethanol 96% 70% 1 L
H2O n/a 371 mL
Total n/a 1,371 mL
Note: When mixing these liquids, you must strictly add ethanol to water, not vice versa.
Laboratory supplies
Centrifuge tubes with flat cap, 15 mL (JetBioFil, catalog number: CFT550150)
Centrifuge tubes with flat cap, 50 mL (JetBioFil, catalog number: CFT500500)
Microcentrifuge tubes, 1.5 mL (JetBioFil, catalog number: CFT000015)
Microcentrifuge tubes, 2.0 mL (JetBioFil, catalog number: CFT000020)
Laboratory glass jar 100 mL, with divisions, with screw lid, dark glass (MiniMed, catalog number: 10007205)
Vial 2 mL, dark glass (ALWSCI Technologies, catalog number: C0001177)
Vial 10 mL, clear glass, 22 mm × 52 mm (ALWSCI Technologies, catalog number: C0000053)
Dialysis bag M-Cel, pore diameter 14 kDa (Viscase, catalog number: 2141-1425)
Dialysis bag clamp (Scienova GmbH, catalog number: 40329)
Laboratory beaker, clear glass, 2 L (MiniMed, catalog number:10003807)
Cellulose acetate membrane filter, 0.45 μm (Sartorius, catalog number: 11106-37-N)
Membrane filters PP, 10 μm (Gluvex, catalog number: MLPP1001000)
Glass for microslides (MiniMed, catalog number: 12003421)
Cover glass for microslides (MiniMed, catalog number: 12003309)
Syringe 1 mL (BD Micro-Fine Plus, catalog number: 320935)
96-well V-bottom plate, conical bottom, non-treated, no lid (Costar, catalog number: 3897)
Pipette microtips, 2–20 μL (JetBioFil, catalog number: PPT100020)
Pipette microtips, 10–200 μL (JetBioFil, catalog number: PPT000200)
Pipette microtips, 100–1,000 μL (JetBioFil, catalog number: PPT000000)
Petri dishes, D 9.0 cm (JetBioFil, catalog number: MCD000090)
Adson microsurgical tweezers, 130 mm (Medical Equipment, catalog number: MF-2000)
Tissue tweezers, 130 mm (Medical Equipment, catalog number: MF-2102)
Equipment
Ultrasonic homogenization and Bandelin Sonopuls HD 2070 homogenizer at a frequency of 20 kHz and a power density of 1 W/cm2 (Germany)
Amicon® Stirred Cell 50 mL (Merck Millipore)
Magnetic stirrer (IKA)
Magnetic stirring bar (IKA, model: IKAFLON® 15)
Multifunctional refrigerated centrifuge (Eppendorf, model: 5810R)
Microplate reader (BMG Labtech, model: CLARIO Star Plus)
Drybath thermo shaker (Thermo Scientific)
Inverted microscope with a 40× objective (Olympus IX73)
Water purification system for ultrapure water (Merck Millipore, model: Milli-QTM Advantage A10TM)
Single-channel variable volume dispenser 100–1,000 mL, 10–100 mL, 20–200 mL, 5–50 mL, and 1,000–5,000 mL (Thermo Fisher Scientific)
Software and datasets
ClarioStar MARS 4.01 R2 (BMG LabTech, Germany)
ImageJ software 1.51j8 (National Institutes of Health, USA) (the open-source version is available online for free for download)
OriginPro 2018 SR1 (OriginLab Corporation, USA)
Excel 2019 (Microsoft Cooperation, USA)
Procedure
Conjugation of WPI by FITC
Prepare a solution of 5% WPI in PBS (0.1 M, pH 8.3). Dissolve 1 g of WPI in 20 mL of PBS.
Prepare a 2.5 mL FITC solution in DMSO (concentration 5 mg/mL) in a dark glass bottle.
Mix WPI and FITC solutions in a dark glass bottle. Stir the resulting solution using an IKA magnetic stirrer for 24 h in a dark place at 4 °C.
It is highly important to wash the obtained conjugated FITC-WPI solution out from the unreacted FITC molecules. This can be performed by using various methods such as dialysis, gel-filtration, retraining, or chromatography. In this work, we used dialysis against water. For this, place the prepared FITC-WPI solution in a dialysis bag (pore size 12–14 kDa). Secure the dialysis bag tightly with clamps at both ends to avoid leakage of the solution. Immerse the dialysis bag with FITC-WPI solution in 2 L of deionized water and keep it under stirring for 48 h in a dark place at 4 °C.
Store the freshly prepared FITC-conjugated WPI in a refrigerator without access to light.
Caution: Fluorescent dyes are sensitive to light. Therefore, all procedures with fluorescently labeled protein are carried out in dark glass bottles or covered with aluminum foil.
Note: The reaction of FITC-WPI conjugation can proceed faster if the process takes place at room temperature. In this case, the conjugation takes 8 h. However, the WPI solution cannot be stored for a long time at temperatures above 8 °C. For this reason, we highly recommend performing the conjugation at 4 °C for 24 h. The use of magnetic stirrer IKA MINI is suitable for such a long stirring.
Figure 1. Dark glass bottle with FITC-WPI conjugate solution
Formation of mucoadhesive emulsion microgels
Place 2 mL of 5% WPI-FITC solution in a 20 mL glass beaker.
Add 0.3 mL of linseed oil to 2 mL of WPI-FITC solution. This corresponds to a 1:3 ratio of the WPI solution:oil (wt:wt). A detailed scheme for the synthesis of emulsion microgels is presented in Figure 2 (upper panel).
Caution: It is necessary to strictly observe the proportions of the WPI phase:oil phase, since the particle size of the resulting emulsion microgels will be strongly dependent on this ratio. The higher the water:oil ratio, the larger the resulting diameter of the emulsion microgels particles. A smaller amount of aqueous phase may result in a deficiency of WPI molecules in the system and, as a result, fewer protein-stabilized emulsions. The water:oil ratio also influences the loading capacity of emulsion microgels and their rates of degradation and release of encapsulated substances. To obtain larger emulsion microgels particles in their diameter and number, it is necessary to proportionally increase the volume of oil. If the concentration of the initial WPI solution is changing, the volume of oil also changes according to Table 1 below.
Table 1. Ratios of WPI solutions and oil (w:w) for preparing emulsion microgels
WPI concentration Ratio WPI solution:oil (w:w)
1:1 1:3 1:5
2.5% [V (WPI), mL:V (oil), mL] (2.0:0.05) (2.0:0.15) (2.0:0.25)
5% [V (WPI), mL:V (oil), mL] (2.0:0.1) (2.0:0.3) (2.0:0.5)
7.5% [V (WPI), mL:V (oil), mL] (2.0:0.15) (2.0:0.45) (2.0:0.75)
Immerse the probe of the ultrasonic homogenizer into the resulting mixture of WPI solution and oil. Apply ultrasound at a frequency of 20 kHz and a power density of 1 W/cm2 for 1 min to obtain the emulsion microgel.
Note: During the process of ultrasonic homogenization, it is possible to use additional cooling measures to prevent localized heating. However, this is not a necessary step due to the short duration of exposure of the mixture to the ultrasound.
Filter the obtained emulsion microgels using a filtration cell Amicon® stirred cell 50 mL and a filter membrane with 0.45 μm pore size.
Note: You can learn how to work with a Amicon® stirred cell using the user guide on the official website of Amicon: https://www.merckmillipore.com/RU/ru/product/Amicon-Stirred-Cell-50mL,MM_NF-UFSC05001?ReferrerURL=https%3A%2F%2Fwww.google.com%2F#anchor_UG.
Wash the emulsion microgels using the filtration cell with 10 mL saline (0.9% NaCl) and a filter membrane surface with a 0.45 μm pore size using the 1,000 mL single-channel dispenser until the supernatants become clear.
After filtration, dilute the obtained sedimented emulsion microgels at the 0.45 μm filter membrane up to 5 mL with saline (0.9% NaCl). Then, carefully collect diluted microgels from the membrane surface using a pipette and place it in a separate tube for a while until the next step.
Pass the obtained emulsion microgels through the 10 μm filter membrane in order to separate the large oil droplets. Collect the filtered emulsion microgels in the glass vial.
Store the freshly prepared emulsion microgels in the capped glass vial in a refrigerator at 4 °C. Photographs of the obtained samples of emulsion microgels are presented in Figure 2A–2C (middle panel).
Figure 2. Preparation procedure and overview of the resulting emulsion microgels. Upper panel: Schematic process of emulsion microgels preparation. A–C: Photographs of prepared emulsion microgels with different ratios of WPI solution–oil and different concentrations of WPI solutions used for sample preparations. D–F: Optical images of emulsion microgels with different ratios of WPI solution–oil and fixed concentration of WPI solution (5%). The average diameters of particles of emulsion microgels are given as mean ± standard deviation.
Determination of emulsion microgel sizes
Diluted the initial emulsion microgel sample 1,000 times with saline (0.9% NaCl). Place 5 μL of diluted emulsion microgel onto a glass slide and cover with a coverslip.
Obtain optical images of emulsion microgels with the Olympus IX73 inverted microscope and a 40× objective (Figure 2D–2F).
Measure the diameters of emulsion microgels particles using optical images (Figure 2D–2F). Perform measurements using the ImageJ software. To calculate the average diameter of emulsion microgel particle size, at least 100 measurements and 10 images for each sample were analyzed. The average diameter of particles was presented as the mean ± standard deviation.
Note: The Olympus IX73 inverted microscope contains a fluorescent module. However, to calculate the number of particles in this work, brightfield images were used.
In vitro release study
Place 1 mL of emulsion microgel sample in a 2 mL Eppendorf tube. Add 1 mL of the artificial urine and mix using the vortex for 1 min. Prepare three independent samples for each time point of probing (24, 48, 72, 96, and 120 h). The whole route of release study is represented at Figure 3 (upper panel).
Place samples in a Drybath thermo shaker at 22 °C with continuous shaking (600 rpm).
At specific time points (10 min, 24, 48, 72, 96, and 120 h), take the samples from the Thermo shaker and centrifuge at 14,500× g for 3 min until the separation of oil and water phases occurs.
After centrifugation, the aqueous and oil phases should be separated (oil phase layer covers the aqueous phase on top). Probe the 1 mL of aqueous phase of the sample using a syringe needle.
Add 1 mL of fresh medium to the sample and mix it thoroughly with a vortex until the emulsion becomes homogeneous (usually it takes 3–5 min). Then, return it to the thermo shaker.
D1. Preliminary preparation of obtained probes for spectrophotometric analysis
Dilute 1 mL of obtained probes of aqueous phase with 1 mL of ethanol 70% in order to sediment the residual protein and dissolve the residual oil. Centrifuge the mixture at 14,500× g at room temperature for 3 min.
Separate the aqueous phase from sediment. The obtained probes are ready for spectrophotometric analysis.
Note: A dilution of obtained probes may be necessary before the spectrophotometric measurements. For this purpose, a stock solution of artificial urine is needed.
Spectrophotometric determination of FITC-WPI amount encapsulated and released from the emulsion microgels
Place 200 μL of each probe in triplicates (prepared as described above in section D) in a Costar® 96-cell microplate.
Analyze absorbance spectra in a range of 200–1,000 nm with the CLARIO Star Plus microplate reader (Figure 3A).
The absorbance maximum for FITC in artificial urine solution is registered at 478 nm at FITC concentration ranging from 5 to 50 μg/mL (Figure 3A).
The amount of FITC-WPI in probes is determined on the basis of optical density value at 478 nm using the corresponding calibration curves (Figure 3B). In order to obtain calibration curves, absorption spectra of solutions with various FITC concentrations in media were measured. For this, a solution of FITC in DMSO with a concentration of 1 mg/mL was prepared. Then, a series of solutions of known FITC concentrations ranging from 5 to 50 μg/mL were made (5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg/mL). These solutions were obtained by diluting a 1 mg/mL fluorescent dye solution with a stock solution of artificial urine (see step D1, note). Then, 200 μL of solutions of known concentrations were placed into the wells of the Costar® 96-cell microplate and measured spectrophotometrically. Based on the optical density at the fluorescent dye absorption maximum and known concentrations of FITC in solutions (Figure 3A), calibration curves were constructed (R2 = 0.998) (Figure 3B).
Figure 3. Overview of experiment on the model drug release from emulsion microgels. Upper panel: schematic process of release experiment. Bottom panel: (A) Characteristic adsorption spectra of FITC in artificial urine for different FITC amounts; (B) calibration curve of FITC in artificial urine; (C) kinetics of FITC-WPI release from emulsion microgels in artificial urine. Data are presented as the mean ± standard error calculated from three measurements for each sample.
To obtain the release kinetics curves, the calculated amounts of FITC-WPI in probes received during the experiment at specific time points (10 min, 24, 48, 72, 96, and 120 h) (see section D) were plotted against the time of incubation of the emulsion microgels in model medium (artificial urine). The dependence of the released FITC-WPI concentration from the emulsion microgels on the incubation time is shown in Figure 3C. The most stable and prolonged release of WPI-FITC is observed for the sample (7.5%, 1–3). The maximum released amount is observed after 96 h and is ~58% of total FITC amount loaded in the microgels. In the case of the sample (5%, 1–3), the release kinetics profile reaches a maximum level after 48 h. In the case of the sample (2.5%, 1:3), a statistically significant increase in FITC-WPI concentration was detected after 10 min of incubation.
Quantification of FITC-WPI loading capacity in emulsion microgels
The amount of FITC-WPI in filtrates obtained during the process of sample preparation (Msolution) (as described in section D) was measured in order to determine the FITC-WPI loading capacity in emulsion microgels. The filtrates were processed as described in step D1. The amount of FITC in filtrates was determined spectrophotometrically as described in section E.
Note: In this case, the stock solution used in probes preparation and dilutions was the mixture of ethanol 70%: saline NaCl 0.9% (at a 1:1 ratio).
The loading capacity of FITC-WPI in emulsions was calculated as:
L
C
(
%
)
=
M
i
n
i
t
i
a
l
-
M
s
o
l
u
t
i
o
n
M
i
n
i
t
i
a
l
×
100
%
where Minitial is the amount of FITC in the initial conjugated FITC-WPI solutions, which were used in the preparation of emulsions; Msolution is the amount of FITC-WPI in solutions obtained after emulsion filtration.
Mucoadhesion properties and retention of the emulsion microgels on porcine urinary bladder tissues
Porcine bladder tissues were obtained from the Saratov State Vavilov Agrarian University (Saratov, Russia). The freshly extracted urinary bladders were immediately packed with dry ice and transported in a cold box. The experiment started immediately after the urinary bladder was received.
Carefully cut a bladder tissue to samples with size 1 cm × 1 cm and wash with 3 mL of artificial urine solution. Cut samples with meticulous precision and accuracy to keep the upper urothelial layer intact.
Perform background fluorescence microscopy imaging on each tissue sample before application of the emulsion microgels.
Apply 100 μL of emulsion microgels solution to the mucosal surface of urinary bladder samples with a pipette, maintaining its uniform spreading on the bladder surface, and keep for 10 min. After that, remove non-absorbed emulsion microgels by washing with 6 mL of the artificial urine solution three times. For this, intensively pour the artificial urine from the pipette tip on the surface of the bladder tissue sample, which is placed in the Petri dish (9 cm in diameter). After that, collect spent artificial urine from the Petri dish. The experiment was conducted in triplicate for each type of emulsion microgels.
Place porcine bladder tissues on the microscope stage of inverted microscope Olympus IX73 with a 40× objective (Figure 4A). The fluorescence of WPI-FITC was excited at the wavelength range 470–495 nm, and the emission was detected at the wavelength range 510–550 nm.
Caution: It is important to begin the experiment promptly after obtaining a fresh porcine bladder since the mucosal layer of the urothelium begins to degrade almost immediately.
Figure 4B shows a fluorescent image of the surface of porcine bladder tissue treated with the emulsion microgels sample (5%, 1:3) for 10 min and after a single wash with artificial urine.
Figure 4. Study of fluorescence signals from FITC-labeled emulsion microgels adsorbed on porcine urinary bladder tissue. (A) Porcine urinary bladder tissue on the microscope stage. (B) Optical fluorescent images of the urothelium surface of porcine bladder after its incubation with emulsion microgels samples and after its one-time washing with artificial urine. Exposure time, 50 ms. Scale bar: 100 μm.
Quantitative estimation of WPI-FITC content in bladder mucosa
Incubate urinary bladder tissues in 1 mL of trypsin solution in Tris-HCl buffer for 60 min at 37 °C and continuous shaking at 500 rpm. After that, centrifuge obtained homogenates at 400× g for 1 min at room temperature. After that, collect the liquid phase with a pipette.
Add 200 μL of obtained liquid phase to the Costar® 96-cell microplate (in triplicate). Emission spectra (475 nm excitation, 490–510 nm emission range with 1 nm step) were recorded with the CLARIOstar Plus microplate reader.
Intact urinary bladder tissues homogenates mixed with known FITC amounts (40, 30, 20, 10, 5, and 2.5 μg/mL) were used as solutions for obtaining the calibration curve and prepared in the same approach.
The emission intensity at 500 nm was used in the calibration and calculation of the WPI-FITC content in bladder tissues.
Data analysis
Calculation of droplet sizes of emulsion microgels
For calculating the average emulsion droplet size, at least 100 measurements and 10 images for each sample were analyzed. The ImageJ software was used for image processing and statistics. At the first step, upload the optical image of the emulsion microgels into the ImageJ software. Before measurement, calibrate the scale of this image in length units (set scale). Then, measure the diameter of an emulsion droplet directly at the image by means of available tools of measuring linear dimensions in this program (for example, a segment). Based on 100 measurements, calculate the average particle diameter as mean ± standard deviation using statistical methods in Excel. ANOVA can be used to compare average diameters of particles of different types of emulsion microgels. A p-value of < 0.05 is considered as statistically significant.
Quantitative measurement of FITC-WPI amount
The amounts of FITC-WPI, which were (i) encapsulated in emulsion microgels, (ii) released during incubation model media, and (iii) retained on the urothelium of the porcine bladder, were measured spectrophotometrically based on their absorption spectra.
All experiments were carried out in triplicate to obtain statistically significant results. Based on three measurements, average amounts of FITC-WPI were calculated and presented as mean ± standard deviation.
ClarioStar MARS software was used to obtain calibration lines and calculate the FITC-WPI average amount.
The Origin software was used to obtain the release kinetics curves.
Validation of protocol
The whole procedure is validated in our recent work and supplemental information.
Saveleva et al. [9]. Mucoadhesive Emulsion Microgels for Intravesical Drug Delivery: Preparation, Retention at Urothelium, and Biodistribution Study. ACS Applied Materials and Interfaces (Figure 1, panels B–D, H–J; Figure 3, panel D, Figure S-3, panel “2.5% 1:3, 1 wash”).
General notes and troubleshooting
General notes
We assume that the presented protocols for characterizing and testing WPI-based emulsion microgels can also be adapted (with minor modifications) to various other types of emulsion-based carriers containing mucoadhesive components (e.g., polysaccharides, proteins, and biopolymers).
This protocol, with minimal adaptive modifications, could also be applied to the studies on other model mucous tissues containing a mucosal layer, for example, mucous membranes of the intestines, eyes, nose, and throat, to study the mucoadhesive ability of various drug carriers (considering the fluorescent labeling of these carriers).
Troubleshooting
To maintain better stability and long-term storage of emulsion microgels, it is recommended to add a preserving agent (for example, sodium azide) to the WPI solution at the preparation stage. Store the prepared emulsion microgels in the refrigerator at 4 °C.
During long-term storage, slight phase separation and the formation of a layer of large oil droplets on the surface are possible. To bring samples of emulsion microgels to their initial condition, subject them to ultrasonication (frequency of 20 kHz and a power density of 1 W/cm2) with the ultrasound probe for 1 min. This will allow re-homogenization of the emulsion microgels.
The absence of a fluorescent signal from emulsion microgels after application to the surface of the bladder is possible at a low concentration of the encapsulated fluorescent dye. In this case, it is necessary to optimize the settings of the fluorescent microscope. It is worth considering that living tissues have their own autofluorescence, in which case it is necessary to adjust the maximum level of fluorescent signal for samples with the maximum dye content.
Acknowledgments
This study was supported by the Russian Science Foundation (project No. 21-75-10042), https://rscf.ru/project/21-75-10042. This protocol was adapted from Saveleva et al. [9]. We also acknowledge all contributors to prior work [9] in which this protocol is based.
Competing interests
The authors declare no competing interests.
Ethical considerations
No human subjects are involved in this work.
References
Kolawole, O. M., Lau, W. M., Mostafid, H. and Khutoryanskiy, V. V. (2017). Advances in intravesical drug delivery systems to treat bladder cancer. Int J Pharm. 532(1): 105–117.
GuhaSarkar, S. and R Banerjee, R. (2010). Intravesical Drug Delivery: Challenges, current status, opportunities and novel strategies. J Control Release. 148(2): 147–159.
Ognenovska, S., Mukerjee, C., Sanderson-Smith, M., Moore, K. H. and Mansfield, K. J. (2022). Virulence Mechanisms of Common Uropathogens and Their Intracellular Localisation within Urothelial Cells. Pathogens. 11(8): 926.
Karavana, S., Ay Şenyiğit, Z., Çalışkan, Ã., Sevin, G., İlem Özdemir, D., Erzurumlu, Y., Şen, S. and Baloğlu, E. (2018). Gemcitabine hydrochloride microspheres used for intravesical treatment of superficial bladder cancer: a comprehensive in vitro/ex vivo/in vivo evaluation. Drug Des Devel Ther. 1959–1975.
Tamura, K., Kikuchi, E., Konno, T., Ishihara, K., Matsumoto, K., Miyajima, A. and Oya, M. (2015). Therapeutic effect of intravesical administration of paclitaxel solubilized with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) in an orthotopic bladder cancer model. BMC Cancer. 15(1): 317.
Frangos, D. N., Killion, J. J., Fan, D., Fishbeck, R., Von Eschenbach, A. c. and Fidler, I. J. (1990). The Development of Liposomes Containing Interferon Alpha for the Intravesical Therapy of Human Superficial Bladder Cancer. J Urology. 143(6): 1252–1256.
Rajaganapathy, B. R., Chancellor, M. B., Nirmal, J., Dang, L. and Tyagi, P. (2015). Bladder Uptake of Liposomes after Intravesical Administration Occurs by Endocytosis. PLoS One. 10(3): e0122766.
Tyagi, P., Chancellor, M. B., Li, Z., de Groat, W. C., Yoshimura, N., Fraser, M. O. and Huang, L. (2004). Urodynamic and Immunohistochemical Evaluation of Intravesical Capsaicin Delivery Using Thermosensitive Hydrogel and Liposomes. J Urology. 171(1): 483–489.
Saveleva, M. S., Lobanov, M. E., Gusliakova, O. I., Plastun, V. O., Prikhozhdenko, E. S., Sindeeva, O. A., Gorin, D. A. and Mayorova, O. A. (2023). Mucoadhesive Emulsion Microgels for Intravesical Drug Delivery: Preparation, Retention at Urothelium, and Biodistribution Study. ACS Appl Mater Interfaces. 15(21): 25354–25368.
Article Information
Publication history
Received: Mar 21, 2024
Accepted: Jun 4, 2024
Available online: Jun 24, 2024
Published: Jul 5, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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5,028 | https://bio-protocol.org/en/bpdetail?id=5028&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis and Quantification of the Mitochondrial–ER Lipidome
AD Alexis Diaz-Vegas
AD Anthony S. Don
JB James G. Burchfield
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5028 Views: 858
Reviewed by: Marc-Antoine SaniSneha RayRITU SOM
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Original Research Article:
The authors used this protocol in eLIFE Nov 2023
Abstract
Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress.
Key features
• Analysis and quantification of lipids in mitochondria–ER fraction through liquid chromatography–tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics).
• LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems.
• LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.
Keywords: Mitochondria Endoplasmic reticulum Subcellular fractionation Lipidomics Ceramides Cardiolipin
Graphical overview
Background
Mitochondria are essential cellular components with intricate structures and functions vital for life. Comprising two distinct membranes with unique lipid compositions, these dynamic organelles play multifaceted roles in maintaining cellular homeostasis. While many of these lipids originate from the endoplasmic reticulum (ER) and are subsequently transported to the mitochondria, the precise mechanisms governing the maintenance of mitochondrial membrane lipid composition and its response to environmental stressors remain incompletely understood.
Recent advances in detection methodologies, particularly in lipidomics, have revolutionized our understanding of lipid physiology by uncovering previously unrecognized lipid functions, such as how certain subgroups of lipid species respond to metabolic perturbations and their association with human diseases [1–3]. Although lipidomics has predominantly been applied to study changes in lipid composition at the whole tissue or cell level, less attention has been devoted to elucidating the lipid composition of subcellular compartments and how this evolves in response to cellular stress. Lipidomics analysis in subcellular fractions, such as mitochondria, enables the specific determination of lipid changes within organelles without interference from lipid signatures of other regions such as lipid droplets, nucleus, plasma membrane, and endosomal/lysosomal compartments [2,4,5].
We have implemented a mitochondria isolation protocol capable of fractionating mitochondria and mitochondria–ER fractions in the skeletal muscle cell line L6 myotubes. These fractions are then subjected to mass spectrometry (MS)-based lipidomics, enabling lipid composition dissection in these specific subcellular regions. This approach can shed light on how lipid dynamics within mitochondria and their associated ER compartments are influenced by cellular stressors [2].
Materials and reagents
Biological materials
L6 myotubes (L6, CRL-1458, ATCC, or other suitable cell line or tissue)
Reagents
DMEM high glucose (Gibco, catalog number: A5256701)
Fetal bovine serum (FBS) (Gibco, catalog number: 11965092)
GlutaMax (Gibco, catalog number: 35050061)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 9048-46-8)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417)
EGTA (Thermo, catalog number: E1219)
HEPES (Gibco, catalog number: 11560496)
Bovine serum albumin (BSA) fatty acid–free (Sigma, catalog number: A8806)
Protease inhibitor Complete Mini, EDTA-free (Roche, catalog number: 11836170001)
Mannitol (Millipore, catalog number: 63560)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771)
Tris (VWR, catalog number: 0826)
Sodium chloride (NaCl) (VWR, catalog number: 27810.364)
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M2670)
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016)
Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: AM9640G)
Tween-20 (Thermo Fisher Scientific, catalog number: 28320)
d18:1/17:0 ceramide standard (Avanti Polar Lipids, catalog number: 860517)
d18:1/17:0 sphingomyelin standard (Sapphire Bioscience, catalog number: 25592)
d17:1 sphingosine standard (Avanti Polar Lipids, catalog number: 860640)
d17:1 sphingosine 1-phosphate (S1P) standard (Sapphire Bioscience, catalog number: 22498)
18:1/15:0 d7-diacylglycerol standard (Avanti Polar Lipids, catalog number: 791647)
14:0/14:0/14:0/14:0 cardiolipin standard (Avanti Polar Lipids, catalog number: 710332)
Methyl-tert-butyl ether (MTBE) (Sigma-Aldrich, catalog number: 650560)
Methanol (Sigma-Aldrich, catalog number: 34860)
Water HPLC grade (Sigma-Aldrich, catalog number: WX0004)
Formic acid (Merk, catalog number: 1.00264)
Ammonium formate (Merk, catalog number: 70221)
Acetonitrile (Sigma-Aldrich, catalog number: 34851)
2-propanol (Sigma-Aldrich, catalog number: 270490)
Percoll (Thermo Fisher, Catalog number: B22095.09)
Solutions
Cell culture media (see Recipes)
Basal media (see Recipes)
DPBS (see Recipes)
Stock solutions (see Recipes)
Lysis buffer 1 (see Recipes)
Percoll gradient (see Recipes)
Lysis buffer 2 (see Recipes)
Sample buffer (see Recipes)
Mobile phase A (see Recipes)
Mobile phase B (see Recipes)
Internal standard (see Recipes)
Recipes
Cell culture media
DMEM high glucose
1 mM GlutaMax
10% FBS
For 500 mL of DMEM high glucose, add 5.5 mL of Glutamax and 50 mL of FBS.
Basal media
DMEM high glucose
1 mM GlutaMax
0.2% BSA with fatty acid
For 500 mL of DMEM high glucose, add 5.5 mL of Glutamax and 0.2 g of BSA.
DPBS
PBS
1 mM MgCl2
1 mM CaCl2
For 100 mL of PBS, add 1 mL of MgCl2 (stock 1 M) and 1 mL of CaCl2 (stock 1 M).
Lysis buffer 1
250 mM Mannitol
5 mM HEPES pH 7.4
0.5 mM EGTA (see Note 1)
0.1% BSA with fatty acid
Adjust pH to 7.4 with KOH
On the day of use: 1/100 volume of protease inhibitor Complete Mini.
This buffer has to be prepared fresh from stock solutions, and powder BSA needs to be added at the end.
Keep this buffer on ice throughout the whole protocol.
Percoll gradient
250 mM mannitol
5 mM HEPES pH 7.4
0.5 mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid], also known as egtazic acid (INN, USAN)
0.1% BSA with fatty acid
18% Percoll
Use Lysis buffer 1 (see Note 6) to dilute Percoll to the desired concentration.
Lysis buffer 2
250 mM mannitol
5 mM HEPES pH 7.4
0.5 mM EGTA
Adjust pH to 7.4 with KOH
On the day of use: 1/100 volume of protease inhibitor cocktail set III
This buffer has to be prepared fresh from stock solutions.
Keep this buffer on ice throughout the whole protocol.
Lipid extraction solvent
For lipid extraction, each solvent is added individually to the homogenate (see methods) with a ratio of 10:3:2.5 (v:v:v) of methyl-tert-butyl ether (MTBE):Methanol:Water
Resuspension solvent
80% MeOH
20% deionized water
0.2% formic acid
2 mM ammonium formate
LC-MS/MS mobile phase A
0.1% formic acid
10 mM ammonium formate
60% acetonitrile
40% water
LC-MS/MS mobile phase B
0.1% formic acid
10 mM ammonium formate
90% 2-propanol
10% acetonitrile
Internal standard
50 µL is added to each sample
40 µM d18:1/17:0 sphingomyelin (2 nmol/sample)
10 µM d18:1/17:0 ceramide (0.5 nmol/sample)
4 µM d17:1 sphingosine (0.2 nmol/sample)
4 µM d17:1 sphingosine 1-phosphate (0.2 nmol/sample)
40 µM 18:1/15:0 d7-diacylglycerol (2 nmol/sample)
80 µM 14:0/14:0/14:0/14:0 cardiolipin (4 nmol/sample)
100 µM d7-cholesterol (5 nmol/sample)
Additional internal standards may be added for the measurement of other lipid classes.
The internal standard mixture can be stored at -30 °C.
Laboratory supplies
50 mL conical tube (Corning, catalog number: 001634)
Polypropylene tube, 14 mL, 14 mm × 95 mm (Beckman Coulter, catalog number: 331374)
2 mL tubes, 13 mm × 25 mm (Beckman Coulter, catalog number: 357329)
Micro tube 3810X, 1.5 mL (Eppendorf, catalog number: EPPE0030125.150)
Safe-lock tubes 2.0 mL (Eppendorf, catalog number: 0030120.094)
1 mL syringe
5 mL glass tubes
HPLC tubes
Corning® tissue-culture treated culture dishes (Corning, catalog number: CLS4305990)
Micropipettes
Equipment
Refrigerated microcentrifuge
2.1 100 mm Waters Acquity UPLC C18 column (1.7 μm pore size) (Thermo, model: 186002352)
Sonicator water bath (Thermoline, model: 505)
SavantTM SpeedVacTM high-capacity concentrators (Thermo Fisher, catalog number: SC210A-230)
TSQ Altis triple quadrupole mass spectrometer coupled to a Vanquish HPLC system (Thermo Fisher Scientific, model: TSQ03-11002)
Cell homogenizer with 18 μm ball (ISOBIOTEC)
Thermo centrifuge, ultra-speed (Thermo Fisher, model: WX100, catalog number: 096-247028)
TLA-110 fixed-angle rotor (Beckman Coulter, catalog number: 366735)
SW 40 Ti swinging-bucket rotor (Beckman Coulter, catalog number: 331301)
JA-12 fixed-angle aluminum rotor (Beckman Coulter, catalog number: 360992)
Precision balance FZ-i Series 320 g × 0.001 g (Australian Scientific, catalog number: FZ-300i)
Electrophoresis chamber (Life Technologies, catalog number: EI0002)
Transfer apparatus (Life Technologies, catalog number: EI0002)
Sponge pads for XCell II blotting (Life Technologies, catalog number: EI9052)
Cell scraper
Odyssey® DLx imaging system (LI-COR, model: BX41-PH-B)
Vortex mixer (Ratek, model: VM1)
Software and datasets
Image StudioTM (LI-COR)
TraceFinder software (Thermo Fisher)
Microsoft Excel
Procedure
We describe below the step-by-step procedure for performing a mitochondria–ER isolation followed by lipidomic analysis and quantification [6]. This procedure has been applied to both L6 myotubes and HeLa cells and published in Diaz-Vegas et al. [2]. For a different cell line, further optimization might be necessary (e.g., starting material). Store all buffers at 4 °C and perform the procedure on ice. Room temperature is defined as 22 °C throughout this protocol. Washing steps are provided for each step throughout the protocol. For this protocol, 15 fully confluent dishes of 15 cm were pulled together to obtain enough material to isolate the different fractions (equal to one biological replicate).
Mitochondria–ER isolation
Before starting
Cool cell homogenizer to 4 °C.
Cool down DPBS.
Cool down rotors (TLA110, sw40Ti, JA-12).
Cool down centrifuges.
Cool down 50 and 1.5 mL tubes.
Basal period
Cultivate L6 cells to 100% confluency in 15 cm diameter tissue culture plates (15 dishes per condition).
Remove the media and wash cells with room-temperature PBS (three times, 6 mL each time).
Add 10 mL of basal media and incubate at 37 °C for 2 h in the incubator (95% O2, 5% CO2).
Harvesting and cell lysis
Transfer the dishes to an ice tray.
Immediately, wash the cells five times with ice-cold DPBS by gently adding the buffer against the wall of the dish to prevent cell loss.
After the last wash, add 4 mL of lysis buffer 1 to one plate.
Using a cell scraper, remove the cells (see Note 2).
Tilt the plate to allow cells to accumulate in one region. When the first plate is ready, remove DPBS from a second plate and transfer the entire volume from plate 1 to plate 2.
Repeat the scrapping process in the new dish. Continue this procedure until all plates have been scraped, collecting the entire sample (see Note 3).
Transfer the collected sample to an ice-cold 50 mL conical tube.
Spin down cells at 1,500 rpm (300× g) for 5 min in a JA-12 rotor at 4 °C.
Discard supernatant and resuspend cells in 5 mL of lysis buffer 1.
Assemble the cell homogenizer (see Note 4).
Equilibrate the cell homogenizer with 1 mL of lysis buffer 1. Discard this buffer after equilibration.
Transfer cell suspension using a 1 mL syringe and process the samples using the cell homogenizer.
Pass 10 times back and forth through the cell homogenizer (see Note 5).
Transfer lysate to a new ice-cold 50 mL Falcon tube. Repeat step A3m until all samples are collected in this new Falcon tube (see Note 6).
Spin down nuclei for 10 min at 1,810 rpm (600× g at rmax) in a JA-12 rotor at 4 °C.
Transfer supernatant (~4 mL) to a new ice-cold 50 mL Falcon tube and discard pellet.
Divide the supernatant into two ice-cold 1.5 mL Eppi® tubes for isolation of crude mitochondria (reserve 1 mL as whole-cell lysate for further experiments, e.g., lipidomics in whole lysate, western blotting, etc., and store at -80 °C).
Crude mitochondria isolation
Transfer the 1.5 mL Eppi® tubes to a refrigerated microcentrifuge and spin at 8,500 rpm (10,300× g at rmax) for 10 min in a JA-12 rotor at 4 °C. This will separate crude mitochondria (pellet) from post-mitochondrial fraction (supernatant).
Transfer supernatant containing post-mitochondrial fraction to a new 1.5 mL Eppi® tubes and store at -80 °C.
Resuspend pellet in 1 mL of lysis buffer 1 by gently pipetting up and down (see Note 7).
Isolation of mitochondria–ER fractions
Carefully, layer the suspension into a polycarbonate tube containing 7.9 mL of 18% Percoll gradient (see Note 8). Add 3 mL of Lysis buffer 1 on top with a p1000 to calibrate the tubes (see Note 9).
Weigh each polycarbonate tube and balance with Lysis buffer 1 (± 0.001 g) (Note 10).
Centrifuge at 95,000× g at rmax for 30 min at 4 °C using a sw40Ti rotor.
With a 1 mL syringe, aspirate the band at the top portion of the tube and transfer it (~2 mL) into two Beckman 1.4 mL tubes (1 mL per tube). This first band will contain the mitochondria–ER fraction.
With another 1 mL syringe, aspirate the band at the bottom of the tube and transfer it (~2 mL) into four Eppi® tubes (500 µL per tube). This will contain the pure mitochondrial fraction.
Weigh each Beckman 1.4 mL tube and balance with Lysis buffer 1 (± 0.001 g).
Spin down the mitochondrial-associated membranes (MAM) fraction at 60,000 rpm for 1 h using the TLA 110 rotor at 4 °C.
Mitochondria–ER will be a mucous pellet on a clear Percoll sediment at the bottom of the tube. Aspirate out Mitochondria–ER carefully with a micropipette and transfer it to an Eppi® tube (~200 µL) (see Note 11).
Wash the mitochondria–ER pellet three times with lysis buffer 2 (max speed for 15 min in JA-12 rotor at 4 °C).
Add 50 µL of 5× sample buffer and adjust the volume to 300 µL with 1× sample buffer for western blotting or store at -80 °C for lipid extraction.
Add 1 mL of lysis buffer 1 to each tube with pure mitochondria and mix (see Note 7).
Spin down the mitochondrial fraction at 10,000 rcf for 10 min at 4 °C.
Mitochondria will form a loose pellet. Carefully aspirate most of the supernatant (~1.2 mL) and replace it with more lysis buffer 1.
Spin down the mitochondrial fraction at 10,000 rcf for 10 min at 4 °C.
Aspirate supernatant and resuspend pellet in 300 µL of 1× sample buffer (100 µL per tube) or store at -80 °C for lipid extraction. Resuspension requires repeated up and down pipetting.
Analysis of protein content is determined by the BCA method. In these fractions, cytochrome content can be evaluated using the method of Vanneste [7] and Nicholls [8]. These mitochondrial preparations can be assayed for CoQ content by LC-MS/MS after extraction [9].
Lipid extraction and lipidomics analysis
Quality control samples
Blank solution without internal standards.
Blank solution with internal standards (see Recipes for internal standards).
Set up pooled quality control samples (replicates) to be observed for variations throughout the analytical run. This may involve preparing a whole-cell lysate. For this experiment, we used a pool sample from all conditions as the control.
Lipid extraction
This procedure was adapted from Matyash et al. [10].
Prepare internal standard mixture containing 2 nmol d18:1/17:0 sphingomyelin, 0.5 nmol d18:1/17:0 ceramide, 0.2 nmol d17:1 sphingosine, 0.2 nmol d17:1 sphingosine-1-phosphate (S1P), 2 nmol 18:1/15:0 d7-diacylglycerol, 2 nmol 14:0/14:0/14:0/14:0 cardiolipin, and 5 nmol d7-cholesterol per sample, diluted in 50 µL of methanol per sample. Additional internal standards may be included for other lipid classes (see Note 12).
Use 30 μg of mitochondrial protein for extraction.
Add 200 μL of MeOH (MS grade) and 50 μL of internal standard stock to safe-lock tubes (2.0 mL) (see Note 13).
Add 850 μL of MTBE to the tube and sonicate in the bath sonicator in the cold room for 30 min. Check every 10 min to ensure the bath remains cold (see Note 15).
Add 212 μL of MilliQ water to each tube to induce phase separation (see Notes 15–16).
Vortex the sample at max speed for 10 sec and spin at 2,000× g for 5 min to complete phase separation.
Transfer the upper organic phase into 5 mL glass tubes (see Note 15).
Repeat steps B2b–f twice to re-extract the lower phase. Add 2 mL of the solvent mixture, whose composition is equivalent to the expected composition of the upper phase (obtained by mixing MTBE/methanol/water 10:3:2.5, v/v/v) for each re-extraction (see Note 15).
Collect the upper phases and combine them in the 5 mL glass tube.
Dry the extract overnight in a Speedvac under a low-heat setting.
Reconstitute the extract in 400 μL of 0.1% FA/1 mM ammonium formate in 80% MeOH (cover the sample vials with parafilm). Vortex for 10 s at max speed at room temperature (see Note 16).
Centrifuge the 5 mL glass tubes carefully at 2,000× g for 10 min to pellet insoluble material, and then transfer 300 μL of the methanol extract to a glass HPLC vial.
Store vials at -30 °C for further analysis.
Lipid quantification by targeted lipidomics
Lipids are separated on a 2.1 × 100 mm Waters Acquity UPLC C18 column (1.7 μm pore size) using a flow rate of 0.28 mL/min.
Total run time is 25 min with a binary solvent gradient, starting at 20% of mobile phase B (80% A) and holding for 3 min, ramping up to 100% B from 3 to 14 min, holding at 100% B from 14 to 20 min, returning to 20% B at 20.5 min, and holding at 20% B for a further 4.5 min.
Lipids are detected on a triple quadrupole or quadrupole-ion trap instrument operating in positive ion mode. Each lipid species is detected as a distinct precursor-product ion pair using selected ion monitoring.
Ceramides, sphingomyelin, sphingosine, and sphingosine 1-phosphate are identified as the [M+H]+ precursor ion, with m/z 262.3 (sphinganine), 264.3 (sphingosine), or 266.3 (sphinganine) product ion, and m/z 184.1 product ion in the case of sphingomyelin. Diacylglycerols (DAGs) are identified as the [M + NH4]+ precursor ion and product ion corresponding to neutral loss of a fatty acid. Cardiolipins are identified as the [M + H]+ precursor ion and product ion corresponding to a constituent DAG [M + H - H2O]+ ion. Cholesterol is detected using precursor m/z 369.4 and product m/z 161.1.
Quantification of lipid profile in whole lysate and mitochondrial fraction is shown in Figure 1.
Figure 1. Representative results from targeted lipidomic for ceramides. A. L6 myotubes were incubated with the fatty acid palmitate (150 µM for 16 h) and ceramide levels were measured in whole lysate or mitochondrial–ER fraction. B. Abundance of the different ceramide species in lysate and mitochondrial–ER fraction. N = 3–4 biological replicates, **p < 0.001, ****p < 0.0001.
Data analysis
Lipids are quantified as the area under the peak for a specific precursor–product ion pair. It is recommended that each peak comprises a minimum of 8–10 scans for that specific precursor–product ion pair. Peak detection and integration is performed with vendor-specific software or freeware such as Skyline [11]. For our analyses, TraceFinder was used. The amount of each lipid is determined first as the ratio to its class-specific internal standard. This is then multiplied by the amount of internal standard added to estimate the nmol of each lipid in the sample, then divided by the amount of protein used for lipid extraction, so that lipid levels are expressed as nmol lipid/mg protein. Levels of individual lipid species may then be compared between sample groups or treatment conditions using statistical analyses appropriate to the experimental design.
Validation of protocol
This protocol has been used and validated in the following research article(s):
Diaz-Vegas et al. [2], Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle, eLife 12, RP87340 (Figure 2, panel C, D; Figure 3, panel C, D).
Ceramide species were quantified using targeted lipidomics. N = 3 biological replicates. Control conditions refer to cells with vehicle control for each experiment. Student’s t-test was utilized for comparing two groups, whilst ordinary one-way ANOVA followed by Dunnett’s multiple-comparison test was employed for comparing multiple groups.
General notes and troubleshooting
General notes
The use of EGTA is essential to avoid mitochondrial calcium overload during isolation.
Keep the other 14 dishes on ice with 10 mL of DPBS during this process.
Volume will rise to ~12 mL.
It is possible that different ball sizes are necessary for different cell lines. In this protocol, we used an 18 μm ball size for cell lysis.
It is critical to keep consistency in speed to obtain consistent results without damaging mitochondria structure.
Wash and dry the homogenizer after the whole 5 mL of sample has been passed (once with tap water and then with distilled water).
Use a 1 mL blunt tip to prevent mechanical mitochondrial damage during this step.
Use a 1 mL syringe for better results.
This volume can vary depending on the polycarbonate tube size.
Dry out the surface of the polycarbonate tube; otherwise, it will stick to the centrifuge and it will be almost impossible to take it out.
Do not aspirate the supernatant, as mitochondria–ER pellet is loose.
Prior to extraction, thaw internal standards to room temperature for 1 h and then vortex.
Additional internal standards should be included if you wish to measure other lipid classes. Internationally accepted conventions (Lipidomics Standards Initiative, https://lipidomicstandards.org/) recommend the inclusion of at least one internal standard for each lipid class of interest. See for example [12]. You may use deuterated internal standards for sphingolipids, in place of the d17:1 and d18:1/17:0 variants.
Place a mixture of water and ice inside the sonicator to ensure samples are cold during this step. Remove excess liquid and add ice if multiple runs of sonication are required.
MTBE, when mixed with air, can cause a hazardous/flammable atmosphere. Always use this chemical in a fume hood.
Subtract from this 212 μL of the volume associated with the sample. For instance, if the 30 μg of mitochondrial proteins were in 50 μL of buffer, here we only need to add 162 μL of MilliQ (212 − 50 = 162). This is critical to maintain the MeOH:MTBE:H2O ratio.
Thoroughly wrap each tube with parafilm before vortexing.
Acknowledgments
A.D.V., J.G.B., D.E.J., National Health and Medical Research Council, 2013621; A.D.V., Australian Research Council, DP210102099; D.E.J., Australian Research Council, FL200100096; National Health and Medical Research Council, GNT1120201; GNT1061122; A.S.D., National Health and Medical Research Council, GNT112613; A.D.V., Diabetes Australia, Y22G-DIAA; A.D.V., Mitochondrial Foundation, G057. We have adapted this protocol Bui et al. [6].
Competing interests
The authors have no competing interests to declare.
References
Chaurasia, B., Tippetts, T. S., Mayoral Monibas, R., Liu, J., Li, Y., Wang, L., Wilkerson, J. L., Sweeney, C. R., Pereira, R. F., Sumida, D. H., et al. (2019). Targeting a ceramide double bond improves insulin resistance and hepatic steatosis. Science. 365(6451): 386–392.
Diaz-Vegas, A., Madsen, S., Cooke, K. C., Carroll, L., Khor, J. X., Turner, N., Lim, X. Y., Astore, M. A., Morris, J. C., Don, A. S., et al. (2023). Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle. eLife. 12: e87340.
Wittenbecher, C., Cuadrat, R., Johnston, L., Eichelmann, F., Jäger, S., Kuxhaus, O., Prada, M., Del Greco M., F., Hicks, A. A., Hoffman, P., et al. (2022). Dihydroceramide- and ceramide-profiling provides insights into human cardiometabolic disease etiology. Nat Commun. 13(1): 936.
Hammerschmidt, P., Ostkotte, D., Nolte, H., Gerl, M. J., Jais, A., Brunner, H. L., Sprenger, H. G., Awazawa, M., Nicholls, H. T., Turpin-Nolan, S. M., et al. (2019). CerS6-Derived Sphingolipids Interact with Mff and Promote Mitochondrial Fragmentation in Obesity. Cell. 177(6): 1536–1552.e23.
Turner, N., Lim, X. Y., Toop, H. D., Osborne, B., Brandon, A. E., Taylor, E. N., Fiveash, C. E., Govindaraju, H., Teo, J. D., McEwen, H. P., et al. (2018). A selective inhibitor of ceramide synthase 1 reveals a novel role in fat metabolism. Nat Commun. 9(1): 3165.
Bui, M., Gilady, S. Y., Fitzsimmons, R. E., Benson, M. D., Lynes, E. M., Gesson, K., Alto, N. M., Strack, S., Scott, J. D., Simmen, T., et al. (2010). Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties. J Biol Chem. 285(41): 31590–31602.
Vanneste, W. H. (1966). The Stoichiometry and Absorption Spectra of Components a and a3 in Cytochrome c Oxidase*. Biochemistry. 5(3): 838–848.
Nicholls, P. (1976). The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain. Biochim Biophys Acta-Bioenerg. 430(1): 13–29.
Burger, N., Logan, A., Prime, T. A., Mottahedin, A., Caldwell, S. T., Krieg, T., Hartley, R. C., James, A. M. and Murphy, M. P. (2020). A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo. Free Radical Biol Med. 147: 37–47.
Matyash, V., Liebisch, G., Kurzchalia, T. V., Shevchenko, A. and Schwudke, D. (2008). Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 49(5): 1137–1146.
Pino, L. K., Searle, B. C., Bollinger, J. G., Nunn, B., MacLean, B. and MacCoss, M. J. (2017). The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics. Mass Spectrom Rev. 39(3): 229–244.
Lee, J. Y., Harney, D. J., Teo, J. D., Kwok, J. B., Sutherland, G. T., Larance, M. and Don, A. S. (2023). The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus. Mol Neurodegener. 18(1): 63.
Article Information
Publication history
Received: Feb 20, 2024
Accepted: Jun 12, 2024
Available online: Jun 24, 2024
Published: Jul 5, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Biochemistry > Lipid > Lipid isolation
Cell Biology > Organelle isolation > Mitochondria
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Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis
ZL Ziqiang P. Li
JH Jennifer Huard
EB Emmanuelle M. Bayer
VW Valérie Wattelet-Boyer
Published: Vol 14, Iss 13, Jul 5, 2024
DOI: 10.21769/BioProtoc.5029 Views: 588
Reviewed by: Raniki Kumari Anonymous reviewer(s)
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Abstract
CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880 and at1g51570), using one or two sgRNA per gene.
Key features
• A simple method for Arabidopsis edited lines establishment using CRISPR-Cas9 technology
• Versatile cloning strategy combining various technologies for convenient cloning (Gateway®, Golden Gate)
• Multigene targeting with high efficiency
Keywords: Plant genome editing CRISPR-Cas9 Arabidopsis thaliana sgRNA multiplexing Gateway® cloning Golden Gate cloning
Background
CRISPR-Cas9 technology, which serves as a powerful genome editing tool, is based on the bacterial RNA-guided CRISPR-Cas9 system [1]. The editing process involves two main actors: the Cas9 nuclease and a unique guide RNA (sgRNA) that will direct the Cas9 protein to the DNA target for genome editing. Plants, like other organisms, need these elements to form the CRISPR-Cas9 complex, which will be directed to the targeted sequence depending on the crRNA part of the sgRNA [2]. Plant transformation with binary vectors containing sequences for Cas9 expression and sgRNA synthesis remains one of the primary and widely used methods for the production of these actors in cells. Numerous vectors and editing strategies have been developed, such as strategies to target multiple genes simultaneously through sgRNA multiplexing. This approach addresses challenges such as gene redundancy and enhancing editing efficiency, eliminating the need for time-consuming multiple crossings [3–6]. Other ways of improvement using various promoters have been explored to optimize the expression of the Cas9, especially in the context of multitargeting but also to avoid mosaic mutations. Indeed, while promoter UBQ10 enhances mutation efficiency in comparison to the 35S promoter, ubiquitous promoters driving the expression of Cas9 could lead to mosaic mutation patterns [7]. To fix this problem, egg cell–specific promoters like EC1.2, embryo sac, embryo, and endosperm, and pollen-specific promoters like YAO or NUC1 can be used. In these conditions, Cas9 expression induces homozygous or biallelic mutants in the early generations removing the problem of mosaicism [8–10]. Taking advantage of previously published works, we chose efficient elements and strategies and merged them to develop a very versatile cloning system that combines MultiSite Gateway® [11] and Golden Gate [12] technologies.
In this protocol, we describe the step-by-step procedure for generating the binary vector that contains the endogenous tRNA-processing system, previously described to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system [4], and the egg cell–specific promoter EC1.2 associated to EC1.1 enhancer to drive the expression of Cas9 [8] (Figure 1). For precise genome editing, this protocol can be readily adjusted to accommodate various designs of binary vectors. This includes the utilization of different tissue-specific promoters, inducible promoters, or alternative nucleases such as SaCas9, Cas12a, or zCas9i, which can enhance both the efficiency and specificity of editing [13–17].
Figure 1. Cloning strategy. Cloning strategy to express zCas9 under the control of egg cell promoter (EC1.1p) fused to enhancer sequence (EC1.2en) and to target DNA using several sgRNA cloned in the form of a polycistronic gene. (R1, R2, R3, R4, L1, L2, L3 and L4 represent recombination sites.)
Materials and reagents
Biological materials
pHEE401E plasmid (Addgene, catalog number: 71287)
pGTR plasmid (Addgene, catalog number: 63143)
pFRm43GW plasmid (Addgene, catalog number: 133748)
p4P1R_EC1.2en-EC1.1p plasmid (Addgene, catalog number: 213912)
pDONR221_zCas9 plasmid (Addgene, catalog number: 213913)
pDONRTM P2R-P3 (Thermo Fisher Scientific, catalog number: 12537-023)
One ShotTM TOP10 chemically competent E. coli (Thermo Fisher Scientific, catalog number: C404010)
List of primers common to all strategies
Order common and specific primers according to the following features:
Synthesis scale: 0.05 μmol; purification: cartridge; format: in solution (water); concentration: 100 μM.
(Supplier: Merck, but could be another one)
Fw-Primer 1: 5′- GGGGACAGCTTTCTTGTACAAAGTGGAACGACTTGCCTTCCGCACAATAC - 3’
Rev-Primer 2: 5′- atggtctcaTGTTAATCACTACTTCGACTCTAG - 3′
Fw-Primer 3: 5′- taggtctccAACAAAGCACCAGTGGTCTAGTGG - 3′
Rev-Primer 12: 5′- atggtctcaAAGCACCGACTCGGTGCCACTTTTTC - 3′
Fw-Primer 13: 5′- taggtctccGCTTTTTTTTGCAAAATTTTCCAGTCG - 3′
Rev-Primer 14: 5′- GGGGACAACTTTGTATAATAAAGTTGATATTGGTTTATCTCATCGGAAC - 3′
M13-Fw (–20): 5′- GTAAAACGACGGCCAG - 3′
M13-Rev: 5′- CAGGAAACAGCTATGAC - 3′
Reagents
Enzymes for molecular biology
Q5® high-fidelity DNA polymerase (NEB, catalog number: M0491)
OneTaq 2× Master Mix with standard buffer (NEB, catalog number: M0482)
BsaI-HF restriction enzyme (NEB, catalog number: R3733)
T4 DNA ligase (Thermo Fisher Scientific, catalog number: 15224025)
GatewayTM BP ClonaseTM II (Thermo Fisher Scientific, catalog number: 11789020)
GatewayTM LR ClonaseTM II (Thermo Fisher Scientific, catalog number: 11791020)
Reagents for molecular biology
Cutsmart buffer (NEB, catalog number: B6004)
dNTP solution MIX 10 mM (NEB, catalog number: N0447)
TAE (Tris-Acetate-EDTA buffer) (Euromedex, catalog number: EU0202)
SYBRTM Safe DNA gel stain (Thermo Fisher Scientific, catalog number: S33102)
Agarose (Euromedex, catalog number: LE-8200)
DNase-free water (autoclaved at 110 °C for 30 min)
Reagents for microbiology
LB Miller (Euromedex, catalog number: AE-0103)
Bacteriological agar (Euromedex, catalog number: 1330)
Kanamycin bisulfate (Euromedex, catalog number: EU0420)
Spectinomycin dihydrochloride pentahydrate (Merck, catalog number: S4014)
Kit for molecular biology
Monarch® PCR & DNA Cleanup kit (NEB, catalog number: T1030)
NucleoSpin® Plasmid kit (Macherey Nagel, catalog number: 740588)
Solutions
Kanamycin bisulfate stock solution (25 mg/mL) (see Recipes)
Spectinomycin dihydrochloride pentahydrate stock solution (50 mg/mL) (see Recipes)
LB Miller media (see Recipes)
Recipes
Kanamycin bisulfate stock solution (25 mg/mL)
Reagent Final concentration Amount
Kanamycin bisulfate 25 mg/mL 0.25 g
ddH2O n/a 10 mL
Total n/a 10 mL
Filter with a 0.22 μm filter, aliquot by 1 mL, and store at -20 °C.
Spectinomycin dihydrochloride pentahydrate stock solution (50 mg/mL)
Reagent Final concentration Amount
Spectinomycin dihydrochloride pentahydrate 50 mg/mL 0.5 g
ddH2O n/a 10 mL
Total n/a 10 mL
Filter with a 0.22 μm filter, aliquot by 1 mL, and store at -20 °C.
LB Miller media
Reagent Final concentration Amount
LB Miller 25 g/L 25 g
Bacteriological agar 20 g/L 20 g
ddH2O n/a 1,000 mL
Total n/a 1,000 mL
Autoclave at 110 °C for 30 min
Wait until the temperature of the media is at approximately 60 °C and add the antibiotic (final concentration: kanamycin bisulfate 25 μg/mL or spectinomycin dihydrochloride pentahydrate 50 μg/mL).
Laboratory supplies
Reaction tubes, 0.5 mL, PP (SARSTEDT, catalog number: 72699)
Reaction tubes, 1.5 mL, PP (SARSTEDT, catalog number: 72690001)
Petri dishes, Ø 90 mm (VWR, catalog number: 391-0556)
Wooden toothpicks length 80 mm (DUTSCHER, catalog number: 505802)
Glass beads, Ø 5 mm (DUTSCHER, catalog number: 068503)
10/20 μL XL graduated TipOne® tips (STARLAB, catalog number: S1110-3700-C)
200 μL UltraPoint® graduated TipOne® tips (STARLAB, catalog number: S1113-1700-C)
1250 μL XL graduated TipOne® tips (STARLAB, catalog number: S1112-1720-C)
Microtubes 0.2 mL + flat caps (DUTSCHER, catalog number: 010208)
Equipment
Thermocycler (BIORAD, C1000 Touch Thermal Cycler, catalog number: 1851148)
Complete Vortex Genie 2 (DOMINIQUE DUTSCHER, catalog number: 079008)
Electrophoresis chamber Mupid ONE (DOMINIQUE DUTSCHER, catalog number: 088900)
Water bath (VWR, catalog number: IKAA20004382)
Shaking incubator, digital, benchtop, ES-20 (VWR, catalog number: 444-0936)
Oven (DOMINIQUE DUTSCHER, catalog number: 485134)
NanoDrop 2000 (THERMOFISHER SCIENTIFIC, catalog number: ND-2000)
Software and datasets
Web application tool CRISPR-P (v1.0, 2014) (http://crispr.hzau.edu.cn/CRISPR/) [18]
Procedure
Design SgRNA sequences and primers
Use the web application tool CRISPR-P to choose sgRNA (http://crispr.hzau.edu.cn/CRISPR/).
Select start design and choose the target genome [Arabidopsis thaliana (TAIR10)].
Enter the locus tag (e.g., At3g57880 for AtMCTP3).
Choose a sequence guide (protospacer) according to several criteria:
Choose the best location to target your gene of interest. Refer to the sgRNA target on the gene map (highlighted in yellow, Figure 2a).
The guide score must be as close to 100% as possible (the higher the score, the more effective and specific the sgRNA is) (Figure 2b); we recommend a 98% cutoff guide score value for an effective and specific sgRNA.
Avoid sgRNA with possible off-target effects; if this is not possible, proscribe the potential off-target in exon (refer to the number of off-target sites, Figure 2c).
Identify the 20-nucleotide sequence of the selected protospacer [refer to guide sequence, Figure 2c; the PAM (protospacer adjacent motif) sequence is highlighted in green].
Figure 2. CRISPR sgRNA design using the web application tool CRISPR-P [18]. Use the web application tool CRISPR-P to choose the best guides according to their location (a), specificity (b), and possible off-target effects (c). Here, we present data corresponding to the protospacer used in sgRNA3 designed to target the AtMCTP3 gene, described in Appendix 1.
Construction of the polycistronic gene
Note: The protocol described here is inspired by the already published sgRNA multiplexing system coupled with the endogenous tRNA processing system [4].
The polycistronic gene is made of the same repeated modules (t-RNA, sgRNA scaffold) regularly interspaced by several guide sequences (spacers) designed with the CRISPR-P web application tool. In this protocol, we describe how to produce a molecular construction allowing the assembly of four sgRNAs (Figure 3); however, the design is the same to clone more sgRNA, and numbers of t-RNA and sgRNA scaffold modules just have to be implemented.
Figure 3. CRISPR sgRNA multiplexing in tRNA-sgRNA polycistronic gene. Multiple PCR reactions are necessary to obtain all the elements of the polycistronic gene (promoter, fusions of tRNA-guide-sgRNA scaffold, terminator). After purification, all the elements are assembled by digestion/ligation according to the Golden Gate cloning method to form the polycistronic gene, which is then ready to clone in entry vector pDONRTM P2R-P3 using Gateway® Technology.
Primers design
All modules are assembled according to the Golden Gate cloning system. Spacers are the only unique sequences in the polycistronic gene. Their sequences are used to design primers containing overhang extremities to perform the Golden Gate reaction (Figure 4; refer also to [4] Sup. Data Figure S6). In addition, the final Golden Gate reaction product is then cloned in an acceptor vector according to the Gateway® Technology Cloning. For that, recombination sequences are required at the extremities.
See Appendix 2 for details of amplified elements and primer sequences required for the assembly of the polycistronic gene targeting AtMCTP3, AtMCTP4.
Note: The primer design method described here is from the previously published sgRNA multiplexing system coupled with the endogenous tRNA processing system (Xie et al., PNAS 2015, Sup. Data Figure S6). Adaptations were made to include the AtU6-26 promoter, U6-26 terminator, and attB sequences at the ends of the polycistronic gene for recombination into the entry vector pDONRTM P2R-P3.
Figure 4. Primers design and example of PCR product. A. The polycistronic gene consists of a succession of pre-tRNA sequences, guide sequences used for targeting, and gRNA scaffolds. The guide sequences used for the Golden Gate cloning multiplexing strategy have to be included in the primers allowing the amplification of the sgRNA scaffold and pre-tRNA modules. An example of an amplification product is shown in the red box. B. Structure of the forward and reverse primers designed for the construction of a guide sequence (spacer n). A BsaI site is added at 5' of each primer for Golden Gate cloning. Nucleotides 9–12 of the guide sequence will serve as the sticky end after digestion with BsaI (green box). The 3' end of the forward primer is complementary to the start of the sgRNA scaffold, while the 3' end of the reverse primer is inversely complementary to the end of the pre-tRNA module.
Part of the primers shown in Figure 3 will be common to all editing strategies; see below:
(attB2r and attB3 recombination sites: bold; BsaI restriction sites: underline; overhang site for Golden Gate cloning: italics)
Fw-Primer 1: 5′- GGGGACAGCTTTCTTGTACAAAGTGGAACGACTTGCCTTCCGCACAATAC - 3′
Rev-Primer 2: 5′- atggtctca TGTT AATCACTACTTCGACTCTAG - 3′
Fw-Primer 3: 5′- taggtctcc AACA AAGCACCAGTGGTCTAGTGG - 3′
Rev-Primer 12: 5′- atggtctca AAGC ACCGACTCGGTGCCACTTTTTC - 3′
Fw-Primer 13: 5′- taggtctcc GCTT TTTTTTGCAAAATTTTCCAGTCG - 3′
Rev-Primer 14: 5′- GGGGACAACTTTGTATAATAAAGTTGATATTGGTTTATCTCATCGGAAC - 3′
The other primers (from 4 to 11 in this example, Figure 3), which are necessary for the synthesis of the guide sequence, must be designed according to the indications below:
(N1 to N20 correspond to the 20 nucleotides of the gRNA spacer)
Note: Be careful to avoid any guide sequence containing a BsaI restriction site. This would make polycistronic gene assembly impossible.
Forward primer: Fw_n, containing BsaI restriction site and the second half part of the guide sequence [nucleotides 9 to 20 (forward sequence)] fused to the 5′ end sequence of the gRNA scaffold (forward sequence).
5′- taggtctcn-N9N10N11N12N13N14N15N16N17N18N19N20-gttttagagctagaa-3’
Reverse primer: Rev_n, containing BsaI restriction site and the first half part of the guide sequence [nucleotides 1 to 12 (reverse complement sequence)] fused to the 3’ end sequence of the pre tRNA (reverse complement sequence).
5′- cgggtctcn-N12N11N10N9N8N7N6N5N4N3N2N1-tgcaccagccggg-3’
Cloning of the polycistronic gene
The polycistronic gene will be synthesized by assembling various PCR products whose ends are made cohesive after digestion. The assembly of these modules is done in an orderly manner according to the cohesive ends designed for the Golden Gate cloning strategy.
The steps consist of the amplification of each module by PCR, the purification of the PCR products, the assembly by a succession of digestions and ligations according to the Golden Gate cloning method, and then the cloning into an entry vector using Gateway® Technology.
These steps are detailed below. We take the example of a molecular construction allowing the assembly of 4 sgRNAs (Figure 3).
Amplification of each module by PCR (See Table 1 for the list of modules that will be amplified, refer to Table 2 and Table 3 for the PCR protocol and program).
Table 1. List of modules to amplify. For each module, the template, the primers, and the size of the PCR product are detailed. See Appendix 2 for the sequence of amplified elements and primers used.
Module ID Template Forward primer Reverse primer PCR product size
U6-26 promoter pHEE401E (Addgene, catalog number: 71287) Fw-Primer1 Rev-Primer2 465 bp
Pre-tRNA(1) pGTR (Addgene, catalog number: 63143) Fw-Primer3 Rev-Primer4 107 bp
sgRNA(1)-pre-tRNA(2) pGTR (Addgene, catalog number: 63143) Fw-Primer5 Rev-Primer6 195 bp
sgRNA(2)-pre-tRNA(3) pGTR (Addgene, catalog number: 63143) Fw-Primer7 Rev-Primer8 195 bp
sgRNA(3pre-tRNA(4) pGTR (Addgene, catalog number: 63143) Fw-Primer9 Rev-Primer10 195 bp
sgRNA(4) pGTR (Addgene, catalog number: 63143) Fw-Primer11 Rev-Primer12 108 bp
U6-26 terminator pHEE401E (Addgene, catalog number: 71287) Fw-Primer13 Rev-Primer14 230 bp
Material needed
10 μM diluted primers
Q5® High-Fidelity DNA Polymerase
dNTP solution MIX 10 mM
pHEE401E plasmid
pGTR plasmid
DNase-free water
Agarose
TAE (Tris-Acetate-EDTA buffer)
SYBRTM Safe DNA gel stain
Monarch® PCR & DNA Cleanup kit
Table 2. PCR protocol for one reaction
Components Volume
Template (4 ng/µL) 1 μL
5× Q5 reaction buffer 10 μL
dNTP (10 mM) 1 μL
Forward primer (10 μM) 1 μL
Reverse primer (10 μM) 1 μL
Q5 DNA polymerase 0.5 μL
DNase-free water 35.5 μL
Table 3. PCR program (inspired by NEB’s instructions)
Temperature Time Nb of cycles
98 °C 30 s 1×
98 °C 10 s 25×
TM °C 30 s
72 °C 1 min/kb
72 °C 2 min 1×
12 °C Hold
Troubleshooting: These reaction conditions are optimal, but if despite everything no amplification is obtained, it is recommended to use new solutions (buffer, dNTP), to re-dilute the primers, and to use a new aliquot of DNase-free water.
PCR product purification
i. 5 μL of each PCR product is checked by electrophoresis on agarose gel (1%) – TAE 0.05% – SYBRTM Safe 0.005%.
ii. If the amplification is specific and leads to a single band on an agarose gel, the PCR products can then be purified using a suitable purification kit (e.g., Monarch® PCR & DNA Cleanup kit).
iii. Elute in 10 µL of elution buffer; samples with higher DNA yield can be diluted after PCR purification.
Assembly of purified PCR products using the Golden Gate cloning system
The protocol consists of a succession of digestions and ligations of the purified PCR products in order to construct the polycistronic gene with the attB2r and attB3 recombination sites at its ends (See Table 4 and 5 for Golden Gate reaction and incubation conditions)..
Material needed
Purified PCR products
BsaI-HF restriction enzyme
10× Cutsmart buffer
T4 DNA ligase
DNase-free water
Table 4. Golden Gate reaction
Components Volume
Purified PCR products 1 2 μL
Purified PCR products 2 2 μL
Purified PCR products 3 2 μL
Purified PCR products 4 2 μL
Purified PCR products 5 2 μL
Purified PCR products 6 2 μL
Purified PCR products 7 2 μL
10× Cutsmart buffer 2 μL
BsaI-HF 1 μL
T4 DNA ligase 1.5 μL
DNase-free water 1.5 μL
Use a thermocycler to perform successive digestion and ligation according to the program below:
Table 5. Golden Gate incubation conditions
Temperature Time Nb of cycles
40 °C 10 min 3×
16 °C 10 min
50 °C 10 min 1×
80 °C 20 min 1×
Cloning of the polycistronic gene into the pDONRTM P2R-P3 vector
The recombination sites flanked Golden Gate reaction product are integrated into the pDONRTM P2R-P3 vector according to the Multisite Gateway® Technology protocol and various recombination protein activities (Figure 5)(See Table 6 for BP reaction conditions).
Note: Some minor modifications were made to the Multisite Gateway® Technology protocol. Namely, BP recombination reaction volume is reduced from 10 to 5 μL, we do not work with an equimolar ratio between insert and entry vector but systematically add 3 μL of Golden Gate reaction product regardless of the concentration, and no Proteinase K treatment is necessary after the BP recombination reaction.
Figure 5. Generating the entry clone containing the polycistronic gene. Figure adapted from the user guide “MultiSite Gateway® Three-Fragment Vector Construction Kit.” A BP recombination reaction is performed between the attB2r and attB3-flanked polycistronic gene and pDONR™ P2R-P3 to generate an entry clone.
Material needed
attB2r-Golden Gate reaction product-attB3
Purified plasmid DNA pDONRTM P2R-P3 (150 ng/μL)
GatewayTM BP ClonaseTM II
One ShotTM TOP10 chemically competent E. coli
LB Miller media
Kanamycin bisulfate
Bacteriological agar
Table 6. BP reaction conditions
Components Volume
Golden Gate reaction product 3 μL
pDONRTM P2R-P3 1 μL
GatewayTM BP ClonaseTM II 1 μL
Note: Be sure to keep the BP Clonase®II enzyme mix on ice while preparing the recombination reaction. Return the enzyme mix to -20 °C immediately after use.
Incubate overnight at 25 °C.
The day after, use all the BP reaction to transform chemically competent cells:
i. Add 5 μL of BP reaction to 50 μL of One ShotTM TOP10 chemically competent E. coli (e.g.).
ii. Incubate for 30 min on ice.
iii. Heat shock for 1 min 30 s at 37 °C.
iv. Transfer on ice for 2 min.
v. Add 1 mL of LB Miller media.
vi. Shake for 45 min at 37 °C.
vii. Spread with glass beads on LB Miller agar plate containing kanamycin (25 µg/mL).
viii. Incubate the plate overnight at 37 °C.
Troubleshooting: If no colony is obtained after transformation, repeat the reaction under the same conditions. If the problem persists, this is probably due to the enzyme mixture, which may have been stored on ice for too long before being returned to -20 °C during the previous use.
Screening positive colonies by PCR
Prepare a PCR reaction mix (conditions for one reaction are described in Table 7. See also Table 8 for PCR program) and use colonies as templates to check whether or not they contain the construct.
Material needed
10 μM diluted primers
Use universal primers (see below) or specific primers for the polycistronic gene:
M13 Forward (-20): 5'-GTAAAACGACGGCCAG-3'
M13 Reverse: 5'-CAGGAAACAGCTATGAC-3'
OneTaq 2× Master Mix with standard buffer
Table 7. PCR Protocol
Components Volume
OneTaq 2× Master 12.5 μL
Forward primer (10 μM) 1 μL
Reverse primer (10 μM) 1 μL
DNase-free water 10.5 μL
i. Use a toothpick to pick a colony and spread it on an LB Miller agar (25 g/L) with kanamycin (25 μg/mL) plate.
ii. Directly afterward, dip the toothpick into the PCR reaction to transfer a very small number of cells (this step is critical, too many cells could inhibit the PCR reaction).
iii. Proceed in this way for each colony to be tested
iv. Incubate the plate overnight at 37 °C and run the PCR in the thermocycler using the following conditions:
Table 8. PCR program (adapted from NEB’s instructions)
Temperature Time Nb of cycles
94 °C 30 s 1×
94 °C 30 s 25×
TM °C 30 s
68 °C 1 min/kb
68 °C 5 min 1×
12 °C Hold
v. 20 μL of each PCR product are checked by electrophoresis on agarose gel (1%) – TAE 0.05% – SYBRTM Safe 0.005%.
vi. Pick corresponding positive colonies on the plate with a toothpick and inoculate 5 mL of LB Miller + kanamycin (25 μg/mL) for each.
vii. Incubate culture and shake overnight at 37 °C.
viii. Perform plasmid extraction and purification for each clone using a suitable kit (e.g., NucleoSpin® Plasmid kit, Macherey Nagel).
ix. Elute plasmid DNA according to the supplier’s instructions.
x. Using a spectrophotometer, determine the sample concentration (A260 nm) and check the DNA purity (A260/A280 nm ratio should be comprised between 1.8 and 2.0).
xi. Perform sequencing using the universal primers below and verify the integrity of the polycistronic gene sequence.
M13 Forward (-20): 5'-GTAAAACGACGGCCAG-3'
M13 Reverse: 5'-CAGGAAACAGCTATGAC-3'
Troubleshooting: If no amplification is obtained after PCR reaction, repeat the reaction under the same conditions but taking care to resuspend only a very small quantity of bacteria in the reaction medium because too many cells could inhibit the reaction.
Assembly in destination vector
The promoter, ZCas9, and the polycistronic gene cloned independently into entry vectors are then assembled using Gateway® Cloning technology to form a final plasmid ready for plant transformation (Figure 6). All these elements, flanked by compatible recombination sequences, will assemble in an orderly manner thanks to the integrase and excisionase activity of a mixture of enzymes according to the Multisite Gateway® Technology protocol (see Table 9 for LR reaction conditions).
Note: Some minor modifications were made to the Multisite Gateway® Technology protocol. Namely, LR recombination reaction volume is reduced from 10 to 5 μL, we do not take into account the molarity but the concentration of the plasmids in ng/µL, and no Proteinase K treatment is necessary after the LR recombination reaction.
Figure 6. Generating the expression clone containing the promoter, zCas9, and the polycistronic gene (adapted from the user guide “MultiSite Gateway® Three-Fragment Vector Construction Kit”).
Homologous recombination reaction
Material needed
Purified plasmid DNA of your attL4 and attR1-flanked entry clone (supercoiled, 80 ng/μL)
Purified plasmid DNA of your attL1 and attL2-flanked entry clone (supercoiled, 80 ng/μL)
Purified plasmid DNA of your attR2 and attL3-flanked entry clone (supercoiled, 80 ng/μL)
Purified plasmid DNA pFRm43GW (150 ng/μL)
GatewayTM LR ClonaseTM II
One ShotTM TOP10 chemically competent E. coli
LB Miller media
Spectinomycin dihydrochloride pentahydrate
Bacteriological agar
Table 9. LR reaction conditions
Components Volume
attL4 and attR1-flanked entry clone 1 μL
attL1 and attL2-flanked entry clone 1 μL
attR2 and attL3-flanked entry clone 1 μL
pFRm43GW 1 μL
GatewayTM LR ClonaseTM II 1 μL
Note: Be sure to keep the LR Clonase® II enzyme mix on ice while preparing the recombination reaction. Return the enzyme mix to -20 °C immediately after use.
Incubate overnight at 25 °C.
The day after, use all the LR reaction to transform chemically competent cells:
i. Add 5 μL of LR reaction to 50 μL of One ShotTM TOP10 chemically competent E. coli (e.g.).
ii. Incubate for 30 min on ice.
iii. Heat shock for 1 min 30 s at 37 °C.
iv. Transfer on ice for 2 min.
v. Add 1 mL of LB Miller media.
vi. Shake for 45 min at 37 °C.
vii. Spread on LB Miller agar plate containing spectinomycin (50 μg/mL).
viii. Incubate the plate overnight at 37 °C.
Troubleshooting: If no colony is obtained after transformation, repeat the reaction under the same conditions. If the problem persists, this is probably due to the enzyme mixture, which may have been stored on ice for too long before being returned to -20 °C during the previous use.
Screening positive colonies by PCR
Perform PCR screening as described in step B2e. Use various couples of primers to verify the integrity of the vector (Table 10); see Figure 7 for primers details.
Troubleshooting: If no amplification is obtained after PCR reaction, repeat the reaction under the same conditions but taking care to resuspend only a very small quantity of bacteria in the reaction medium, because too many cells could inhibit the reaction.
Table 10. List of primers needed for the PCR screening
Primer ID Primer sequence 5’ > 3’ Size of the PCR product
EC1.2en-fw TTGCGTTTGGTTTATCATTGCG 400 bp
EC1.2en-rv AGTGTTGTCGATGTGTCATGT
Zcas9-fw GGATGATGATGACAAGATGG 450 bp
Zcas9-rv GTGGTAGGCAACCTCGTC
U6-26p-fw GATTAGGCATCGAACCTTC 750 bp
U6-26t-rv GTTTATCTCATCGGAACTGC
Figure 7. Performing PCR to check the integrity of the vector. Little arrows represent primers used to check that the promoter, zCas9, and the polycistronic gene were cloned into the destination vector.
For colonies that were positives for the three PCR reactions:
Perform plasmid extraction and purification as described in step B2e [use LB Miller + spectinomycin (50 µg/mL) to inoculate positive colonies in order to perform plasmid extraction and purification].
Sequencing
Perform sequencing using the primers below and verify the integrity of the destination vector (Table 11); see Figure 8 for primers details.
Table 11. List of primers needed for the destination vector sequencing
Primer ID Primer sequence 5’ > 3’
pEC1.2en-seq-F GGAGCGCTACTGATTCAAC
Cas9Seq_658_678 GTTGACAAGCTGTTCATCCAG
Cas9Seq_1520_1539 AGTCAGAGGAGACGATCACG
Cas9Seq_2330_2349 TGCAGACCGTGAAGGTTGTG
Cas9Seq_3163_3186 TACGATGTGAGGAAGATGATCGCC
U6-26p-seq-F TGTCCCAGGATTAGAATGATTAGGC
Figure 8. Performing sequencing to check the integrity of the vector. Little arrows represent primers used to check by sequencing that promoter, zCas9, and the polycistronic gene were assembled into the destination vector.
After sequencing and checking, the plasmid is ready to transform Agrobacterium tumefaciens (strain C58C1). The transformed strain will be used for Arabidopsis thaliana transformation by the floral dip method [19]. Transformants are then selected according to seed tagRFP fluorescence (red seed coat selection) [20].
Validation of protocol
AtMCTP3 and AtMCTP4 genes belong to the multiple C2 domains and transmembrane region protein (MCTP) multigenic family. Associated proteins are key regulators of cell-to-cell signaling in plants and act as ER-PM tethers specifically at plasmodesmata. The previously characterized Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects and impaired plasmodesmata function and composition [21]. This mutant is strongly affected in its development; thus, AtMCTP3 and AtMCTP4 genes are excellent candidates to validate a multi-targeting genome editing protocol.
The protocol we described allowed us to edit simultaneously AtMCTP3 and AtMCTP4 genes using two guides per targeted gene and to obtain the Atmctp3/Atmctp4 double mutant–associated phenotype (Figure 9B). We compare also with the same cloning strategy using only one guide per targeted gene. Our strategy allowed us to obtain at least 15% of biallelic CRISPR Atmctp3/Atmctp4 mutants using one guide per gene, and 20% CRISPR Atmctp3/Atmctp4 mutants using two guides per gene (either homozygous or biallelic mutants depending on the guide) (Table 12). Most of the time, the "two guides per gene" strategy led to big deletions for AtMCTP3 gene (around 440 bp deletion depending on the line), while only 1 or 2 bp indels were obtained for AtMCTP4 gene.
Figure 9. AtMCTP3 and AtMCTP4 genes targeting and associated phenotype. (A) SgRNA targeting zone in AtMCTP3 and AtMCTP4 genes. (B) CRISPR double mutant Atmctp3/Atmctp4 associated phenotype (yellow arrow) compared to wild type on the side. Both black and red SgRNA were used for two guides per gene strategy. Only red guides were used for the single guide per gene strategy.
Table 12. roportion of CRISPR Atmctp3/Atmctp4 mutants for both strategies and type of mutation obtained for each sgRNA. NA = not applicable.
Atmctp3,
Atmctp4
phenotype
Atmctp3 homozygous mutation
Atmctp3
biallelic mutation
Atmctp4 homozygous mutation Atmctp4 biallelic mutation
SgRNA4 SgRNA3 SgRNA4 SgRNA3 SgRNA2 gRNA1 SgRNA2 SgRNA1
1 guide strategy n = 19
3/19
15.78%
NA 0/3 NA 3/3 NA 0/3 NA 3/3
2 guides strategy n = 24
5/24
20.83%
5/5 4/5 0/5 0/5 0/5 2/5 2/5 3/5
Highlighting versatility and adaptability to inducible or cell type-specific editing
Here, we described a cell type–specific editing system that avoids mosaicism by using the egg cell promoter to control zCas9 expression. Depending on the biological question, other cell type–specific editing strategies may be necessary (e.g., if one wants to target only the quiescent center, cortex in the root). These questions can be assessed using cell type–specific promoters. These promoters can also be used in an inducible version in association with the XVE module to allow induction with β-estradiol. To do this, the promoter of interest must be cloned in pDONRTM P4-P1R vector according to Thermo Fisher’s recommendations.
Briefly, the promoter should be amplified with primers containing attB4 and attB1r recombination sites (see below for primer sequences). The PCR product is then cloned into the entry clone via a BP recombination reaction (with the same conditions as described in section B2d for cloning the polycistronic gene into pDONRTM P2R-P3 vector). Classical steps of E. coli transformation, screening, and sequencing are carried out as described previously.
Primer sequences for promoter amplification:
attB4 5’ - GGGG-ACA-ACT-TTG-TAT-AGA-AAA-GTT-GNN--(template-specific sequence)-3’
attB1r 5’ – GGGG-AC-TGC-TTT-TTT-GTA-CAA-ACT-TGN--(template-specific sequence)-3’
Finally, the newly designed vector is used for final LR recombination in association with the attL1 and attL2-flanked entry clone (encoding for the Cas9) and the attR2 and attL3-flanked entry clone (encoding for the polycistronic gene pre-tRNA/sgRNA) (following the conditions described previously).
Acknowledgments
This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (project 772103-BRIDGING to E.M.B.).
Competing interests
The authors declare no competing interests.
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Lei, Y., Lu, L., Liu, H. Y., Li, S., Xing, F. and Chen, L. L. (2014). CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants. Mol Plant. 7(9): 1494–1496. https://doi.org/10.1093/mp/ssu044
Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6):735-43. https://doi.org/10.1046/j.1365-313x.1998.00343.x.
Shimada, T. L., Shimada, T. and Hara‐Nishimura, I. (2010). A rapid and non‐destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61(3): 519–528. https://doi.org/10.1111/j.1365-313x.2009.04060.x
Brault, M. L., Petit, J. D., Immel, F., Nicolas, W. J., Brocard, L., Gaston, A., Fouché, M., Hawkins, T. J., Crowet, J. M., Grison, M. S., et al. (2019). Multiple C2 domains and Transmembrane region Proteins (MCTPs) tether membranes at plasmodesmata. 20(8):e47182. https://doi.org/10.15252/embr.201847182.
Supplementary information
The following supporting information can be downloaded here
Appendix 1. List of protospacers chosen for AtMCTP4 and AtMCTP3 genes targeting.
Appendix 2. Details of amplified elements required for the polycistronic gene assembly.
Article Information
Publication history
Received: Apr 4, 2024
Accepted: Jun 11, 2024
Available online: Jul 2, 2024
Published: Jul 5, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Category
Plant Science > Plant molecular biology > Genetic analysis
Molecular Biology > DNA > Gene expression
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5,030 | https://bio-protocol.org/en/bpdetail?id=5030&type=0 | # Bio-Protocol Content
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This is a correction notice. See the corrected protocol.
Peer-reviewed
Correction Notice: Live-Cell Imaging and Analysis of Germline Stem Cell Mitosis in Caenorhabditis elegans
RZ Réda M. Zellag
YZ Yifan Zhao
AG Abigail R. Gerhold
Published: Jun 20, 2024
DOI: 10.21769/BioProtoc.5030 Views: 155
Reviewed by: Gunar FabigDemosthenis Chronis Anonymous reviewer(s)
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After official publication in Bio-protocol (https://bio-protocol.org/e4272), we realized that an error in the Background section occurred during the editorial process, which caused certain text to be duplicated. The published text “Like other stem cells, C. elegans GSCs are kept in a stem-like state by signaling from a somatic niche (the distal tip cell, Kimble and White, 1981; Figure 1A and 1E). Like several types of mammalian stem cells, the size of the C. elegans GSC pool is maintained according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions to maintain a relatively constant number of stem cells. according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions, thus maintaining a relatively constant number of stem cells (Morrison and Kimble, 2006; Joshi et al., 2010)” has been corrected to “Like other stem cells, C. elegans GSCs are kept in a stem-like state by signaling from a somatic niche (the distal tip cell, Kimble and White, 1981; Figure 1A and 1E). The size of the GSC pool is maintained according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions that make more stem cells (Morrison and Kimble, 2006; Joshi et al., 2010).”
This correction is purely textual and does not change the content of the Background section or anything relating to the published procedure.
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Workflow of Genome-Wide Mediation Analysis (GMA) by Incorporating Intermediate Omics Data as Mediators
ZY Zhikai Yang
QZ Qi Zhang
JY Jinliang Yang
Published: Jul 20, 2024
DOI: 10.21769/BioProtoc.5031 Views: 27
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Abstract
Linking genotype to phenotype is a long-standing research topic in quantitative genetics and is essential for biological sciences. As a causal inference method, genome-wide mediation analysis (GMA) has proven to be a powerful tool to complement the widely used genome-wide association study (GWAS) and transcriptome-wide association study (TWAS). GMA can identify single nucleotide polymorphisms (SNPs) that directly affect the final phenotype or do so indirectly through intermediate omics data mediation. When considering transcriptomics data as the intermediate trait, this method can pinpoint specific genes as transcriptional mediators connecting genotype with phenotype. The results from GMA, therefore, provide explicit causation hypotheses for downstream functional studies. This protocol provides code and a workflow to demonstrate the mediation analysis procedure using a simulated dataset. The demo analysis can be conducted in R using a laptop or desktop computer.
Keywords: Genome-wide mediation analysis RNA-seq Genomics Omics Causation
Graphical overview
The input and output datasets of genome-wide mediation analysis (GMA) in identifying intermediate mediators to connect genotype with phenotype.
Background
Phenotypic traits, especially complex agronomic traits, are usually determined by a large number of genetic loci. The penetrance of the genetic variants often occurs through different layers of intermediate molecular processes, i.e., gene regulation [1], spatiotemporal gene expression [2], and translation [3]. Identifying the genetic loci and revealing the related intermediate molecular processes are, therefore, critical for biological understanding and crop improvement.
Genome-wide association study (GWAS) establishes the association between genotype [i.e., single nucleotide polymorphisms (SNPs)] and traits of interest [4]. Similarly, transcriptome-wide associated study (TWAS) connects SNP with gene expression levels [5]. However, none of these methods bridges the three variables of genotype, intermediate molecular processes, and conventional phenotype in a single analysis. Mediation analysis, as a statistical causal inference method, establishes a causal chain for these three variables, i.e., from genotype as the exposure to the intermediate molecular process (i.e., gene expression) as the mediator and eventually to the conventional phenotype as the outcome. Our previous work has laid down the statistical foundation of the high-dimensional mediation analysis [6] and applied it to a maize diversity panel to identify a number of mediator genes [7]. By modeling genotype to phenotype through two pathways, GMA can detect SNPs 1) directly affecting phenotype without any mediation through the intermediate variables under investigation or 2) indirectly through intermediate processes, i.e., gene expression or other intermediate omics data.
In this workflow, we use simulated transcriptome data as the intermediate molecular traits to demonstrate the GMA procedure. We provide input data formats and output results interpretation. It is worth noting that GMA is not limited to treating transcriptome data as the intermediate variable; other genome-wide variables, such as metabolome or proteome, can also be considered as intermediate variables. These intermediate variables identified through GMA can be further validated through biological functional analyses.
Equipment
Laptop or desktop computer running a Windows or Linux system (i.e., a MacBook Pro computer)
Running time: To run a small dataset (i.e., in this example, approximately 300 different genotypes with 5,000 SNPs and RNA-seq data of 1,200 genes), 5 GB RAM and 4 CPU cores with 0.5 h running time should be sufficient. However, for a larger dataset (i.e., 300 genotypes with approximately 1 million SNPs and 8,000 genes), at least 160 GB RAM and 16 CPU cores with 1 h running time are recommended.
Software and datasets
R v4.1.3 (https://cran.r-project.org/)
R packages:
data.table_1.14.8
glmnet_4.1-8
MASS_7.3-58.2
rrBLUP_4.6.3
parallel_4.1.3
doParallel_1.0.17
CompQuadForm_1.4.3
circlize_0.4.16
dplyr_1.1.0
RcolorBrewer_1.1-3
Input data
To conduct GMA, three input datasets are needed: a SNP matrix as the exposure, the phenotypic values as the outcome, and the omics matrix as the mediator, as well as one optional input dataset (i.e., a matrix of principal components to control population structure). The formats of the input datasets are described below.
Exposure (“input/Z_matrix.txt”): a numerical matrix of bi-allelic SNP data (Table 1). It is coded as “-1, 0, 1” to represent homozygous reference, heterozygous, and homozygous alternative genotypes. For missing data, it can simply be imputed by averaging the genotypic value or using SNP imputation software such as Beagle [8], fastPHASE [9], or LinkImpute [10].
Table 1. SNP genotype matrix (or the Z matrix).
In the matrix, the row represents the plant genotype, and the column represents the SNP site.
[SNP1] [SNP2] … [SNPM]
[genotype 1] -1 0 -1
[genotype 2] 0 1 1
… … … … …
[genotype n] 1 -1 … 0
Confounder (“input/X0_matrix.txt”): optional principal components of the Z matrix (Table 2). This matrix can be calculated from the Z matrix to control for the population structure; confounders can also be anything that may affect mediators and outcomes, like environmental factors that can affect gene expression (mediator) and phenotype (outcome). It is optional for the GMA.
Table 2. Principal components of the Z matrix (or the X0 matrix). In this matrix, the row represents the plant genotype, and the column represents each principal component.
[PC1] [PC2] [PC3]
[genotype 1] 37.67511 -2.86529 0.788754
[genotype 2] 62.41023 -85.0894 16.63496
… … … ..
[genotype n] 65.67986 -96.6183 9.525744
Mediator (“input/X_matrix.txt”): the intermediate omics dataset (Table 3). For the omics input data, population-wide measurement of each variable is required, for example, a population-wide RNA-seq data for each gene [11].
Table 3. Intermediate omics data matrix (or the X matrix). Here, normalized RNA-seq data were employed as the intermediate trait. The row represents the plant genotype, and the column represents each normalized read count of each gene.
[gene 1] [gene 2] … [gene q]
[genotype 1] 2.215721 -0.74665 … 0.037008
[genotype 2] -0.96617 0.433468 … 0.441124
… … … … …
[genotype n] 0.297719 -0.63026 … 0.009335
Outcome (“input/y_matrix.txt”): the phenotypic trait of interest (Table 4).
Table 4. Phenotype matrix (or the y matrix).
The row represents the plant genotype, and the column represents a trait of interest (BLUP/BLUE are recommended over raw phenotype).
[trait1]
[genotype 1] 4.214806
[genotype 2] 3.828688
… …
[genotype N] -3.691982
Procedure
In this demo case, we simulated a maize diversity panel composed of n = 271 different inbred lines. In the population, 5,000 SNP markers were genotyped, and the gene expression levels of 1,200 genes were considered the intermediate traits. Five of these 1,200 genes were simulated as the mediator genes, each of which was controlled by 10 indirect SNPs. The heritability of the phenotype is simulated as 0.75.
Case study
Calculate the confounding effect (or the X0 matrix):
Several principal components can be used as the fixed effects to control population structure as the confounding effects if using a structured population.
Open the R console and then run the following script. Note that in the analysis, the first p=3 principal components were used.
source('lib/utils.R')
Z <- fread("input/Z_matrix.txt", header=T, data.table=FALSE)
Z = as.matrix(Z)
X0 <- getpca(Z, p=3) # here the first p=3 PCs were extracted
Conduct GMA using different methods:
library(glmnet)
library(MASS)
library(rrBLUP)
library(parallel)
library(doParallel)
library(CompQuadForm)
source('lib/highmed2019.r')
source('lib/fromSKAT.R')
source('lib/MedWrapper.R')
source('lib/reporters.R')
subX = X[, 1:100]
# run the fixed effect model that assign equal penalty on the two data types (time: ~1min).
run_GMA(y, X0, X, Z, ncores=10, model="MedFix_eq", output_folder="output/")
# run the fixed effect model that minimizes BIC (time: ~1min).
run_GMA(y, X0, X, Z, ncores=10, model="MedFix_fixed", output_folder="output/")
# run the random effect model using linear kernel, and extract the model that minimizes BIC (time: ~5min).
run_GMA(y, X0, X, Z, ncores=10, model="MedMix_linear", output_folder="output/")
# run the random effect model using shrink_EJ kernel, and extract the model that minimizes BIC (time: ~5min).
run_GMA(y, X0, X, Z, ncores=10, model="MedMix_shrink", output_folder="output/")
Visualization of results (this step is optional):
Note that only outputs of “Med_Fixed” methods can be used to generate circos plot, as these methods provide dSNPs and iSNPs results. Here, we demonstrate the visualization results using the output data from “Med_Fixed_BIC”.
library(circlize)
library(dplyr)
library("RColorBrewer")
gwas <- qGWAS(y, Z, plot=FALSE)
fwrite(gwas, "output/gwas_results.csv", sep=",", row.names = FALSE, quote=FALSE)
source("lib/circosplot.R")
circos_med(gwas_res="output/gwas_results.csv",
med_res="output/mediators_fixed_bic_trait_V1.csv",
dsnp_res="output/dsnps_fixed_bic_trait_V1.csv",
isnp_res="output/isnps_fixed_bic_trait_V1.csv",
chrlen="input/Chromosome_v4.txt",
gene_position= "input/gene_pos.csv",
out_tiff = "graphs/circos.tiff")
Result interpretation
Summary file (Table 5):
Table 5. Overall summary of GMA results. In the table: pmed.pure, proportion of the variance mediated; v.tot, variance of total effect; v.med, variance of indirect effect; v.dir, variance of direct effect; n.med, number of significant mediators after adjustment to control for the false discovery proportion (FDP) in mediator selection; pval.cut, the threshold for deciding significance; n.direct, number of direct SNPs.
pmed.pure v.tot v.med v.dir n.med pval.cut n.direct
0.685373 737.4137 505.4035 156.9284 6 0.05 9
Mediator file (Table 6):
Table 6. Identified mediators from GMA results. In the table: id, mediator gene id; e2m, p-value of effect from exposure to mediator; m2y, p-value of effect from mediator to outcome; e2m2y, maximum value between e2m and m2y; padj, adjusted p-value; coef, product of effect from exposure to mediator and effect from mediator to outcome.
id e2m m2y e2m2y padj coef
V1 1.11E-101 0.617229 0.617229259 0.617229259 0.004557
… … … … … …
V237 5.36E-145 0 5.36E-145 2.68E-144 -0.32756
dSNP file (Table 7):
Table 7. Output of direct SNPs (or dSNPs). Note that only “Med_Fix” methods will report direct SNPs. In the table: snp, direct SNP ID; pval, p-value of effect from exposure to outcome; coef, effect from exposure to outcome.
snp pval coef
4-102613116 1.20E-171 -0.20507
… … …
4-184962925 1.20E-171 -0.14465
iSNP file (Table 8):
Table 8. Output of indirect SNPs (or iSNPs). Note that only “Med_Fix” methods will report indirect SNPs. In the table: medi, name of the mediator gene; snps_for_medi, indirect SNPs for the corresponding mediator; coef, effect from exposure to mediator.
medi snps_for_medi coef
V178 1-61707925 0.118978
… … …
V178 1-207329129 -0.24462
Circos Plot (Figure 1):
Figure 1. Visualization of genome-wide mediation analysis (GMA) results using a circos plot. In this circos plot, the outermost circular track represents the 10 maize chromosomes; the next inner track shows the genome-wide association study (GWAS) results, with two circular blue dashed lines indicating -log(p-value) of 5 and 10 and the red lines denoting the position of direct single nucleotide polymorphisms (SNPs); the next inner track shows the relative positions of identified mediator genes (in this case, seven significant mediators were identified) with different mediator genes represented by different colors; the lines in the innermost circle connects mediators with their corresponding indirect SNPs.
Acknowledgments
This work is supported by the US Department of Energy, Grant No. DE-SC0023138 and the USDA-NIFA Agriculture and Food Research Initiative Grant No. 2019-67013-29167 to Jinliang Yang and the University of New Hampshire Start-up fund to Qi Zhang. This protocol is modified from a previous publication (Yang et al., 2022).
Competing interests
The authors declare that they have no competing interests.
References
Carthew, R. W. (2021). Gene Regulation and Cellular Metabolism: An Essential Partnership. Trends Genet. 37(4): 389–400. https://doi.org/10.1016/j.tig.2020.09.018
Schoenfelder, S. and Fraser, P. (2019). Long-range enhancer–promoter contacts in gene expression control. Nat Rev Genet. 20(8): 437–455. https://doi.org/10.1038/s41576-019-0128-0
Ingolia, N. T., Hussmann, J. A. and Weissman, J. S. (2018). Ribosome Profiling: Global Views of Translation. Cold Spring Harbor Perspect Biol. 11(5): a032698. https://doi.org/10.1101/cshperspect.a032698
Hayes, B. (2013). Overview of Statistical Methods for Genome-Wide Association Studies (GWAS). Methods Mol Biol. 1019: 149–169. https://doi.org/10.1007/978-1-62703-447-0_6
Wainberg, M., Sinnott-Armstrong, N., Mancuso, N., Barbeira, A. N., Knowles, D. A., Golan, D., Ermel, R., Ruusalepp, A., Quertermous, T., Hao, K., et al. (2017). Transcriptome-wide association studies: opportunities and challenges. Nat Genet. 51(4):592–599. https://doi.org/10.1038/s41588-019-0385-z
Zhang, Q. (2021). High-Dimensional Mediation Analysis with Applications to Causal Gene Identification. Stat Biosci. 14(3): 432–451. https://doi.org/10.1007/s12561-021-09328-0
Yang, Z., Xu, G., Zhang, Q., Obata, T. and Yang, J. (2022). Genome-wide mediation analysis: an empirical study to connect phenotype with genotype via intermediate transcriptomic data in maize. Genetics. 221(2): e1093/genetics/iyac057. https://doi.org/10.1093/genetics/iyac057
Browning, S. R. and Browning, B. L. (2007). Rapid and Accurate Haplotype Phasing and Missing-Data Inference for Whole-Genome Association Studies By Use of Localized Haplotype Clustering. Am J Hum Genet. 81(5): 1084–1097. https://doi.org/10.1086/521987
Scheet, P. and Stephens, M. (2006). A Fast and Flexible Statistical Model for Large-Scale Population Genotype Data: Applications to Inferring Missing Genotypes and Haplotypic Phase. Am J Hum Genet. 78(4): 629–644. https://doi.org/10.1086/502802
Money, D., Gardner, K., Migicovsky, Z., Schwaninger, H., Zhong, G. Y. and Myles, S. (2015). LinkImpute: Fast and Accurate Genotype Imputation for Nonmodel Organisms. G3 Genes|Genomes|Genetics 5(11): 2383–2390. https://doi.org/10.1534/g3.115.021667
Kremling, K. A. G., Chen, S. Y., Su, M. H., Lepak, N. K., Romay, M. C., Swarts, K. L., Lu, F., Lorant, A., Bradbury, P. J., Buckler, E. S., et al. (2018). Dysregulation of expression correlates with rare-allele burden and fitness loss in maize. Nature. 555(7697): 520–523. https://doi.org/10.1038/nature25966
Supplementary information
Data and code availability: All data and code have been deposited to GitHub: https://github.com/Bio-protocol/high_dim_mediation/tree/main
Article Information
Publication history
Accepted: Jun 5, 2024
Published: Jul 20, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages
JS Juan C. Sanchez
JB Joseph K. Baumgardt
EW Elizabeth R. Wright
Published: Vol 14, Iss 14, Jul 20, 2024
DOI: 10.21769/BioProtoc.5032 Views: 1022
Reviewed by: Munenori IshibashiElena A. Ostrakhovitch Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Journal of Bacteriology Feb 2021
Abstract
A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications.
Key features
• Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods.
• High-throughput cryo-EM data collection of filamentous objects.
• Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.
Keywords: Cryo-EM Helical reconstruction Bacterial flagellum Polymers Purification Vitrification
Graphical overview
Graphical overview of flagellar filament purification and cryo-EM imaging and analysis. Following bacterial propagation of C. crescentus, cells are pelleted, and a series of centrifugation steps are used to collect ejected flagellar filaments. The filament quality is assessed before the sample is vitrified for cryo-EM data collection. Cryo-EM data is processed generating an electron potential map that is used for building an atomic model of the filament.
Background
The flagellum is an appendage that supports motility in many bacterial species. The flagellum, an extracellular helical propeller, provides cells with a means to navigate through viscous environments. At the cell membrane, an ion-powered motor complex generates torque to rotate the helical filament. Although critical for cellular function, flagellum synthesis is an energetically expensive process that requires the regulated expression of over 60 genes to form a fully functional flagellum. These genes encode both structural and regulatory proteins [1,2]. Bacterial flagella are complex nanomachines, which have evolved to support the lifestyles and ecological niches of the associated species. It has been determined that ~45% of bacterial species possess multiple flagellin proteins, the structural proteins that comprise the flagellar filament [3,4]. In some species, only a single flagellin is synthesized and incorporated into the nascent filament due to phase variation, while others generate filaments comprised of multiple different flagellins [5–8]. Previously, studying the structure of these flexible helical polymers would require the introduction of point mutations to lock the flagellar filament into straightened forms [9,10]. Through these studies, it was determined that the flagellum was organized into 11 protofilaments made up of many flagellin monomers, each of which contains four domains, denoted D0, D1, D2, and D3 (Figure 1A). However, these studies were limited because little information could be gleaned about biologically relevant molecular interactions that impact flagellin subunit packing and filament architecture, i.e., without the imposed straightening mutations.
Our studies focus on the dimorphic bacterium, Caulobacter crescentus. The C. crescentus genome contains six flagellin genes that are expressed and incorporated into the wild-type filament [3,5]. Our structural analyses have shown that C. crescentus flagellins lack the D2 and D3 domains (Figure 1B) [11]. Interestingly, as C. crescentus undergoes cell division, a cell will eject the polar flagellar filament to allow for the synthesis of a polar stalk. The filament purification method presented here concentrates ejected filaments through a series of centrifugation and washing steps. The process of filament ejection can be artificially stimulated in other species when cells exhaust nutrients and enter the stationary phase [12,13]. Incorporating this step during bacterial propagation could allow our workflow to be applied to many other bacteria. The purified flagellar filaments were assessed by SDS-PAGE analysis and negative stain transmission electron microscopy (TEM) before cryo-EM studies. Although single-particle cryo-EM structure determination has exploded in the past decade, tools specific to iterative real-space helical reconstruction have lagged [14]. Fortunately, several new tools have become available that accelerate the reconstruction of helical polymers to high resolution [15–20]. Here, we demonstrate the application of those tools to determine the structures of both straight and curved helical polymers below 3.0 Å resolution. The protocol encompasses (1) methods to purify flagellar filaments from exhausted media, (2) detailed cryo-EM reconstruction workflows that use either symmetrized or asymmetrical reconstruction methods to resolve straight or highly curved filaments, and (3) an outline for model building and validation of large multimeric protein models based on the reconstructed maps. The protocols can be applied to studies of flagellar filament structures across multiple species. Additionally, the general cryo-EM workflow is not limited to flagellar filaments and can be applied to other helical biopolymers.
Figure 1. Comparison of bacterial flagellin models. A. Canonical flagellin model from Salmonella enterica serovar Typhimurium with D0, D1, D2, and D3 domains (PDB: 1UCU) [9]. B. Caulobacter crescentus FljM flagellin model with D0 and D1 domains, and lacking D2 and D3 domains (PDB: 8UXN) [21].
Materials and reagents
Biological materials
Caulobacter crescentus ΔfljJKLNO (FljM only); C. crescentus strain with all flagellins deleted and the fljM flagellin gene introduced on a plasmid with kanamycin resistance (ERW2301) [21]
Caulobacter crescentus ΔfljJLMNO (FljK only); C. crescentus strain with genome deletions to all flagellins except for the fljK gene (TPA2353) [3]
Reagents
Peptone (Fisher Scientific, catalog number: BP1420-500)
Yeast extract (Fisher Scientific, catalog number: BP1422-500)
Bacto agar (BD Difco, catalog number: DF0140010)
Magnesium sulfate heptahydrate (Millipore Sigma, catalog number: MX0070)
Calcium chloride dihydrate (Sigma-Aldrich, catalog number: C3306-500G)
Kanamycin sulfate (Gibco, catalog number: 11-815-024)
Protease inhibitor cocktail (Millipore Sigma, catalog number: 539137)
Phosphate-buffered saline (1× PBS) (Corning, catalog number: 21-040-CV)
AnyKD Mini-PROTEIN TGX stain-free protein gels (Bio-Rad, catalog number: 4568126)
PageRuler Plus prestained protein ladder (Thermo Fisher, catalog number: 26619)
Coomassie Brilliant Blue (Sigma-Aldrich, catalog number: B7920-50G)
2% uranyl acetate (UA) (EMS, catalog number: 22400-2)
Solutions
1 M MgSO4 (see Recipes)
1 M CaCl2 (see Recipes)
Peptone yeast extract (PYE) agar (see Recipes)
Peptone yeast extract (PYE) liquid media (see Recipes)
1% Uranyl Acetate (see Recipes)
Recipes
1 M MgSO4
Reagent Final concentration Amount
Magnesium sulfate heptahydrate 1 M 61.62 g
H2O n/a 250 mL
Total n/a 250 mL
Dissolve the MgSO4 in water while stirring.
Filter the solution through a 0.22 µm filter sterile bottle top filter.
1 M CaCl2
Reagent Final concentration Amount
Calcium chloride dihydrate 1 M 36.75 g
H2O n/a 250 mL
Total n/a 250 mL
Dissolve the CaCl2 in water while stirring.
Filter the solution through a 0.22 µm filter sterile bottle top filter.
Peptone yeast extract (PYE) agar
Reagent Final Concentration Amount
Peptone 0.2% (w/v) 2 g
Yeast extract 0.1% (w/v) 1 g
Bacto agar 1.5% (w/v) 15 g
1 M MgSO4 1 mM 1 mL
1 M CaCl2 0.5 mM 0.5 mL
H2O n/a Fill up to 1 L
Total n/a 1 L
Combine peptone, yeast, Bacto agar, and H2O in a flask and autoclave at 121 °C, 15 PSI for at least 20 min.
Allow the media to cool to ~55 °C before adding the salts.
Note: For antibiotic-resistant strains, the media is supplemented with kanamycin at a concentration of 50 µg/mL.
From 1 L of media, pour approximately 25 mL per plate into approximately 40 Petri plates (100 mm).
Peptone yeast extract (PYE) liquid media
Reagent Final concentration Amount
Peptone 0.2% (wt/v) 2 g
Yeast extract 0.1% (wt/v) 1 g
1 M MgSO4 1 mM 1 mL
1 M CaCl2 0.5 mM 0.5 mL
H2O n/a Fill up to 1 L
Total n/a 1 L
Combine peptone, yeast, and H2O in a flask and autoclave at 121 °C, 15 PSI for at least 20 min.
Allow the media to cool to ~55 °C before adding the salts.
Note: for antibiotic-resistant strains, the media was supplemented with kanamycin at a concentration of 50 µg/mL.
1% uranyl acetate (UA)
Reagent Final concentration Amount
2% uranyl acetate 1% (v/v) 250 µL
H2O n/a 250 µL
Total n/a 500 µL
Mix 1 part sterile water with 1 part 2% UA stain (EMS) and filter through a Spin-X centrifuge tube with a 0.22 µm filter (Costar).
Laboratory supplies
100 mm × 15 mm Petri dishes (Fisher Scientific, catalog number: S33580A)
250 mL vacuum filtration system (VWR, catalog number: 10040-464)
1.7 mL centrifuge tubes (Denville, catalog number: C2170)
0.22 µm Spin-X centrifuge tube filter (Costar, catalog number: 8160)
200 mesh carbon film, copper grids (EMS, catalog number: CF200-CU)
Parafilm (Bemis, catalog number: PM996)
Whatman #1 filter paper (Whatman, catalog number: 1001-090)
Quantifoil R2/1 200 mesh, copper grids (Quantifoil Micro Tools GmbH, catalog number: Q210CR1)
Standard Vitrobot Filter Paper, Ø55/20 mm, Grade 595 (Ted Pella, catalog number: 47000-100)
Equipment
NanoDrop spectrophotometer (Thermo Fisher, catalog number: ND2000)
Incubator with shaker (Labnet, catalog number: I-5311-DS)
500 mL centrifuge bottles (Nalgene, catalog number: 3141-0500)
70 mL polycarbonate bottle assembly with aluminum caps (Beckman Coulter, catalog number: 355622)
RC-5B Plus centrifuge (Sorvall, catalog number: SO-RC5B)
GS-3 rotor (Sorvall, catalog number: SO-GSA3)
Optima XL-80K ultracentrifuge (Beckman Coulter, catalog number: 8043-30-1211)
Type 45 Ti fixed angle titanium rotor (Beckman Coulter, catalog number: 339160)
Harvard Trip 1400/1500 Series balance (Ohaus, catalog number: 80000005)
5424 R microcentrifuge (Eppendorf, catalog number: 05-400-005)
KS 260 basic shaker (IKA, catalog number: Z341835)
Plasma cleaner (Harrick Plasma Inc., catalog number: PDC-32G)
Static dissipator (Mettler Toledo, catalog number: UX-11337-99)
PTFE well plate (custom made; similar product: Kibron, catalog number: 6344)
Style N5 reverse pressure tweezers (Dumont, catalog number: 0202-N5-PS-1)
Talos L120C 120 kV transmission electron microscope (TEM) (Thermo Fisher Scientific), or equivalent
Cryo grid box (Sub-Angstrom, catalog number: SB)
Vitrobot Mark IV vitrification robot (Thermo Fisher Scientific)
Titan Krios G3i 300 kV transmission electron microscope (TEM) (Thermo Fisher Scientific)
K3-GIF direct electron detector with energy filter (Gatan Inc., AMETEK)
Supermicro server (KingStar) with two Xeon E5-2640 processors (Intel), 256 GB memory, and 4× 1080Ti GPU (NVIDIA), or equivalent setup.
Software and datasets
CryoSPARC (https://cryosparc.com/)
IMOD (https://bio3d.colorado.edu/imod/)
AlphaFold2 (https://alphafold.ebi.ac.uk)
UCSF ChimeraX (https://www.cgl.ucsf.edu/chimerax/)
PHENIX (https://phenix-online.org/)
Coot (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/)
Procedure
Bacterial propagation
Using aseptic techniques, streak out the bacterial strains of interest onto the appropriate media to generate isolated colonies. Here, we isolated colonies of the Caulobacter crescentus ΔfljJLMNO (FljK only, no antibiotic resistance) and ΔfljJKLNO (FljM only, kanamycin resistance) strains. Cells were grown at 30 °C on PYE agar plates; for kanamycin-resistant strains, the media was supplemented with kanamycin at a concentration of 50 µg/mL.
Note: These two strains only synthesize one out of the six flagellin structural proteins, resulting in mono-flagellin filaments. Additionally, the ΔfljJLMNO (FljK only, no antibiotic resistance) strain produces curved filaments, while the ΔfljJKLNO (FljM only, Kan+) strain produces straighter filaments.
Inoculate 5 mL of PYE liquid media with a single colony and incubate overnight at 30 °C shaking at 200 rpm.
Using the overnight culture, inoculate 4 L of liquid media (split into three 1.3 L preps) and grow cells to exponential phase (OD600 = 0.6–0.8) at 30 °C with shaking at 200 rpm. In general, inoculating a 1.3 L culture with 300 µL of a C. crescentus overnight culture will take roughly 20–24 h to grow to exponential phase. We advise checking the OD after 18 h and then checking every 90 min until the desired OD is reached.
Note: For example, for C. crescentus FljM filaments, 300 μL of overnight culture at an OD600 of 0.278 was used to inoculate the 1.3 L of PYE media, which resulted in 1.3 L of culture at an OD600 of 0.674 after 22 h and 30 min of incubation at 30 °C with shaking.
Flagellar filament purification
Pellet cells at 10,000× g for 15 min using an RC-5B Plus centrifuge and GS-3 rotor with 500 mL centrifuge bottles or an equivalent setup.
Decant the supernatant (containing ejected filaments) into clean flasks and discard the cell pellet. Ensure no visible cellular debris is collected with the supernatant. Repeat this step until all 3 L of bacterial culture are centrifuged. If needed, you can store the supernatant at 4 °C overnight or continue to the next step.
Note: Although it is possible to store the supernatant beyond 24 h (i.e., over a weekend) before continuing to step B3, we have not studied the effects of prolonged supernatant storage on the integrity of the bacterial filaments.
Collect the flagellar filaments from the supernatant by centrifuging the supernatant at 35,000 rpm (~96,000× g) for 35 min at 4 °C using an Optima XL-80K ultracentrifuge and a Type 45 Ti fixed angle titanium rotor or a similar setup. For this procedure, we used 70 mL polycarbonate bottle assemblies with aluminum caps. Using a Harvard Trip balance, balance two tubes against each other and load them opposite of each other in the ultracentrifuge; repeat for additional tubes. Alternatively, a digital lab scale can be used here. Always ensure any tube loaded into the ultracentrifuge is filled to the top and balanced before centrifugation.
Discard the supernatant and save the filament-containing pellet. Repeat the previous step with additional volumes of the supernatant until all the flagellar filaments are pelleted. Increase the size of the filament-containing pellet by adding fresh supernatant to the same tubes with the pellet after each spin. For efficiency, use two sets (six tubes per set) of tubes, ensuring one set is balanced and ready to load into the ultracentrifuge. This step takes approximately 6 h.
Once all the supernatant is centrifuged and discarded, cover the filament-containing pellet with 10 mL of pre-chilled PBS buffer and incubate overnight at 4 °C with shaking at 100 rpm using a KS 260 basic shaker or an equivalent setup. This will help to resuspend the pellet without rigorous mechanical disruption to the filaments. While shaking, ensure the pellet is always suspended in the PBS buffer.
Combine the resuspended pellets from previous steps. If you have two sets of 70 mL polycarbonate bottle assemblies with aluminum caps (12 tubes total), you should combine everything into two tubes containing ~60 mL of material each. Top off tubes with PBS and balance them.
Centrifuge tubes at 13,500 rpm (~14,000× g) for 15 min at 4 °C using an Optima XL-80K ultracentrifuge and a Type 45 Ti fixed angle titanium rotor, or a similar setup, to pellet any large debris still present in the prep.
Collect the supernatant by decanting the supernatant into a clean 70 mL polycarbonate bottle assembly and discard the cellular debris pellet.
To collect flagellar filaments, centrifuge the supernatant at 35,000 rpm (~96,000× g) for 35 min at 4 °C using an Optima XL-80K ultracentrifuge and a Type 45 Ti fixed angle titanium rotor, or a similar setup.
Discard the supernatant and save the filament pellet.
Cover the pellet with 500 μL of PBS buffer and incubate overnight at 4 °C with shaking at 200 rpm using a KS 260 basic shaker or an equivalent setup. Arrange the tubes so the pellet is always suspended in the PBS buffer.
Transfer the filament suspension to a clean 1.7 mL centrifuge tube. Cool the 5424 R microcentrifuge to 4 °C and centrifuge the tubes at 17,500× g for 15 min. Repeat once.
Collect the supernatant in a clean 1.7 mL centrifuge tube and centrifuge at maximum speed (~21,000× g) for 2 h using the 5424 R microcentrifuge. Increase the temperature to 6 °C to prevent excessive ice buildup in the centrifuge.
Discard the supernatant. Gently and quickly wash the pellet with 1 mL of PBS buffer and repeat once. Specifically, add 1 mL of PBS buffer to the side of the tube above the filament pellet; then, remove the PBS buffer without disturbing the pellet.
Add 50 μL of pre-chilled PBS buffer and store the sample overnight at 4 °C under static conditions.
Note: The purified sample may be stored for up to four weeks at 4 °C before loss of filament integrity. However, we encourage using the sample for the remaining experiments within two weeks of preparation, if possible. We have not characterized the effects of freeze/thaw cycles on sample integrity.
Run SDS-PAGE and stain with Coomassie Brilliant Blue stain or other staining reagent such as silver stain to assess the quality of the purification. Using an AnyKD Mini-PROTEIN TGX stain-free protein gel, we observed a band at ~25 kDa indicative of flagellin monomers for FljM and FljK filaments (Figure 2A and 2B, respectively). Lane 1 is a 4 µL aliquot from step B11 and lane 2 is a 2 µL aliquot from step B15. These additional steps greatly increase the filament concentration in the final pellet, as seen in the differences between lanes 1 and lanes 2 (Figure 2). The difference in protein quantity between strains is attributed to differences in filament lengths (Figure 2A compared to Figure 2B). The FljM strain produces smaller filaments (~3 µm) as compared to the FljK strain (~5.5 µm). Additionally, since the purification yields intact flagellar filaments, we observed bands above 70 kDa, which are likely hook-associated proteins (Figure 2).
Figure 2. SDS-PAGE analysis of purified flagellar filaments. A. Lane M is an aliquot of the PageRuler Plus protein ladder, lane 1M is an aliquot of the flagellar pellet after step B11, and lane 2M is an aliquot of the final cell pellet for the C. crescentus FljM strain. B. Lane M is the protein ladder, lane 1K is the flagellar pellet after step B11, and lane 2K is the final cell pellet for the C. crescentus FljK strain. Arrows indicate the flagellin band at ~25 kDa for each strain.
Negative stain
Analysis by SDS-PAGE provides information about the concentration and purity of flagellin monomers. However, it does not inform us about the quality of the intact filaments. There is no biochemical assay to determine whether the filaments are intact. For this step, we use negative-stain TEM to assess the quality of the filaments and to determine a proper concentration of filaments to deposit on EM grids for vitrification.
Glow-discharge 200 mesh carbon film, copper EM grids (EMS) with a plasma cleaner PDC-32G or an equivalent system (Figure 3, step 1).
Line a PTFE well block (custom-made or Kibron) with a piece of parafilm and demagnetize with a static dissipater (Figure 3, step 2).
Fill two wells of the PTFE well block with 50 µL of sterile, Nanopure water and two wells with 50 µL of 1% UA (Figure 3, step 3).
Place 5 µL of the sample onto the EM grid and allow it to incubate for 1 min (Figure 3, step 4).
Blot away the excess liquid by touching the blotting paper to the edge of the EM grid (Figure 3, step 5).
Wash the EM grid in water by touching the face of the EM grid to a water droplet and blotting away excess liquid. Repeat once (Figure 3, step 6).
Wash the EM grid once in a drop of 1% UA and blot away excess stain (Figure 3, step 7).
Hold the EM grid face to a drop of UA stain for 15 s and blot away excess liquid (Figure 3, step 8).
Allow the EM grid to dry for at least 5 min before storing for imaging (Figure 3, step 9).
Repeat this process for additional sample dilutions to assess which concentration of filaments is best for cryo-EM analysis. We typically image a range of dilutions including 2×, 5×, and 10× sample dilutions in PBS buffer, as well as a concentrated sample.
Image negative-stain EM grids on a Talos L120C 120 kV TEM (or equivalent) with a total electron dose of 20–30 e-/Å2 (Figure 4).
Figure 3. Negative-stain workflow. Step-by-step workflow for preparing negative stain grids of the purified C. crescentus flagella as detailed in the negative stain section. Numbering in the figure corresponds to numbering in Section C. All blotting steps are conducted as in step C5, by touching the edge of the grid to the filter paper. The procedure is repeated for replicate grids or different sample concentrations to be imaged by electron microscopy.
Figure 4. Negative-stain EM analysis of purified flagellar filaments. Micrograph of C. crescentus FljM flagellar filaments stained with 1% UA. Arrows indicate flagellar filaments; asterisks indicate extracellular vesicles that persist during the purification but do not inhibit cryo-EM data collection. Scale bar, 100 nm.
Sample vitrification
Sample vitrification using the Vitrobot Mark IV has become routine in the cryo-EM field. Below is a brief outline of the vitrification process with suggested blotting parameters that yield grids suitable for cryo-EM data collection.
Add 60 mL of distilled water into the water reservoir.
Turn on the Vitrobot Mark IV and set the relative humidity to 95% and the temperature to 20 °C.
Attach standard Vitrobot filter paper to the blotting pads and allow time for the system to equilibrate (~15 min).
Glow discharge R2/1 200 mesh, copper grids using a plasma cleaner or an equivalent system.
Cool the Vitrobot foam dewar, ethane cup, and metal spider with LN2. Once the setup has cooled, condense the ethane in the ethane cup. Monitor ethane and LN2 levels throughout the entire vitrification process and top off accordingly.
On the Vitrobot screen, set the drain time to 0.5 s and the wait time to 60 s. We freeze grids with blot times ranging from 2–4 s and a blot force of 0–6. It may be necessary to test a range of blotting conditions to determine what works best for your specific setup.
Use the Vitrobot tweezers to pick up a grid, mount the tweezers onto the Vitrobot, and select Continue on the screen. Mount the foam dewar and follow the on-screen process.
Apply 4 µL of your sample to the carbon side of the grid and select continue to proceed with the blotting conditions specified in step D6.
Allow the system to plunge the specimen into the cryogen; then, transfer the vitrified grid to a labeled grid box for storage.
Repeat steps D6–9 for the desired number of grids and ensure you test a range of blotting conditions and/or sample concentrations.
Cryo-EM data collection
Data collection parameters are specific to the microscope and detector used and should be adjusted according to the equipment available. Here, imaging was performed on a Titan Krios G3i FEG-TEM operated at 300 kV. Movies were collected on a Gatan K3 direct electron detector with a BioQuantum energy filter set to 20 eV. Dose-fractionated micrographs were collected in correlated-double sampling (CDS), counting mode spanning a relative defocus range of -0.5 to -2.5 µm with increments of 0.25 µm. A nominal magnification of 105,000× with a pixel size of 0.834 Å was used to acquire micrographs with a total dose of 45-55 e-/Å2 (1 e-/Å2/frame) (Figure 5). On average, ~250 movies were collected per hour using EPU/AFIS, acquiring three shots per hole and multiple holes per stage movement.
Figure 5. Representative cryo-EM micrographs of C. crescentus flagellar filaments. A. Motion-corrected micrograph of C. crescentus FljM flagellar filaments (straight). B. Motion-corrected micrograph of C. crescentus FljK flagellar filaments (curved). Scale bar, 100 nm.
Cryo-EM data processing of straight helical polymers (symmetrized refinement)
CryoSPARC was used to process C. crescentus flagellar filaments with an average diameter of 130 Å. C. crescentus flagellins range from 25 to 29 kDa and lack both D2 and D3 domains (Figure 1B). Only custom parameters are outlined below. If not specified, then default parameters were used. It is important to note that all datasets are unique, and one set of parameters may not be suitable for all needs; however, the steps below will greatly improve the user’s ability to differentiate between and analyze both straight and curved filaments. Further optimization may be needed on a case-by-case basis. The raw micrographs for both the straight FljM filaments and the curved FljK filaments are available on the EMPIAR database (entries EMPIAR-12122 and EMPIAR-12076, respectively) [22].
As with any software program, it is best to complete the designed tutorial before processing unique datasets. We encourage new users to first complete cryoSPARC’s single-particle tutorial (https://guide.cryosparc.com/processing-data/get-started-with-cryosparc-introductory-tutorial) to become acquainted with the software [20].
The preprocessing, particle picking, 2D classification, initial 3D refinement (Helical Refinement), and 3D variability analysis (steps F1–15 and G1–15) are the same for both the symmetrized (straight filaments) and asymmetrical (curved filaments) reconstruction workflows (Figure 6 and Figure 8). After completing these initial steps, it will be evident as to which path to continue. We have added a note after step F15 to help the user decide which workflow to use. For clarity, we have included all steps for both workflows, although the first 15 steps are the same, and we have provided a workflow diagram listing the steps performed for each reconstruction method (Figure 6 and Figure 8). Additionally, we provided movies with all the parameters for both reconstruction workflows to supplement the steps below (Video 1 and Video 2).
Figure 6. Workflow diagram of the helical (symmetrized) reconstruction method. The cryoSPARC jobs are provided here as a quick reference for processing data. These steps correspond to the steps in section F titled Cryo-EM data processing of straight helical polymers (symmetrized refinement) and to those in Video 1. Critical results are provided in Figure 7.
Video 1. Cryo-EM data processing of straight helical polymers following symmetrized refinement procedures in cryoSPARC
Figure 7. Cryo-EM data processing of straight helical polymers using symmetrized refinement yields high-resolution structures. A. Results of 2D classification after training the filament tracer neural net (step F11). Green boxes indicate classes that were selected for additional processing (step F12) (scale bar, 180 Å). B. 3D volume from the first helical refinement using a featureless cylinder as an initial reference (step F13). The blue arrow indicates α-helices that are present in the flagellin D1 domain. C. A Gaussian distribution of the particle tilt angles indicates proper symmetry was imposed in the 3D reconstruction, further verified by the high-resolution details present in the 3D volume (step F13). D. 3DVA helps to sort particle heterogeneity in the initial particle set. Volume 4 contains 51% of the particles and contains high-resolution features (blue arrow). Volume 1 also displays this quality but contains far fewer particles. The green box indicates Volume 4 was selected for further processing. E. Local resolution map of the final 3D volume along the helical axis F. Top view of the local resolution map of the final 3D volume. The key indicates a map range from 1.75 to 2.75 Å (step F25). G. The tilt angle plot from the final reconstruction displays a Gaussian distribution (step F24). H. The GS-FSC curve of the final map to a global resolution of 2.1 Å. The blue curve indicates the GS-FSC without masking, the green curve indicates the GS-FSC when a soft solvent mask extending 40 Å from the edge of the map is applied, the red curve indicates the GS-FSC when a tight mask extending only 12 Å from the edge of the map is applied, and the purple curve indicates the GS-FSC of the structure with a tight mask and correction by noise substitution. Scale bars for the 3D volumes are 25 Å.
Import movies:
We will start by assigning the proper path for movies data and gain reference. The movies data is the directory where raw micrographs are stored. Ensure the gain file is applied correctly by providing the proper flips and rotations. The gain parameters below are specific to our data collection. We first flip the gain file along the x-axis (left to right) and then rotate the file counterclockwise twice at 90°. You may need to seek advice from cryo-EM facility personnel where the data was collected for proper gain handling. To ensure micrograph dimensions are correct set Skip Header Check to false. Parallelize the job over multiple CPUs to speed up the processing.
Flip gain ref & defect file in X: True
Rotate gain ref: 2
Raw pixel size (Å): 0.834
Accelerating voltage: 300
Spherical aberration (mm): 2.7
Total exposure dose (e-/Å2): 45
Skip header check: False
CPUs to parallelize: 10
We used 10 CPUs for a total run time of 55 min for 9,780 movies.
Patch motion correction:
Set the input to Imported movies from the Import Movies job (step F1). Run the default parameters and parallelize over multiple GPUs. We used four GPUs for a run time of 9 h and 17 min for 9,780 micrographs.
Patch CTF:
Set the input to Micrographs from the Patch Motion Correction job (step F2). Run the default parameters and parallelize over multiple GPUs. We used four GPUs for a total run time of 5 h for 9,780 micrographs.
Curate exposures:
Depending on the number of micrographs collected and the quality of the data, curating the dataset based on motion correction and CTF statistics may improve downstream results and decrease processing times. For this job, set the input to Micrographs processed from the Patch CTF job (step F3). Do not set auto thresholds. Instead, eliminate any outliers during the interactive session based on average intensity, average defocus, astigmatism, CTF fit resolution, defocus range, defocus tilt angle, relative ice thickness, total full-frame motion distance, and full-frame motion curvature. Here, we rejected 2,976 exposures and accepted 6,804 exposures for further processing.
Filament tracer (template-free tracing):
To improve automated picking, first generate a set of templates (2D classes) by running template-free tracing on a subset of your data (50–100 micrographs). We will then use these templates to retrain the Filament Tracer and apply the neural network to our entire dataset. For this job, set the input to Exposures accepted from the Curate Exposures job (step F4).
Filament diameter: 130 Å
Separation distance between segments (diameters): 0.5
Minimum filament length (diameters): 3
Number of mics to process: 50
Number of mics to plot: 20
Minimum filament diameter: 120
Maximum filament diameter: 140
This job took 4 min to run. For initial estimates of filament diameter, open motion-corrected micrographs in IMOD or other visualization software to manually measure the filament diameter [23].
Extract from micrographs:
Here, we will set the box size for extracting particles. The box size should be set to at least 1.5× the filament diameter. Too small of a box size will result in no obvious curvature in the reconstruction of curved filaments. We use a box size spanning ~430 Å to observe the presence or lack of curvature in our reconstructions. Users may need to optimize this parameter during processing. Set the inputs to All particles and Micrographs from the Filament Tracer job (step F5). Parallelize over multiple GPUs to increase processing speeds.
GPUs to parallelize: 2
Extraction box size: 512
This job took 1 min to extract 7,112 particles from 50 micrographs.
2D classification:
Now, we will classify the particles to help remove suboptimal particles (non-flagella) before retraining the picking neural net. Set the input to Particles extracted from the Extract From Micrographs job (step F6).
Number of 2D classes: 50
Maximum resolution (Å): 6
Initial classification uncertainty factor: 10
Align filament classes vertically: True
GPUs to parallelize: 2
Parallelizing over two GPUs resulted in a run time of 10 min for 7,112 particles.
Select 2D classes:
Set the inputs to All particles and 2D class averages from the 2D Classification job (step F7). The interactive interface will allow the user to manually select the best-looking classes. Of the 7,112 particles, 4,817 particles (68%) from 19 classes were selected and 2,295 particles (32%) from 31 classes were excluded.
Filament tracer (with templates and a larger inter-box distance):
We will use the best 2D classes from step F8 for template-based filament tracing. We will also increase the inter-box distance, i.e., the separation distance between particles, to avoid oversampling in our reconstruction. We aim for a separation of ~200 Å; however, in practice this value could be as small as one helical repeat or the unique asymmetrical unit in the helical polymer. Set the inputs to Templates selected from the Select 2D Classes job (step F8) and Exposures accepted from the Curate Exposures job (step F4).
Filament diameter (Å): 130
Separation distance between segments (diameters): 1.6
Minimum filament length to consider (diameters): 3
Standard deviation of Gaussian blur (diameters): 0.2
This job resulted in 290,598 particles from 6,804 micrographs and took 10 h and 36 min to complete. It may be helpful to optimize these parameters on a small subset of micrographs prior to picking on the whole data set. To do so, provide an input to setting number of micrographs to process and number of mics to plot. For new datasets, we set both values to 50 to test picking parameters on only 50 micrographs and to view the picking quality in the log. Rerun the job with the optimized parameters on all the micrographs by removing the setting number of micrographs to process input value.
Extract from micrographs:
Binning particles during early steps improves particle alignment for 2D classification and 3D reconstruction jobs because this eliminates noise found at high-resolution frequencies that may lead to inaccuracies in particle alignments. Additionally, binning particles increase processing speeds during early iterations when particle sets are large due to the presence of suboptimal particles. Set the inputs to All particles and Micrographs from the latest Filament Tracer job (step F9).
GPUs to parallelize: 2
Extraction box size (pix): 512
Fourier crop to box size (pix): 256
With the larger inter-box distance, we extracted 231,432 particles from 6,798 micrographs. This job took 1 h and 12 min to complete.
2D classification:
This step allows for the removal of suboptimal particles in the data set. Additionally, because automated picking is not perfect, many times the hook structure or other high-contrast features may also be picked and need to be removed from the particle set. Set the input to All particles from the latest Extract From Micrographs job (step F10).
Number of 2D classes: 50
Maximum resolution (Å): 6
Initial classification uncertainty factor: 10
Number of online-EM iterations: 40
GPUs to parallelize: 2
This job took 25 min to complete (Figure 7A).
Select 2D classes:
Set the inputs to All particles and 2D class averages from the previous 2D Classification job (step F11). From the interactive screen, select the classes with expected flagellar filament features. We selected 19 classes with 180,872 particles (78%) for further processing and excluded 31 classes with 50,560 particles (22%) (Figure 7A).
Helical refinement (cylindrical initial model):
For helical refinement, an initial estimate of helical symmetry parameters is necessary for the reconstruction of the correct volume. Initial estimates of helical symmetry parameters can be calculated from the averaged power spectrum of particles from one 2D class in a process called Fourier Bessel indexing. CryoSPARC has implemented an Average Power Spectra job that will compute the power spectrum for a selected 2D class, which can then be imported into programs such as HELIXPLORER, PyHI, and HI3D for indexing [15,18]. Alternatively, symmetry parameters from a homologous structure can be used as an initial estimate and refined during reconstruction. We have chosen the latter option for reconstructing flagellar filaments in this workflow. Set the input to Particles selected from the Select 2D Classes job (step 12).
Helical twist estimate (°): 65.9
Helical rise estimate (Å): 4.8
Maximum out-of-plane tile angle (°): 45
Limit shifts along helical axis: True
Override outer filament diameter for search (Å): 140
Override inner filament diameter for search (Å): 10
Resolution to begin real-space symmetrization: 6
Resolution to begin local searches of helical symmetry: 4
Minimum helical twist to search over (°): 62.9
Maximum helical twist to search over (°):68.9
Twist grid size (number of grid points): 128
Minimum helical rise to search over (Å): 4.32
Maximum helical rise to search over (Å): 5.28
Rise grid size (number of grid points): 128
Initial lowpass resolution (Å): 20
Generate a cylindrical initial model: True
Filament outer diameter (Å): 140
Filament inner diameter (Å): 10
This job took 1 h and 7 min to complete. The gold standard Fourier shell correlation (GS-FSC0.143) reached the Nyquist limit of 3.4 Å for binned particles with a pixel size of 1.668 Å. Typically, for straight filaments, we observe secondary structures (α-helices) at this point in the reconstruction process (Figure 7B). Additionally, if the correct symmetry was imposed, we would expect to see a Gaussian distribution for the particle tilt angles (Figure 7C).
3D variability:
We have found that results from 3D classification are suboptimal in cryoSPARC and instead prefer to run a 3D Variability job to better sort heterogeneity within the particle set. Set the inputs to All particles and Mask from the Helical Refinement job (step F13). Ensure particles with 3D alignments are used for 3DVA, i.e., particles from any 3D refinement job. Additionally, memory issues are a concern when running 3DVA with large box sizes (> 500 pixels); thus, we will keep the particles binned during these steps.
Number of modes to solve (eigenvectors): 3
Filter resolution (Å): 3.5
This job took 1 h and 20 min to complete.
3D variability display:
Next, we will display 3DVA results based on component coordinates and group similar particles into clusters. The user can define the number of clusters to generate; here, we have chosen five clusters. Set the inputs to All particles and 3D Variability volumes from the 3D Variability job (step F14).
Output mode: cluster
Number of frames/clusters: 5
This job took 2 min to complete. Use ChimeraX to analyze the results by first downloading the “Volume series” output file. Ideally, you will observe one class that has many particles and a structure with well-resolved secondary structures. Here, cluster 4 contained 77,284 particles and we could see α-helices with side chain densities (Figure 7D). The map from cluster 1 also contained a well-resolved volume but had only 16,339 particles. We chose cluster 4 particles for further processing; alternatively, we could combine both cluster 1 and cluster 4 particles for further processing.
Note: This is where the processing workflows split between straight and curved filaments. As mentioned in step F13, if secondary structures are well resolved and the tilt angle plot displays a Gaussian distribution, then symmetrized (helical) reconstruction is best for your data set. However, if you are confident in the symmetry parameters, but the tilt angle plot displays a bimodal distribution, then there may be too much curvature in the particle set for adequate reconstruction via symmetrized (helical) refinement methods. The initial 3D volume (step F13) with helical symmetry imposed most likely has ill-defined secondary structures and few to no obvious side chain densities (i.e., “tubes” of density), and the GS-FSC may even reach the Nyquist limit for binned particles. Furthermore, 3D volumes from the 3DVA (steps F14 and 15) may appear curved along the helical axis as opposed to the symmetrized model from step F13. If these results are observed, then it is best to move on to the asymmetrical reconstruction workflow below (part G).
Helical refinement:
We will rerun a refinement job after discarding suboptimal particles. Since we observed a Gaussian distribution for the tilt angles and a well-resolved map, we will continue to use the Helical Refinement jobs in this workflow (symmetrized reconstruction). We will use the refined helical parameters from the Helical Refinement job (step F13) as our initial estimates for helical symmetry. These values can be found at the end of the event log from step F13. Set the inputs to the volume and particles of your chosen cluster, in this case Volume 4 and Particles 4 from the 3D Variability Display job (step F15).
Helical twist estimate (°): 65.9
Helical rise estimate (Å): 4.732
Maximum out-of-plane tile angle (°): 10
Limit shifts along helical axis: True
Resolution to begin real-space symmetrization: 6
Resolution to begin local searches of helical symmetry: 4
Minimum helical twist to search over (°): 62.9
Maximum helical twist to search over (°):68.9
Twist grid size (number of grid points): 512
Minimum helical rise to search over (Å): 4.259
Maximum helical rise to search over (Å): 5.205
Rise grid size (number of grid points): 512
Initial lowpass resolution (Å): 10
Mask (dynamic, static): static
This job took 23 min to complete. We observed a Gaussian distribution for tilt angles and the GS-FSC0.143 reached the Nyquist limit (3.4 Å) for this binned particle set. Symmetry parameters were refined resulting in a twist of 65.923° and a rise of 4.734 Å.
Extract from micrographs:
We curated our dataset to a point where resampling the particles to their original pixel size would lend to an increase in resolution and improve side-chain densities. Here, we will extract particles to the original pixel size without setting a value for the Fourier crop to box size (pix) parameter. Set the inputs to All particles from the previous Helical Refinement job (step F16) and to Micrographs from the Extract From Micrographs job (step F10).
Extraction box size (pix): 512
Fourier crop to box size (pix): Not set
Recenter using aligned shifts: True
Using two GPUs, extracting 77,283 particles took 1 h and 42 min to complete.
Volume tools (map resampling):
In addition to resampling particles to the original pixel size, we must do the same for the map volume and mask generated in step F16. Set the input to Refined volume from the Helical Refinement job (step F16) and resample to the same box size as the final particles set (step F17).
Resample to box size (pix): 512
Type of input volume: map
Type of output volume: map
This job takes only seconds to run.
Volume tools (mask resampling):
Repeat the previous job for the mask volume. Set the input to Mask from the Helical Refinement job (step 16).
Resample to box size (pix): 512
Type of input volume: mask
Type of output volume: mask
This job takes only seconds to run. At this point, we have resampled the particles, map, and mask to the original pixel size of 0.834 Å for additional processing.
Local motion correction:
Perform per particle motion correction to improve particle quality. Set the inputs to Micrographs and Particles extracted from the Extract From Micrographs job (step F17).
Extraction box size (pix): 512
Using 2 GPUs this job took 2 h to complete.
Helical Refinement:
Set the inputs to Particles extracted from the Local Motion Correction job (step F20), Volume from the Volumes Tools (map resampling) job (step F18), and Mask from the Volumes Tools (mask resampling) job (step F19). We will use the refined helical parameters from the latest Helical Refinement job (step F16) as initial estimates of helical twist and rise. These values are stored at the end of the event log from step F16.
Helical twist estimate (°): 65.923
Helical rise estimate (Å): 4.734
Maximum out-of-plane tile angle (°): 10
Limit shifts along helical axis: True
Resolution to begin real-space symmetrization: 6
Resolution to begin local searches of helical symmetry: 4
Minimum helical twist to search over (°): 62.923
Maximum helical twist to search over (°):68.923
Twist grid size (number of grid points): 512
Minimum helical rise to search over (Å): 4.261
Maximum helical rise to search over (Å): 5.207
Rise grid size (number of grid points): 512
Initial lowpass resolution (Å): 10
Mask (dynamic, static): static
This job took 2 h and 40 min to complete and resulted in a map with improved resolution (GS-FSC0.143 2.23Å) and map quality.
Global CTF refinement:
To further improve the map, we will refine CTF parameters that were initially estimated at the start of our processing. Set the inputs to All particles, Refined volume, and Mask from the latest Helical Refinement job (step F21).
Number of iterations: 5
Fit Tilt: True
Fit Trefoil: True
Fit Spherical Aberration: True
Fit Tetrafoil: True
Fit Anisotropic Magnification: True
This job took 13 min to complete.
Local CTF refinement:
Next, we will refine per particle defocus values. Set the inputs to All particles from the Global CTF Refinement job (step F22) and Refined volume and Mask from the latest Helical Refinement job (step F21). We will use default parameters for this job. This job took 5 min to complete.
Helical refinement:
Now that we have curated the particle set and refined both motion correction and CTF parameters, we are ready to perform the final refinement job. Set the inputs to All particles from the Local CTF Refinement job (step F23), and Refined volume and Mask from the latest Helical Refinement job (step 21). We will use the refined helical parameters from step F21 as initial estimates for helical symmetry parameters.
Helical twist estimate (°): 65.923
Helical rise estimate (Å): 4.734
Maximum out-of-plane tile angle (°): 10
Limit shifts along helical axis: True
Resolution to begin real-space symmetrization: 6
Resolution to begin local searches of helical symmetry: 4
Minimum helical twist to search over (°): 62.923
Maximum helical twist to search over (°):68.923
Twist grid size (number of grid points): 512
Minimum helical rise to search over (Å): 4.261
Maximum helical rise to search over (Å): 5.207
Rise grid size (number of grid points): 512
Initial lowpass resolution (Å): 10
Mask (dynamic, static): static
The final refinement job took ~2 h and 50 min to complete and further increased the maps’ global resolution from 2.23 to 2.10 Å; the tilt angle plot displayed a Gaussian distribution (Figure 7G, 7H). Our final helical parameters were a twist of 65.923° and a rise of 4.732 Å. Multiple volumes are available for download under the Refined volume output. These include both half maps, a refined map, a sharpen map, a symmetrized map, and a sharpen symmetrized map. We find that building a model into the sharpen symmetrized map works best. However, the user may choose to run additional Sharpening Tools jobs and manually set a B-factor for sharpening either the map or the symmetrized map.
Local resolution estimation:
As a final post-processing step, we generate a local resolution volume to better understand the map quality using cryoSPARC’s BlocRes program. Set the inputs to Refined volume and Mask from the final Helical Refinement job (step F24).
FSC threshold: 0.143
Use GPU or CPU for computation: GPU
Enable FSC weighting: True
Alternatively, the user can set Enable MonoRes to True to perform local resolution estimation with MonoRes instead of BlocRes. This job took 27 min to complete. The output file “map_locres”, along with the final map from step F24, can be opened in ChimeraX to generate a local resolution map (Figure 7E, 7F). In ChimeraX, go to Volume > Surface Color, set Color surface to the final map, set by to volume data value, and set using map to the local resolution map. Select Options to set the number of colors and the coloring palette desired. Select Key to add and edit parameters for the local resolution map key. For a detailed tutorial on coloring surface maps, see ChimeraX’s YouTube page (https://youtu.be/2EDZQs7lGNs?si=47v77qnzxLABJnBf).
Cryo-EM data processing of curved helical polymers (asymmetrical refinement)
Since some steps are similar for both workflows, we have noted steps where more details can be found in the previous section (symmetrized refinement).
Figure 8. Workflow diagram of the asymmetrical reconstruction method. The cryoSPARC jobs are provided here as a quick reference when processing data. These steps correspond to the steps in section G titled Cryo-EM data processing of curved helical polymers (asymmetrical refinement) and to those in Video 2. Key results are detailed in Figure 9.
Video 2. Cryo-EM data processing of curved helical polymers following asymmetrical refinement procedures in cryoSPARC
Import movies:
Assign the proper paths to movies data and gain reference. See step F1 on handling gain files.
Flip gain ref & defect file in X: True
Rotate gain ref: 2
Raw pixel size (Å): 0.834
Accelerating voltage: 300
Spherical aberration (mm): 2.7
Total exposure dose (e-/Å2): 55
Skip header check: False
CPUs to parallelize: 10
We used 10 CPUs for a total run time of 1 h for 8,813 movies.
Patch motion correction:
Set the input to Movies from the Import Movies job (step G1). Running the default parameters on four GPUs resulted in a total run time of 10 h and 8 min for 8,813 micrographs.
Patch CTF:
Set the input to Micrographs from the Patch Motion Correction job (step G2). Running the default parameters on four GPUs resulted in a total run time of 4 h and 54 min for 8,813 micrographs.
Curate exposures:
Set the input to Micrographs processed from the Patch CTF job (step G3). Here, we manually rejected 2,835 exposures and accepted 5,978 exposures for further processing.
Filament tracer (template-free tracing):
Set the input to Exposures accepted from the Curate Exposures job (step G4). For more details, see step F5.
Filament diameter: 130 Å
Separation distance between segments: 0.5
Minimum filament length: 3
Number of mics to process: 50
Number of mics to plot: 20
Minimum filament diameter: 120
Maximum filament diameter: 140
This job took 3 min to complete.
Extract from micrographs:
Set the inputs to Micrographs and All particles from the Filament Tracer job (step G5).
GPUs to parallelize: 4
Extraction box size: 512
This job took 1 min to extract 8,966 particles from 50 micrographs. See step F6 on box size selection.
2D classification:
Set the input to Particles extracted from the Extract From Micrographs job (step G6).
Number of 2D classes: 50
Maximum resolution (Å): 6
Initial classification uncertainty factor: 10
Align filament classes vertically: True
GPUs to parallelize: 4
Parallelizing over four GPUs resulted in a run time of 10 min for 8,966 particles.
Select 2D classes:
Set the inputs to All particles and 2D class averages from the 2D Classification job (step G7). Manually select the best-looking classes. Here, we selected 6,795 particles from 28 classes.
Filament tracer (with templates and a larger inter-box distance):
Set the inputs to Templates selected from the Select 2D Classes job (step G8) and Exposures accepted from the Curate Exposures job (step G4).
Filament diameter (Å): 130
Separation distance between segments (diameters): 1.6
Minimum filament length to consider (diameters): 3
Standard deviation of Gaussian blur (diameters): 0.2
This job took 12 h and 21 min to complete and resulted in 372,299 particles from 5,978 micrographs. See step F9 for details on optimizing the filament tracer neural network.
Extract from micrographs:
Set the inputs to All particles and Micrographs from the Filament Tracer job (step G9). See step F10 for details on binning data. Here, we will resample the data from a pixel size of 0.834 Å to a 2× binned pixel size of 1.66 Å.
GPUs to parallelize: 2
Extraction box size (pix): 512
Fourier crop to box size (pix): 256
This job took 1 h and 12 min to complete, resulting in 334,330 particles from 5,977 micrographs.
2D classification:
Set the inputs to All particles from the previous Extract From Micrographs job (step G10).
Number of 2D classes: 50
Maximum resolution (Å): 6
Initial classification uncertainty factor: 10
Align filament classes vertically: True
Remove duplicate particles: True
Minimum separation distance (for duplicate particles) (Å): 50
Number of online-EM iterations: 40
GPUs to parallelize: 2
This job took 28 min to complete (Figure 9A). We set remove duplicate particles to true as curved filaments will use Homogeneous Refinement jobs in later steps. If the sample is of a straight filament (symmetrized refinement), remove duplicate particles can be set to false, since cryoSPARC accounts for neighboring particles during the gold-standard split in Helical Refinement jobs.
Select 2D classes:
Set the inputs to All particles and 2D class averages from the 2D Classification job (step G11). We manually selected 31 classes with 310,135 particles (93%) and excluded 19 classes with 24,195 particles (7%) (Figure 9A).
Helical refinement (cylindrical initial model):
Set the input to Particles selected from the Select 2D Classes job (step G12). See step F13 for details on determining helical parameters.
Helical twist estimate (°): 65.5
Helical rise estimate (Å): 4.8
Maximum out-of-plane tile angle (°): 45
Limit shifts along helical axis: True
Override outer filament diameter for search (Å): 140
Override inner filament diameter for search (Å): 10
Resolution to begin real-space symmetrization: 6
Resolution to begin local searches of helical symmetry: 4
Minimum helical twist to search over (°): 62.5
Maximum helical twist to search over (°):68.5
Twist grid size (number of grid points): 128
Minimum helical rise to search over (Å): 4.32
Maximum helical rise to search over (Å): 5.28
Rise grid size (number of grid points): 128
Initial lowpass resolution (Å): 20
Generate a cylindrical initial model: True
Filament outer diameter (Å): 140
Filament inner diameter (Å): 10
This job took 1 h and 32 min to complete and resulted in a map resolution (3.4 Å) that reached the Nyquist limit for the 2× binned data. For curved helical polymers, secondary structures are not as clear as straight filaments at this stage (Figure 9B). Additionally, we observe a bimodal tilt distribution in the Helical Refinement job (Figure 9C). These two data points suggest that an asymmetrical reconstruction may be necessary. We will run 3DVA to tease out whether curvature is inhibiting high-resolution reconstruction.
Note: In some cases, it may be useful to repeat this step to improve map quality. Instead of using a featureless cylinder, use the resulting refined volume as in the initial model for a second round of helical refinement.
3D variability:
Set the inputs to All particles and Mask from the Helical Refinement job (step G13). See step F14 for details on 3DVA.
Number of modes to solve (eigenvectors): 3
Filter resolution (Å): 3.5
This job took 2 h and 29 min to complete.
3D variability display:
Next, set the inputs to All particles and 3D Variability volumes from the 3D Variability job (step G14).
Output mode: cluster
Number of frames/clusters: 5
This job took 3 min to complete. Open the Volume series in ChimeraX to analyze the results of the 3DVA. Identify the best class, specifically looking at the quality of secondary structures. We determined Volume 0 to be the best map with a slight curve to the filament along the helical axis.
Homogeneous refinement:
Next, we will rerun the 3D refinement, 3D variability, and 3D variability display jobs with the curved filament reconstruction from the 3D Variability Display job (Volume 0, step G15) as the initial reference map. Set the inputs to Particles 0, Particles 1, Particles 2, Particles 3, Particles 4, and the best map (Volume 0) from the 3D Variability Display job (step G15).
Initial lowpass resolution (Å): 10
This job took 15 min to complete. We observe much higher map quality in this map as compared to the Helical Refinement job (step G13), although both resolve to a GS-FSC0.143 of 3.4 Å. Several subunits from the Homogeneous Refinement map display D1 domains with α-helices as opposed to a “tube” of volume observed in the Helical Refinement map (step G13) (Figure 9D). Bulky side chains and glycosylation sites are also better resolved.
3D variability:
Although the quality of the map, with 2× binned particles, has greatly improved, it is important to further curate our particle set to further improve our results. Set the inputs to All particles and Mask from the Homogeneous Refinement job (step G16).
Number of modes to solve (eigenvectors): 3
Filter resolution (Å): 3.5
This job took 2 h and 25 min to complete.
3D variability display:
Next, set the inputs to All particles and 3D Variability volumes from the 3D Variability job (step G17).
Output mode: cluster
Number of frames/clusters: 5
This job took 3 min to complete. The highest quality map was Volume 0 with 45,319 particles. Volume 1 through Volume 4 lacked obvious side chain densities, had discontinuities to the carbon backbone density, or lacked clear α-helices (Figure 9E). In our experience, some datasets contain multiple “good” volumes; the user may decide to combine particles from the “good” classes for further processing or pick the single class with the highest particle count for further processing. We typically proceed with the latter option.
Homogeneous refinement:
Set the inputs to the best particle set(s) and volume(s) from the 3DVA. We set the inputs to Particles 0 and Volume 0 from the 3D Variability Display job (step G18).
Initial lowpass resolution (Å): 10
This job took 4 min to complete. We observe a slight improvement from the Volume 0 map due to the automated sharpening as part of the Homogeneous Refinement job.
Extract from micrographs:
Now that we have curated our data to a final particle set, we will resample our pixel size to the original size (0.834 Å) for further processing. Set the inputs to All particles from the previous Homogeneous Refinement job (step G19) and to Micrographs from the Extract From Micrographs job (step G10).
Extraction box size (pix): 512
Fourier crop to box size (pix): Not set
Recenter using aligned shifts: True
Using 1 GPU, extracting 44,978 particles took 3 h and 4 min to complete.
Volume tools (map resampling):
Next, we will resample the pixel size of the latest map to match the particles. Set the input as the Refined volume from the Homogeneous Refinement job (step G19) and resample the map to the same size as the particles in the previous step.
Resample to box size (pix): 512
Type of input volume: map
Type of output volume: map
This job takes only seconds to run.
Volume tools (mask resampling):
Now resample the Mask from the Homogeneous Refinement job (step G19) to the original pixel size.
Resample to box size (pix): 512
Type of input volume: mask
Type of output volume: mask
This job takes only seconds to run. Now, all the inputs for subsequent processing will have a pixel size of 0.834 Å and a box size of 512 pixels.
Local motion correction:
Now, we will perform per-particle motion correction to improve the quality of the particles. Set the inputs as the Micrographs and Particles extracted from the Extract From Micrographs job (step G20).
Extraction box size (pix): 512
Using two GPUs, this job took 4 h and 9 min to complete.
Homogeneous refinement (with global CTF refinement):
Unlike helical refinement jobs, we can run global CTF refinements directly in the Homogenous Refinement job rather than running a stand-alone job. Set the inputs to Particles extracted from the Local Motion Correction job (step G23), Volume from the Volume Tools job (step G21), and Mask from the Volume Tools job (step G22).
Initial lowpass resolution (Å): 10
Minimize over per-particle scale: True
Optimize per-group CTF params: True
Fit Tilt: True
Fit Trefoil: True
Fit Spherical Aberration: True
Fit Tetrafoil: True
Fit Anisotropic Mag: True
This job took 31 min to complete, resulting in a map with a GS-FSC0.143 of 3.05 Å.
Note: If the resolution does not improve, run a stand-alone global CTF refinement job as in step F22 followed by a homogenous refinement job.
Homogeneous refinement (with local CTF refinement):
Again, unlike helical refinement jobs, we can run local CTF refinement (defocus refinement) along with the Homogenous Refinement job. Set the inputs to All Particles, Refined volume, and Mask from the latest Homogenous Refinement job (step G24).
Initial lowpass resolution (Å): 10
Optimize per-particle defocus: True
This job took 34 min to complete, and we observed a slight increase in the map’s global resolution to a GS-FSC0.143 of 2.82 Å.
Note: If the resolution does not improve, run a stand-alone local CTF refinement as in step F23 followed by a homogenous refinement job.
Local refinement:
As a final step, we perform a Local Refinement job to push our map quality a bit further. Set the inputs to All Particles, Refined volume, and Mask from the previous Homogenous Refinement job (step G25). Alternatively, the user may use ChimeraX to define a custom mask for this step. A step-by-step guide can be found on cryoSPARC’s website (https://guide.cryosparc.com/processing-data/tutorials-and-case-studies/mask-selection-and-generation-in-ucsf-chimera). Running the default parameters took 54 min and resulted in a slight increase in map quality and a GS-FSC0.143 of 2.72 Å (Figure 9H).
Symmetry search utility:
Although we are not applying helical symmetry to the final reconstruction, understanding the estimated helical arrangement of the subunits is useful information for comparison with the straight, symmetrized structure. This job takes a volume and provides the best local minima for helical parameters over a range of values defined by the user. The ranges for twist and rise will be unique for the helical polymer; see step F13 for details on estimating helical symmetry parameters. Set the inputs to the Refined volume and Mask from the Local Refinement job (step G26).
Search over pitch/number of subunits, or rise/twist: rise
Search min and max over helical rise (Å): 4,5.5
Search min and max over helical twist (°): 55,75
Override outer filament diameter for search (Å): 200
Override inner filament diameter for search (Å): 10
Which map to search: map_sharp
This job took 2 min to complete and resulted in a table, found in the event log, that details the 20 best local minima of helical symmetry parameters. The values are arranged by mean squared error (MSE), a measurement of how close the experimental data is to the predicted model. A lower MSE suggests there is higher confidence in the estimated symmetry parameters. A twist of 65.51° and a rise of 4.812 Å best detail the arrangement of subunits in this flagellar filament, having the lowest MSE value of the local minima examined.
Local resolution estimation:
Finally, we will generate a local resolution map to detail the quality of our final reconstruction. Set the inputs to the Refined volume and Mask from the Local Refinement job (step G26).
FSC threshold: 0.143
Use GPU or CPU for computation: GPU
Enable FSC weighting: True
This job took 54 min to complete (Figure 9F, 9G). See step F25 for further details on using ChimeraX to apply the local resolution plot to the final map.
Figure 9. Cryo-EM data processing of curved helical polymers by asymmetrical reconstruction methods improves map quality. A. 2D classes from particles that were picked after training the filament tracer neural net (step G11). Green boxes highlight classes that were selected for further processing (step G12) (scale bar, 180 Å). B. Initial 3D volume with imposed helical symmetry results in the blurring of secondary structures. The red arrow indicates an expected α-helix that is blurred and more “tube-like” (step G13). C. Bimodal distribution of particle tilt angles suggests that the high curvature observed in the micrographs impedes high-resolution structure determination when imposing helical symmetry (step G13). D. Curved 3D volumes are observed after 3DVA. The best volume is used as a reference for one round of homogeneous refinement (no helical symmetry imposed) using all particles. This process results in a map with much higher quality and improved secondary structure features (blue arrow). Black line indicates the center of the 3D volume. E. A second round of 3DVA helps to sort heterogeneity within the particle set. Volume 0 has the best-resolved α-helices (blue arrow) and is selected (green box) for further processing. F. The selected particle set is used for additional rounds of homogeneous reconstruction, CTF refinement, and local motion correction, resulting in a final map with improved map quality displaying secondary structures and side-chain densities. The local resolution map shows that the highest resolved density is along the center of the helical axis. G. A top view of the filament shows lower resolution density at the end of the reconstruction. The key indicates a map range from 1.75 to 3.75 Å (step G26). H. The GS-FSC reports a global resolution of 2.72 Å. Scale bars for the 3D volumes are 25 Å.
Model building and validation
To accelerate model building, a predicted model or a previously solved structure can be used as an initial starting point. Alternatively, the user may manually build the protein chain using Coot. Here, we will outline how we use AlphaFold to generate a predicted model that is then manually and automatically refined into the experimental data using ChimeraX, Coot, and Phenix, in an iterative fashion (Figure 10) [24–27]. With the growing number of structure predictions already stored in the AlphaFold database, it is best to first search for your protein of interest in the AlphaFold database (https://alphafold.ebi.ac.uk). If your structure prediction is available, download the PDB file and move to step H3. If not, follow all the steps below.
Additionally, there are many workflows that combine various software packages for model refinement. For less-experienced users, DiNunno et al. [28] provided a detailed protocol centered on modeling and validation.
Figure 10. Model building and validation workflow using AlphaFold2, ChimeraX, Coot, and PHENIX
Obtain the protein sequence using Uniprot or another database of choice.
Go to the AlphaFold Colab notebook (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb), follow the steps for generating an initial prediction, and download the PDB file.
Open the final cryo-EM map and the AlphaFold PDB file in ChimeraX and fit the model into the map using Tools > Volume Data > Fit in Map. First, you will need to manually align the model to the map followed by rigid-body fitting the model into the map using the Fit in Map tool. Save the PDB file ensuring you select Save relative to model: and choose the correct map from the drop-down menu. Your model is now aligned relative to your cryo-EM map.
Open the aligned model and cryo-EM map in Coot. Click on the Map button (right-hand side) and then click Estimate to automatically estimate the weight for map fitting. Next, select Refine > Chain Refine to run an all-atom real space refinement on the model. Inspect the model to ensure the carbon backbone fits into the map density. The user can manually drag atoms into the corresponding density. Once satisfied, accept the refinement results.
Run a validation analysis to identify outliers that need to be fixed. Select Validate > Ramachandran Plot > ModelName.PDB, followed by Validate > Rotamer Analysis > ModelName.PDB, and Validate > Geometry analysis > ModelName.PDB. Fix outliers on these plots by using the Real Space Refine Zone tool, Auto Fit Rotamer tool, or other model refinement tools on the right-hand panel. Once outliers are fixed, save the file coordinates.
Open PHENIX to start a real-space refinement job. In the Input/Output tab, provide the proper map (MRC format) and model (PDB format) files. Also, provide a global resolution in the Resolution box. Under the Refinement settings tab, in addition to running the default parameters, select Use secondary structure restraints. Click on the Rotamers button and for Fit select outlier_or_poormap from the drop-down menu. For Nproc, define how many CPUs to run to speed up calculations; we typically set this to 4. Click Run at the top of the window. Once the job is complete, inspect the Validation tab to review the results.
Sometimes, it may be necessary to repeat step H4 and/or step H5 to improve atoms or residues that were flagged in the validation report. Repeat these steps as necessary until the validation statistics are satisfactory.
We can now use the refined model to fit additional protein chains into the cryo-EM map by repeating step H3. In the symmetrized maps, we confidently built 44-mer models; in the asymmetrical maps, we built approximately 36 chains per map. Once all the chains are placed into the cryo-EM density map, we must combine the separate chain models into one multimeric model containing all the protein chains. From the ChimeraX command line, run “combine #1-44” where the integers after the “#” dictate the model IDs in ChimeraX. This command will combine 44 separate chains into one model and rename the chains, ensuring each one has a unique chain ID. Programs such as PHENIX require all chains to be in one model for refinement.
Note: As an optional step, we can repeat steps H3, H4, and H5 for a chain from each protofilament to improve auto-refinement of the multimeric structure. This is useful for curved filaments where the D1 domain is in a different confirmation in each of the 11 protofilaments. After refining one chain per protofilament, ensure that you fit additional chains into the corresponding protofilament. We typically fit 3–4 chains per protofilament. Repeat for each protofilament and combine the many chains into one model as described above.
Run PHENIX’s real-space refinement as in step H5 ensuring the multimeric model is supplied in the Input/Output tab. Also, if the model was fit into a symmetrized map, you can select NCS constraints in the Refinement Settings tab to apply non-crystallographic symmetry during refinement. Review the validation results.
These large flagellar filament models encompass tens of thousands of atoms and, although automatic refinement greatly improves the model, manual intervention is usually necessary. Use Coot to touch up any outliers that may appear in the PHENIX validation log. Save the model coordinates once you are satisfied with the model.
In PHENIX, run the comprehensive validation (cryo-EM) tool. In the Input/Output tab, supply the model (PDB format), the final map (MRC format), and both half-maps (MRC format). Provide the map resolution and click Run. Once completed, review the model validation statistics.
If there are any outliers, repeat step H9 and/or H10 until results are satisfactory.
As an additional validation measure, submit the results to the PDB validation system (https://validate-rcsb-1.wwpdb.org).
Data analysis
Data processing for any cryo-EM project is dependent on the sample, data collection instrumentation, and computational hardware and software. The protocol and workflow presented here were used to generate cryo-EM maps and atomic models for C. crescentus FljM and FljK flagellar filaments. Atomic models have been deposited in the Protein Data Bank under accessions 8UXN and 8UXJ for FljM and FljK, respectively. Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accessions EMD-42770 and EMD-42766 for FljM and FljK, respectively.
Validation of protocol
This protocol or parts of it has been used and validated in the following research articles:
Montemayor et al. [11]. Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage φ CbK Predation of Multiflagellin Bacteria. Journal of Bacteriology. https://doi.org/10.1128/jb.00399-20 (Figure 5, panel A).
Sanchez et al. [21]. Atomic-level architecture of Caulobacter crescentus flagellar filaments provide evidence for multi-flagellin filament stabilization. bioRxiv. https://doi.org/10.1101/2023.07.10.548443 (Figure 1, panel A–I).
General notes and troubleshooting
Limitations
This protocol is for the isolation and purification of flagella ejected from bacterial cells, cryo-EM imaging of the purified flagella, and single particle helical reconstruction of flagella using cryoSPARC software. We have tested the protocol on other filamentous polymers, including bacterial pili. However, we have not exhaustively tested the protocol on a large range of helical polymers because this may require additional optimization of steps associated with sample purification and/or cryo-EM data processing.
Troubleshooting
The procedures and workflows described in the protocol are starting points for individuals new to cryo-EM data collection and data analysis of filamentous targets that are straight or exhibit curvature. Samples are themselves variable and the protocol may require changes for optimal production and purification. These could include bacterial strain, growth medium, growth time, pH and salt concentrations of buffers, and types and timing of centrifugation and resuspension steps; each of these may need assessment and optimization to produce samples suitable for cryo-EM data collection. In addition, the hardware and software solutions for cryo-EM data collection and data processing may change over time, thus improving throughput and overall outcomes.
Acknowledgments
This work was supported in part by the University of Wisconsin, Madison, the Department of Biochemistry at the University of Wisconsin, Madison, and public health service grants R01 GM104540 and U24 GM139168 to E.R.W. from the NIH. This work was supported in part by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Numbers DE-SC0018409. J.C.S. was supported in part by the Biotechnology Training Program at the University of Wisconsin, Madison, T32 GM135066, the Steenbock Predoctoral Graduate Fellowship administered by the University of Wisconsin-Madison Department of Biochemistry, and the SciMed Graduate Research Scholars Fellowship with support for this fellowship provided by the Graduate School, part of the Office of Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison, with funding from the Wisconsin Alumni Research Foundation and the UW-Madison. We are grateful for the use of facilities and instrumentation at the Cryo-EM Research Center in the Department of Biochemistry at the University of Wisconsin, Madison. We are grateful for the computational resources supplied through the SBGrid Consortium [29]. We are grateful to Eric J. Montemayor and Nicoleta T. Ploscariu for valuable discussions on flagella purification and helical reconstruction [11].
Data deposition. Atomic models have been deposited in the Protein Data Bank under accessions 8UXN and 8UXJ for FljM and FljK, respectively. Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accessions EMD-42770 and EMD-42766 for FljM and FljK, respectively. The raw micrographs for both the straight FljM filaments and the curved FljK filaments are available on the EMPIAR database (entries EMPIAR-12122 and EMPIAR-12076, respectively).
Competing interests
The authors declare no competing interests.
References
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Driks, A., Bryan, R., Shapiro, L. and DeRosier, D. J. (1989). The organization of the Caulobacter crescentus flagellar filament.J Mol Biol. 206(4): 627–636.
Luo, Y., Wang, J., Gu, Y. L., Zhang, L. Q. and Wei, H. L. (2023). Duplicated Flagellins in Pseudomonas Divergently Contribute to Motility and Plant Immune Elicitation. Microbiol Spectr. 11(1). Doi: 10.1128/spectrum.03621-22
Nuijten, P. J., van Asten, F. J., Gaastra, W. and van der Zeijst, B. A. (1990). Structural and functional analysis of two Campylobacter jejuni flagellin genes.J Biol Chem. 265(29): 17798–17804.
Lederberg, J. and Iino, T. (1956). Phase Variation in Salmonella. Genetics. 41(5): 743–757.
Yonekura, K., Maki-Yonekura, S. and Namba, K. (2003). Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy.Nature. 424(6949): 643–650.
Wang, F., Burrage, A. M., Postel, S., Clark, R. E., Orlova, A., Sundberg, E. J., Kearns, D. B. and Egelman, E. H. (2017). A structural model of flagellar filament switching across multiple bacterial species.Nat Commun. 8(1): 1–13.
Montemayor, E. J., Ploscariu, N. T., Sanchez, J. C., Parrell, D., Dillard, R. S., Shebelut, C. W., Ke, Z., Guerrero-Ferreira, R. C. and Wright, E. R. (2021). Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage varphi CbK Predation of Multiflagellin Bacteria.J Bacteriol. 203(5). Doi: 10.1128/jb.00399-20.
Ferreira, J. L., Gao, F. Z., Rossmann, F. M., Nans, A., Brenzinger, S., Hosseini, R., Wilson, A., Briegel, A., Thormann, K. M., Rosenthal, P. B., et al. (2019). γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures. Plos Biol. 17(3): e3000165.
Zhuang, X. Y. and Lo, C. J. (2020). Construction and Loss of Bacterial Flagellar Filaments. Biomolecules. 10(11).
Egelman, E. H. and Wang, F. (2021). Cryo-EM is a powerful tool, but helical applications can have pitfalls.Soft Matter. 17(12): 3291–3293.
Zhang, X. (2022). Python-based Helix Indexer: A graphical user interface program for finding symmetry of helical assembly through Fourier-Bessel indexing of electron microscopic data.Protein Sci. 31(1): 107–117.
Bepler, T., Morin, A., Rapp, M., Brasch, J., Shapiro, L., Noble, A. J. and Berger, B. (2019). Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat Methods. 16(11): 1153–1160..
Wagner, T., Merino, F., Stabrin, M., Moriya, T., Antoni, C., Apelbaum, A., Hagel, P., Sitsel, O., Raisch, T., Prumbaum, D., et al. (2019). SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM.Commun Biol. 2(1): 218.
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Article Information
Publication history
Received: May 6, 2024
Accepted: Jun 14, 2024
Available online: Jun 30, 2024
Published: Jul 20, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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5,033 | https://bio-protocol.org/en/bpdetail?id=5033&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis of Guard Cell Readouts Using Arabidopsis thaliana Isolated Epidermal Peels
RP Rosario Pantaleno
PS Paula Schiel
CG Carlos García-Mata
DS Denise Scuffi
Published: Vol 14, Iss 14, Jul 20, 2024
DOI: 10.21769/BioProtoc.5033 Views: 674
Reviewed by: Wenrong HeVenkatasalam Shanmugabalaji Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Physiology Feb 2018
Abstract
Stomata are pores surrounded by a pair of specialized cells, called guard cells, that play a central role in plant physiology through the regulation of gas exchange between plants and the environment. Guard cells have features like cell-autonomous responses and easily measurable readouts that have turned them into a model system to study signal transduction mechanisms in plants. Here, we provide a detailed protocol to analyze different physiological responses specifically in guard cells. We describe, in detail, the steps and conditions to isolate epidermal peels with tweezers and to analyze i) stomatal aperture in response to different stimuli, ii) cytosolic parameters such as hydrogen peroxide (H2O2), glutathione redox potential (EGSH), and MgATP-2 in vivo dynamics using fluorescent biosensors, and iii) gene expression in guard cell–enriched samples. The importance of this protocol lies in the fact that most living cells on epidermal peels are guard cells, enabling the preparation of guard cell–enriched samples.
Key features
• Isolation of epidermal peels as a monolayer enriched in guard cells.
• Measurement of cytosolic guard cell signaling component dynamics in isolated epidermal peels through fluorescent biosensor analysis.
• Gene expression analysis of guard cell–enriched isolated tissue.
Keywords: Guard cells Epidermal peels Stomatal aperture Biosensors RNA Arabidopsis thaliana
Graphical overview
Created with biorender.com
Background
Stomata are microscopic pores located in the epidermis of aerial tissues of most land plants, delimited by specialized cells known as guard cells. Given that the plant epidermis is covered by the cuticle, an external impermeable layer, almost 95% of gas exchange occurs through stomatal pores. Thus, the regulation of the stomatal pore area controls the uptake and release of CO2 and O2 and the maintenance of hydric homeostasis by the regulation of H2O vapor loss, through the transpiration stream [1].
Guard cells continuously sense internal and external cues and integrate them into a complex signaling network that produces modifications in guard cell volume to control the stomatal pore width [2,3]. The isolation of epidermal peels, cellular monolayers mainly formed by functional guard cells, has become a widely used and validated experimental system to study signaling mechanisms in plants [4].
Several protocols have been developed in order to study guard cell physiology [5–7]. Although all of them converge in the isolation of epidermal peels, there are different approaches including the use of adhesive tape [8–10], resin imprints where leaf surface is copied [11–13], blender and filters [14–16], tape and razor blade [17–19], and striping with fine-tip tweezers [20–22].
For the last twenty years, our research group has reproducibly employed fine-tip tweezers to prepare epidermal strips, where almost 90% of living epidermal cells are guard cells (Figure 1A and B) [20,23–27]. Isolation of epidermal peels allowed us to perform stomatal aperture assays and in vivo measurements of endogenously encoded fluorescent proteins (biosensors) that specifically detect hydrogen peroxide (H2O2,), the glutathione redox potential (EGSH), and the biologically relevant portion of adenosine 5'-triphosphate (MgATP2-) [25,27]. In addition, the methodological procedure presented here has been employed to obtain guard cells–enriched RNA (Figure 1B) to analyze transcript levels of different target genes [23,26]. Although peeling produces mechanical stress, evidenced by an increase in reactive oxygen species (ROS) in guard cells, we set up the conditions to recover the strips to basal redox status levels without affecting guard cells’ viability or their physiological responses to different signals [27]. In this protocol, we describe how to prepare isolated epidermal peels excised from the abaxial side of Arabidopsis thaliana leaves and how to perform different experiments using this biological system to study real-time and single-cell physiological processes.
Figure 1. Living cells in epidermal peels. A. Arabidopsis thaliana wild-type ecotype Col-0 epidermal peels were incubated in opening buffer (OB, 5 mM MES pH6.1, 50 mM KCl) under light for 30 min and then loaded with 5 μM of the viability fluorescent dye fluoresce in diacetate (FDA) for 5 min. Peels were washed three times with OB and then mounted on a coverslip, and analyzed by epifluorescence microscopy. Fluorescent cells were quantified, and the total cell/fluorescent cell percentage was calculated for each cell type. PC: pavement cells, GC: guard cells. B. Representative image of an epidermal peel stained with FDA taken with a 40 × objective. Scale bar: 10 μm. C- RT-PCR analysis using guard cell–specific gene ECIREFERUM2 (CER2) (930 bp) and mesophyll cell–specific gene carbonic anhydrase 1 (Canh1) (1,000 bp) was performed in RNA prepared from guard cell–enriched (GC-e) or mesophyll cell–enriched (MC-e) extracts. The amplification of ACTIN (651 bp) transcripts under identical conditions was used as a constitutive expression control. Data from Scuffi et al. [26].
Materials and reagents
Biological materials
4–6 weeks-old wild-type Arabidopsis thaliana (ecotype Col-0) and transgenic Arabidopsis thaliana lines expressing the following biosensors: roGFP2-Orp1 [28], Grx1-roGFP2 [29], and ATeam1.03-nD/nA [30]. See Table 1 to find detailed information regarding the fluorescent biosensor features.
Table 1. Biosensor characteristics
Biosensor name roGFP2-Orp1 Grx1-roGFP2 ATeam 1.03-nD/nA
Parameter detected H2O2 EGSH MgATP2-
Fluorescent protein EGFP EGFP mse CFP, cp173-mVenus
Promoter 35S (CaMV) UBQ10 (from Arabidopsis) 35S (CaMV)
Plasmid pH2GW7 pBinCM pH2GW7
Selection in plants Hygromycin Kanamycin Hygromycin
Rationing principle Dual excitation, Single emission Dual excitation, Single emission Single excitation, Dual emission (FRET)
Emission maximum 511 nm 511 nm 475 nm (mseCFP), 527 nm (cp173-mVenus)
Microscopy type Epifluorescence CLSM Epifluorescence CLSM Epifluorescence CLSM
Excitation wavelength/filter 450–490 nm, 385–425 nm 405 nm, 488 nm 450–490 nm, 385–425 nm 405 nm, 488 nm 426–446 nm 458 nm
Emission range 505–530nm 505–530 nm(*) 505–530 nm 505–530 nm(*) 467–499 nm, 528.5–555.5 nm(**) 465–500 nm, 526–561 nm(*)
References (15) (14), (22) (15) (11): (22) (15) (3)
(*) A 650–695 nm filter can be used for chlorophyll fluorescence collection.
(**) Use a dichroic 510–440 nm mirror for simultaneous acquisitions.
CSLM: Confocal scanning laser microscopy.
Reagents
Soil (Antoniucci, https://viveroantoniucci.mitiendanube.com/productos/tierra-negra/)
Vermiculite (Terrafertil, https://www.terrafertil.com/productos_jardin/acondicionadores_vermiculita.html)
Perlite (Terrafertil, https://www.terrafertil.com/productos_jardin/acondicionadores_vermiculita.html)
2-(N-Morpholino) ethane sulfonic acid (MES) (Sigma, CAS: 4432-31-9)
Potassium chloride (KCl) (Sigma, CAS: 7447-40-7)
Potassium hydroxide (KOH) (Sigma, CAS: 1310-58-3)
Trizol (Invitrogen, catalognumber: 15596026)
Solutions
Opening buffer (see Recipes)
Recipes
Opening buffer
Reagent stock Final concentration Amount
MES (50 mM, pH 6.1)* 5 mM 10 mL
KCl (1M) 50 mM 5 mL
H2O n/a 85 mL
Total n/a 100 mL
(*) MES is adjusted to pH with KOH
Laboratory supplies
Precision tweezers, Dumont #5 (Fine Science Tool, catalog number: 11251-10)
Scalpel n° 4 (KLS Martin, catalog number: 10-100-04)
1 mL syringe with needle with the tip bent, n° 27 g (Bremen Seiseme, catalog number: 7791914003029)
Pipette tips [Deltalab, catalog number: 327-34 (2-200 μL), 200070 (1,000 μL)]
Pipettes [Gilson, Pipetman, catalog number: F144056M (P20), F144058M (P200), F144059M (P1000)]
24-well plate (BIOFIL, catalog number: TCP001024)
Petri dish 55 × 14 mm (Deltalab, catalog number: 200201)
1.5 mL tubes (Deltalab, catalog number: 200400)
Microscope slides (Deltalab, catalog number: D100010)
Coverslips 18 × 18 mm nº 1 (Deltalab, catalog number: D101818)
Neubauer chamber (BOECO Germany, catalog number: BOE01)
Mortar (Günther Argentina, catalog number: 14660) and pestle (Günther Argentina, catalog number: 154200)
Liquid nitrogen
Equipment
Brightfield microscope (Olympus, model: CKX53) coupled to a digital camera (AmScope, model: MU1000) and 40 × objective (LUC Plan FLN, numerical aperture: 0.6)
Confocal laser scanning microscope (CLSM) (Carl Zeiss Microscopy, model: LSM980) with a 40 × water immersion objective (C-Apochromat, 1.2 numerical aperture)
Epifluorescence microscope (Nikon, model: Ti-E) coupled to a double digital camera (Hamamatsu, model: ORCA-D2 Dual CCD) and 60 × oil immersion objective (CFI Plan APO Lambda, 1.4 numerical aperture)
Software and datasets
NIS-Elements AR (https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research)
Fiji analysis software (ImageJ), National Institutes of Health, Version 1.53t [31]
Procedure
Stratificate Arabidopsis thaliana seeds in water at 4 °C and in darkness for 48 h. Sow the seeds in a proportion of soil:vermiculite:perlite (3:1:1, v/v/v) and grow them for 4–5 weeks (for stomatal aperture and biosensor experiments) or 5–6 weeks (for RNA extraction) under short-day conditions (8/16 h light/dark photoperiod, 200 µmol photons m-2s-1).
Stomatal aperture
Before removing the epidermal peels, prepare the 24-well plate by adding 500 μL of opening buffer (OB) in as many wells as treatments to be performed, including the control. Then, pipette 50 μL of OB drops onto a clean flat surface, previously washed with deionized water (surface sterilization is not needed) (Figure 2A). Excise epidermal peels from the abaxial side of fully expanded leaves of Arabidopsis thaliana plants. Place the leaf on the index finger and hold it with the thumb and the middle finger. Detach the epidermis layer piercing the leaf with tweezers and slowly pull upwards at a 45° angle (Figure 2B).
Immediately after, place the peels outside up on the OB drops pipette in step A1 (Figure 2C) and remove the mesophyll tissue with a scalpel (Figure 2D).
Note: An epidermal layer of approximately 4 mm2 is enough to image stomata in different microscope fields.
Lift the epidermal peels with a 1 mL syringe with a needle bent (at an approximate 120° angle) (Figure 2E) and put them in each well of the plate prepared in step A1 (Figure 2F). Put about four peels per well to have enough tissue for documentation. Maintain the epidermal peels in OB under white light for 3 h to promote the maximal opening of most of the stomata.
Pipette the respective treatments into each well from a 100 × stock solution and incubate the peels for the desired time. Then, take the peels from each well and mount them in a microscope slide with an OB drop. Observe stomata in an optical brightfield microscope with a 40 × objective (LUC Plan FLN, numerical aperture: 0.6) (Figure 2G).
Take a photo of the 50 um2 squares from a Neubauer chamber, or a calibrated slide, in each experiment to have a reference scale.
Figure 2. Epidermal peeling protocol. A. Opening buffer (OB) drops. B. Peeling with tweezers. C. Epidermal peel on an OB drop. D. Mesophyll dissection. E. Lifting peels with the needle. F. Incubation in a 24-well plate. G. Brightfield microscopic image of stomata, taken with a 40× objective, showing the stomatal aperture values measured as the maximal distance between the inner walls of guard cells. H. Fluorescence image of stomata expressing roGFP2-Orp1, taken with a 40× objective, where the nucleus, inner cell walls, and a portion of the cytosol from guard cells are indicated by arrows. Scale bars: 10 μm.
Biosensors
Excise epidermal peels with tweezers from the abaxial side of Arabidopsis thaliana leaves expressing the genetically encoded proteins roGFP2-Orp1, Grx1-roGFP2, or ATeam 1.03-nD/nA (as described in steps A1–A3).
Place epidermal peels in a 24-well plate containing OB under white light in the growing chamber for at least 7 h and up to a maximum of 12 h [27].
Add desired treatments to epidermal peels directly in the incubation well.
Note: For the redox-sensitive roGFP2-Orp1 and Grx1-roGFP2 sensors, include 10 mM H2O2 and 20 mM DTT treatments for 10 min to fully oxidize or reduce the sensors [22,27]. On the other hand, to avoid the effects of active photosynthesis on the ATP levels detected, ATeam 1.03-nD/nA peels should be pre-incubated for at least 1 h in the dark, before treatment addition [30,32].
Image peels in a CLSM or an epifluorescence microscope with 40× (C-Apochromat, 1.2 numerical aperture) or 60× (CFI Plan APO Lambda, 1.4 numerical aperture) objectives, respectively, and take photos. For roGFP2-Orp1 and Grx1-roGFP2 imaging using a CLSM, excite samples sequentially at 488 nm and 408 nm (line-switching mode) and collect fluorescence at 505–530 nm. For epifluorescence microscopy, excite roGFP2-Orp1 and Grx1-roGFP2 sequentially with 450–490 nm and 385–425 nm and collect the emissions using a 505–530 nm band pass filter (GFP-specific filter). For ATeam 1.03-nD/nA imaging with CLSM, excite samples at 458 nm and collect the fluorescence at 465–500 nm (mseCFP) and 526–561 nm (cp173-mVenus). For epifluorescence microscopy, excite ATeam 1.03-nD/nA using a 426–446 nm filter and collect fluorescence with 467–499 nm (mseCFP) and 528.5–555.5 nm (cp173-mVenus) filter with a dichroic 510 nm mirror (Hamamatsu Photonics) for simultaneous acquisitions.
Analyze fluorescence intensity in the cytosol of guard cells. Define the region of interest (ROI) using Fiji software, as described in Data Analysis section, avoiding the cytosolic portion adjacent to the inner cell walls, since these structures usually emit significant autofluorescence (Figure 2H).
RNA
For guard cell–enriched (GC-e) RNA extraction, collect epidermal peels from the abaxial side of fully expanded Arabidopsis leaves (as described in step A3) and place them in an OB-containing Petri dish until the surface of the dish is fully covered with peels. Incubate the peels for 3 h in white light and then perform the desired treatments.
Note: Use twelve 5–6-week–old Arabidopsis plants for each treatment to collect enough epidermal peels to completely cover the surface of a Petri dish with 15 mL of OB (diameter: 9 cm, surface: 56,7 cm2).
For mesophyll cell–enriched (MC-e) RNA extraction, incubate the rest of the leaf, after detaching the abaxial epidermal peel, in an OB-containing Petri dish (as described in step C1). The leaf surface without epidermis must be in direct contact with OB.
Note: Obtain these MC-e samples just if you are interested in comparing gene expression in different cell types.
Remove the buffer with a pipette and harvest the peels with a needle until obtaining a “peel pellet” and dry it with a blotting paper to remove the remaining buffer.
Following the manufacturer instructions, grind the tissue samples (CG-e and MC-e) in a mortar with liquid nitrogen and extract RNA with the TriZol method. For qRT-PCR, synthesize cDNA from 1 ug of total RNA. Represent data in a graph (Figure 3).
Note: AtACT2 (At3g08510) gene is recommended as a housekeeping gene for GC-e and MC-e samples.
Figure 3. Analysis of the expression levels of DES1 gene in guard cell–enriched (GC-e) and mesophyll cell–enriched (MC-e) RNA extracts of wild-type plants in response to abscisic acid (ABA). Epidermal peels (GC-e) and mesophyll tissue (MC-e) prepared from Arabidopsis thaliana wild-type ecotype Col-0 were pre-incubated for 3 h in opening buffer (OB, 5 mM MES, pH 6.1, 50 mM KCl) and then treated with or without 50 μM of ABA under light. After 90 min of treatment, qRT-PCR analysis of DES1 gene expression was performed. The values are expressed as relative units (RU) to the control treatment. Data from Scuffi et al. [26].
Data analysis
Stomatal aperture
Open the file from the Neubauer chamber in Fiji (ImageJ) software to set the scale. Using the Straight Line tool, measure the length of the squares (50 μm) by clicking on Analyze > Measure, or with the M key. Repeat at least ten times to reduce the error. Copy this data into a spreadsheet and average all the measurements. Set the scale to this value by clicking on Analyze > Set Scale. Complete the field Distance in pixels with the averaged pixel number and fill the Known distance with the grid known size of the Neubauer chamber. Click the option Global to set the same scale for all images.
Once the scale is set, open the image files of the epidermal peels. Measure the width of the stomatal pore by drawing a straight line between the inner wall so the guard cells at the middle of the stomatal pore (Figure 2G and H). Press M after the line is drawn.
Copy the values obtained in the Results window into a spreadsheet and represent data in a plot (Figure 4).
Figure 4. Stomatal closure response to abscisic acid (ABA). Epidermal peels were excised from the abaxial side of leaves from 5-week-old Arabidopsis thaliana wild-type ecotype Col-0 plants. Strips were subsequently floated in opening buffer (OB, 5 mM MES, pH 6.1, 50 mM KCl) for 3 h under light. Then, they were treated (ABA) or not (Control) for 90 min with 20 μM of abscisic acid (ABA). Stomatal aperture values are expressed as absolute values in micrometers and are represented as points in the boxplots, where the boxes are bound by the 25th to 75th percentile. The line in the middle is the median, the darker point is the mean, and the whiskers span from 10th to 90th percentile. The asterisk indicates statistical differences between treatments (t-test, p < 0.05).
Biosensors
Open files in Fiji (ImageJ) software. Both channels will appear.
Open the menu Analyze > Set Measurements and select Mean grey value.
Transform each channel to a 32-bit image.
Open Region of Interest (ROI) manager: Analyze > Tools > ROI Manager.
Select the Freehand Line, delimit the ROI to the cytosol of one guard cell avoiding the cytosol portion adjacent to the inner cell walls (Figure 2H), and press Add in the ROI Manager. Using a circle, draw a ROI in a region without stomata to obtain the background fluorescence value that is then going to be subtracted from guard cells’ fluorescence.
Activate the window of one of both channels and press Measure in the ROI Manager to obtain the mean grey value as an expression of fluorescence intensity from all delimited regions. Repeat the procedure for the other channel.
Copy the values obtained in the Results window into a spreadsheet.
Subtract background values to guard cells’ cytosolic fluorescence for each channel analyzed.
Calculate the 408/488 nm or the Venus/CFP ratio dividing the guard cell fluorescence intensity from each channel and represent results in a plot (Figure 5).
Figure 5. Cytosolic roGFP2-Orp1 oxidation in guard cells. Epidermal peels were prepared from Arabidopsis thaliana wild-type ecotype Col-0 leaves expressing the cytosolic biosensor roGFP2-Orp1, which detects H2O2. Peels were incubated in opening buffer (OB, 5 mM MES, pH 6.1, 50 mM KCl) for 7 h under light and then treated with 20 mM DTT or 1 mM H2O2 for 10 min, to fully reduce or oxidize the sensor, respectively, or with 20 µM of abscisic acid (ABA) for 10 min. Guard cells’ images were obtained using an epifluorescence microscope. The ratio 405/488 nm was calculated for each guard cell, normalized to the control, and represented as individual points. Box plots are bound by the 25th to 75th percentile. The line in the middle is the median, the darker point is the mean, and the whiskers span from 10th to 90th percentile. Black points represent the outliers. Red and blue dotted lines and bands indicate the mean and the 25th to 75th percentile of maximum and minimum ratio values obtained or full oxidation (1 mM H2O2) and full reduction (20 mM DTT) of the sensor. Data was taken from at least three independent experiments for each treatment. The asterisk indicates statistical differences between treatments (t-test, p < 0.05). Ratiometric images are false-colored and represent the mean of different treatments. Scale bar: 10 μm.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article (s):
Stomatal aperture
García-Mata, C. and Lamattina, L. (2010). Hydrogen sulphide, a novel gasotransmitter involved in guard cell signalling. New Phytol.
Distéfano, A. M. et al. (2012). Phospholipase D δ is involved in nitric oxide-induced stomatal closure. Planta.
Scuffi, D. et al. (2014). Hydrogen Sulfide Generated by l-Cysteine Desulfhydrase Acts Upstream of Nitric Oxide to Modulate Abscisic Acid-Dependent Stomatal Closure. Plant Physiol.
Scuffi, D. et al. (2018). Hydrogen Sulfide Increases Production of NADPH Oxidase-Dependent Hydrogen Peroxide and Phospholipase D-Derived Phosphatidic Acid in Guard Cell Signaling. Plant Physiol.
Pantaleno, R. et al. (2023). Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity. Plant Physiol.
Biosensors
Scuffi, D. et al. (2018). Hydrogen Sulfide Increases Production of NADPH Oxidase-Dependent Hydrogen Peroxide and Phospholipase D- Derived Phosphatidic Acid in Guard Cell Signaling. Plant Physiol.
Pantaleno, R. et al. (2023). Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity. Plant Physiol.
GC-eRNA
Distéfano, A. M. et al. (2012). Phospholipase D δ is involved in nitric oxide-induced stomatal closure. Planta.
Scuffi, D. et al. (2014). Hydrogen Sulfide Generated by l-Cysteine Desulfhydrase Acts Upstream of Nitric Oxide to Modulate Abscisic Acid-Dependent Stomatal Closure. Plant Physiol.
General notes and troubleshooting
General notes
Double-blind testing for both image acquisition and analysis is highly recommended.
Use leaves with a similar stage of development to ensure that the stomata responses are comparable among them. For example, leaves 8–13 of Arabidopsis are usually of similar size and stage.
Take approximately three photos per peel and make sure to capture different sample fields.
Use at least four epidermal peels from different leaves per condition.
Keep the same objective and camera settings to take all the photos within an experiment.
Perform at least three or four independent assays for each type of experiment.
Competing interests
The authors declare no competing interests.
References
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Daloso, D. M., Antunes, W. C., Pinheiro, D. P., Waquim, J. P., AraÚJo, W. L., Loureiro, M. E., Fernie, A. R. and Williams, T. C. R. (2015). Tobacco guard cells fix CO2 by both Rubisco and PEP case while sucrose acts as a substrate during light-induced stomatal opening: Isotope labeling kinetics in guard cells. Plant Cell Environ. 38 (11): 2353–2371. https://doi.org/10.1111/pce.12555
Kwak, J. M. (2003). NADPHoxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J. 22 (11): 2623–2633. https://doi.org/10.1093/emboj/cdg277
Su, J., Zhang, M., Zhang, L., Sun, T., Liu, Y., Lukowitz, W., Xu, J. and Zhang, S. (2017). Regulation of Stomatal Immunity by Interdependent Functions of a Pathogen-Responsive MPK3/MPK6 Cascade and Abscisic Acid. Plant Cell. 29 (3): 526–542. https://doi.org/10.1105/tpc.16.00577
Thor, K. and Peiter, E. (2014). Cytosolic calcium signals elicited by the pathogen‐associated molecular pattern flg22 in stomatal guard cells are of an oscillatory nature. New Phytol. 204 (4): 873–881. https://doi.org/10.1111/nph.13064
Ye, W., Munemasa, S., Shinya, T., Wu, W., Ma, T., Lu, J., Kinoshita, T., Kaku, H., Shibuya, N. and Murata, Y. (2020). Stomatal immunity against fungal invasion comprises not only chitin-induced stomatal closure but also chitosan-induced guard cell death. Proc Natl Acad Sci U.S.A. 117 (34): 20932–20942. https://doi.org/10.1073/pnas.1922319117
Zhang, J., Zhou, M., Ge, Z., Shen, J., Zhou, C., Gotor, C., Romero, L. C., Duan, X., Liu, X., Wu, D., et al. (2020). Abscisic acid‐triggered guard cell L‐cysteine desulfhydrase function and in situ hydrogen sulfide production contributes to hemeoxygenase‐modulated stomatal closure. Plant Cell Environ. 43 (3): 624–636. https://doi.org/10.1111/pce.13685
Garcı́a-Mata, C. and Lamattina, L. (2001). Nitric Oxide Induces Stomatal Closure and Enhances the Adaptive Plant Responses against Drought Stress. Plant Physiol. 126 (3): 1196–1204. https://doi.org/10.1104/pp.126.3.1196
Flütsch, S., Wang, Y., Takemiya, A., Vialet-Chabrand, S. R. M., Klejchová, M., Nigro, A., Hills, A., Lawson, T., Blatt, M. R. and Santelia, D. (2020). Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis. Plant Cell. 32 (7): 2325–2344. https://doi.org/10.1105/tpc.18.00802
Rodrigues, O., Reshetnyak, G., Grondin, A., Saijo, Y., Leonhardt, N., Maurel, C. and Verdoucq, L. (2017). Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA- and pathogen-triggered stomatal closure. Proc Natl Acad Sci U.S.A. 114 (34): 9200–9205. https://doi.org/10.1073/pnas.1704754114
Distéfano, A. M., Scuffi, D., García-Mata, C., Lamattina, L. and Laxalt, A.M. (2012). Phospholipase D δ is involved in nitric oxide-induced stomatal closure. Planta. 236 (6): 1899–1907. https://doi.org/10.1007/s00425-012-1745-4
García‐Mata, C. and Lamattina, L. (2010). Hydrogen sulphide, a novel gasotransmitter involved in guard cell signalling. New Phytologist. 188 (4): 977–984. https://doi.org/10.1111/j.1469-8137.2010.03465.x
Pantaleno, R., Scuffi, D., Costa, A., Welchen, E., Torregrossa, R., Whiteman, M. and García-Mata, C. (2023). Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity. Plant Physiology. 191 (3): 2001–2011. https://doi.org/10.1093/plphys/kiac591
Scuffi, D., Álvarez, C., Laspina, N., Gotor, C., Lamattina, L. and García-Mata, C. (2014). Hydrogen Sulfide Generated by l-Cysteine Desulfhydrase Acts Upstream of Nitric Oxide to Modulate Abscisic Acid-Dependent Stomatal Closure. Plant Physiology. 166 (4): 2065–2076. https://doi.org/10.1104/pp.114.245373
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Article Information
Publication history
Received: Mar 4, 2024
Accepted: Jun 16, 2024
Available online: Jul 3, 2024
Published: Jul 20, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell signaling > Second messenger
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Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy
ST Stephan Tetenborg
EM Elizabeth Martinez-Soler
JO John O`Brien
Published: Vol 14, Iss 14, Jul 20, 2024
DOI: 10.21769/BioProtoc.5034 Views: 342
Reviewed by: Philipp Wörsdörfer Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Biological Chemistry Nov 2023
Abstract
Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein–protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed “whorls” because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.
Keywords: Connexin 36 ER retention trafficking HEK293T cell PDZ
Background
Gap junctions are clusters of intercellular channels that directly connect the cytoplasm of adjacent cells, providing a conduit for the exchange of metabolites and ions. In chordates, gap junctions are formed by connexins, a diverse family of membrane proteins that have the ability to oligomerize into dodecameric channels. Mutations in connexin genes are the cause of a variety of inherited diseases. In many cases, these pathologies have been linked to trafficking defects that compromise the ability of the channel to form functional gap junctions [1,2]. Among the 21 connexins that have been identified in humans, only a few variants have been studied in terms of trafficking through intracellular compartments [2–4]. To study basic transport mechanisms of gap junction proteins, researchers take advantage of expression systems, such as HeLa or HEK293T cells, and transfect these cells with recombinant expression vectors. In this article, we describe a detailed protocol that can be used to determine endoplasmic reticulum (ER) retention defects for connexin 36 (Cx36). In a previous study, we described a transport defect that prevented the functional ER export of Cx36, causing the connexin to accumulate in the ER [5]. This retention mechanism promoted the formation of gap junction-like aggregates that reshaped the ER into concentric multi-membrane vesicles (Figures 1B and D), which we termed connexin whorls. These structures were characterized by several distinct features: 1) Each sheet within the whorl exhibited ultrastructural features that were indistinguishable from an actual gap junction; 2) whorls are hollow inside and their diameter varied, ranging from 0.3 to 3 µm; 3) whorls colocalized with ER-phagy receptors Tex264 (Testis-expressed protein 264) (Figure 1C) and p62; 4) whorl formation requires docking interactions of extracellular loops in Cx36 facing the ER lumen. Substituting the extracellular loop cysteines (C55 or C62) via site-directed mutagenesis prevents whorl formation. Similar whorl-like structures were reported for the lens connexin Cx50 in a previous study by Lichtenstein et al., [6], which suggests that ER-derived whorls are formed by many connexins that oligomerize in ER. Therefore, the protocol we describe here is not only applicable for Cx36 but might be used for other connexin isoforms. However, two important aspects have to be considered: 1) Some connexins, for instance Cx43, oligomerize in the Golgi [7], which would make the docking of Cx43 containing connexons in the ER impossible; and 2) whorls have to be tested for the presence of ER proteins, for instance Tex264, to determine the compartment they originated in (Figure 1C).
Figure 1. Membrane topology of Cx36. Protein structure of Cx36 is illustrated. The two extracellular loops of Cx36 (indicated by upper circle and red arrow) contain three cysteines. The C-terminal tail contains the PDZ binding motif consisting of the following amino acid sequence: SAYV.
Rationale for experimental design
The rationale for the design of connexin mutants we have described in Tetenborg et al., (2023) was based on the observation that truncation of the C-terminal tip in Cx36 prevents functional ER export leading to the intracellular formation of connexin whorls. To test if these multimembrane vesicles are formed by a gap junction-like docking mechanism of opposing hemichannels in the ER, we substituted cysteines in the extracellular loop of Cx36 with serines via site-directed mutagenesis. These mutations were previously shown to block gap junction formation [8] and served as a control experiment in our study to confirm that ER retention causes opposing Cx36 channels to dock intralumenally via the extracellular loops.
Materials and reagents
Biological materials
Anti Cx35/Cx36 antibody (depends on the connexin that is studied); our study focused on Cx36 (Sigma-Aldrich, catalog number: MAB3045)
Anti Testis expressed protein 264 (Tex264) (Novus Biologicals, catalog number: NBP1-89886)
Anti Golgin97 (Thermo Fisher Scientific, catalog number: PA5-30048)
Human embryonic kidney 293T cells T/17 (ATCC, catalog number: CRL-11268)
Connexin 36 expression vector (or any other connexin), map shown in Figure 1
Normal donkey serum [9] (Jackson Immunoresearch, catalog number: 017-000-121)
Donkey anti-mouse Cy3 (Jackson Immunoresearch, catalog number:715-165-160)
Donkey anti-rabbit Alexa Fluor 488 (Jackson Immunoresearch, catalog number:715-545-152)
Reagents
Dulbecco’s modified Eagle’s serum (DMEM) (Thermofisher, catalog number: 12800017)
Fetal bovine serum (FBS) (Thermofisher, catalog number: A5209401)
2.5% Trypsin 10× (Thermofisher, catalog number: 15090-046)
Geneporter 2 (Amsbio, catalog number: T20200)
Paraformaldehyde (PFA) 20% (Electron Microscopy Sciences, catalog number: 15712)
Poly-l-lysine (PLL) 0.01% (Millipore, catalog number: A-005-C)
Triton-X 100 (Fisher Scientific, catalog number: BP151-100)
50 mL Falcon tube (Falcon, catalog number: 352070)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P3813)
Vectashield PLUS mounting media, DAPI (Vector Laboratories, catalog number: H-2000)
Tissue Path SuperfrostTM Plus gold (Thermofisher, catalog number: 1518846)
Normal donkey serum (NDS) (Jackson Immunoresearch, catalog number: 017-000-121)
Laboratory supplies
75 cm2 cell culture flask (Corning, catalog number: 430725U)
35 mm × 10 mm style cell culture dish (Corning, catalog number: 430165)
Coverslips (Fisher Scientific, catalog number: 12541000)
24-well plate (Corning Incorporated, catalog number: 3526)
AxygenTM Maxyclear Snaplock microtubes, 1.5 mL (Fisher Scientific, catalog number: 14-222-158)
Nail polish, L.A colors, rapid dry (Electron Microscopy Sciences, catalog number: 72180)
Software and datasets
Fiji, open source [10]
Equipment
Biological safety cabinet, 1300 Series A2 (Thermofisher, catalog number: 72180)
Incubator (Eppendorf, model: Galaxy 170 S)
Eppendorf research plus pipette (Eppendorf, catalog number: 3123000012)
Confocal laser scanning microscope (Zeiss, model: LSM800)
Procedure
Day 1: Seed HEK293T cells
Culture HEK293T cells in DMEM supplemented with 10% FBS and 1% penicillin and 1% streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Grow cells to confluence prior to the experiments.
Transfer 2–3 coverslips to 35 mm dishes and incubate in 0.01% PLL for 30 min at RT.
Remove PLL and briefly wash the coverslips in sterile water.
Prepare 10 mL of a 0.25% Trypsin solution in serum-free DMEM (9 mL of DMEM + 1 mL of 2.5% trypsin).
Remove the media from the cell culture flask and briefly wash the cells with serum-free DMEM.
Apply 10 mL of the trypsin solution and incubate the cells at 37 °C for 10 min in the incubator.
Transfer the dissociated cells to a 50 mL Falcon tube and centrifuge at 1,500× g for 10 min at room temperature.
Remove the supernatant and resuspend the cells in 5–10 mL of serum-free DMEM.
Determine the number of cells per milliliter using a hemocytometer.
Apply 450,000 cells to coated coverslips. Apply 2 mL of DMEM containing 10% FBS and incubate overnight.
Transfect HEK293T with the Cx36 expression vector
Twenty-four hours after seeding, remove the media and briefly wash the cells with serum-free DMEM. Apply 900 µL of serum-free DMEM and place the cells back in the incubator.
Prepare the transfection reaction consisting of mix A and mix B.
Mix A: Combine 5 μL of Geneporter 2 with 43 μL of serum-free DMEM.
Mix B: Combine 1 μg of the Cx36 expression vector (Figure 2) with 50 μL of DNA diluent (Diluent A or B).
Figure 2. Connexin whorls. A. Transiently transfected HEK293T cells expressing Cx36. Cx36 is concentrated at gap junctions, indicated by the long arrow. Cx36 is also visible in perinuclear structures resembling the Golgi apparatus. Indicated by the short arrow. Scale: 5 μm. B. Transfected HEK293T cell expressing the trafficking deficient Cx36/S318ter mutant. Cx36 is retained in the ER and accumulates in ER whorls, which are labeled with short arrows. Gap junctions are still formed by the Cx36/S318ter mutant. Labeled with the long arrow. Scale: 5 μm. C. Example of Cx36 whorls colocalizing with the ER phagy receptor Tex264. Scale: 5 μm. Magnified inset: 2.5 μm. D. Cartoon illustrating the composition of ER whorls. E. Whorls are seen for different connexins. Confocal scan of Halo-tagged zebrafish Cx34.7, an orthologue of Cx36. Scale: 10 μm.
Combine Mix A and Mix B and incubate for 15 min at RT.
Apply the transfection mix to HEK293T cells and incubate for 2 h.
Two hours after step B4, apply 1 mL of 20% FBS DMEM and incubate the cells overnight.
Prepare cells for confocal microscopy
Nineteen to twenty-four hours after transfection, transfer the coverslips to a 24-well plate and briefly wash in PBS.
Fix the cells in 2% PFA in PBS for 15 min at RT.
Wash the cells 3 × 10 min with PBS at RT.
Dilute the Cx36 antibody 1:500 in PBS containing 0.5% Triton-X 100 and 10% NDS.
Additionally, the Cx36 antibody can be combined with a Tex264 (1:200) and a Golgin97 antibody (1:100) for double-labeling experiments.
Apply the antibody solution to the well containing the coverslip and incubate overnight.
The next day, wash the coverslips 3 × 10 min with PBS.
Dilute the secondary Cy3-conjugated antibody in PBS containing 0.5% Triton-X and 10% NDS. When Tex264 and Golgin97 are double labeled with Cx36, a secondary conjugated with Alexa488 is needed.
Incubate for 1 h under light-protected conditions at RT.
Wash the coverslips 3 × 10 min with PBS at RT.
Apply 5 μL of mounting media containing DAPI to an object slide. Mount the coverslips with the cell side facing the mounting media.
Seal the coverslips with nail polish.
Confocal microscopy and image analysis
Image Cx36-transfected cells containing whorls using the Zeiss LSM800 confocal laser scanning microscope (or any other model with sufficient resolution) and 63× oil objective. Choose the correct laser settings to detect Cy3 (labeling Cx36) and DAPI. Use 2–3× zoom and a pixel size of ~50 nm × 50 nm. Scan a sufficient number of cells for each experimental condition.
Images can be acquired as stacks of 1 with a spacing of 0.2 µm.
Open your confocal scans in ImageJ (Fiji) and select individual Cx36 whorls using the rectangular selection tool (Figure 4).
Figure 3. Sketch illustrating the experimental workflow
Duplicate the region of interest (Click shift + D).
Draw a straight line using the line tool and use the measure function to determine the diameter of the whorl.
Determine the number of whorls per cell
Based on the previous measurements, determine a size threshold to exclude smaller vesicles from quantification. As described in Tetenborg et al., [5], vesicles with a diameter smaller than 0.3 μm were not considered whorls.
Open the cell counter plugin (Figure 5) and click initialize.
Select a type of counter and count the whorls in your stack via left clicks.
Figure 4. Measurement of whorl diameter using ImageJ. A. 1) A region of interest (ROI) surrounding the whorl can be selected using the rectangular selection tool. The ROI can be duplicated (shift + D). 2) To measure the diameter of a selected whorl, draw a line using the line selection tool and 3) use the measure function (Ctrl + M). B. The length will be displayed in the last column.
Figure 5. Quantification of ER whorls using the cell counter function in ImageJ. Confocal scans can be opened in ImageJ. Before the quantification, several scans of the experiment should be inspected to determine a size threshold for whorls (see Figure 3). Every vesicle with a diameter under the size threshold is excluded from quantification. The cell counter plugin has to be initialized in order to select vesicles that are counted. Each vesicle that is considered a whorl based on shape and diameter can be selected by a mouse click.
Validation of protocol
This protocol or parts of it has been used and validated in the following research article(s):
Tetenborg, S. et al. (2023). Intralumenal docking of connexin 36 channels in the ER isolates mistrafficked protein. J Biol Chem. (Figure 1, panel C-E, Quantification of 11-30 vesicles, from 9-11 cell clusters)
Acknowledgments
We would like to thank Ya-Ping Lin and Nikki Brantley for the excellent technical assistance. This project was supported by NIH grant R01EY012857 (J.O.). S.T. was supported by the Deutsche Forschungsgemeinschaft (DFG) (TE 1459/1-1, Walter Benjamin stipend).
Data availability
The expression constructs we have described in this article will be made available on Addgene. Until then, vectors are provided upon request by Dr. Tetenborg and Dr. O’Brien: [email protected] and [email protected]
Competing interests
The authors declare no competing interests.
References
Laird, D. W. (2006). Life cycle of connexins in health and disease. Biochem J. 394(3): 527–543. https://doi.org/10.1042/bj20051922
Yum, S. W., Kleopa, K. A., Shumas, S. and Scherer, S. S. (2002). Diverse Trafficking Abnormalities of Connexin32 Mutants Causing CMTX. Neurobiol Dis. 11(1): 43–52. https://doi.org/10.1006/nbdi.2002.0545
Wang, H. Y., Lin, Y. P., Mitchell, C. K., Ram, S. and O'Brien, J. (2015). Two-color fluorescent analysis of connexin 36 turnover: relationship to functional plasticity. J Cell Sci. 128(21): 3888–3897. https://doi.org/10.1242/jcs.162586
Jordan, K., Solan, J. L., Dominguez, M., Sia, M., Hand, A., Lampe, P. D. and Laird, D. W. (1999). Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells. Mol Biol Cell. 10(6): 2033–2050. https://doi.org/10.1091/mbc.10.6.2033
Tetenborg, S., Liss, V., Breitsprecher, L., Timonina, K., Kotova, A., Acevedo Harnecker, A. J., Yuan, C., Shihabeddin, E., Ariakia, F., Qin, G., et al. (2023). Intralumenal docking of connexin 36 channels in the ER isolates mistrafficked protein. J Biol Chem. 299(11): 105282. https://doi.org/10.1016/j.jbc.2023.105282
Lichtenstein, A., Gaietta, G. M., Deerinck, T. J., Crum, J., Sosinsky, G. E., Beyer, E. C. and Berthoud, V. M. (2009). The cytoplasmic accumulations of the cataract-associated mutant, Connexin50P88S, are long-lived and form in the endoplasmic reticulum. Exp Eye Res. 88(3): 600–609. https://doi.org/10.1016/j.exer.2008.11.024
Koval, M. (2006). Pathways and control of connexin oligomerization. Trends Cell Biol. 16(3): 159–166. https://doi.org/10.1016/j.tcb.2006.01.006
Placantonakis, D., Cicirata, F. and Welsh, J. P. (2002). A dominant negative mutation of neuronal connexin 36 that blocks intercellular permeability. Mol Brain Res. 98: 15–28. https://doi.org/10.1016/s0169-328x(01)00306-0
Nufer, O., Guldbrandsen, S., Degen, M., Kappeler, F., Paccaud, J. P., Tani, K. and Hauri, H. P. (2002). Role of cytoplasmic C-terminal amino acids of membrane proteins in ER export. J Cell Sci. 115(3): 619–628. https://doi.org/10.1242/jcs.115.3.619
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): 676–682. https://doi.org/10.1038/nmeth.2019
Article Information
Publication history
Received: Feb 7, 2024
Accepted: Jun 11, 2024
Available online: Jul 2, 2024
Published: Jul 20, 2024
Copyright
© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
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Category
Cell Biology > Cell imaging > Fluorescence
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